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THE ROAD TO AN HIV VACCINE

tejas sudarshan sathe

advisor: adel a.f. mahmoud, m.d., ph.d.

a senior thesis submitted to the department of molecular biology, princeton university in partial fulfillment of the requirements for the degree of bachelor of arts

princeton, new jersey april 25, 2013


Tejas Sudarshan Sathe: The Road to an HIV Vaccine, A Senior Thesis Submitted to the Department of Molecular Biology, Princeton University in Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts, Š April 2013


DECLARATION This thesis represents my own work in accordance with University regulations.

tejas sudarshan sathe

I authorize Princeton University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purposes of scholarly research.

tejas sudarshan sathe

Princeton, New Jersey, April 2013


Dedicated to my loving family, and the many unsung heroes at benches and bedsides around the world working tirelessly to bring an end to the AIDS pandemic.


PUBLICATIONS Some ideas and figures have appeared previously in the following publications: Sathe, T. S. The Path to an HIV Vaccine: The Role of Broadly Neutralizing Antibodies in Discovering Putative Antigenic Sites on HIV-1. (2012). Sathe, T. S. The Hero We Need: An Epidemiological Model of HIV Vaccination. ENV304 Poster Presentation, Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ (2012).

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If I have seen further it is by standing on the shoulders of giants. — Isaac Newton

ACKNOWLEDGMENTS If I were to give due credit to everyone who got me through this endeavor, there would be little room for the thesis. I have had the great opportunity and privilege to work under scientists who are leaders in their fields, but still found time to help me begin my own journey into science. To my thesis advisor, Adel Mahmoud, thank you for your patience and guidance over the past two years, and for challenging and training me to think analytically about such a massive global challenge. To Dennis Burton, thank you for inviting me to your laboratory at Scripps. In my pursuit to understand the challenges of an HIV vaccine, I can think of no better place and no better mentor. To everyone in the Burton Lab, thank you for making my stay in San Diego both intellectually rewarding and exceptionally fun. I am very thankful to two graduate students, Devin Sok and Ruthie Birger, who took time out of their busy schedules to provide me with guidance and feedback. Devin, thanks for patiently guiding me through the tumultuous ride of laboratory research, and for teaching me the value of doing everything in triplicate. From our conversations, I learned as much about science as I did about life. Ruthie, thank you for helping me to develop the mathematical model from an idea for a class project into an integral part of this work. Many thanks go to my peers, who have not only made this thesis possible, but also enriched my Princeton experience beyond measure. I would especially like to acknowledge Santhosh Balasubramanian, Philip Oasis, and Kishan Shah for assistance with the analytical portion of this work. In addition, I would like to thank Rachita Jain, George Maliha, and Adam Kravietz for proofreading. Your willingness to engage with my words and ideas has certainly made this work better. I owe all of you a coffee for the hours of sleep you have lost on my behalf. I cannot thank enough the members of my family who put up with me throughout this process and helped me with every aspect of this work. To my cousin Anuraag Parikh ‘08, thank you for your (usually) constructive criticism. To my cousin Aditya Trivedi ‘16, thank you for sticking with me during the final push to the finish line. Finally, to my mother Swati, my father Sudarshan, and brother Ojas, thank you for following me on this journey, word by word, page by page. Mom, when you wrote your thesis, you thanked me for my “enduring patience and support". I’m not sure how much help I could have been as an infant, but twenty two years later, to call the support of my family “enduring" would be an understatement. Thank you so much.

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CONTENTS 1

2

introduction 1 1.1 The Plague of Our Time 1 1.2 Epidemiology of AIDS 3 1.3 An Unknown Syndrome & A New Virus 5 1.4 The Molecular Biology of HIV 7 1.5 Infection By Deception: Viral Entry 8 1.6 An Invisible Intruder: Provirus Formation 12 1.7 Out of One, Many: Transcription of HIV Genes 1.8 The Global Diversity of HIV 20 1.9 The Course of Infection 22 a tale of two strategies 25 2.1 The Antiretroviral Revolution 25 2.2 The Current Arsenal of Preventions 2.3 The Failure of Preliminary Vaccines

13

29 31

3

the 3.1 3.2 3.3 3.4

impact of an hiv vaccine: a mathematical model The Need for a Vaccine 35 Methodology 38 Results 49 Conclusions 54

4

the 4.1 4.2 4.3 4.4

v3 epitope: a template for vaccine design Introduction 59 Materials & Methods 66 Results 75 Conclusions 84

5

discussion

references

90 104

vii

59

35


LIST OF FIGURES Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10

Structure of the HIV Virion 9 HIV Binding, Fusion, and Entry 11 Expression of HIV genes 15 The HIV Life Cycle 21 Clinical Progression of HIV Infection 24 Clinical Progression of HIV Infection 40 A Compartmental Epidemiological Model for HIV Transmission 40 Model Simulation At Various Treatment Levels 50 Model Simulation With Hypothetical Vaccines 52 Model Simulation with Simultaneous Treatment and Vaccination 54 PGT128 Binding to gp120 is Mediated by N301 and N332 65 Pseudovirus Production 72 Neutralization Assay 74 Optimization of Broadly Neutralizing Antibodies 76 Antibody Score Plot 77 Single Mutant Neutralization Curves 80 Neutralization of N332A and WT Virus by PGT Antibodies 81 Double Mutant Neutralization Curves 82 N301A Inhibits Neutralization When Combined With N332A 83 Structural Analysis of N301A and N332A Mutation 87

LIST OF TABLES Table 3.1 Table 3.2 Table 3.3 Table 3.4 Table 3.5 Table 3.6 Table 3.7 Table 3.8 Table 3.9

Crude Birth and Death Rates 41 Multipliers For the Force of Infection 42 Multipliers for the Progression of Infection 43 Treatment Guidelines 45 Vaccine Efficacy and Duration 47 Initial Conditions 48 Treatment Reduces Incidence and Prevalence 49 Vaccines Reduce Incidence and Prevalence More Than Treatment 53 Vaccination and Treatment is More Effective than Aggressive Treatment 55

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Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table 4.7

Experimental Virus Panel 68 Single Mutations in Env 69 Double Mutations in Env 69 Nucleotide Changes for Site-Directed Mutagenesis 70 Ranking of Antibody Scores 78 Fold Difference in IC50 for N332A and WT Virus 81 Fold Difference in IC50 of N301 N332 Double Mutant 83

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ABSTRACT Acquired Immunodeficiency Syndrome (AIDS) and its etiological agent Human Immunodeficiency Virus (HIV) have caused a global pandemic for which an comprehensive elimination strategy remains elusive. Treatment, which does not clear HIV infection, but rather delays the progression to AIDS, remains economically and socially inaccessible in the places where the burden of disease is greatest, and to date, no vaccine has been developed. Thus, there is some question about how to end the AIDS pandemic. Here, we develop a compartmental epidemiological model for HIV and show that 1) a highly efficacious vaccine would be more effective in reducing incidence and prevalence of disease than aggressive treatment, and 2) a partially efficacious vaccine can also be highly effective if coverage is high. While previous attempts to develop a vaccine have failed, the discovery of broadly neutralizing antibodies in a number of infected individuals as well as the characterization of the viral epitopes these antibodies target has renewed hopes for a vaccine. Here, we demonstrate that variable loop 3 (V3) on the outer domain of the HIV surface protein presents a potentially attractive template for vaccine design. Furthermore, we present a conserved specificity for antibody mediated neutralization at the V3 epitope that is dependent on two glycans. The ability of broadly neutralizing antibodies to instruct the development of a HIV vaccine would validate the paradigm of rational vaccine design and fundamentally alter the way in which we approach vaccines to present and future diseases.

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FOREWORD Well into the thirty first year on the road towards an HIV vaccine, the fact remains that we don’t have one. The aim of this thesis is to answer three fundamental questions about the challenges and opportunities of developing such a vaccine. Why have we failed to develop an HIV vaccine? Do we still need an HIV vaccine? What novel approaches can we use to successfully develop a HIV vaccine? The first question is the subject of the first two chapters of this work. The second two questions are explored in the third and fourth experimental chapters, respectively. In Chapter 1, we review the epidemiology and molecular biology of HIV and AIDS. We show that HIV is one of the most complex viruses we have encountered by discussing its genomic organization, proteomic repertoire, pathogenesis, and infection cycle. In Chapter 2, we discuss current strategies for treatment and prevention of HIV. First, we review advances in antiretroviral therapy as well as limitations to its widespread use. Next, we delve into the failed clinical trials of HIV vaccine candidates, explaining the thought process behind these trials and the reasons they were unsuccessful. In Chapter 3, we develop a compartmental epidemiological model for HIV and use it to test various treatment strategies and hypothetical vaccines. The work in Chapter 3 stemmed from a poster presentation given at the culmination of the course Disease Ecology, Economics, and Policy co-taught by Professors Bryan T. Grenfell and Ramanan Laxminarayan. We show that a high efficacy vaccine could reduce incidence and prevalence of disease by 100% compared to an aggressive treatment scenario. Furthermore, we demonstrate that reductions in vaccine efficacy or duration of protection can be offset with high vaccine coverage and simultaneous pursuit of vaccination and treatment. In Chapter 4, we explore novel approaches to vaccine design. The work in this chapter was conducted at The Scripps Research Institute in La Jolla, CA under the guidance of Dr. Dennis Burton. The recent discovery that broad and potent antibodies to HIV develop in a number of infected individuals has suggested that particular components of the virus may be vulnerable to immune recognition and neutralization. We analyze the quality of the broadly neutralizing antibodies discovered thus far to determine which epitope produces the antibodies that could best protect against infection and be elicited de novo. From this analysis, we conclude that variable loop 3 (V3) in the outer domain of the HIV envelope

xi


surface protein is the best epitope to instruct immunogen design. Furthermore, we analyze neutralization by one V3-specific broadly neutralizing antibody and propose a conserved specificity for antibody neutralization at the V3 loop that is dependent on two glycan residues. While the previous three questions are specific to HIV, the answers to them have far broader applications. In the final chapter of this work, we contemplate the following: Can we reverse engineer a vaccine from an immunological response? In Chapter 5, we explore reverse vaccinology and hypothesize how an epitope identified from an immune response can be successfully turned into an immunogen that can recreate that immune response de novo. We also critically assess rational vaccine design, examining the assumptions it makes about antigenic drivers of vaccine-mediated immunity. We also question whether we can develop vaccines to diseases in which natural immunity does not provide a sufficient guide, such as HIV. HIV is the most studied virus in all of human history, yet the road to a vaccine still has to go through uncharted territory. Whether this effort, like many before it, is another dead end, or the final stretch towards ending the pandemic of AIDS remains to be seen. If successful, the effort will instruct vaccines for diseases now in our midst as well as those as yet unencountered.

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1

INTRODUCTION

1.1 the plague of our time For times immemorial, we have lived amongst and been shaped by pathogens. For most of our history, their world has been invisible to us. Only in recent times have we been able to catch a glimpse that barely scratches the surface, and the more we learn about their world, the more we are humbled by our place in it. Since long before we could see them or begin to comprehend them, we have felt their presence when their species caused waves of widespread destruction among our own. Today, we stand in the middle of one of these great tides, faced with a plague that targets the very mechanisms that evolution has built to destroy it. The Acquired Immunodeficiency Syndrome (AIDS) is the plague of our time. The history of our plagues is a story with many chapters. Both through the robust evolution of self-defense mechanisms and a penchant for innovation and discovery, we have concluded each chapter the victor. The chapter on AIDS remains to be written, and the nature of its conclusion will depend on how well we are able to understand the disease and fashion a mechanism that can conquer it. The word plague evokes images of ancient peoples wasting away in Medieval villages. Indeed, the pandemic of Yersinia pestis that gave plague its name brought colossal loss of life to much of the world between the years 1347 and 1353, killing an estimated one third of the population of Europe 1 . With time, “plague" was adopted into our common vernacular to signify any disease that was widespread and led to a large loss of life. The exploration of the New World by European explorers unleashed plagues of smallpox and measles unto vulnerable native populations. Smallpox killed every one in three people infected, and killed

1


1.1 the plague of our time

millions of individuals each year until its eventual eradication. Waves of Cholera epidemics were responsible for tens of millions of deaths in the nineteenth century. As the world reeled from the First World War, a deadly strain of pandemic influenza virus killed more people than the war itself. At the turn of the twentieth century, poliomyelitis, too, reached pandemic proportions. Improved understanding of the vectors and agents of disease, massive improvements in public health and hygiene, and the development of potent drugs and vaccines largely stemmed the tide of these diseases. Though ancient plagues have existed for centuries and even millennia, cases of AIDS have been documented for little over thirty years. In this short time span, it has led to a staggering loss of life and ravaged entire nations. Far from being a relic of history confined to an oft forgotten corner of the globe, its presence has been felt as severely in some American cities as countries in Sub-Saharan Africa. Besides its epidemiological connotation, the word plague evokes an emotional response–a sense of alarm and fear that arises when we are faced with a disease we do not completely understand and cannot effectively control. This is also true of AIDS, for it arrived when we least expected it. On the eve of the epidemic, the battle against infectious diseases was thought to be mostly won, at least in the developed world. In 1967, William Stewart, then Surgeon General of the United States, stated “The time has come to close the book on infectious diseases. We have basically wiped out infection in the United States" 2 . While this remark may have been apocryphal, the sentiment it expresses was widespread at the time, and without the benefit of hindsight, this view was patently defensible. Over the next decade, victories against infectious disease would only substantiate this notion. Following a massive and successful vaccination campaign, smallpox was declared eradicated in 1979 by the World Health Organization 3 . In the same year, the last naturally occurring cases of poliomyelitis in the United States had been diagnosed 4,5 . The discovery and production of antibiotics in the mid twentieth century abrogated the death sentence of once lethal bacterial infections. It was

2


1.2 epidemiology of aids

in this historical context, that we were first introduced to a disease that would baffle scientific intuition for years to come. 1.2

epidemiology of aids

AIDS has left an enormous global footprint of infection and mortality, and recent progress, while promising, is far from sufficient. In 2011, roughly 34 million people were living with HIV worldwide. The number of infected individuals has increased by 17% over the last decade due to increased access to treatment and the longevity it provides. Likewise, the number of new infections has fallen by 20% over the last decade and was 2.5 million in 2011. 1.7 million individuals died from AIDS related causes in 2011, a decrease of 24% from just five years ago. These trends are promising: fewer people are getting infected from AIDS and those who have it are living longer, healthier lives 6 . Yet to declare victory would be premature. While the trend is in the right direction, the magnitude of this pandemic remains formidable and demonstrates that AIDS remains a modern plague. AIDS today is the seventh-leading cause of death in the world. The causes that precede are either non-communicable diseases that can be addressed through changes in behavior or lifestyle, such as ischemic heart disease or stroke, or broad classes of infectious diseases caused by a myriad of pathogens for which preventive or curative therapies already exist, such as diarrheal diseases and lower respiratory infections. HIV is the singular infectious pathogen responsible for the most death in the world 7 . Global statistics about AIDS belie the geographic complexity of the disease, for numbers vary significantly from region to region. Sub-Saharan Africa is the global epicenter of the epidemic, and while promising trends show that both new infections and deaths have decreased in recent years, one in twenty adults in South Africa is living with the virus today. In fact, seven out of ten infected individuals are from South Africa. Decreasing trends in

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1.2 epidemiology of aids

South Africa and other regions hit hard by AIDS such as the Caribbean are offset by increases in new infections by up to 35% in North Africa and the Middle East, suggesting that global pockets endure in which treatment is either inaccessible or poorly implemented or where political and cultural barriers prevent an effective response. We now know that AIDS can be transmitted sexually, through contact with blood, or vertically, from mother to child 8 . The probability of sexual transmission has been difficult to calculate accurately. While the disease was first discovered in homosexual men and inaccurately stereotyped as a homosexual disease, the majority of worldwide transmissions today are heterosexual. The estimated risk of transmission from males to females was 19 per 10,000 unprotected sex acts, while the risk of transmission from female to males is 10 per 10,000 unprotected sex acts 9 . Thus suggests that transmission occurs primarily from males to females. These statistics vary widely based on the nature of the partnership, stage of infection and viral load in the infected individual, and broad socioeconomic and cultural factors 10 . The transmission probability of HIV from needle sharing by intravenous drug users is 67 per 10,000 injections while the risk from a percutaneous needle stick in a healthcare setting is 30 per 10,000 sticks. Though the risk of HIV transmission from contaminated blood transfusion is significant (90%), there has been considerable progress in screening the blood and thereby eliminating it as a source of new infection, particularly in the developed world 11 . The risk of mother-to-child transmission of HIV ranges from 15 to 45%. While mother to child transmission is the leading cause of HIV in young children, breast feeding may be responsible for over a third of childhood infections in the developing world, with a transmission risk of 15 to 25% 12,13 . These risk factors can be reduced through antiretroviral therapy, condom use, and male circumcision 11 . Surprisingly, even with apparently low rates of transmission, the pandemic has been able to affect a large number of people globally 6 .

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1.3 an unknown syndrome & a new virus

1.3 an unknown syndrome & a new virus Despite the size of this pandemic, it has only been known to us for less than three decades. The historical developments that elucidated the existence of a novel syndrome and led to the discovery of the virus that caused it highlight both the initial optimism that followed early success as well as the the challenges that occurred once the unparalleled complexity of the virus became clear. In 1981, in New York and California, a number of patients presented with rare opportunistic infections such as Pneumocystis pneumonia, mucosal candidisas, and Kaposi sarcoma. The patients were homosexual men or intravenous drug users with deteriorated lymphatic systems, and exhibited a deficiency in T cells designed to fight such infections. Specifically, the T cells these individuals lacked harbored the CD4 cell-surface receptor 14,15 . A year later, what had begun as a smattering of cases on both coasts turned into an epidemic. With no understanding of the cause of the disease, it was given discriminatory names associated with those perceived to be at high risk for transmission, such as gay-related immunodeficiency (GRID) or the “4H disease� due to the populations of Haitians, homosexuals, hemophiliacs, and heroin users affected. In a 1982 meeting of the Centers for Disease Control and Prevention, the disease was given its present title: acquired immunodeficiency syndrome (AIDS). Still, the causative agent behind AIDS remained a mystery 16,17,18 . The discovery of two human T-lymphotropic viruses, HTLV-1 (1979) and HTLV-2 (1982), by Robert C. Gallo’s group at the National Institutes of Health marked the starting point in searching for the AIDS agent. Both of these viruses were retroviruses that infected CD4+ T cells and caused leukemia in patients. Furthermore, both were transmitted either by sexual or blood contact and could be transmitted from mother to child, much like HIV. Thus, Gallo and colleagues concluded that the new syndrome was caused by a retrovirus of the HTLV family 19 . Across the Atlantic, a group at Institut Pasteur led by Luc Montagnier confirmed the presence of Reverse Transcriptase (RT), an enzyme common to retroviruses, in samples

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1.3 an unknown syndrome & a new virus

from AIDS patients but also found that antibodies against HTLV could not bind the AIDS virus 20,21 . Finally, pictures of the virus showed conical cores that distinguished it from HTLV. Montagnier named the new virus Lymphadenopathy-Associated Virus (LAV). The discovery of this new virus was later validated by Jay Levy and colleagues 22 . The search for what came to be known as the human immunodeficiency virus (HIV) was competitive and at times, controversial. The French group was excluded from a United States conference attended by Gallo to unveil the the viral determinant of AIDS. In addition, Francoise Barre-Sinousi and Luc Montagnier shared a Nobel Prize, while Gallo was left out. Nevertheless, scientific collaboration between the two groups led to the discovery of the causative virus and a number of early victories in preventing its transmission. As previously mentioned, the development of a blood test two years after the discovery of HIV was instrumental in cleaning the blood supply of developed countries of HIV contamination and reducing the risk to zero of transmission by blood transfusion 15,21,23 . Recent advances in phylogeny have illuminated the true origins of AIDS, which date several decades prior to its first observed clinical manifestations on both coasts of the United States. HIV in humans arose through the zoonotic transfer of Simian Immunodeficiency Virus (SIV) in West and Central Africa from non-human primates 24,25 . The hunting and butchering of primates for bushmeat, the under preparation of primate meat, and the domestication of monkeys as pets represent possible scenarios in which blood containing SIV could have come into contact with the cutaneous or mucosal membranes of humans 26 . SIV may have crossed from primates to humans several times; however, the strain of HIV that is responsible for the global epidemic is thought to have arisen from one transfer of SIVcpz from the chimpanzee Pan troglodytes troglodytes. This SIVcpz strain arose from recombination of SIVdcm from red-capped mangebeys (Cercocebus torquatus and SIVgsn from greater spotnosed monkeys (Cercopithecus nictitans). P. t. troglodytes chimpanzees living in the Southern

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1.4 the molecular biology of hiv

part of Cameroon harbored the SIV reservoirs from which HIV originated. From Cameroon, the disease travelled to Kinhasa, Democratic Republic of the Congo (Zaire), now considered the cradle of the pandemic 25,27 . The time of this crossover is estimated between 1853 and 1900, a century before the first recorded cases. Perhaps whole pandemics came and went while our generation’s plague brewed beneath the surface of detection. 1.4

the molecular biology of hiv

HIV belongs to the retroviridae (retrovirus) family of RNA viruses. The hallmark feature of retroviruses is their use of a specialized RNA-dependent DNA polymerase called Reverse Transcriptase (RT) to transcribe their RNA genome into DNA that can be integrated into the genomes of the host cells they infect. The relative biochemical instability of RNA and the lack of proofreading mechanisms within RT itself endow HIV with a high degree of mutability. 28 . Within the retroviridae family, HIV belongs to the subfamily lentivirinae (lentiviruses). This category, originally inhabited by the visna-maedi virus that affects sheep, came to include HIV due to its evasion of the host immune system, slow progression of infection, and prolonged clinical latency 29,30 . As the virus has been studied at depth over the last three decades, we now have characterized all of the components of HIV. While HIV borrows heavily from the retroviral playbook, it has also generated novel mechanisms to enhance pathogenesis and survival. HIV contains the three main genes common to retroviruses: gag, pol, and env. gag encodes the major structural components of HIV. A spherical matrix forms the outer core of the virus while a conical capsid forms the inner core. Housed within the capsid are two copies of the RNA genome, making HIV a diploid virus, and the products of the pol gene: Protease (PR), Reverse Transcriptase (RT), and Integrase (Int). First, PR facilitates viral assembly and maturation by cleaving polyproteins into individual units that can assemble into configura-

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1.5 infection by deception: viral entry

tions that allow HIV to replicate and infect competently. Second, RT transcribes the viral genome into DNA. Finally, Int facilitates the entry of HIV DNA into the nucleus and the incorporation of HIV genes in the host genome, allowing HIV to form a provirus that can maintain a prolonged latent reservoir in host cells. Interestingly, HIV is able to transmit from cell to cell using an exocytic pathway rather than a lytic one, meaning that HIV can leave cells without destroying them. A consequence of this mechanism is the incorporation of host lipid membrane into the outer boundary of the virus. Studded in this membrane are the products of the env gene, Envelope spike proteins that mediate HIV entry into cells and are critical for infection to occur (Fig. 1.1). We will now review the mechanisms by which HIV enters cells, establishes a covert presence, and hijacks our own cellular machinery in order to propagate. These mechanisms rely heavily on the genomic organization of the virus and the array of structural and functional proteins they encode. Indeed, the components of the virus and the inherent ability to refine them allows the virus to optimize its pathogenic capability. 1.5

infection by deception: viral entry

Perhaps the most insidious feature of HIV is that infects the very cells that are designed to detect invading substances and prime the immune effectors to mount a response. The breach of host cells by HIV is mediated by the Env spike that is studded in the viral membrane of infective virions. The env gene of HIV transcribes a single protein: gp160. gp160 undergoes extensive N-linked glycosylation with high-mannose sugars in the endoplasmic reticulum. This glycosylation is responsible for forming a barrier around Env that prevents immune detection. gp160 glycoproteins trimerize and are subsequently cleaved in the host Golgi apparatus by a furin protease into gp120, the outer component, and gp41, the transmembrane component. Additional remodeling of sugars also occurs in the Golgi apparatus. Thus, mature Env spikes are trimers of gp120-gp41 heterodimers. Trimeric Env complexes are embedded

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1.5 infection by deception: viral entry

Env Matrix

Capsid

Integrase RNA genome

Protease

Reverse Transcriptase

Figure 1.1: Structure of the HIV Virion Here, we present the authors adaptation of The HIV virion (not drawn to scale). The major structural and functional elements, such as the envelope, capsid, and nucleocapsid are shown. In addition, the RNA genome is represented.

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1.5 infection by deception: viral entry

within the host cell membrane, and a portion of these complexes are incorporated into budding virions. Concurrently, some gp120 molecules dissociate from gp41 and are shed into the extracellular space, leaving gp41 embedded in the membrane. Virions incorporating shed Env spikes are defective 31 . gp120 contains five variable regions (V1 to V5) as well as five conserved regions. The conserved regions fold into a core while variable regions V1 through V4 form exposed loops. The core consists of both an inner domain which interacts with gp41, and an outer domain that forms the base of the variable loops. gp41 contains four major regions. The N-terminal fusion peptide, the N-terminal and C-terminal heptad repeat regions, the membrane proximal external region (MPER), and the transmembrane region which embeds gp41 within the viral membrane 31,32 . Early after its discovery, AIDS was found to deplete a subset of T lymphocytes bearing the cell surface receptor CD4 (CD4+ T cells). We now know that the tropism for CD4 is due to the high affinity of gp120 for this receptor the necessity of HIV to bind it in order to infect and enter cells. CD4 binds to a recessed cavity in between the inner and outer domains. Phenylalanine-43 of CD4 occupies a deep cavity within gp120 creating a contoured fit between the two surface proteins. The binding of CD4 results in a conformational change that exposes the co-receptor binding site (CR-bs). HIV binds several co-receptors, though the two most common are CCR5, which predominates early in infection and CXCR4, which predominates late in infection. The V3 loop is heavily involved in co-receptor binding. Co-receptor binding again results in conformational changes in gp41 that allow the N-terminal fusion peptide to insert into the host membrane and bring the host and viral membranes close together. The MPER creates a fusion pore that allows the two membranes to merge and the contents of the virion to become continuous with the host cytoplasm. The structure and mechanism of gp41 resembles hemmaglutinin (HA) of the Influenza virus 31,33,34,35,36 (Fig. 1.2).

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1.5 infection by deception: viral entry

A

gp41

B

C

CD4bs

gp120 CD4 Receptor Co-receptor

V3 loop V1/V2 loops

Fusion Peptide

D

E

F

Figure 1.2: HIV Binding, Fusion, and Entry (A) gp120 binds the CD4 receptor on T cells, initiating pathogenesis and resulting in a conformational change that exposes the co-receptor binding site on variable loop 3 (V3). (B) Co-receptor binding initiates further conformational changes that expose the fusion peptide of gp41 to the host cell membrane. (C) The fusion peptide inserts into the host membrane. (D) Along with the tethering of gp41 to the viral membrane via the transmembrane region, the fusion peptide results in the formation of a six helix bundle that facilitates the apposition of host and viral membranes. (E) Membrane fusion occurs, making the viral interior continuous with the cytoplasm. (F) Successful viral entry occurs. Figure adapted from Willen et. al. (2012) 37

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1.6 an invisible intruder: provirus formation

1.6 an invisible intruder: provirus formation Once HIV has entered cells, the structural components of the virus disassemble, exposing the contents of the viral capsid to the host cell. One of these elements is the RNA genome of the virus, the molecular repository of genetic information that encodes the many viral proteins. In other words, this genome contains the recipe to make new viruses. Unfortunately, this genome cannot produce new proteins or new viruses in its current form. So, HIV transfers this repository into the host genome, where it can be permanently stored and direct the production of new viruses long after the initial infection. This mechanisms requires two enzymes, a Reverse Transcriptase (RT) that can turn the RNA genome of the virus into a DNA genome, and Integrase (Int) which can insert that DNA genome into the existing genomic structure of the host. RT is a heterodimeric protein. The larger of its subunits contains both a Reverse Transcriptase and RNase H domain, each containing one catalytic active site. The smaller unit is genetically similar to the larger one, but it provides structural rather than catalytic support. RT initially synthesizes a complementary strand of DNA using genomic RNA as a substrate. This produces an RNA-DNA duplex. The RNA template is subsequently degraded by the RNase functionality of Reverse Transcriptase. It is believed that distinct enzymes can perform each of these two functions, though each harbors both catalytic sites. The degradation of template RNA frees the newly synthesized DNA to be used as a substrate for the novel synthesis of a complementary strand, producing a complete DNA duplex that can be incorporated into host genomes 38 . Int transports the newly synthesized DNA into the nucleus, where it mediates the insertion of proviral DNA into the host genome. Int binds viral proteins such as the Matrix protein, Vpr (an accessory protein), and the Nucleocapsid protein to form preintegration complexes that facilitate the entry of proviral DNA into the nucleus. Like RT, Int has two enzymatic

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1.7 out of one, many: transcription of hiv genes

functionalities, the processing of the 3’ ends of each strand to ready it for integration and the strand transfer of proviral DNA. 3’-OH processing is mediated by dimers of Int that form at each end of the newly synthesized DNA duplex and remove two nucleotides from the 3’ end of each strand. The two ends of the molecule then come together to form an Int tetramer which can bind both processed ends of the proviral DNA as well as a short segment of genomic DNA at the integration site in the presence of the metallic cofactor Mg2+ . Proviral DNA is then covalently bound to genomic nucleotides, completing the integration process 39 . 1.7 out of one, many: transcription of hiv genes After embedding proviral dsDNA into the host genome, HIV exploits the host cell machinery to express its genes and facilitate the production of new virions. The first thing that HIV does is increase its own expression. The HIV genome transcribes poorly without the presence of positive regulatory factors. Thus, a doubly spliced 2 kb early transcript encodes the regulator or virion expression protein (Rev), trans-activator of transcription (Tat), and negative regulatory factor (Nef). Tat and Rev are imported back into the nucleus. Tat is a regulatory protein that binds a RNA hairpin loop called the Transactivation Response Element (TAR) in the 5’ Long Terminal Repeat upstream of HIV genes. There, it can dock onto the HIV provirus and upregulate the expression of viral genes. Without transcriptional activation, host RNA polymerases generally fall off before complete transcripts can be produced. Tat allows for processive transcription of late transcripts that produce the structural and functional HIV proteins. Thus, Tat is important for HIV replication. 40,41 . Using traditional nuclear export pathways, HIV genes cannot be translated without splicing in the nucleus. However, effective production of HIV proteins is dependent on alternative splicing mechanisms. To overcome this obstacle, Rev binds a site in the coding region of the env gene called the Rev Response Element (RRE). Rev is able to override the classical

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1.7 out of one, many: transcription of hiv genes

splicing pathway and facilitates the export of the singly spliced 4 kb and the non spliced 9 kb late transcripts that encode structural, functional, and accessory genes of HIV 41 . The T cell-mediated immune response constitutes one arm of the adaptive immune system. T cell receptors (TCR) bind foreign peptides presenting in the context of Major Histocompatibility Complex (MHC) proteins on the surface of antigen presenting cells. HIV Nef disrupts the recognition of foreign peptides by CD4 T cell receptors and is greatly enhances the progression of infection. Nef is a myristoylated protein between 27 Ă? 35 kDa in size. Nef downregulates the CD4 receptor by trafficking CD4 receptors at the cell surface to lysosomes. In addition, Nef also down regulates the MHC Class I peptide which prevents TCR-mediated signaling and destruction, allowing HIV to evade immune detection 41,42 . In addition, Nef has targets in the TCR signaling pathway. For example, Nef binds Lck and recruits it to recycling endosomes and the trans-golgi network. Because Lck is the primary kinase that activates TCR-mediated signaling, its absence from the plasma membrane disrupts effective signaling. More distal in TCR signaling, Nef also interrupts actin remodeling and cell spreading and sequesters LAT, a downstream effector of TCR signaling, into intracellular endosomes 42,43 . The products of the early transcript allow for the synthesis of a singly spliced 4 kb late transcript that encodes the HIV envelope protein (Env), viral protein U (Vpu), viral infectivity factor (Vif), viral protein R (Vpr), and additional Tat, as well as an unspoiled 9 kb late transcript that encodes the Gag protein. A slippery element at the end of the gag gene signals a ribosomal frameshift into the -1 reading frame which allows for co-translation of the Gag-Pro polyprotein 5% of the time. The regulation of HIV RNA splicing is reviewed in 44 and 45 . Thus, three different transcripts based off the same genome sequentially produced to efficiently optimize the production of genomic RNA and functional proteins 46 (Fig. 1.3). Once translated, Gag and Gag-Pol perform several functions. First, they capture and traffic themselves as well as HIV genomic RNA transcripts to the cell membrane. Second, they

14


1.7 out of one, many: transcription of hiv genes

Tat

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p1 NC

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Gag-Pol Polyprotein p2

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Figure 1.3: Expression of HIV genes HIV genes are transcribed in a sequential order. Regulatory genes are transcribed first to optimize conditions for further transcription. Next, structural and functional proteins are produced that form the basis of new virions. Additional accessory proteins enhance conditions for viral budding and exit and overpower host anti-viral defenses

15


1.7 out of one, many: transcription of hiv genes

regulate HIV assembly and budding at the membrane. Third, they comprise the structural components of the mature HIV virion. Gag and Gag-Pol proteins are transported to the cell membrane by a Rev-dependent pathway where they arrange radially, creating a spherical protrusion that initiates the budding of immature virions. The N-terminus of Gag associates with the inner leaflet of the host cell membrane while the C-terminus is directed radially inward 44,46 . The Gag protein is a molecular machine that mediates viral assembly prior to budding and comprises the structural components of mature virions. Gag has four major domains, Matrix (MA), Capsid (CA), Nucleocapsid (NC), and p6. Surrounding the Nucleocapsid domain are two spacer regions p2 and p1 46 . Interestingly, Gag is considered an autonomous machine because it can self-associate with membranes and self-assemble into spherical particles 47 . After viral budding is complete, the radial array of Gag and Gag-Pol proteins undergoes structural changes that produce mature virions. Autocatalytic cleavage of Gag-Pol polyproteins embedded in budding virion releases viral proteases which further cleave the Gag complex into the constituent structural proteins: MA, CA, NC, and p6 and the Pol complex into the constituent enzymes RT and Int. The tethering of Pol to Gag in immature virions ensures the inclusion of essential enzymes within viral virions 38 . The HIV protease (PR) is a vastly important protein because HIV pathogenicity is dependent on the cleavage of poly-proteins necessary for viral assembly into proteins required for maturation and infection. The active protease is a dimer of two identical subunits, and the active site of the active enzyme is in the interface between the two dimers. Two flexible loops on either side of the active side act as "gates" that open to allow substrate access 47 . PR allows the various domains of the Gag complex that together mediate assembly of the virus to perform new structural functions as independent proteins in mature virions.

16


1.7 out of one, many: transcription of hiv genes

The Matrix protein (MA) resides at the N-terminus of Gag during assembly. The Nterminus of MA/Gag is myristolyated, allowing it to target and associate with the inner leaflet of the host plasma membrane. Hydrophobic residues adjacent to the N-terminal myristoyl group enhance interaction with hydrophobic elements of membrane lipids while the C-terminal of MA contains positively charged basic residues that interact with the negatively charged acidic heads of membrane lipids. Some also believed that MA plays an important role in the incorporation of Env into virions, possibly by interacting with the cytoplasmic domain of gp41. In mature virions, the mature Matrix Protein (MA), also called p17, trimerizes and in some cases forms hexamers that remain associated with the viral membrane forming the spherical outer core of the virion and stabilizing the membrane 46,47 . The Capsid (CA) domain forms the structural inner core of the virus. The mature capsid consists of roughly 1200 units of the CA protein. Following cleavage from Gag during maturation, CA units form hexagonal and pentagonal rings that close to form a mature capsid. The mature capsid forms a conical geometric structure called a fullerene cone and houses the genomic RNA of the virus as well as the Pol-derived proteins RT and Int. In addition to its contribution to the viral structure, CA may also function in viral defense by competitively binding Cyclophorin A which blocks the binding of TRIM5Îą, an anti-viral host protein that recruits intruding virions to proteasomes prior to disassembly 46,47 . The first spacer peptide (p2) is also important for Capsid formation. While the linkage between CA and p2 is important for the formation of the spherical immature capsid, while the cleavage of these two domains late during maturation assist in the formation of mature conical capsids 46 . The Nucleocapsid (NC) domain of Gag contains two zinc finger motifs that each coordinate with a zinc ion. In the immature virion, NC links Gag proteins with one another. Furthermore, it binds ALIX, a protein involved in the endosomal sorting complex (ESCRT) pathway that

17


1.7 out of one, many: transcription of hiv genes

promotes viral budding. Finally, the zinc finger motifs on NC are important in binding genomic RNA and ensuring that it is correctly packaged during assembly. In mature virions, NC remains associated with genomic RNA and notably facilitates the annealing of DNA to RNA during reverse transcription 46,47 . The second spacer peptide (p1) is found between NC and p6, the C-terminal domain of Gag. C-terminal mutations in p6 result in hybrid proteins such as p8 (p1-p6) and p15(NC-p1p6) result in virions of reduced competency while mutation of conserved proline residues appears to decrease the stability of genomic RNA in mature virions, reducing their infectivity. The p6 domain of Gag contains binding sites for TSG101, another protein involved in late stage budding via the ESCRT pathway. In mature virions, p6 also binds the accessory protein Vpr 46 . While the 9 kb late transcript encodes the structural and enzymatic proteins of HIV, common to retroviruses. What makes HIV unique is the array of additional proteins that it employs to actively antagonize the host defenses while enhancing its own infectivity. These are the products of the 4 kb late transcript, which encodes the Envelope protein that is critical for infection as well as the accessory proteins: viral infectivity factor (Vif), viral protein U (Vpu), viral protein R (Vpr), and additional Tat. The Viral Infectivity Factor (Vif) is required for the competent replication of HIV and functions in antagonizing an antiviral host factor. Vif targets the host protein APOBEC3G. APOBEC3G is a cytosine deaminase that introduces deoxycytosine to deoxyuracil mutations in the negative strand DNA of HIV during reverse transcription. These mutations are lethal to HIV and abrogate infectivity. If Vif is not present, then APOBEC3G is incorporated into budding virions and exercises its mutagenic ability in the next round of reverse transcription. Vif contains two important domains; the N-terminal domain binds APOBEC3G while the

18


1.7 out of one, many: transcription of hiv genes

C-terminal domain binds proteins involved in a poly-ubiquitination that marks APOBEC3G for rapid degradation by host cellular proteasomes 48 . Viral Protein R (Vpr) enhances viral infectivity by facilitating nuclear import of HIV proviruses, inducing cell cycle arrest, and upregulating HIV gene expression. Vpr binds importin-Îą and nuclear pore proteins. Through this mechanism, Vpr traffics the preintegration complex (PIC) into the nucleus to allow HIV proviral DNA to embed within the host genome. Once in the nucleus, HIV binds to the long terminal repeat (LTR) promoter, enhancing the expression of viral genes. Vpr also arrests the cell in G2 through the binding of DNA binding protein 1 and Cullin 4a-associated factor 1, which possibly acts by ubiqitination of downstream targets and leads to cell cycle arrest. Transcriptional upregulation of HIV genes and cellular arrest increase viremia from T cells and macrophages, even though they are are non-dividing. Thus, Vpr is able to prolong the infective capability of integrated proviruses without either cell division or lysis, highlighting its importance in maintaining late stage infection through a depleted pool of CD4 T cells 49 . Viral protein U (Vpu) is an integral membrane protein that has several functions important for HIV proliferation and release. First, Vpu degrades the CD4 receptor, promoting cellular apoptosis, and mediates viral budding. gp160 precursors can be prematurely sequestered within the endoplasmic reticulum (ER) by binding nascent CD4 molecules being co-expressed by the cell. Vpu induces the polyubiquitination of CD4 in the ER and recruits the receptor to cellular proteosomes for degradation. Interestingly, Vpu protects itself from ubiquitinmediated-degradation by masking its own ubiquitination sites. Second, Vpu inhibits the transcription factor NF-ÎşB, which prevents the activation of the anti-apoptotic Bcl2 pathway and promotes apoptosis via caspase-3. While the cytoplasmic domain is involved in CD4 degradation, the N-terminal domain mediates virion release. Vpu binds Gag and promotes transport to the cell membrane and possibly increases the affinity of the MA domain of Gag

19


1.8 the global diversity of hiv

for membrane lipids. Vpu also prevents the function of the host protein tetherin, which binds viral membranes to the host cell in order to prevent release. In addition, the transmembrane domain of Vpu forms an ion channel that promotes the release of budding virions 25,41,50 . Evolution of the HIV structure and assembly mechanism has endowed HIV with two efficient strategies. First, the use of repeating units allows for the efficient production of a lot of protein from one gene. Considering that this biological scale is achieved exclusively through host cellular mechanisms, the efficiency of HIV structure is apparent. For example, one domain of the gag gene of HIV encodes all 1200 repeating CA proteins that form the inner capsid of the virion. Second, many of HIV’s proteins perform one function when complexed together and another function when cleaved in mature virions. This duality of function is enabled by a viral protease. While MA plays a critical role in membrane targeting and assembly when it constitutes the N-terminal domain of Gag, it plays an alternative role in maintaining the viral structure and stabilizing the membrane as a mature standalone protein. Thus, HIV is not only able to leverage a small nucleotide footprint to generate a large peptide repertoire, it is able to efficiently utilize that repertoire as both engineer and raw material in its production. A complete overview of the HIV life cycle is provided in Fig. 1.4. 1.8

the global diversity of hiv

HIV is the most diverse pathogen that we have encountered. The source of this diversity is the multiplicity of zoonotic transmissions of SIV from non human primates to humans. HIV Type 1 viruses are divided into four groups: M, N, O, and P, while HIV Type 2 viruses are divided into eight groups: A - H. HIV Type M and N arose from the cross-transmission from SIV from the chimpanzee P. t. troglodytes in the Southern part of Cameroon while types O and P originated from cross-transmissions from the gorilla (Gorilla gorilla gorilla) in Western Cameroon. HIV-1 type M is responsible for the global pandemic, while the other types have

20


1.8 the global diversity of hiv

9 Assembly Env

1 Entry

CD4

8 Budding co-receptor RNA RT

7 Transport

2 Reverse Transcription Integrase

DNA Genomic RNA

Gag-Pol

3 Integration HIV provirus

Gag

Golgi

Genomic DNA

5’

3’

4 Transcription 6 Processing

5 Expression

Nucleus Endoplasmic Reticulum

Figure 1.4: The HIV Life Cycle Overview of the life cycle of HIV, showing the infection of a cell, establishment of a provirus and production of new virions. A detailed look at how and when viral genes are expressed is shown in Fig. 1.3.

21


1.9 the course of infection

left a much smaller infectious footprint, limited to a handful of cases in Western and Central Africa. (HIV-1 is used interchangeably with HIV in this work) 25 . While zoonotic transmission provided the initial driving force for viral diversity, the rapid and error-prone replicative potential of the virus has significantly expanded it. This potential is highlighted by the fact that viral sequences show variation of up to 10% in infected individuals. Despite this variation in infected individuals, the majority of new infections are caused by a single founder virus. Inflammatory genital infections have been shown to increase susceptibility to infection by multiple variants, possibly by introducing legions in mucosal membranes that provide multiple viral variants exposure to susceptible cells 25,51 . Nevertheless, this would suggest that a large number of variants die out from person-toperson transmission. Yet, the global diversity of HIV is staggering. The diversity of viruses in one individual rivals the diversity of a global pandemic of influenza 52 . The vast diversity of global HIV strains has prompted the further division of HIV-1 Type M into subgroups, or clades AĂ?D, FĂ?H, J and K. Clade B viruses predominate in North America and Europe while Clade C viruses comprise the majority of infections in Sub-Saharan Africa and thus predominate globally. In addition to these clades, a number of circulating recombinant forms (CRF) have also arisen, predominating in West and Central Africa, Southeast Asia, and China. The ability of phylogenetic tools to elucidate the degree to which HIV has and continues to refine its genetic toolkit highlight the challenges the disease possesses from a global health standpoint 25 . 1.9

the course of infection

HIV can infect CD4+ T lymphocytes, dendritic cells (DC), and macrophages in the mucosa. Immediately after transmission, viruses enter the cross the mucosal epithelium via transcytosis pathways. As stated earlier, local inflammation of these epithelia through infection

22


1.9 the course of infection

can cause ulcerations that make penetrating the mucosal epithelia easier for infecting virions. These epithelia preferentially uptake virions tropic for the CCR5 co-receptor, causing these viruses to predominate early during infection. Mucosal immune cells bearing CD4 and the CCR5 co-receptor, namely CD4+ lymphocytes, DC, and macrophages can then be bound by virions, initiating infection. Dendritic cells use a specialized C-type lectin receptor (CLR) and the CCR5 co-recptor capture and internalize virions and present them to CD4+ T-lymphocytes, facilitating rapid infection of these cells. HIV is thus able to hijack a critical immune function, the internalization of foreign bodies by DC and presentation to T lymphocytes in order to efficiently reach and attack its target cells. After infection, the virus spreads through progressive infections of HIV target cells to the lymph nodes where it is able to establish a latent reservoir. After the initial infection, characterized by peak viremia and rapid depletion of CD4+ cells, HIV enters a latent phase where viremia can remain undetectable for a number of years. By the time the cellular immune response mediated by CD8+ T lymphocytes has been activated, a lymphatic reservoir has been established, precluding viral clearing 53 . Ultimately, infected lymphocytes are depleted and the lymphoid tissue deteriorates over time, leading to the wasting that characterizes AIDS and leaves the body susceptible to opportunistic infections 15 . In this chapter, we have introduced the basic epidemiology of the AIDS epidemic that has continued for over three decades and led to massive loss of life. We have also described the basic molecular biology of the virus, from its characterization, structure, and method of pathogenesis. We have followed the path of the virus from infecting a new cell to producing new virions. While these descriptions are in no way meant to be exhaustive, they provide a primer from which we will draw upon in the later part of this work, which will shift focus from describing the disease to developing a solution to end it.

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1.9 the course of infection

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Figure 1.5: Clinical Progression of HIV Infection The blue line shows the population of CD4+ T lymphocytes while the red line shows the viral load detectable in serum. HIV is characterized by three phases of infection. Initial viremia immediately following infection is followed by an indefinite period of clinical latency that eventually leads to the onset of AIDS and the depletion of CD4+ T cells. This figure was modified from the Internet and is based on Figure 1 of a paper titled Immunopathogenesis of Human Immunodeficiency Virus Infection in The New England Journal of Medicine 54

24


2

A TALE OF T WO STRATEGIES

2.1

the antiretroviral revolution

When the renowned HIV physician, scientist and policymaker Anthony S. Fauci delivered a lecture on AIDS in the Woodrow Wilson School at Princeton University, he showed a picture of himself, three decades younger, in a room of physicians performing rounds on one of the first AIDS patients. Physicians at that time neither understood this disease nor had any means of treating it. All they could do was treat the opportunistic infections that mark advanced AIDS, an experience Dr. Fauci compared to “putting a bandaid on a hemorrhage." Those patients had an average prognosis of six to eight years. He then recounted the heartwarming experience of performing rounds the very morning of the lecture. In the same room shown in the photograph, he confidently stated that a twenty year old diagnosed with HIV could expect to live well into his seventies. The awe in the lecture hall was palpable. The drugs that Dr. Fauci and physicians all across the world routinely prescribe HIV patients have revolutionized the management of the disease and dramatically reduced morbidity and mortality. However, whether they, along with other mechanisms of treatment and prevention so far developed, constitute a strategy to end the epidemic remains an open question. To prevent the spread of any malevolence, be it war, wildfire, or plague, we must aggressively and simultaneously advance two strategies: to extinguish it in those places where it exists and to preclude its existence in any another place or another time. HIV requires us to follow the same principles. And to that end, both treatment and prevention have been attempted. Thus far, success with the former has exceeded and in some cases supplanted the later.

25


2.1 the antiretroviral revolution

Immediately after HIV was established as the causative agents of AIDS, the aim of research in the field shifted to finding a way to stop its spread. The scientific community now had a target, and all intuition and present knowledge of biology was employed in finding a vulnerability that could stop the virus in its tracks. The first major development in HIV therapy came from zidovudine (AZT), a drug that had been developed against retroviral cancers but never reached primetime. It was shown to be effective in reducing mortality and the onset of opportunistic infections in late stage AIDS patients and was rapidly licensed in 1987 15 . Later testing demonstrated its effectiveness in forestalling the progression of disease in earlier-stage patients as well, leading to its widespread use and its establishment as the inaugural tool in an otherwise empty arsenal against HIV. Zidovudine, also known as AZT blocks the action of RT by mimicking a nucleotide and terminating the elongation of proviral transcripts while allowing host polymerases to function normally. While groundbreaking, Zidovudine was far from an ideal therapeutic. It caused a substantial amount of toxicity and side-effects such as anemia. Nevertheless, it demonstrated that the interruption of exclusively viral mechanisms by drugs could reduce the mortality and morbidity of AIDS, and led to a burgeoning of antiretroviral therapies (ART) that attempted to improve the quality of life and prognosis of AIDS patients, even while as harbored the virus 55 . Zidovudine was followed by a slew of novel drugs that improved inhibition of Reverse Transcriptase and targeted other viral proteins and their mechanisms as well, such as protease inhibitors, which interfered with the production of mature viral peptides, integrase inhibitors, which prevented the formation of proviruses, and entry inhibitors, which bound gp120 and prevented binding of the CD4 receptor or affiliated coreceptors. These discoveries were facilitated by parallel advances in structural biology, which allowed for the viewing of the crystalline structures of HIV proteins, allowing scientists to better understand the viral

26


2.1 the antiretroviral revolution

elements they were attempting to target and designed tailored therapeutics to those targets. The most recent innovation in antiviral drugs is a series of fusion inhibitors that target the main protein involved in fusing viral and host membranes, gp41. The prototypic drug in this class, Enfuvirtide was approved for use in 2003. Newer generation ARTs continue to be developed in order to improve effectiveness and reduce toxicity. Yet, these drugs do not cure HIV and cannot stop its eventual progression into AIDS 15,56,57 . Still, they have been largely responsible for transforming HIV into a chronically manageable condition among the populations that can access them, use them correctly, and importantly, afford them. A constant fear that accompanies the use of drugs against infectious agents is the threat of resistance. The same viral mechanisms that allow HIV to escape immune detection allow it to render drugs obsolete. Thus, resistance, which has already proven to be a problem for the more stable pathogens that cause malaria and influenza, could pose an immense challenge to individuals who rely on ART against HIV. One solution to this problem is the use of combination therapies. These combination therapies, known as highly active antiretroviral therapy (HAART), use multiple drugs to ensure that pathogenic variants that mutate resistance to one drug are subdued by others. Through the cooperative pressure exerted by multiple drugs, the course of viral evolution and escape can be slowed. Combination therapies of three drugs have been shown to reduce viremia to undetectable levels. As a result of HAART, global resistance of HIV has been declining, but it remains a problem that must be responded to with constant vigilance. While HAART has shown considerable success in the developed world, it remains inaccessible in the resource poor settings in which HIV wreaks the most havoc 58 . Even HAART is not without challenges. Patients on these therapies are now subject to the toxicities of several drugs rather than one, and the need to remember several dosages and instructions makes adherence more problematic. Innovations in drug design have alleviated some of these burdens by creating combination pills that release drugs

27


2.1 the antiretroviral revolution

at different rates, allowing patients to reduce the logistical burden of receiving treatment. HAART shows promise in stemming the tide of HIV infection at least in the developed world. Still, treatment is fundamentally an imperfect solution to the problem of HIV. Data from modeling in Chapter 3 shows that a vaccine must be part of an elimination strategy. A small amount of research has been conducted on possible cures for HIV, but to date no significant progress has been made on this front. Successful HIV cures have been extremely limited to special scenarios that cannot be easily replicated on a mass scale. In 2007, a man named Timothy Brown from Berlin, Germany who was diagnosed with both acute myeloid leukemia and HIV-1 was given a haematopoietic stem cell transplant from a donor who had homozygous deletions in both of his alleles for the CCR5 receptor. Because HIV requires the CCR5 surface co-receptor in order to bind CD4 and infect helper T cells, replacement of Brown’s bone marrow with donor marrow lacking a CCR5 receptor replenished his body with white blood cells that HIV could no longer infect. Since 2007, the virus has not been detected in his body and he is the only person in the world considered to be cured of HIV 59,60 . While his story does show that it is possible to cure HIV, a fortuitous set of circumstances allowed Brown to achieve this outcome. Bone marrow donors are not readily available, and certainly the prospect of finding enough compatible bone marrow donors with a mutation that very few people have to transplant in to every HIV positive individual is unrealistic. Furthermore, a complex medical intervention is not a sustainable control strategy given the widespread nature of the epidemic, especially in resource-poor settings. Recently, a toddler that was born with the virus from his HIV positive mother was functionally cured of the disease after receiving combination ART 30 hours after birth. The virus was undetectable two years later, suggesting that immediate treatment with ART can prevent the formation of an infection reservoir such that that the virus can be completely cleared from the body after a period of time. While this result are promising, again it is important to consider

28


2.2 the current arsenal of preventions

the scalability of this approach. First, it can be applied to the prevention of mother-to-child transmission because HIV testing of the mother can help determine whether the child is at risk. However, it would prove difficult to use in the majority of heterosexual transmissions that make up a majority of the disease spread because HIV is usually untreated in the early stage of infection. Stories of individuals cured of HIV are sure to make the front page, but they are not effective strategies to deploy on the front lines of the epidemic. Nevertheless, they are instructive in determining vulnerabilities of the virus that may be used by other treatment and preventive mechanisms. 2.2 the current arsenal of preventions Interventions aimed at preventing new infections of HIV have also been developed. The two major behavioral initiatives that have been attempted thus far are condom use and sex education. Condom use has been shown to significantly reduce the incidence of new infections by up to 80% 61 . Furthermore, risky behavior such as intravenous drug use can be reduced through education 62 . Education programs may also be employed to encourage sexual activity at a later age, monogamous relationships, and frequent testing among those at risk. All of these ideas are valuable and should be coordinated into policy planning against AIDS. Male circumcision has also been attempted to reduce the transmission of HIV. A systematic review and meta-analysis of 27 studies was completed by Weiss et. al. in 2000. This analysis found that male circumcision reduced the risk of HIV among sub-Saharan African men by 44%, and reduced the risk of infection for the highest risk men by 71% . From a biological standpoint, the evidence for circumcision seems to be convincing. The Centers for Disease Control and Prevention (CDC) indicates that circumcision reduces the risk of transmission, while the World Health Organization recommends it as an integral part of the HIV prevention

29


2.2 the current arsenal of preventions

toolkit. Some view the reduction of risk in excess of 70% as a sign that circumcision could be as effective as a vaccine. Despite these encouraging numbers, circumcision, like condom use and sex education, remains a controversial practice. Given ethnic, cultural, and religious tensions that have festered in HIV endemic regions long before the epidemic began, the coordination of these efforts may be difficult when policymakers stand in their way. While circumcision is a possible strategy when performed in children, it can cause complications and be painful to adult recipients and can also be associated with negative stigmatization. Furthermore, the risk of transmission immediately after circumcision in seropositive males increases due to the exposed wound surface 63 . Recently, even treatment has waded into the prevention arena. The idea that treatment can subdue the virus to undetectable levels, and thus vastly prevent the transmission of disease has given way to the paradigm of Treatment as Prevention, where treatment has assumed the dual distinction of both reducing individual morbidity and mortality while also reducing population incidence of disease. In fact, individuals receiving ARTs are only 4% as likely to transmit HIV as individuals not being treated 64 . Moreover, pre-exposure prophylaxis (PrEP), the administration treatment even before the acquisition of infection, has been tested and has led to mixed interpretations in the scientific community. In the now famous iPreX Study, roughly 2500 HIV-free men or transgender women who have sex with men were given either an oral combination therapy of two ARTs, emtricitabine and tenofovir, or placebo. Both of these drugs block HIV RT. Individuals were followed for a little over a year, and it was reported that PrEP reduced the incidence of HIV by 44% 65 . Furthermore, the investigators reported that not all of the individuals in the treatment group adhered to treatment, and that the actual risk reduction among individuals who adhered to the regimen was even higher. In addition, the Centre for the AIDS Program of Research in South Africa (CAPRISA 004) trial, the PrEP potential of topical ARTs was assessed in women. In a double-blind, randomized

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2.3 the failure of preliminary vaccines

control trial, over 400 women were given either a 1% vaginal tenofovir gel or placebo gel. The reduction in incidence in women who used tenofovir was reported to be 39%, and the reduction in women with high gel adherence was said to be 54% 66 . Unfortunately, there are severe limitations to the broad application of PrEP, at least given the data currently available. In the iPreX trial, PrEP was only shown to be effective in reducing transmission in homosexual relationships and with two drugs. Furthermore, the reduction in transmission was heavily dependent on adherence, which continues to be problematic. In fact, poor adherence to a two drug therapy that results in infection could lead to the development of resistance, a possible consequence of PrEP overuse. 2.3

the failure of preliminary vaccines

After successful vaccines against a slew of diseases both viral and bacterial, intuition suggested that a methodology had been developed to design vaccines, involving passaging pathogens through cell cultures in vitro, determining which part of the pathogen was able to produce a sterilizing immune response, and finally to introduce that pathogen in a manner that was immunogenic, perhaps by conjugation. HIV proved to be a far more complicated virus than was previously imagined, which resulted in the inadequacy of our current toolset, an observation that was painfully learned through three clinical trials of HIV vaccine candidates. The first attempt at an HIV vaccine was to immunize individuals with a recombinant gp120 protein. After gp120 exhibited a protective effect in non-human primate models and was determined to be both safe and immunogenic in early stage clinical trials, the drug was advanced to two Phase III clinical trials, one based predominantly in North America and the other in Thailand. The North American vaccine trial used a monovalent vaccine composed of two gp120 subunits from distinct Clade B viruses. The Thai vaccine trial used a bivalent vaccine composed of one gp120 subunit from a Clade B virus and the other

31


2.3 the failure of preliminary vaccines

from a Clade E virus. In both trials, gp120 failed to protect individuals from infection or slow disease progression. It is believed that monomeric gp120 was unable to protect against infection because neutralizing antibodies could not be produced against this immunogen. One possible reason for the lack of a neutralizing humoral response is due to the exposure of immunodominant non-neutralizing epitopes on monomeric gp120 that are usually shielded by trimerization 67,68,69 . It is now widely believed that the native trimer is the most instructive structure with respect to vaccine design. The absence of a neutralizing antibody response led to a search for a vaccine candidate that could initiate a cell-mediated immune response to destroy already infected cells that could establish the lymphatic reservoir that is characteristic of irreversible infection. Non-human primate studies suggested that T cell responses may correlate with protection. From 2003 to 2005, A Phase III clinical trial in Thailand was advanced to assess the possible protective effects of simultaneous stimulation of the humoral and cell-mediated immune responses. The vaccine candidate in this trial was a live replicating canarypox vector prime followed by two boosts of recombinant gp120 from Clade B and Clade E viruses. The rationale for a prime boost system in vaccine development is to longitudinally guide the immune response over time by first priming with an antigen that stimulates the correct initial response and subsequently boosting with an antigen that tailors the initial response towards the desired correlates of protection. The study reported that the vaccine reduced the risk of HIV by 31.2% in the Thai population studied. Many in the scientific community celebrated the results, however modest, of this trial, suggesting that for the first time the ability to prevent HIV through vaccination had been demonstrated. In reality, however, the methodology used in this trial was questionable. Under the Intention-to-Treat analysis and Per-protocol analysis, the standards of clinical trials, the protective effects of the vaccine were statistically insignificant (p = 0.16 & p = 0.08, respectively). Only under the modified intention-to-treat analysis

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2.3 the failure of preliminary vaccines

were the results significant. However, this methodology tends to exclude the sickest cases 70 . Furthermore, the vaccine itself was based on shaky science. The canarypox vector used to elicit a T cell response was shown to be poorly immunogenic in early phase clinical trials and the boost that was used was the same recombinant gp120 that had failed to produce a protective effect in the earlier Thai trial 71 . What the Thai trial constituted was a straw victory for a scientific community desperately in need of one. In 2005, a trivalent vaccine containing replication adenovirus vectors (Ad5) carrying the HIV gag, pol, and nef genes was advanced to a Phase IIb proof-of-concept trial cosponsored by Merck and the United States National Institutes of Health. The adenovirus vector was chosen because it was considered harmless and shown to be more immunogenic in non-human primates. Preclinical studies and early trials had demonstrated a cell-meidated response. However, the STEP trial was suspended two years later when the vaccine candidate showed no protection from infection or reduction in disease progression. In fact, the vaccine actually appeared to be deleterious – increasing the likelihood of infection – in individuals that possessed neutralizing antibodies against the Ad5 vector, which turns out to be a significant proportion of the North American and European populations 69,72,73,74 . Unfortunately, our record on HIV vaccines has been disappointing. But it is important to use these failures as learning experiences and discern what went wrong so that we can maximize the chances of future success. It is also important to remember that HIV has been a particularly traumatic pandemic and that each failure has the potential to erode confidence in the scientific community’s ability to solve what appears to be biology’s gordian knot 75 . It is important to consider the specific challenges that HIV poses in terms of vaccine development. The primary difficulty of HIV is that natural infection cannot be used as a model for vaccine design. The founding principle with which Jenner and Pasteur heralded revolutions in vaccinology was that the sterilizing immunity that accompanies natural infection could

33


2.3 the failure of preliminary vaccines

be activated by a non-infectious pathogen. But natural infection with HIV is lifelong, so future infection cannot be prevented. Moreover, natural infection with HIV only results in sterilizing immunity in 20% of those infected, and this too only occurs after the virus has already established a reservoir. Logistically, HIV poses a number of challenges to successful vaccine development as well. First, HIV is the fastest mutating pathogen known in nature, which means that immune responses directed towards particular epitopes of the virus are likely to become obsolete over the course of viral evolution. As stated earlier, a minority of these individuals are able to generate a broad and potent antibody response, but the determinants of this ability remain a mystery. Second, the virus is built in a way to avoid immune detection. The Env spikes on the viral surface form a barrier to immune detection and protect many of the viral epitopes from detection by antibodies. Third, the virus attacks the cells that assist in the cell-mediated immune pathway, harming the ability of cell-mediated immunity to function normally. Finally, the virus establishes a lymphatic reservoir in hosts, meaning that a vaccine must act quickly to prevent infection 73 . The immense challenges in HIV development combined with the large-scale failures in overcoming these challenges have caused some to return to treatment as the gold standard of HIV policy. The two major questions that arise are whether an HIV vaccine is “worth it" and how we will be able to make one given our past history. Answering these questions is the subject of the following two chapters.

34


THE IMPACT OF AN HIV VACCINE: A MATHEMATICAL MODEL

3

3.1 the need for a vaccine In the previous chapter, the two major strategic interventions against HIV, treatment and prevention, were discussed. The realization that ART can significantly reduce viremia in infected individuals has given rise to “treatment as prevention", a new paradigm that blurs the distinction between these two strategies. The ability to prevent disease through ART combined with the failure to produce an effective vaccine has prompted a questioning of the continued relevance of a vaccine and the impact it can have 76 . Another issue of interest is how much impact a preliminary vaccine with reduced efficacy or duration of protection may have on the trajectory of the epidemic. Mathematical modeling can be illustrative in projecting the course of a disease under various initial conditions and interventions. While several models of HIV have been developed, there remains a paucity of available data on the relative impact of vaccination over treatment. It is difficult to model the impact of vaccination accurately because, to date, no successful vaccine has been developed against HIV. However, historically vaccination has been shown to be the most effective strategy in eliminating the spread of disease and is the strategy responsible for the only known eradications of disease in humans and other animals. Whether the same is true for AIDS is the subject of this chapter. In order to model the impact of a vaccine, the existing landscape of HIV treatment must be utilized as a baseline. Initially, treatment guidelines for HIV infection suggested that individuals should begin treatment when their CD4+ T cell count dropped below 200 cells/mm3 , due to the demonstrable increase in mortality when treatment was was initiated below this level 77 . This guideline was updated to recommend commencement of therapy at a CD4+

35


3.1 the need for a vaccine

count of 350 cells/mm3 . Currently, the merits of expanding treatment to individuals with CD4+ counts between 350 cells/mm3 and 500 cells/mm3 are being assessed 77,78,79 . This would constitute an aggressive treatment guideline, as 500 cells/mm3 marks the lower end of the normal range for CD4+ T cells. While these guidelines are based on the benefits to patient morbidity and mortality, the idea that treatment can be used as prevention has led some to abandon a clinical start point for treatment altogether and view treatment exclusively through the context of population benefit 80 . For example, a proposal by Granich and colleagues (2008) at the World Health Organization advocates universal annual testing of individuals starting at the age of 15, followed by immediate ART. Their modeling suggests that a majority of infected individuals would receive ART within five years and that within a decade, the pandemic would be well on its way to elimination. The approach of early and aggressive treatment is based on the observation that viremia peaks early during infection. Thus, reducing the viremia at this critical point yields the most gains in using treatment to prevent disease 81 . Regardless of the biological merits of treatment as prevention, the logistical feasibility of scaling treatment up to the required scale is dubious. First, the Granich model estimates that the universal test and treat policy will achieve a 90% coverage by 2016, which is an unrealistic assumption. A look at the present statistics in the United States is revealing. About 20% of individuals living with HIV are not even diagnosed. Roughly 67% of infected individuals have access to care, and less than half of them adhere to their treatment regimens. Overall, about 25% of individuals affected are able to achieve viral suppression 82 . Certainly, the statistics are bound to be worse in resource poor settings where generalized epidemics occur (Ruthie Birger, personal communication). In fact, ART coverage in parts of Northern Africa and the Middle East is only 15% (World Health Organization data). Furthermore, optimizing treatment to prevent disease may adversely affect patient morbidity, the original intention of

36


3.1 the need for a vaccine

treatment, and reduce adherence. While early treatment may prevent disease transmission, there is a considerable reduction in quality of life from drug toxicity. Whether people who have disease but do not feel ill will adhere to therapy better than the sick individuals for which adherence has been calculated is doubtful 80 . While treatment as prevention may be biologically sufficient to control the AIDS pandemic in the long term, its scalability in practice is questionable. On the other hand, a vaccine can confer true protection without many of the biological and logistical complications of ART. First, the vaccine can be given prophylactically and does not require burden of testing individuals that is required for treatment. Second, because a vaccine exploits the host’s own immune response to prevent infection, protection is not mediated by a foreign therapeutic, and there is little chance of drug toxicity or side effects after the initial vaccination. While the nature of the vaccine may require a series of vaccinations or booster vaccinations to prevent waning immunity, the burden of vaccination will be far less than that of treatment, in which one or more medications must be taken daily, often at various times of the day and with special requirements 83,84 . Finally, vaccination tends to be more cost effective than treatment, both due to the reduced cost of the vaccine and the health gains from the reduced burden on health systems 85 . In addition, the reduced cost of vaccination can help increase access. If we can demonstrate that a vaccine can be as effective or more effective in preventing transmission of disease, then we can conclude that a vaccine presents the best realistic chance of ending the AIDS pandemic. The lack of an effective vaccine has resulted in a paucity of data regarding its potential impact. To fill this void, mathematical modeling has been used to estimate the impact of a possible vaccine. The modest reported efficacy of the vaccine candidate from the RV144 Thai Trial, since shown not to be efficacious, prompted an interest in modeling the impact of imperfect vaccines with partial efficacy and waning immunity. These studies were applied

37


3.2 methodology

to local populations. These vaccine models suggested that lowly efficacious vaccines with waning immunity would be poorly effective in the long term without subsequent boosting, suggesting that high efficacy and duration of protection must be cornerstones of a highly effective vaccine 86,87 . Additionally, a model for a partially efficacious vaccine in the United States showed that significant prevention of disease could be acheived 88,89 . While these vaccines may show limited effectiveness in local or national settings, a larger analysis at the global scale is required to understand the true impact of a vaccine. In this study, we develop a compartmental model of HIV transmission and analyze the effect of various vaccine candidates on the long term progression of the pandemic. We analyzed three possible vaccines: an ideal vaccine, a vaccine with 70% efficacy, and a vaccine whose protection lasted for ten years. Each of these hypothetical vaccines are better than any vaccine that has been advanced through clinical trial thus far. We show that an ideal vaccine can lead to reductions in incidence and prevalence of disease of up to 100% over aggressive treatment. Furthermore, we show that even a partially efficacious vaccine can reduce incidence and prevalence of disease over 95% from aggressive treatment, given high vaccine coverage. Finally, we show that an ideal vaccine can lead to even further reductions in incidence and prevalence of disease if used in conjunction with treatment. 3.2

methodology

3.2.1 Compartments of the Model We developed a simple mathematical model to determine the effects of various interventions on the progression of the AIDS pandemic over time. The model contained one compartment for susceptible, uninfected individuals (S). Succeptible individuals can be infected by other infected individuals during any stage of the disease. Transmission is highest during the early stage of disease and lowest during clinical latency, following a complicated trajectory that

38


3.2 methodology

corresponds to the level of viremia (shown by the red line in Fig. 3.1). We simplified this trajectory into three discrete stages and probabilities of transmission, primary infection (I0 ), latent infection (I1 ), and late stage AIDS (A), based on a previous model of HIV transmission 90 . Individuals in the primary infection stage (I0 ) produce high titers of virus due to the rapid replication of viruses and the establishment of a viral reservoir in the lymphatic tissue 15 . This results in an initial depletion of CD4+ T cells as well as a peak in viremia and transmission potential. Latent infection (I1 ) is characterized by a reduction in viral replication for a number of years as well as a stabilization in the CD4+ T cell count. Finally, AIDS (A) occurs when the CD4+ count begins to diminish and is clinically characterized by a high degree of immunosupression and the onset of opportunistic infections. Clinically, this stage of infection is marked by increased susceptibility to opportunistic infections and death 54 . In addition to infectious compartments, we developed two additional compartments to model the two main strategic interventions discussed in Chapter 2. A treatment compartment (T) to model the effects of antiretroviral therapy given after infection, as well as a vaccination compartment (V) to model the effects of a prophylactic vaccine. Individuals in the treatment compartment have the infection and can transmit the disease to susceptible individuals, albeit with a much lower probability than individuals not receiving treatment. Individuals who receive vaccination do so prior to infection and bypass the susceptible compartment entirely. In order to model the effects of vaccines with differential duration of protection and efficacy, we included pathways from the vaccinated compartment (V) back to susceptibility and infection, respectively (Fig. 3.2). The sum of the individuals in each compartment constitutes the total population N (Eq. 1).

S + I0 + I1 + A + T + V = N

39

(1)


107

1200 1100 1000

106

900 800

105

700 600 500

104

400 300

103

200 100 0

0

3

6

Weeks

9

1

12

2

3

4

5

6

7

Years

8

9

10

11

HIV RNA (copies per mL Plasma)

CD4+ Lymphocyte Count (cells/mm3)

3.2 methodology

102

Figure 3.1: Clinical Progression of HIV Infection This figure was modified from the Internet and is based on Figure 1 of a paper titled Immunopathogenesis of Human Immunodeficiency Virus Infection in The New England Journal of Medicine 54 .

µ (1 − p)Λ

S δ

µ β

I0

µ γ1

I1

µ γ2 γ3

η

(1 − ε)β

V

T

µ

µ

ν

A θ

Figure 3.2: A Compartmental Epidemiological Model for HIV Transmission Individuals enter either a susceptible (S) or vaccinated (V) compartment, depending on their vaccination status. Infection progresses through three stages with differential probability of transmission to susceptible individuals: primary infection (I0 ), latentinfection(I1 ), and AIDS (A)). Finally, treatment (T) sequesters infected individuals into a compartment that prolongs life and reduces the probability of transmission to susceptible individuals.

40


3.2 methodology

Variable

Description

Estimated Value

Λ µ

Crude birth rate Crude death rate

20 per 1000 individuals (0.02) 20 per 1000 individuals (0.02)

Table 3.1: Crude Birth and Death Rates In this model, we assumed that the crude birth and death rate was the same in order to isolate the effects of HIV infection on survival. Modeling with a growing population can lead to instability that makes the data more difficult to interpret.

3.2.2 Differential Flows Between Compartments We generated a series of differential equations to model the flow of individuals between compartments. Individuals progress sequentially from susceptibility to primary infection and then into latent infection and AIDS. Individuals can enter a vaccinated compartment through prophylactic vaccination and bypass the susceptible compartment entirely. Imperfect vaccines allow exit from vaccination back into susceptibility or primary infection. Furthermore, we assume that individuals can enter the treatment compartment from either latent infection or AIDS due to the difficulty in diagnosing HIV during primary infection. Finally, outflows from the treatment compartment go directly to the AIDS stage, as treatment symptomatically resembles prolonged latency. We use the variable Λ to represent the crude birth rate of the population and the variable µ to represent the crude death rate of the population. In order to isolate the effects of AIDS, we made a general assumption that the background birth rate and death rates were equal, and set them both to be 20 per 1000 people. We also assumed the the general birth rate corresponds to the rate at which individuals reach sexual maturity. Thus, we justified using Λ to model entry into S. Furthermore, we assumed that the probability of death from non AIDS-related causes is the same during every stage of infection, vaccination, or treatment. As a result, we justified an outflow of µ from each compartment (Table 3.1).

41


3.2 methodology

Variable

Value

ω0 ω1 ωA ωT

2.7 0.1 0.7 0.004

Table 3.2: Multipliers For the Force of Infection The data for these multipliers is based on proportional infectivity in each of the stages of infection, as described by Hollingsworth et. al. (2008) 90 . The infectiousness of individuals who are being treated ωT is 4% of that of latent infection 64 .

The initial flow from susceptibility to primary infection (S → I0 ) is the most critical for HIV dynamics, as it is the fundamental link that allows for the propagation of infection throughout the population. This flow is proportional to the number of susceptible individuals and the force of infection for HIV (β). We model the force of infection by estimating the contribution of each of the infected compartments to the transmission of infection to susceptible individuals. This contribution is based on both the probability of transmission from each compartment (ω0 , ω1 , ωA , and ωT , respectively) as well as the proportion of the infected individuals in that compartment out of the total population ( IN0 ,

I1 IA , , N N

and

IT , N

respectively) 90 (Eq. 2,

Table 3.2).

β = ω0

I0 I1 A T + ω1 + ωA + ωT N N N N

(2)

While progression from S to I0 requires a force of infection, progression from I0 to I1 (γ1 ) and I1 to A (ν) and A to AIDS-related death (γA ) occur chronologically without external

42


3.2 methodology

Variable

Flow

Value

Duration (Years)

γ1 γ2 ν

I0 → I1 I1 → IA A → Death

4.16 0.119 0.05

0.24 8.38 0.75

Table 3.3: Multipliers for the Progression of Infection The rate at which individuals progress from one compartment of infection to the next is based on the inverse of the duration of the initial stage of infection. These durations were obtained from Hollingsworth et. al. (2008) 90 . The parameter ν is used to show the rate at which individuals die from AIDS due to convention. This is also the reciprocal of the duration of the AIDS stage, or about three quarters of a year.

factors. The rate of progression from one compartment to the next is equal to the inverse of the duration of time spent in the first compartment (Eq. 3).

γn = Rate of progression from In → In+1 =

1 Duration of In

(3)

Using data from a study population in Rakai, Uganda, we obtained values for the duration of each stage of HIV infection. These data suggest that primary infection lasts for 0.25 years (roughly thirteen weeks), latent infection lasts for 8.38 years, and the final AIDS stage lasts for 0.75 years 90 . Interestingly, these data correspond well to the time course of infection shown in Fig 3.1 54 . We applied the reciprocal of each of these values to the sequential flow between differential compartments (Table 3.3). While HIV epidemiology has been modeled significantly before, the innovation in our model is the addition of compartments for vaccination and treatment and the comparative analysis of these two interventions. Individuals within the treatment compartment (T) have been tested and diagnosed for infection and are currently taking antiretroviral treatments. We make no distinction here between first generation (ART) and combination therapies

43


3.2 methodology

(HAART). Furthermore, we have assumed universal access to medical care and treatment regimens as well as universal adherence to these treatments. As mentioned previously, due to the difficulty of diagnosing HIV during the primary infection stage, we assume that individuals can only start treatment in I1 and A, and that the rate at which individuals receive treatment from both of these compartments is the same. Furthermore, as treatment does not clear infection, we model an outflow from T to A. 3.2.3 Modeling Treatment and Vaccination To substantiate each of these flows, we apply the same methodology that was used to treat the chronological progression of infection. In this study, we test treatment using two treatment guidelines: the current guideline, which suggests that individuals with CD4+ T cell count below 350 cells/mm3 should commence treatment, and an aggressive treatment guideline, which suggests that individuals should begin treatment earlier, when their CD4+ count drops below 500 cells/mm3 . In order to do so, we mapped these CD4+ counts to time points on the initial chart of disease progression. While HIV infection varies from individual to individual, we concluded that a CD4+ count of 500 would be achieved two years after infection and a CD4+ count of 350 would be achieved five years after infection (Fig 3.1). We applied the inverse of these time values to the flows into the treatment compartment (η, θ) 78,79 (Eq. 4, Table 3.4).

η = θ = Rate of initiating treatment =

1 Duration of infection prior to treatment

(4)

Finally, because ART can prolong the life of individuals infected with HIV, we introduced a flow to delay the progression of individuals in T to A accordingly. Individuals whose disease is detected early and adhere well to HAART can expect to reach an average life expectancy,

44


3.2 methodology

Variable

Flow

Value (CD4+ = 350)

Value (CD4+ = 500)

η θ γ3

I1 → T A→T A → Death

0.2 0.2 0.05

0.5 0.5 0.05

Table 3.4: Treatment Guidelines The values obtained here come from treatment guidelines as well as a study of South African cohorts that was performed to determine the increase in life expectancy provided by antiretroviral treatments to HIV 78,79,91

however each of the aforementioned conditions is difficult to achieve in practice. To determine the increase in life expectancy provided by treatment, we used data from a series of cohort studies performed in South Africa. While the increases in life expectancy varied by gender, age of onset, and CD4+ count at start of treatment, we determined that on average ART provided 20 additional years of life 91 . As a result, we set the outflow from T to A to the reciprocal of this value (Eq. 5, Table 3.4).

γ3 = Rate of AIDS progression with ART =

1 Increased life expectancy from ART

(5)

While the modeling of treatment at various levels can be instructive in determining the effects of treatment as prevention, the true motivation of this work is to determine the impact of a potential HIV vaccine by demonstrating its increased effectiveness over both current and aggressive scenarios. We assume that individuals are vaccinated prophylactically before reaching sexual maturity. As a result, these individuals can forego entry into S entirely. The rate at which individuals enter V is assumed to resemble the crude birth rate Λ. To distinguish individuals who enter S and V, we use the multiplier p to describe vaccine coverage–the

45


3.2 methodology

proportion of individuals who receive vaccination. Thus, at any time t, pΛN are entering V while (1 − p)ΛN are entering S. In this study, we assess the value of particular hypothetical vaccines at different levels of coverage. In addition, we also observed the effects of varying vaccine efficacy and duration of protection. In order to model the former, we added an outflow from V to I0 and modified the force of infection β used in the flow from S to I0 with the multiplier (1 − ε), where ε is the relative efficacy of the vaccine (Eq. 6, Table 3.5).

ε = Relative vaccination efficacy =

Number of individuals protected from infection (6) Number of individuals vaccinated

To model the duration of protection, we include a backwards flow to delay the progression of V to S, somewhat resembling the flow from T to A previously described. This flow models the loss of protection and is given by the inverse of duration of protection (Eq. 7, Table 3.5).

δ = Rate of protection loss =

1 Duration of Protection (Years)

(7)

Together, these flows constitute a system of differential equations that can be solved over a period of time to model the trajectory of the AIDS pandemic under various treatment and preventive interventions.

46


3.2 methodology

Variable p ε δ

Description

Flow

Vaccine Coverage →V Vaccine Efficacy V → I0 Duration of Protection V → S

Table 3.5: Vaccine Efficacy and Duration We plan to modify vaccine coverage, efficacy, and duration of protection in the experimental analysis to measure the impact of various hypothetical vaccines. Partial efficacy is achieved by adding a partial force of infection to vaccinated individuals. Waning immunity is achieved by returning vaccinated individuals to the susceptible compartment over time.

dS dt dA dt dI0 dt dT dt dI1 dt dV dt

= (1 − p)NΛ − (µ + β)S + δV = γ2 I1 − [µ + ν + θ]A + γ3 T = βS − (µ + γ1 )I0 + εV = −(µ + γ3 )T + ηI1 + θA = γ1 I0 − (µ + η + γ2 )I1 = pNΛ − (µ + εβ + δ)V

Initial Conditions In order to test our model, we populated each of the compartments with several initial conditions. We assessed the progression of the epidemic at the global level. We set N(0) to 6.97 × 109 , which was the world population at the time of this analysis. We used the most recent available epidemiological data of the AIDS pandemic. 35 million people are currently living with HIV. We set T (0) to 8 × 106 , reflecting the number of HIV infected individuals

47


3.2 methodology

Variable N S I0 I1 A T V

Description

Initial Value

Total population Number of susceptible Individuals Number of individuals with primary HIV infection (no treatment) Number of individuals with latent HIV infection (no treatment) Number of individuals with AIDS (no treatment) Number of individuals on ART Number of individuals vaccinated

6.97 × 109 6.94 × 109 7.20 × 105 2.41 × 107 2.16 × 106 8.0 × 106 0

Table 3.6: Initial Conditions Initial conditions used to populate the compartments of the HIV model.

currently receiving treatment. We divided the remaining 26 million individuals with HIV and without treatment into three compartments, allocating them based on the proportion of time spent in each compartment to the total time of infection. As no successful vaccine exists, we set V(0) to 0 90 (Table 3.6). 3.2.4

Data Analysis

We wanted to observe the epidemic over the long term. We had to extend the analysis over a period of 100 years in order to observe the different progression of various interventions. We solved the system of differential equations using ode45 in MATLAB. Standard epidemiological definitions were used to calculate the endpoints measured in this study. Survival was measured by the total size of the population at each time point. Incidence was measured by dividing the number of individuals with primary infection at each time point (I0 ) by the total susceptible population (S) at each time point. Prevalence was measured by dividing the total number of individuals with infection (I0 + I1 + IA + and T) by the total population size (N) at each time point. At the model end point (t=100), survival, incidence, and prevalence were recorded for each intervention. The survival, incidence, and prevalence under current and aggressive

48


3.3 results vs. No Treatment Treatment Level

%∆Survival

%∆Incidence

%∆Prevalence

350

8447

-78

-53

500

20344

-91

-78

Table 3.7: Per cent change in survival, incidence, and prevalence using current and aggressive treatment guidelines over no treatment is shown. Favorable values are shown in blue (higher survival, lower incidence, and lower prevalence).

treatment guidelines, CD4+ = 350 cells/mm3 and CD4+ = 500 cells/mm3 , respectively, were reported as a percentage of their corresponding measures under no treatment. Likewise, survival, incidence, and prevalence under various hypothetical vaccines were reported as a percentage of their corresponding measures under no treatment and the two treatment scenarios described. 3.3 results 3.3.1 Aggressive Treatment Leads to Prevention of Disease First, we established a baseline by observing the progression of the AIDS pandemic without any treatment, and with current and aggressive treatment guidelines. Treatment with ART improves survival and reduces both the incidence and prevalence of disease. Specifically, treating individuals at current guidelines leads to a 78% reduction in incidence and 53% in incidence compared to no treatment at the end point of analysis (t = 100). Beginning treatment earlier, as indicated by the aggressive guideline, results in a reduction of incidence by 91% and prevalence by 78% over no treatment. This suggests that early treatment can prevent disease, probably through reductions in viremia during the additional three years of treatment. However, it is important to note that these analyses assume universal access to treatment and universal acceptance and adherence as well (Fig. 3.3, Table 3.7).

49


3.3 results 9

x 10

Incidence

Survival

6

4

2

0.03

0.5

0.025

0.4

Prevalence

8

0.02 0.015 0.01

20

40

60

Years

80

100

0.2 0.1

0.005 0 0

0.3

0 0

20

40

60

Years

80

100

0 0

20

40

60

Years

80

100

Treatment Guidelines No treatment

CD4+ = 350cells/mm3

CD4+ = 500 cells/mm3

Figure 3.3: Model Simulation At Various Treatment Levels The HIV epidemiological model was used to simulate the progression of the epidemic under various treatment conditions to establish a baseline to measure vaccine impact. The model was run without treatment (solid gray line), with treatment starting at a CD4+ count of 350 cells/mm3 (dashed gray line), and CD4+ count of 500 cells/mm3 (dotted gray line). The data indicate that early treatment can improve survival and reduce incidence and prevalence of disease. Furthermore, the model shows improvement in outcomes when treatment is begun earlier, validating the paradigm of treatment as prevention.

3.3.2 A vaccine could be far more effective than aggressive treatment To assess the relative impacts of vaccination, we envisaged three possible vaccines. The first vaccine was “ideal", protecting 100% of the individuals vaccinated and providing life long immunity. A vaccine with 70% efficacy was also tested, protecting 7 out of every 10 individuals vaccinated for life. Finally, a vaccine with waning immunity was analyzed. Individuals vaccinated with the third vaccine would all be protected, though on average the protection would wane after ten years. We compared the trajectories and end points of these vaccines with the treatment guidelines previously discussed. An ideal vaccine can reduce incidence and prevalence of HIV by up to 100% over aggressive treatment with universal coverage. In addition, it can reduce incidence by 46% and prevalence by 80%, even with a modest coverage of 40%. Survival with an ideal vaccine is 60% lower with an ideal vaccine at 40% coverage. This can be explained by a necessary assumption in

50


3.3 results

our model. To compare vaccination and treatment effectively, treatment was removed from the vaccination analysis. Thus, at low levels of coverage, individuals who are not vaccinated cannot recieve treatment. Hence, there is a larger reduction in survival during the time that a reservoir of immunity develops in the population. This issue is resolved at high levels of coverage (Fig. 3.4 Top Row, Table 3.8). A vaccine with partial efficacy (70% in this model) can also be more effective than treatment, depending on coverage. For example, with universal coverage, a 70% efficacious vaccine can lead to a 46% reduction in incidence over aggressive treatment and a 95% reduction in prevalence. Even at 80% coverage, the partially efficacious vaccine would reduce incidence by 54% and prevalence by 92% over current treatment guidelines Fig. 3.4 Middle Row, Table 3.8). A vaccine with waning immunity is unable to control the pandemic in the long term, and is certainly less effective than both current and future treatment guidelines. Even with universal coverage, the vaccine results in an incidence that is 474% higher than aggressive treatment and a prevalence that is 106% higher than aggressive treatment Fig. 3.4 Bottom Row, Table 3.8). 3.3.3 Simultaneous Treatment and Vaccination Is the Most Effective Intervention In order to demonstrate the relative effects of vaccination and treatment, the analyses were conducted independently, meaning that the V compartment was removed when treatment was tested and the T compartment was removed when vaccination was tested. As a result, with partial efficacy or coverage, there was a considerable population of susceptible individuals that could not receive treatment after infection and would continue spreading disease without any reduction in viremia. In addition, a number of years passed in which these individuals spread disease before the vaccinated population reached a critical mass to generate a reservoir of population immunity. As a result, the vaccine alone resulted in reductions in survival

51


3.3 results Ideal Vaccine

9

8

x 10

Ideal Vaccine

0.03

Incidence

Survival

4

2

Prevalence

0.025 6

0.02 0.015 0.01

20

60

80

Years

0 0

100

70% Efficacy

9

8

40

x 10

20

40

60

Years

80

Prevalence

Incidence

Survival

2

0.02 0.015 0.01

20

9

8

x 10

40

60

Years

80

0 0

100

10 Year Protection

20

40

60

Years

80

20

40

60

Years

80

100

100

80

100

70% Efficacy

0.2

20

40

60

Years

10 Year Protection

0.02 0.015 0.01

0.4 0.3 0.2 0.1

0.005 0 0

80

0.5

Prevalence

Incidence

Survival

2

60

Years

0.3

0 0

100

0.025

4

40

0.4

10 Year Protection

0.03

6

20

0.1

0.005 0 0

0.2

0.5

0.025

4

0.3

0 0

100

70% Efficacy

0.03

6

0.4

0.1

0.005 0 0

Ideal Vaccine

0.5

0 0

20

40

60

Years

80

100

0 0

20

40

60

Years

80

100

Vaccine Coverage 40%

80%

60%

100%

Treatment Guidelines No treatment

CD4+ = 350cells/mm3

CD4+ = 500 cells/mm3

Figure 3.4: Various types of vaccines were simulated using the HIV epidemiological model. First, an ideal vaccine with 100% efficacy and lifelong immunity was simulated (Top Row). An ideal vaccine can be even more effective than aggressive treatment, especially with high coverage, with increased survival and lower incidence and prevalence. If the vaccine efficacy is reduced to 70% (Middle Row), then the vaccine is still more effective than the current treatment guideline, given high coverage. On the other hand, a vaccine without lifelong immunity (Bottom Row) is not effective in controlling that AIDS pandemic in the long term. Each of the baseline measurements for treatment is provided for context: no treatment (solid gray line), current treatment guidelines (dashed gray line), and aggressive treatment guidelines (dotted gray line).

52


3.3 results

%∆Survival

%∆Incidence

%∆Prevalence

Coverage

350

500

350

500

350

500

Ideal

40%

8065

-4

-60

-95

-78

-46

-96

-91

-80

Ideal

60%

14269

68

-30

-99

-95

-87

-99

-98

-95

Ideal

80%

19502

129

-4

-100

-99

-98

-100

-100

-99

Ideal

100%

22497

164

11

-100

-100

-100

-100

-100

-100

70%

40%

2352

-71

-88

-58

92

368

-77

-51

9

70%

60%

5606

-33

-72

-77

4

154

-89

-77

-48

70%

80%

10446

23

-48

-90

-54

14

-96

-92

-81

70%

100%

15646

84

-23

-95

-78

-46

-99

-98

-95

10Y

40%

411

-94

-98

-23

249

752

-30

47

224

10Y

60%

830

-89

-95

-32

207

651

-40

26

179

10Y

80%

1468

-82

-92

-40

169

559

-48

9

140

10Y

100%

2397

-71

-88

-48

135

474

-56

-7

106

Table 3.8: Vaccines Reduce Incidence and Prevalence More Than Treatment Per cent change in survival, incidence, and prevalence of various vaccine candidates over current and aggressive treatment guidelines is shown. Values favorable to the vaccine are shown in blue, while values favorable to treatment are shown in red. Positive values are favorable for survival, while negative values are favorable for incidence and prevalence. -100 in the incidence column means that vaccination at the specified coverage (row) results in a 100% decrease in incidence over treatment at the specified level (column).

53


3.4 conclusions 9

−3

x 10

2

Incidence

Survival

6

4

2

0 0

20

40

60

Years

80

100

x 10

0.05

1.5

Prevalence

8

1

0.5

0.04 0.03 0.02 0.01

0 0

20

40

60

80

Years

100

0 0

20

40

60

Years

80

100

Vaccine Coverage 40%

80%

60%

100%

Figure 3.5: Model Simulation with Simultaneous Treatment and Vaccination Concomitant vaccination of susceptible individuals as well as treatment of infected individuals (at current treatment guidelines) dramatically increases survival and leads to the eradication of disease over time. This scenario also suggests that a dual strategy would dramatically reduce incidence and prevalence. The strategy is shown at various levels of vaccine coverage, and the no treatment control is shown for context (solid gray line)

over treatment in the short term. When we repeated the analysis combining treatment with ART at current guidelines and prevention with an ideal vaccine, the results were striking. Incidence and prevalence were both reduced by 100% with universal coverage and by 89% over the aggressive treatment guideline at 40% coverage. While a vaccine is the most effective intervention against HIV, it must be used in conjunction with treatment to ensure that infected individuals do not spread disease while susceptible individuals receive protection from the vaccine (Fig. 3.5, Table 3.9). 3.4 conclusions There is little doubt that a vaccine would have a tremendous impact on the progression of the AIDS pandemic over time. Through mathematical modeling, we demonstrate that this impact would be significantly greater than even the scaling up of antiretroviral therapy prescription and coverage. Here, we show the results of a series of simulations performed on a deterministic, compartmental model of HIV transmission based on a system of differential

54


3.4 conclusions %∆Survival

%∆Incidence

%∆Prevalence

q

350

500

350

500

350

500

0.4

22498

164

11

-99

-95

-89

-98

-95

-89

0.6

24602

189

21

-100

-99

-99

-100

-99

-98

0.8

25307

197

24

-100

-100

-100

-100

-100

-100

1

25617

201

26

-100

-100

-100

-100

-100

-100

Table 3.9: Vaccination and Treatment is More Effective than Aggressive Treatment Per cent change in survival, incidence, and prevalence of simultaneous treatment at current guidelines and ideal vaccine over current and aggressive treatment guidelines is shown. Values favorable to simultaneous vaccination and treatment are shown in blue, while values favorable to treatment alone are shown in red.

equations. We model the two major strategic interventions for HIV/AIDS: treatment with antiretroviral therapy and prevention with a prophylactic vaccine. We used current treatment guidelines (CD4+ = 350 cells/mm3 ) as well as a proposed aggressive guideline (CD4+ = 500 cells/mm3 ) in order to provide a baseline to measure vaccine impact. We subsequently modeled three potential vaccines: an ideal vaccine, a partially efficacious vaccine, and a vaccine with waning immunity. Our model, while simple, has yielded several key insights into intervention strategies for HIV. First, we demonstrate that antiretroviral therapy is effective in reducing the mortality from AIDS and the incidence of HIV, an observation that has been validated by the ability of ART to increase longevity and reduce morbidity. In our analysis, following the aggressive treatment guideline leads to a greater reduction in incidence and prevalence over the current treatment guideline and no treatment. Thus, we were able to validate the paradigm of treatment as prevention. However, it is important to note that this model assumes universal testing and accessibility of treatment as well as universal acceptance of and adherence to treatment. These conditions are not observed under real-world conditions. For example, the rate of testing in a South African cohort, while the percentage of those tested who actually sought

55


3.4 conclusions

treatment was 33%. Refusal of treatment can affect 1 out of 5 individuals affected with HIV, while dropout from treatment programs can affect 1 in 10 individuals 92 . Thus, while treatment may be biologically effective, problems in implementation may undermine its usefulness. While we could have added parameters in our model to compensate for accessibility and adherence, the global effect of leaving them out is to bias the results against the vaccine. Even, so the vaccine proved more effective than treatment in our analysis. Second, our model suggests that a vaccine remains the best hope to control the AIDS pandemic in the long term. An ideal vaccine would be the best possible solution. Without any treatment, it could reduce incidence and prevalence by 100% over aggressive treatment in 100 years. Furthermore, even with 40% coverage, significant reductions in incidence and prevalence could be achieved. A 70% efficacious vaccine would exceed the effectiveness of aggressive treatment as well, so long as coverage exceeded 80%. Finally, while a vaccine with waning immunity would not be as effective as current or aggressive treatment guideline scenarios, it could be a powerful part of an HIV control strategy if used in conjunction with treatment (Figs. 3.3, 3.4, and 3.5, Tables 3.7, 3.8, and 3.9). By that measure, we believe that the vaccine has relatively underperformed in our model due to unrealistic assumptions about treatment accessibility and adherence. It is important to note that these challenges in implementation would be significantly eased with a vaccine. Vaccines are generally far more cost effective than therapeutic treatments; they require fewer doses, and adherence is rarely a problem. Furthermore, vaccines do not cause the same side effects of drug toxicity that ART does because the mechanism of protection is mediated by a system that has already eliminated autoreactive elements and therefore does not harm the host. Finally, vaccines overcome the large problem of resistance because they prevent infection, the major driver of evolutionary change.

56


3.4 conclusions

A number of simplifying assumptions were made in our model that can be refined in future work. Apart from the assumptions regarding treatment, we assume a homogenous mixing of the population. In other words, any two infected people on earth have an equal probability of interacting and transmitting disease. One way around this issue is to perform the analysis among a likely cohort of susceptible individuals within a local population. Modeling accurate interactions between individuals at the global level is an exceptionally difficult task. Still, our results can be broadly applicable to generalized epidemics within smaller localities, although further modeling work will need to be done to demonstrate the specific levels of efficacy and duration required for effectiveness. In addition, in order to achieve a stable steady-state population, we set the crude birth rate and crude death rate equal to one another. In reality, the crude birth rate is almost twice the crude death rate, and the population of the world is rapidly expanding. It would be interesting to observe the effects of a growing population on the spread of disease under these various scenarios. For each compartment, we used infection rates derived from a study of HIV positive individuals in Rakai, Uganda, where the infection rate of HIV is far higher than the global average. We used determinations by the United Nations to determine the CD4 count at which HIV positive individuals would be started on ART and used this number to calculate the rate at which people would be enrolled. Furthermore, the force of infection used in this model calculated the risk of transmission per sex partner, while calculating the transmission per sex act would provide better estimates. Wherever possible, an attempt was made to bias the model against the vaccine and to use worst-case scenarios, in order to give vaccination a true acid test of effectiveness. We present a quantitative rationale for aggressive research in pursuit of an HIV vaccine. The global health impacts of this discovery would be nothing short of epochal in scope. Biologically, a vaccine that was highly efficacious and protected individuals for life would be far superior to aggressive treatment, even if treatment was massively scaled up. Furthermore,

57


3.4 conclusions

a vaccine would ease many of the implementation challenge that have made ART difficult to disseminate and implement around the world, especially in resource poor settings. While the need for a vaccine is clear, the road to develop one is still shrouded in many uncertainties. However, rapid progress and novel approaches are now being applied to this problem. The next chapter of this work discusses one such approach in detail, and its implications for vaccine design.

58


THE V3 EPITOPE: A TEMPLATE FOR VACCINE DESIGN

4.1

4

introduction

Though HIV is the most heavily studied virus in the history of infectious disease, no successful HIV vaccine has been developed to date. The inability of natural infection to confer sterilizing immunity poses a significant roadblock to vaccine development. In most infected individuals, HIV infection results in the production of numerous neutralizing antibodies against autologous virus roughly three months after infection 93 . Unfortunately, rapid mutation under the selective pressure of neutralization drives viral escape and renders these antibodies ineffective. The development of cross-reactive antibodies that can neutralize heterologous viral isolates is more rare and slower. Broadly neutralizing antibodies (bNabs) to HIV are observed in roughly 20% of the sera of infected individuals several years after infection 94,95,96 . The relatively high viral load observed in infected individuals that produce a broadly neutralizing humoral response suggests that the development of these antibodies requires prolonged and sustained exposure to diverse primary viruses 95 . Large screens of infected individuals have revealed that 1% of HIV infected individuals produce exceptionally broad and potent neutralizing responses, and several bNAbs have been isolated from these individuals 97 . The ability of bNabs to elucidate epitopes for immunogen design is an area of intense study. Interestingly, passive administration of bNabs have shown protection against intravenous and vaginal challenge with chimeric simian-human immunodeficiency virus (SHIV) in non-human primate models 98,99 . While this protection does not necessarily indicate that antibody mediated neutralization is a mechanistic correlate of immunity to HIV, it does present an attractive tool to explore in the context of rational vaccine design.

59


4.1 introduction

The aim of rational vaccine design is to identify the epitopes recognized by HIV-specific bNabs and reconstitute those epitopes in safe and effective immunogens, with the hope that these immunogens will elicit bNabs de novo in uninfected individuals. Here, we discuss the identification and characterization of several HIV-specific bNabs, present an algorithm to determine their usefulness for vaccine design, and specifically elaborate on the epitope specificity of a power family of bNabs. A handful of these individuals have produced exceptionally potent neutralizing antibodies, making them elite neutralizers 100 . Unfortunately, most elite neutralizers are very sick, supporting the view that the development of bNabs requires prolonged and sustained exposure to the virus. The ability of bNabs to protect from infection challenge in non-human primate models has been a promising development that has strongly implicated bNabs as mechanistic correlates of immunity against HIV 98,101 . If this is the case, than the ability to elicit neutralizing antibodies prior to infection would exhibit a protective effect. Thus, the discovery and development of bNabs isolated from infected individuals show considerable potential in guiding future vaccine design. 4.1.1 The First Broadly Neutralizing Antibodies The first bNab discovered was b12, which targets the CD4 binding site (CD4bs) on gp120. b12 neutralized 75% of clade B isolates, and fewer isolates from other clades. b12 is able to target the CD4 binding site due to a long "finger-like" protrusion on the CDR H3 that accesses a recessed cleft of the CD4 binding site. Furthermore, protrusions on gp120 fit between CDR’s on b12, resulting in a complementary surface binding that defies the usual “flat" epitopes of antibody recognition. b12 binds trimeric gp120, an essential feature of all bNabs. This suggests that the native trimer shields non-neutralizing epitopes that are exposed on monomeric gp120 units. This is thought to be the reasoning behind the failure of monomeric gp120 to

60


4.1 introduction

show efficacy in previous vaccine trials 67,68 . While b12 is an interesting antibody and certainly provides a feasible epitope to model in immunogen design, structural complications may reduce its effectiveness. For example, the width of the antibody is twice that of the CD4 receptor, restricting the fit and requiring increased precision to achieve neutralization 75,102 . b12 has since been surpassed by several other antibodies with greater breadth and potency. In addition, because the antibody was isolated using surface phage display, the pairings of heavy and light chains may not be correct 103 . Nevertheless, it provided the first conformation that a single antibody could be effective against a variety of viral isolates. The low affinity of peptide-glycan interactions, usually in the micro-molar range, was thought to be too weak to mediate neutralization, compared to the nano-molar affinity of peptide-peptide interactions (Devin Sok, personal communication). Also, because glycosylation is a post-translational mechanism managed by host cellular machinery, it was believed that the immune system would have trouble recognizing glycan epitopes as non-self. Thus, the discovery of the bNab 2G12, which exclusively bound glycan epitopes on the outer domain of gp120, challenged intuition. 2G12-mediated neutralization is dependent upon glycans at positions 295, 332, 386, and 392. Structural data has revealed that 2G12 harbors a unique domain swap between its variable heavy (VH ) regions that results in a crossed-over, vertical configuration. The loss of a ball-and-socket joint that rigidifies the “Y" shape of antibodies gives the VH domains more flexibility to form multivalent interactions with surface glycans. While 2G12 is not an effective bNab by today’s standards, it helped reveal that weakened affinity of peptide-glycan interactions can be partially overcome by interacting with several glycans at once. However, the bizarre structural change of 2G12 foreshadowed very limited potential to elicit a similar antibody de novo 75,104 . In addition to antibodies targeting epitopes on gp120, antibodies that targeted gp41 have been discovered as well. 2F5 and 4E10 both target epitopes on the membrane proximal ex-

61


4.1 introduction

ternal region (MPER) of the gp41 ectodomain. This is the portion of the protein that sits outside of the host derived membrane surrounding the virion and supports the attachment to gp120. Because gp41 is responsible for membrane fusion and viral entry, these antibodies are believed to inhibit the fusion of host and viral membranes as opposed to the binding of CD4 by gp120. Both 2F5 and 4E10 are able to neutralize a diverse panel of HIV strains across various clades suggesting that this MPER epitope is well conserved across HIV subtypes. Additionally, this alpha-helical epitope seems to be structurally simpler, ostensibly making it a more feasible target than the topographically complex CD4bs or glycan shield. Unfortunately, while 4E10 has broad neutralizing ability against almost all virus subtypes, it lacks the potency of stronger bNabs and requires concentrations above the physiological norm to be effective 75,105,106 . Furthermore, 255 and 4E10 exhibit autoreactivity to a host phospholipid, presenting additional problems in re-elicitation due to the deletion of B cell responses against host antigens 107 . 4.1.2

The Next Generation: Increased Breadth and Potency

The discovery of antibodies that had significant but limited ability to neutralize viruses in vitro prompted additional studies that used novel high throughput methods of B cell sorting and antibody detection in order to discover and characterize neutralizating antibodies that were broader and stronger and could serve as better templates for vaccine design 74,108 . The broadly neutralizing antibody problem was approached from both pathogenic and immunological perspectives. Using structural data as a guide, Mascola, Nabel, and colleagues applied computational tools to design a synthetic gp120 core stabilized in a CD4-bound configuration (resurfaced stabilized core 3), and used this probe to search for antibodies with CD4bs specificity. It was through this methodology, that the extremely broad antibody VRC01, and a number of bNabs with CD4bs epitope specificity were isolated 108,109,110 .

62


4.1 introduction

Meanwhile, Walker and colleagues screened the sera of 1800 individuals around the world infected with HIV to select donors for bNab recovery. Immunoglobulin G (IgG) from roughly 30,000 activated memory B cells from one of those donors was analyzed with a high-throughput assay for binding to monomeric and trimeric Env proteins as well as primary viral isolates. From this methodology, the two broad and potent antibodies PG9 and PG16 were isolated. PG9 and PG16 recognize variable loops 1 and 2 (V1/V2), again igniting interest in the glycan shield of HIV as a possible epitope of interest. Because both of these antibodies preferentially bind Env trimer, it is believed that they recognize a quaternary epitope that is stabilized by trimerization 100 . Specifically, both PG9 and PG16 were dependent upon N-linked glycans at positions 160 and 166 for productive neutralization 111 . In the screen performed by Walker and colleagues, 1% of the individuals showing the highest neutralization breadth in sera were designated as “elite neutralizers." The same highthroughput analysis that yielded PG9 and PG6 was applied to the top four donors among the elite neutralizing group. PGT121-123, PGT125-128, 130, 131, PGT135-137, and PGT141-145 were isolated from the four donors, respectfully. A variation in both breadth and potency was observed in these bNabs, but a number of them demonstrated potency that was 10 times higher than observed in current generation antibodies such as VRC01, PG9, and PG16 (PGT121-130,135). Competition with a variable loop 3 (V3)-specific antibody and inability to bind gp120 lacking V3 revealed that PGT antibodies 121-131 were V3-specific. Glycan arrays indicated that PGT 125-128 and 130 bound glycan epitopes, and the elucidation of a crystal structure of PGT128 bound to gp120 has since demonstrated binding with glycans at positions 301 and 332 (Fig. 4.1 32 . Interestingly, PGT121-123 failed to bind glycans on the array, despite requirement of N332 for neutralization. This discrepancy could either indicate that the glycan array poorly resembles glycan binding in vivo or that the glycan at position 332 plays an indirect role in neutralization by PGT121-123. Notably, PGT121 and 128 are the two

63


4.1 introduction

most potent antibodies discovered to date, and both have been shown to require N332 for neutralization. Because these two antibodies developed independently from two donors, the demonstration of a conserved epitope specificity would be promising for vaccine design 100 Now, several bNabs have been already defined and several more are being discovered. A complete review of the bNabs discovered thus far as well as an organizational nomenclature for them is provided by Kwong & Mascola (2012) 108 . Each of them constitutes a data point that can be used to generate a more complete picture of the immune response following HIV infection. While the mechanisms of protection from HIV infection remain unknown, the correlation of protection shown by bNabs through passive administration studies makes them promising tools for immunogen design 98,99 . Using a rational vaccine design approach to HIV, we hope to identify the best possible bNabs produced from infection, characterize the epitopes these bNabs recognize, and reconstitute them as immunogens. Here, we demonstrate an attempt to follow this methodology for the V3 epitope. The aims of our study were two-fold. First, we wanted to conduct a multivariate analysis on the various bNabs discovered in order to determine the epitope specificities most amenable to vaccine design. We determined that the V3 loop was the most promising candidate due to strong performance of V3-specific antibodies, specifically PGT121 and PGT128. Second, we desired to map the epitope of PGT121 and its clonal variants PGT122 and PGT123. Because no crystal structure exists for these antibodies, we employed single and double mutagenesis experiments and determined that PGT121 is dependent upon N332 for neutralization, validating previous studies. Interestingly, we also found a secondary dependency on N301, suggesting a possible conserved epitope specificity with PGT128. The possibility that distinct pathways of bNab development converge on a specific epitope, and that the bNabs produced against this epitope are highly effective are promising steps towards the eventual design of an HIV vaccine.

64


4.1 introduction

A Fab PGT-128

B N301 N301 V3 loop

N332

CDR H3 CDR H2

gp120

N332

Figure 4.1: PGT128 Binding to gp120 is Mediated by N301 and N332 This figure has been adapted from Pejchal et. al. (2011). 32 The authors determined the crystal structure of gp120 bound to the Fab of PGT antibody 128 at 1.29. Ă… . (A) While our experiments used PGT antibodies 121-123, the importance of the glycans at positions 301 and 332 in PGT121 mediated neutralization makes PGT128 an interesting model to study. PGT128 binds N301, N332, and the V3 loop of gp120. All of these sites are important for virus neutralization. (B) A closer look at the binding between the two glycans show that the glycans dock the Fab between them and the V3 loop is studded between the CDR H2 and CDR H3 of PGT128, showing the extensive contour mapping that occurs between antibody and antigen. We believe that a similar method of epitope recognition is utilized by the antibody PGT121.

65


4.2 materials & methods

4.2 materials & methods 4.2.1

Multivariate Antibody Analysis

Antibodies were assessed according to three criteria: breadth, potency, and germ line similarity. These data were obtained from the literature 74,100,108,110 . Whenever discrepancies were found, the most recent data available was utilized. Breadth was measured as a percentage of viral isolates neutralized on an in vitro panel. Potency was assessed by measuring the mean inhibitory concentration (IC50 ) of the antibody. This is the concentration of bNab required to neutralize 50% of the virus. log10 IC50 values were used in order to measure fold differences in potency. Germ line similarity was measured by observing the degree of mutation from a germ line precursor exhibited by each bNab. Per cent mutation in the nucleotides of the variable heavy chain (VH ) was used when available. Otherwise, per cent change in amino acids was used. In order to analyze the combined effects of the three variables to determine their usefulness in vaccine design, an objective function was designed to optimize a score based on the maximization of breadth (B), potency (P), and germ line similarity (M) (Eq. (8)). In order to do this, the objective function maximized the per cent of strains neutralized, minimized log IC50 values (as more potent bNabs require less concentration to neutralize virus), and minimized per cent mutation from the germ line. The disparity in the measurement units of the three variables, as well as the variance of the data precluded optimization without normalization. In order to normalize the data, each of the three variables was divided by the range of that variable for all bNabs studied. This allowed the objective function to measure the contribution of each variable equally.

66


4.2 materials & methods

Xi = c1

Bi log10 Pi Mi − c2 − c3 Range(B) Range(log10 P) Range(M)

(8)

where : B = Neutralization breadth of bNab i P = IC50 of bNab i M = % Mutation from Germline of bNab i The multipliers [c1 , c2 , c3 ] were included to allow users to weight the importance of each variable in the optimization as necessary. In this analysis, each variable was weighed equally. As a result, the multipliers were silenced by setting c1 = c2 = c3 = 1. 4.2.2 Virus Selection and Mutation HIV virus strains were chosen to represent various geographic regions and viral subtypes. For all experiments, the reference strain HxB2 was used to align sequences and define mutations. For single mutation experiments, a three virus panel consisting of the Clade B viruses 92BR020 (92BR) and JRCSF and the Clade C virus IAVI C22 were used. For double mutation experiments, the 92BR and IAVIC22 viral strains were used, along with the Clade A virus 92RW024 (92RW), which is resistant to N332A mutation and serves as a negative control. A description of the Env strains used is provided in Table 4.1. All HIV envelope proteins in plasmids were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (NIH ARRRP). A replication defective plasmid containing all of the viral genes except env (pSG3 ∆env) was obtained from the laboratory of Dennis Burton (The Scripps Research Institute, La Jolla, CA). Single and double mutant Env viruses were created in order to map bNab epitopes. Single mutants were selected using a predictive computational model provided by N. Lance Hepler and colleagues (University of California, San Diego, La Jolla, CA). This model was tested

67


4.2 materials & methods

Name

Segment

Subtype

HXB2 92BR020 IAVI C22 JRCSF 92RW024*

gp160 (env) B gp160 (env) B gp160 (env) C gp160 (env) B gp160 (env) A

Origin

Year

Accession

Reference

France Brazil N/A United States Rwanda

1983 1992 N/A 1986 1992

GM984107 AY669718 N/A AY426125 AY669699

N/A 112

N/A 113 112

Table 4.1: Experimental Virus Panel Viruses from several geographical regions and subtypes were chosen. Single mutation analysis was performed using the viral strains 92BR, IAVI C22, and JRCSF. *Double mutation analysis was performed using the viral strains 92BR, IAVI C22, and 92RW. The HxB2 strain was used as a reference.

using data from a panel of 109 diverse HIV-1 pseudo viruses representing large geographical diversity 114 . 10 mutations predicted by the model were selected for this study. While the mutation N332A was also suggested by this model, it was treated independently as a control given its known implication in PGT antibody neutralization. The mutations selected are shown in Table 4.2. Double mutants were created by introducing mutations into env plasmids already containing an N332A mutation. The mutations introduced were asparagine residues that were sites for N-linked glycosylation and had been implicated in neutralization by other bNab. Each mutation was from asparagine to alanine (N → A) in order to remove the glycan at that position without creating additional perturbations in the structure (Table 4.2). 4.2.3

Preparation of Env Mutants

Single point mutations were introduced into Env plasmids through site-directed mutagenesis. Primers were obtained from ValueGene Inc., and designed to introduce the nucleotide mutations shown in Table 4.4. using the QuickChange protocol (Agilent Technologies) Mutagenized Env plasmids were sequenced in order to confirm that the correct mutation had

68


4.2 materials & methods

Name

Original

Mutant

K46R Lysine Arginine V85V Valine Alanine N229R Asparagine Arginine T244S Threonine Serine H330H Histidine Alanine N302N Asparagine Alanine N325D Asparagine Aspartic Acid R419R Arginine Alanine S440A Serine Alanine N332A Asparagine Alanine

Position

gp120 Location

46 85 229 244 330 302 325 419 440 85

C1 C1 C2 C2 V3 V3 V3 V4/C4 C4 V3/C3

Table 4.2: Single Mutations in EnvVarious mutants predicted by a computational model were selected for this analysis. These mutations span a wide range on the gp120 structure going from constant region 1 (C1) to constant region 4 (C4) and encompassing variable loop 3 (V3) and variable loop 4 (V4). The mutation N332A was used as a positive control because the glycan at position 332 has been implicated in neutralization and binding by PGT antibodies 121-123.

Name

Original

N197A Asparagine N234A Asparagine N241A Asparagine N295A Asparagine N301A Asparagine N362A Asparagine N386A Asparagine N392A Asparagine

Mutant

Position

gp120 Location

Alanine Alanine Alanine Alanine Alanine Alanine Alanine Alanine

197 234 241 295 301 362 386 392

V2/C2 C2 C2 C2 V3 V4 V4 V4/C4

Table 4.3: Double Mutations in Env Mutations were introduced into env plasmids containing the N332A mutation. Mutations were specifically selected to remove N-linked glycans. Mutations spanned a wide range on gp120, spanning constant regions 2 and 4 (C2, C4) and variable loops 2-4 (V2, V3, and V4).

69


4.2 materials & methods Name

Strain

Mutation

Position

Name

Strain

Mutation

Position

H330H

HxB2

GA → GC

988, 989

R419R

HxB2

AG → GC

1255, 1256

K46R

N229R

N302A

92BR020

985, 986

92BR020

1240, 1241

IAVI C22

940, 941

JRCSF

1234, 1235

JRCSF

988, 989

HxB2

HxB2

A→T

730

137

92BR020

730

92BR020

134

IAVI C22

685

JRCSF

137

JRCSF

727

HxB2

A→G

T244S

AA → CG

685, 686

V85V

HxB2

T→C

254

92BR020

685, 686

92BR020

251

IAVI C22

640, 641

IAVI C22

251

JRCSF

682, 683

JRCSF

254

HxB2

AA → GC

904, 905

N332A

HxB2

AA → GC

994,995

92BR020

904, 905

92BR

991,993

IAVI C22

859, 860

IAVI C22

946,947

JRCSF

901, 902

JRCSF

988,989

Table 4.4: Nucleotide Changes for Site-Directed Mutagenesis The following genetic mutations were introduced into the Env plasmids in order to generate the desired mutants. Primers were designed using the QuickChange Primer Design tool by Agilent Technologies and inputting the nucleotide changes in this table.

been introduced (Eton Biosciences Inc). Mutant Env were then transformed into competent E. coli containing a resistance marker to the antibiotic carbenicililn that was rescued by transformation with plasmid. Transformed cells were grown and concentrated plasmid was obtained though plasmid purification (Qiagen). Purified plasmids were stored at 4◦ C. Double mutants were produced by introducing single mutations into plasmids harboring the N332A mutation (previously described). Mutagenesis and plasmid preparation was performed as previously described.

70


4.2 materials & methods

4.2.4 Pseudovirus Production Pseudoviruses were produced by co-transfection of Env plasmids with a pSG3 vector containing the HIV genome lacking Env (pSG3-∆Env) as previously described 115,116,117 . Mammalian 293T cells were cultured in 6cm round circular dish at a concentration of 1.5 × 106 cells per plate. For each mutant, 5µg of Env plasmid containing the desired mutation was mixed with 10µg of pSG3 vector with 50µL of FUGENE Transfection Reagent (Promega) in OptiMEM medium (Invitrogen). This mixture was incubated for 30 minutes before being added drop wise to the 293T cell cultures. Virions in the media were incubated at 37◦ C and harvested after 72 hours. The pseudovirions produced from this procedure were competent for only one replication cycle because the genomic component lacked Env. However, there remains a possibility that mutation of the stop codon in the Env gene of pSG3 could cause reversion into replication competent viruses (Fig 4.2). 4.2.5

Neutralization Assay

In order to determine bNab neutralization of viruses, the monoclonal bNabs PGT121, PGT122, and PGT123 were used in single mutation experiments. PGT121 alone was used to test double mutant viruses. These antibodies were isolated by Walker et. al. (2011), and were provided courtesy of the laboratory of Dennis Burton (The Scripps Research Institute, La Jolla, CA) 118 . Neutralization assays were essentially performed as previously described 115,116,117 . TZM-bl cells were plated in 96-well flat-bottom culture plates at a concentration of 10,000 cells per well. TZM-bl cells express the CD4 cell surface receptor as well as the CXCR4 and CCR5 co-receptors. In addition, the cell line contains a reporter luciferase gene whose expression is activated in the presence of HIV Tat. Antibodies were added to cell cultures at a concentration of 8ng/µL for Env single mutants and 20ng/µL for Env double mutants along with 8 serial dilutions diluted 4-fold and a virus only control. Env mutant viruses were subsequently added

71


4.2 materials & methods

A

env

C Δe HIV nv

Internal proteins

Env Create lipid membranes around plasmids

HIV Δenv env

D

B

Figure 4.2: Pseudovirus Production Replication defective pseudo viruses are produced by cotransfecting a plasmid containing Env with a plasmid backbone containing the HIV genome with Env deleted. The separate expression of Env and the rest of the HIV genes results in pseudovirions that are competent for one round of replication. These virions express trimeric Env with the introduced mutations and can be used to conduct in vitro experiments of various Env mutants.

72


4.2 materials & methods

to antibody treated TZM-bl. Cell-antibody-virus mixtures were incubated at 37◦ C for 72 hours. 10µL of medium from each well was transferred into a new white solid 96-well plate and 10µL of Bright Glo reagent was added to each well. Luminescence was recorded using a luminometer. Per cent neutralization was measured by [Luminescence without antibody − Luminescence with antibody]/Luminescence without antibody × 100 (Fig 4.3). The IC50 was defined as the concentration of antibody (ng/µL) required in order to achieve 50% neutralization. Lower IC50 values correspond to a more potent antibody (Fig. 4.3).

73


4.2 materials & methods

A

C Env

mAbs

92BR 92TH 93IN 94UG IAVI C22 JR CSF

HIV pseudovirion

No neutralization

PGT-121 PGT-122 PGT-123

Antibodies

HIV genes

Plated TZM-bl

Tat

Tat

Nucleus

HIV genome CD4

D

B

CXCR4 CCR5

luc

Neutralization

Nucleus No light CD4

CXCR4 CCR5

luc

Figure 4.3: Neutralization Assay TZM-bl target cells express the genes necessary for HIV infection, such as the CD4 cell surface receptor and CCR5 and CRCX4 co-receptors as well as a luciferase reporter gene activated by HIV Tat. In the presence of HIV infection, these cells luminesce, thus neutralization can be measured by the reduction in luminescene. This reporter system has widely been used to detect neutralization of HIV by antibodies in vitro 115,116,117 .

74


4.3 results

4.3 results 4.3.1 The V3 epitope is an attractive template for immunogen design All of the bNabs against HIV target a limited number of epitope specificities, each of which is a candidate template for immunogen design. We wanted to determine which of these epitopes could make the best template based on the quality of the bNabs directed towards that epitope. Each antibody was plotted first along each variable separately and second as a composite coordinate of all three variables. Antibodies were door coded according to the Env site they target. CD4 binding site (CD4bs) bNabs were colored in green, V3 loop bNabs in blue, MPER bNabs in red, and V1/V2 loop bNabs in orange. The individual comparison of each of these variables shows that no one antibody or group of antibodies predominates in all three variables (Fig. 4.4A). On average, MPER bNabs exhibit the highest breadth, with antibodies 4E10 and 10E8 able to neutralize almost all viral subtypes. However, MPER antibodies exhibit very low potency, with the exception of 10E8, which exhibits moderate potency. CD4bs antibodies exhibit high breadth and moderate to high potency, but are significantly mutated from the germ line. V3 loop specific antibodies show incredibly high potency–up to ten times that of the best antibodies from other specificities. They show low to moderate mutation from the germ line. However, they show a lower breadth than other antibodies. Finally, V1 and V2 loop specific antibodies show both moderate breadth and potency, but are more similar to their germ line counterparts than other classes of antibodies. Overall, these data indicate a trade-off between breadth, potency, and mutation from the germ line. This is intuitive, as refining an antibody to be more broad and potent requires significant affinity maturation, which leads to variation from a germ line precursor. In order to determine which antibody or antibody class was the best, a composite data point was constructed for each bNab using the data from each variable and plotted on a three-dimensional plot, with each

75


4.3 results

A

PGT135 2G12

PGT121

PGT128

b12

20

30

40

50

60

70

80

B

VRC01

VRC−CH31

PGT145 3BNC117

2F5 10

PG9 VRC−PG04

90

4E10 10E8 100

45

PGT135 PG9 PGT128

3BNC117

PGT121

VRC01

2G12

PGT145

VRC−CH31

b12

VRC−PG04 10E8 −2

2F5 4E10

−1

10

0

10

1

10

10

Potency IC50 PGT135 PG9 4E10

PGT128

% Mutation from Germline

Breadth (%)

40 2G12

35

3BNC117

30

VRC−PG04

20

VRC−CH31

PGT135

2F5

PG9

4E10

15 PGT128 10 0

2G12

VRC01

b12

25

PGT145 PGT121 1

10

20

0

10

40 2F5

PGT145 b12

10

15

VRC−CH31

VRC−PG04

10E8

PGT121 20

3BNC117

25

30

35

40

60

Brea

dth ( 80 %)

VRC01 45

−1

10 100

−2

10

Pote

ncy

IC 50

% Mutation from Germline

Figure 4.4: Optimization of Broadly Neutralizing Antibodies (A) Univariate analysis of all bNabs. bNabs targeting the CD4bs are shown in green, those targeting the V3 loop are shown in blue, those targeting the MPER are shown in red, and those targeting the V1/V2 loop are shown in orange. bNabs were ranked according to breadth, potency, and mutation from the germ line. (B) Multivariate analysis of all bNabs. Each bNab was given a composite point containing the three variables previously mentioned and then plotted on a three-dimensional plot containing each variable as an axis. bNabs with similar specificities appear to cluster on this plot.

axis corresponding to one of the variables measured (Fig 4.4B). Interestingly, bNabs directed towards a particular specificity tend to cluster on this plot, despite the wide range of breadth, potency, and mutation they exhibit when each of the variables is considered individually. This plot provides a qualitative analysis of how antibodies are distributed in relation to the three variables discussed. Next, we developed an objective function to quantitatively rank each of the bNabs discovered thus far by optimizing the variables breadth, potency, and mutation from the germ line. We assumed that each variable was equally important and weighted each equally in the model. To analyze fairly across variables with different units, we normalized each variable by dividing each data point by the range of the data. This allowed the optimization function to

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4.3 results

VRC−PG04 2G12

PG9 PGT145

PGT135 b12

VRC01 2F5 3BNC117

−0.8

−0.6

−0.4

−0.2

0

0.2

VRC−CH31 4E10

0.4

PGT121 PGT128

10E8 0.6

0.8

1

Antibody Score

Figure 4.5: Antibody Score Plot This plot demonstrates a quantitative ranking of all bNabs produced by the objective function previously described. Antibodies are plotted here from worst (left) to best (right).

consider units fairly. The optimization function produced the output shown in Figure 4.5 and Table 4.5. PGT128 and PGT121 scored highest, with scores of 0.94 and 0.91, respectively. Interestingly, the top five scoring bNabs were all from the PGT class, and specifically from two donors. PGT121-123 are clonal variants from the same donor, as are PGT125-131. PGT121 and PGT128 are the most potent antibodies in each class, respectively. While V3 loop specific bNabs score highest, they also have the greatest range, with the first generation bNab 2G12 achieving the lowest score of -0.71. The highest MPER specific bNab is 10E8, with a score of 0.68, the highest CD4bs specific bNab is VRC-CH31, with a score of 0.65, and the highest V1/V2 loop specific bNab is PG9, with a score of 0.6. Our model suggests that the V3 epitope on gp120 was able to elicit antibodies with the best combination of breadth, potency, and germ line similarity in two distinct HIV infected individuals. Furthermore, it shows that each of these individuals produced several clonal variants with close-to-optimal scores. While further refinements

77


4.3 results Antibody

Score

Antibody

Score

Antibody

Score

Antibody

Score

PGT128

0.94

PG9

0.60

VRC01

0.33

PGT144

-0.10

PGT121

0.91

PGT142

0.58

VRC-PG04

0.26

VRC03

-0.11

PGT123

0.75

PGT127

0.56

PGT130

0.25

PGT131

-0.12

PGT126

0.74

PGT125

0.55

3BNC117

0.24

b12

-0.40

PGT122

0.73

PG16

0.54

PGT135

0.17

PGT137

-0.56

10E8

0.68

PGT143

0.50

PGV04

0.14

PGT136

-0.62

VRC-CH31

0.65

PGT141

0.48

2F5

0.12

2G12

-0.71

PGT145

0.61

4E10

0.48

NIH45-46

-0.07

Table 4.5: Ranking of Antibody Scores Antibody scores from objective function are shown here, sorted from largest to smallest.

in the optimization algorithm are necessary, these data suggest that the V3 epitope may be the most instructive in effective immunogen design. We hope to further investigate the mechanism through which PGT121-123 bind the V3 loop to observe similarities between PGT128 class antibodies and V3 specific antibodies in general. 4.3.2

PGT121 is dependent on both N332A and N301

Single env mutants were introduced into the viral strains 92BR, IAVIC22, JRCSF. High titers of virus were produced containing these mutations. While mutagenesis experiments were also conducted for the strains 92TH, 93IN, and 94UG, we were unable to produce a high titer of virus in these strains. Neutralization assays were then performed as previously described using an antibody concentration of 5ng/ÂľL along with 8 serial dilutions diluted 4 fold and a virus only control. This experiment was performed with the PGT antibodies 121, 122, and 123. The mutation N332A, which is known to inhibit neutralization in PGT121 was used as a positive control while WT virus which is fully neutralized by PGT121-123 was used as a negative control. The neutralization profiles of all of the mutations were analyzed to observe whether any of the mutations led to viral escape. None of the mutations significantly affected

78


4.3 results

neutralization, with the exception of N332A, which has already been implicated in PGT class antibody mediated neutralization in the literature (Fig 4.6). Thus, we confirmed previous reports that the glycan at position 332 is necessary for PGT121-123 neutralization. Next, we wanted to determine whether N332 is sufficient for PGT121-123 neutralization by increasing the concentration of antibody from 5ng/ÂľL to 20ng/ÂľL. Antibody saturation indicates whether the reduction in neutralization is partial or complete. Residues sufficient for neutralization result in complete loss of neutralization when removed, regardless of antibody concentration. We repeated neutralization assays for wild type (WT) env and N332A env in the strains 92BR, IAVI C22, and 92RW. We used 20ng/ÂľL of the antibodies PGT121-123 with 8 serial dilutions diluted 4-fold and a virus only control in order to determine a complete neutralization curve for the N332A mutation. We calculated the fold difference in IC50 in order to quantify the change in neutralization when the mutation was introduced. The neutralization curves indicate that the removal of the glycan at position 332 partially reduces neutralization by the antibodies PGT121-123. The quantitative data supported this observation. The IC50 of 92BR ranged from roughly 4 to 200 times higher in N332A mutants over the wildtype while the IC50 of IAVI C22 ranged from roughly 70 to a 1000 times higher in N332A mutants over the wild type. On the other hand, the fold-difference between N332A and WT in the 92RW strain ranged from roughly 4 to 50. Thus, we determined that 92RW is an N332A resistant strain and can be used as a negative control in future experiments. Interestingly, we determined that the N332A mutation does contribute to viral neutralization but is not sufficient for neutralization by PGT121. As a result, we sought to determine the remaining elements of the PGT121-123 epitope. Due to the similar effects on N332 exhibited by the antibodies PGT121-123, we chose to focus on PGT121, the most potent clonal variant of that set. We attempted to assess the effect of the simultaneous removal of two glycans on PGT121 neutralization. PGT128 has been

79


92BR

60 40 20

10-4

10-2

100

H330A N229R T244S V85A N332A WT

80 60 40 20 0 10-6

102

10-4

H330A K46R N229R R419A T244S V85V BR N332A WT

60 40 20

10-4

10-2

100

Per Cent Neutralization

Per Cent Neutralization

92BR

80

0 10-6

102

H330A K46R N229R R419A T244S V85V N332A WT

60 40 20

10-2

100

PGT-123 (ng/μL)

102

Per Cent Neutralization

Per Cent Neutralization

92BR

10-4

60 40 20 0 10-6

10-4

H330A N229R T244S V85A N332A WT

80 60 40 20 0 10-6

10-4

10-2

100

H330A N229R T244S V85A N332A WT

60 40 20

10-4

10-2

100

PGT-123 (ng/μL)

102

H330A K46R N229R R419A T244S V85A N332A WT

80 60 40 20 0 10-6

10-4

10-2

100

102

PGT-122 (ng/μL)

80

0 10-6

100

JRCSF

100

102

IAVIC22

100

10-2

PGT-121 (ng/μL)

PGT-122 (ng/μL)

80

0 10-6

H330A K46R N229R R419A T244S V85A N332A WT

80

102

IAVIC22

100

PGT-122 (ng/μL)

100

100

JRCSF

100

PGT-121 (ng/μL)

PGT-121 (ng/μL)

100

10-2

Per Cent Neutralization

0 10-6

IAVIC22

100

Per Cent Neutralization

H330A K46R N229R R419A T244S V85V N332A WT

80

102

Per Cent Neutralization

100

Per Cent Neutralization

Per Cent Neutralization

4.3 results

JRCSF

100

H330A K46R N229R R419A T244S V85A N332A WT

80 60 40 20 0 10-6

10-4

10-2

100

102

PGT-123 (ng/μL)

Figure 4.6: Single Mutant Neutralization Curves Single point mutations were introduced into plasmids containing the HIV Env gene using site-directed mutagenesis. Mutant Env plasmids were then cotransfected with plasmids containing an Env-deleted HIV genome backbone (HIV∆Env). Neutralization of mutant viruses was then assessed after treatment with PGT antibodies 121, 122, and 123 in order to determine which residues were critical in antibody recognition and binding. Here we show that none of the mutants produced led to a knock-out of neutralization with the exception of N332A, a mutation that removes the glycan at position 332. This glycan has already been found to be conformationally important for PGT121-123, even though these antibodies primarily bind peptide epitopes. These data suggest that the computational model will require additional refinement before it can be used as a predictive tool for antibody escape.

80


92BR

100

Per Cent Neutralization

Per Cent Neutralization

4.3 results

PGT-121 WT PGT-122 WT PGT-123 WT PGT-121 N332A PGT-122 N332A PGT-123 N332A

80 60 40 20 0 10-4

10-2

100

102

60 40 20 0 10-4

Fold Difference in IC50

Per Cent Neutralization

100

102

N332A vs. WT

92RW PGT-121 WT PGT-122 WT PGT-123 WT PGT-121 N332A PGT-122 N332A PGT-123 N332A

60 40 20

10-2

10-2

Antibody Concentration

80

0 10-4

PGT-121 WT PGT-122 WT PGT-123 WT PGT-121 N332A PGT-122 N332A PGT-123 N332A

80

Antibody Concentration

100

IAVIC22

100

100

102

1500 1000 500

PGT-121 PGT-122 PGT-123

300 200 100 0

92BR

IAVIC22

92RW

Antibody Concentration

Figure 4.7: Neutralization of N332A and WT Virus by PGT Antibodies The neutralization of N332A and WT virus was observed at high concentration to determine whether N332A mediated neutralization single-handedly. The results suggest that other residues are a part of the PGT epitope as well.

92BR

IAVIC22

92RW

78.95 4.30 1077.94

4.54 21.51 50.18

PGT121 4.29 PGT122 279.86 PGT123 205.02

Table 4.6: Fold Difference in IC50 for N332A and WT Virus Neutralization of WT and N332A mutant viruses was assessed for the three virus strains 92BR, IAVI C22, and 92RW using a high concentration of PGT121. The data indicate that N332A results in partial but incomplete neutralization of 92BR and IAVIC22 while 92RW is resistant to N332A.

shown to interact with glycans at positions 301 and 332, and we wanted to observe whether a two-glycan system was conserved in the PGT121 family. Double mutants were generated by

81


N332A N197A N332A N301A N332A N362A N332A N386A N332A N392A N332A WT

80 60 40 20 0 10-4

10-2

100

102

PGT-121 (ng/μL)

100

IAVIC22 N332A N295A N332A N362A N332A N386A N332A N392A N332A WT

80 60 40 20 0 10-4

10-2

100

PGT-121 (ng/μL)

102

Per Cent Neutralization

92BR

100

Per Cent Neutralization

Per Cent Neutralization

4.3 results 92RW

100

N332A N234A N332A N241A N332A N295A N332A N339A N332A N392A N332A WT

80 60 40 20 0 10-6

10-4

10-2

100

102

PGT-121 (ng/μL)

Figure 4.8: Double Mutant Neutralization CurvesNeutralization of double mutants containing N332A and a secondary glycan removing mutation was compared to N332A alone and WT virus using a high concentration of PGT121. Among all mutants tested, only N301A showed an appreciable reduction in neutralization in the strain 92BR. This mutant was not produced in either IAVI C22 or 92RW.

introducing single mutations into env plasmids containing the N332A mutation. Mutation sites were selectively chosen to be asparagine residues that were sites of N-linked glycosylation in order to analyze the effects of glycan removal on PGT121 binding. Thus, along with position 332, we removed glycans at positions 234, 241, 295, 301, 339, and 392. We used a concentration of 20ng/µL of antibody as well as 8 four-fold serial dilutions and a no-antibody control, as this concentration had shown us a complete neutralizing curve in the previous experiment. Interestingly, while most of the double mutants exhibited a neutralization profile consistant with the single N332A mutant, the N301A N332A double mutant showed a further reduction in neutralization (Fig 4.8). The glycan at position 301 had not previously been implicated in neutralization by PGT121. To resolve this discrepancy, we repeated the experiment in the viral strain 92BR. This time, we generated the N301A N332A double mutants along with N301A and N332A single mutants and WT control. We anticipated that the further reduction of neutralization in the double mutant was the result of a cooperative effect between single mutants, and that each single mutant would partially reduce neutralization. Contrastingly, the data showed that the N301A mutation did not have an appreciable effect on neutralization. There was a negligible fold difference in IC50 between the WT and N301A strains. However, the N332A strain had an IC50 that was roughly 30 times higher than WT virus, and the double mutant N301A N332A

82


Difference in Neutralization vs. WT

92BR

100

WT N301A N332A N301A N332A

80 60 40 20 0 10-4

10-2

100

Fold Difference in IC50

Per Cent Neutralization

4.3 results

102

80 60 40 20 0

N301A

N332A

N301A N332A

PGT-121 (ng/ÎźL)

Figure 4.9: N301A Inhibits Neutralization When Combined With N332A (Right) The N301A N332A double mutant, N301A and N332A single mutants, and WT virus were all assessed for neutralization capability with a high concentration of PGT121. While the N301A mutant resembled WT in neutralization, when placed in conjunction with N332A resulted in reduced neutralization over N332A alone. (Left) The fold difference in IC50 is shown graphically. N301A N332A shows a significantly higher IC50 than both N332A alone and WT virus.

N301A

N332A

N301A N332A

0.653

28.7

60.9

Table 4.7: Fold Difference in IC50 of N301 N332 Double Mutant Fold difference in IC50 over WT is shown. Interestingly, the N301 mutation is only observed when combined with N332.

had an IC50 that was roughly 60 times higher than the WT virus and two times higher than N332A alone, suggesting a significant additional loss of neutralization. The N301A mutation was silent alone, but had an appreciable effect on neutralization by PGT121 in combination with N332A (Fig. 4.9). 4.3.3 The computational model requires additional refinement The initial selection of single mutants was derived from a computational model designed to predict what mutations would allow for viral escape. Out of the many mutations predicted by the model, ten were tested on three strains of virus (92BR, IAVI C22, and JRCSF) using

83


4.4 conclusions

three antibodies (PGT121, PGT122, and PGT123). None of the predicted mutations led to viral escape. We believe that additional refinements in this computational model will be required before it can be used as a suitable replacement to alanine scanning mutagenesis. Our data suggests that computational modeling should take into account the three dimensional distance between epitopes rather than viewing them as individual linear units. Computational modeling shows incredible promise and will probably be a critical component of rational vaccine design. Nevertheless, advances in computer science, such as the ability to solve three dimensional structures from linear sequences in polynomial time may be required before a complete three dimensional epitope analysis can be computationally performed. 4.4 conclusions A recent review by Kwong and Mascola (2012) poses the question: Does the highly effective PGT121, for example, like PG9 and PGT128, also utilize two glycans and a bit of Env-protein surface to affect its broad recognition? If so, then a single mechanism of recognition would be employed by all of the currently identified highly effective glycan-reactive HIV-1 neutralizers 108 . We believe that we can answer this question in the affirmative. Here we show that the broad and potent antibody PGT121 is primarily dependent on the glycan at position 332 and secondarily dependent on the glycan at position 301. Furthermore, we have shown that while the loss of neutralization is significantly higher in the double mutant than either N332 alone or WT, it is not complete. Therefore, there must be other residues involved in the epitope. Because we tested other glycans that PGT121 could possibly bind, our data support that the remaining interactions are peptides. As Kwong and Mascola suggest, our data suggest a conserved specificity for the two most potent families of the PGT class of antibodies, and possibly further glycan specific bNabs.

84


4.4 conclusions

Furthermore, we developed an objective function for the quantitative ranking of all of the bNabs discovered from the sera of infected individuals. Previous work showed that all of these antibodies attracted a limited number of epitope specificites on gp120 and gp41 111 . The goal of rational vaccine design is to use these specificities as templates and eventually present them to healthy individuals as immunogens. We believe that breadth, potency, and germ line similarity are three important characteristics that antibodies must have to ensure both that they will be able to exhibit a protective effect against pathogenic challenge and that they will be able to be re-elicited reasonably well. Even if bNabs have been discovered in infected individuals, there is a need to ensure that their re-elicitation has a high probability of protecting uninfected hosts from infection. Limited breadth, a high required concentration, and extensive hypermutation present limitations that may interfere with their development and ability to protect individuals. It is important to note that the breadth and potency data used in this experiment is derived from a number of in vitro panels, and it is possible that each is more sensitive or resistant to particular antibodies. Efforts to standardize breadth and potency information for the bNabs currently known will help refine this analysis. The results of this analysis suggest that PGT128 and PGT121 are the most effective antibodies, based on an optimization of breadth, potency, and germ line similarity. In addition, PGT clonal variants of these two antibodies make up the top five antibodies in this ranking. These data suggest that the variable loop 3 specificity is the strongest candidate for immunogen design. However, the model that we have used is relatively simple and additional refinements will need to be done in order to determine the relative utility of this epitope. Furthermore, while PGT128 and PGT121 comprise the first two spots on the ranking, V3 specific antibodies such as 2G12 and some of the weaker members of the PGT class comprise antibodies at the bottom. While 2G12 is a unique antibody due to its variable heavy domain swap, the PGT class antibodies show a high degree of variation in their overall effectiveness. Thus, if the

85


4.4 conclusions

V3 epitope is used in future immunogen design, it is important that it is configured to favor the elicitation of highly effective antibodies. The mechanism for PGT121 neutralization of HIV was previously unknown. In order to better elucidate the structural modifications we made, the mutants produced were visually analyzed in PyMol for PGT128 bound to a gp120 engineered outer domain (Fig. 4.10). While we require structural data in order to confirm a dual dependency on glycans at positions 301 and 332 for PGT121 mediated neutralization, the mutagenesis approach can help elucidate a mechanism for antibody mediated binding. We propose a novel mechanism for this neutralization. The eventual binding target of PGT121 is probably a peptide residue on the V3 loop. The glycan at position 332 provides an extension that can interact with the antigen binding fragment (Fab) of PGT121 that stabilizes the incoming antibody. The Fab then has some flexibility which allows it to come into contact with the glycan at position 301. These two interactions then guide the Fab to its ultimate peptide epitope. Without the glycan at 301, the glycan at 332 can mediate this stabilization relatively well. But without the glycan at 332, the Fab cannot achieve the secondary interaction with N301, therefore the N301 mutation is only seen in conjunction with the N332 mutation. Future studies will be required to corroborate our results in related antibodies and diverse viral strains. We were unable to produce high titers of virus in three strains of virus: 92TH021, 93IN905, and 94UG103 in the single mutant analysis. Furthermore, we were unable to produce high titers of virus harboring the N301A N332A double mutation in the IAVI C22 and 92RW strains. The ability to confirm our results in several strains strengthens the potential that the V3 epitope is conserved and significant on a variety of viral subtypes. The role of accessory interactions such as the interaction of PGT121 with N301 require additional elucidation. One interesting approach of study could involve observing the effects of N301 mutation in an N332 resistant strain such as 92RW. We hope to validate our results in the clonal variants of PGT121 and PGT128 (PGT122&123 and PGT125-131, respectively).

86


4.4 conclusions

A

B

WT

N301A mini V3 N301

A301

N332

N332

gp120

C

D

N332A

N301A N332A A301

N301

A332

A332

Figure 4.10: Structural Analysis of N301A and N332A Mutation This crystal structure was obtained from Pejchal et. al. (2011), who determined the crystal structure of gp120 bound to the Fab of PGT128 at a resolution of 1.29Ă… 32 . In the following diagrams, we have removed both the light and heavy chains of the antibodies and oriented the gp120 fragment such that the V3 loop is directly facing us. (A) WT gp120 shows N-linked glycosylation at positions 301 and 302, creating a pocket on the outer domain of gp120 that contains the V3 loop. (B) The mutagenesis of the asparagine residue at position 301 into an alanine removes the potential for glycosylation at this site, thus removing the glycan at this position. (C) The same mutagenesis of the asparagine at position 332 to alanine removes the glycan at position 332. (D) A double mutagenesis of the asparagines at both positions 301 and 332 results in the removal of glycans at positions 301 and 302 on gp120.

87


4.4 conclusions

The ability to leverage advances in computational techniques will be a critical component of future epitope discovery. The predictive model that we used failed to reveal any escape mutants. We believe the the fundamental problem with this model is that it only analyzes residues at a linear amino acid level, whereas epitopes are three dimensional and must be understood in that context. Short of that, we will require structural analysis by x-ray crystallography in order to gain a higher resolution image of antibody-antigen interactions. Unfortunately, structural data only provides a snapshot, whereas mutagenesis can provide additional insights into how antibodies bind antigens and instruct immunogen design. Parts of the PGT121 have not yet been discovered. A number of future directions will need to be pursued in order to better understand the V3 loop epitope and its significance. First, in addition to refining our current hypothesis by using different viral strains and antibodies, we hope to continue characterizing the PGT121 and PGT128 epitope. To begin this process, alanine scanning mutagenesis should be conducted with all of the peptide residues in between N301 and N332. Presumably these two glycans flank the antibody Fab, therefore we can expect significant binding sites to occur in between them. As stated earlier, it is important to consider the epitope in three dimensions, and any residues that could be relevant should be obtained from the available structural data of PGT128 bound to an engineered outer domain of the gp120 V3 loop 32 . After elucidating the epitope, we hope to better understand the contributions of the two glycans in PGT antibody binding. While this study focused on removing glycans to test neutralization escape, we propose the addition or lengthening of glycans as well as enhanced glycan stabilization at the V3 loop to determine if these changes increase either neutralization in vitro or immunogenicity. Finally, the broadest application of Kwong and Mascola’s comment stated earlier in this section is that it may suggest a conserved binding mechanism not only for antibodies directed towards the V3 loop, but also for antibodies targeting surface glycans 108 . Could a 2-glycan

88


4.4 conclusions

peptide epitope be context independent? An epitope that can produce antibodies targeting a variety of specificities is an interesting prospect in vaccine design. For example, if the binding mechanism of the V3 bNab PGT121 and PGT128 and the V1/V2 loop bNab PG9 is similar, then perhaps an general immunogen containing two glycans and a series of peptides could be reconstructed de novo that would be able to elicit multiple V1, V2, and V3 specific antibodies. Overall, we have provided one template for vaccine design and measured its relative effectiveness. Furthermore, we have demonstrated a conserved epitope specificity recognized by two independent B cell lineages. Whether or not the V3 loop will yield an effective immunogen is uncertain; however, we can be reasonably confident that if broadly neutralizing antibodies are found to drive vaccine-mediated immunity, the V3 epitope will be a significant player in vaccine design. A discussion of how vaccines may be reverse engineered from an immunological response, and whether the assumptions of the rational vaccine design attempted in this work are sound, is the subject of the next and final chapter of this work.

89


5

DISCUSSION

The aim of this thesis was to answer three questions about HIV vaccines and a broader question about vaccine development. Why have we failed to develop a vaccine? Do we still need a vaccine? And, what new approaches can we use to develop one? After answering these questions, we can ask Can we rationally design vaccines towards any disease? We have failed to develop a HIV vaccine because HIV is an extraordinarily complex pathogen and because natural immunity does not provide us with a sufficient model with which to develop a vaccine. This complexity can be explained by the molecular biology of the virus. The genomic organization of HIV and the slew of proteins it encodes endow the virus with an extraordinary capacity for mutation and a number of mechanisms to overwhelm host defenses, respectively. Because HIV is a retrovirus, it harbors a RNA genome that is chemically unstable and prone to mutation and recombination 119 . In addition, the enzyme that oversees the replication of this genome is error-prone and lacks proof reading mechanisms 28 . As a result, HIV possesses a mutation rate greater than any other known pathogen. This rapid rate of viral evolution allows the virus to continually evade immune capture and destruction. While the genome itself may be the source of HIV’s diversity, the information it carries provides the virus with additional capabilities. A series of viral accessory proteins degrade antiviral factors, downregulate antiviral mechanisms, and optimize conditions for viral replication and propagation within the host. Even the structural elements of HIV contribute to its complexity. Conserved regions of the Env surface protein of HIV are heavily glycosylated, resulting in a barrier around the surface protein that prevents immune detection. Furthermore, exposed parts of Env are hypervariable in nature, a structural manifestation of genomic instability.

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discussion

Thus, through the brute force of rapid and inaccurate replication, HIV has stumbled upon an elegant mechanism to evade, counter, and conquer the host. In addition to the complex molecular biology of the pathogen, we are perplexed both by what mechanisms of the host defense a vaccine should activate and how a vaccine should activate those mechanisms. The main difficulty in developing an effective vaccine against HIV is that natural infection does not result in sterilizing immunity and thus, cannot serve as an adequate model for vaccine design. Instead of mimicking the immune system, in itself a difficult task, an HIV vaccine must exceed it. For diseases such as smallpox and measles, antibodies have been defined as correlates of protection and can protect individuals from new infection 120 . On the other hand, HIV infection does not produce a reliable and effective humoral or cell-mediated immune response. HIV primarily infects T lymphocytes bearing the CD4 cell surface receptor. As a result the CD4+ T cell response is significantly diminished in infected individuals 94 . CD4 selectively interacts with the Major Histocompatibility Complex (MHC) class II peptide and facilitates T cell receptor recognition of foreign peptides presented on MHC-II by antigen presenting cells (B cells, macrophages, or dendritic cells). This interaction activates the CD4+ T cells and allows them to perform a number of effector functions, notably the activation of naive CD8+ T cells, which allows them to kill cells infected by virus, and the activation of naive B cells, which initiates class switching and antibody secretion 121 . To add immunological insult to injury, HIV preferentially infects HIV-1 specific CD4+ T cells, thus facilitating the demise of the very immune response directed towards it 122 . Much of the earliest parts of HIV infection are mediated by a single founder virus. At peak viremia during early infection, CD8+ T cells are primed to the founder virus and the viral load subsequently decreases. However, soon after, the virus mutates rapidly into a form that can

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escape the cytotoxic lymphocyte response and go on to establish infection 94 . From that point onward, CD8+ activity controls the viral set point but is unable to clear the infection 123,124 . Also, HIV is characterized by the absence of a broadly neutralizing antibody response in a majority of infected individuals. Intial B cell activity occurs a few days after the virus is detectable in serum. It takes another two weeks for free antibodies to be produced, first against gp41 and then against gp120, indicating that class switching and activation by CD4+ T cells occurs during this time. However, none of these antibodies are neutralizing. Only 12 weeks after infection do neutralizing antibodies to autologous virus appear. The onset of these antibodies results in selection of escape mutants and viral evasion of the humoral immune response 125,126,127 . A number of studies indicated that a minority of infected individuals develop broadly neutralizing antibodies after prolonged infection 128 . Finally, HIV rapidly establishes a latent viral reservoir within the host lymphatic system. Soon after crossing the mucosal barrier, infected CD4+ T cells as well as dendritic cells carrying the virus converge at draining lymph node, where high populations of susceptible CD4+ CCR5+ cells allow for rapid dissemination of infection throughout the lymphatic tissue. Specifically, the gut-associated lymphatic tissue (GALT) houses the majority of this lymphatic reservoir of infection and is the site of rapid depletion of CD4+ cells early during infection. Once the virus is maintained in the GALT and other lymphatic tissues, it is all but impossible for a vaccine to clear it. At this point, the best intervention is a reduction of the viral set point, which can be achieved either through treatment or a therapeutic vaccine that activates CD8+ responses 127,129,130 . Thus, in addition to surpassing the natural immune response, a vaccine must act quickly to prevent infection before a reservoir can be established. The second aim of this work was to determine whether a vaccine was still justified given the challenges in developing one and the advent of treatments that could reduce the viral load significantly and even help prevent disease. To that end, we developed a mathematical model

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of HIV transmission and assessed the survival and reduction in incidence and prevalence of disease that a vaccine would confer over treatment. The model was a compartmental, deterministic model that assessed the trajectory of the global AIDS pandemic for a period of 100 years. Our results strongly advocate the pursuit of a prophylactic vaccine for HIV. When considering interventions for the control and prevention of diseases, it is important to consider the merits from both a scientific and the logistical perspective. A vaccine is superior to treatment on both fronts. To compare the relative effectiveness of vaccination, we used two treatment scenarios, the current treatment guideline and an aggressive treatment guideline that has been proposed. We assumed universal access and adherence to treatment. Our model demonstrated that a perfectly efficacious vaccine could reduce both incidence and prevalence by 100% compared to the aggressive treatment scenario. Furthermore, even if only 40% of the population was vaccinated, the reduction in incidence and prevalence would be 46% and 80%, respectively, over the aggressive treatment scenario. A partially efficacious vaccine could show dramatic reductions in incidence and prevalence if a large proportion of the population was vaccinated. Furthermore, a vaccine, even with waning effectiveness, could be an important part of an elimination strategy if used in conjunction with treatment at current guidelines. Beyond scientific credentials, a policy must be feasible as well, taking into account real world conditions. Today, testing for HIV, access to treatment at the current guideline, and adherence to therapies is far from universal. Pursuing an even more aggressive treatment guideline would mean additional costs, increased toxicity, potentially increased morbidity, and possible rise in viral resistance. Thus, it is important to note that a vaccine is necessary to end the pandemic, is more effective than treatment, and is far more feasible to implement (Chapter 3). Given the challenges in developing an HIV vaccine and the exigent need for one, the third aim of this work was to explore novel strategies for vaccine development. The biological

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complexities of the pathogen make immune recognition and clearance difficult. The logical step is to search in nature for examples in which the immune system has been able to achieve any success at all against HIV, and work backwards to pinpoint vulnerabilities on the virus. The task of vaccine development then moves to turning those vulnerabilities into a compound that is safe, antigenic, and immunogenic in humans. Thus far, two groups of people with interesting responses to HIV infection have been identified. One group of HIV infected individuals is able to maintain viremia below a detectable level. Some individuals in this group have been able to control infection in this manner for over 25 years, earning the group the moniker "elite controllers". The mechanism for this astounding level of control remains a mystery, though several correlates of immunity have been defined that implicate a cell-mediated immune response, such as an increase in HIV specific CD8+ T cells and class I human leukocyte antigen (MHC-I). Interestingly, while the cytotoxic lymphocyte response specific to HIV is elevated, the overall T cell response remains quiescent. Elite controllers demonstrate what would be a favorable clinical and epidemiological outcome for a vaccine: the control of infection to a low level that significantly reduces the morbidity, mortality and potential for transmission. Elite controllers demonstrate a clinical outcome without a known correlate. On the other hand, about 20% of individuals infected with HIV develop cross-reactive neutralizing antibodies that are effective against heterologous strains of virus 95,96,125,126,127 . 1% of these individuals produce extremely potent neutralizing antibodies (bNabs) and are referred to as “elite neutralizers". Surprisingly, effective control of viremia is inversely related to the development of broadly neutralizing antibodies, possibly because bNabs require persistent exposure to heterologous virus in order to develop 95 (Devin Sok, personal communication). Thus, the clinical outcome associated with these correlates of protection are unfavorable. The ability of bNabs to protect against subsequent challenge with chimeric simian human immunodefi-

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ciency virus (SHIV) after passive transfer in non-human primate models has supported their role as mechanistic correlates of immunity 99,127 . Ultimately, we used the latter of these two groups as a guide, and chose to explore bNabs in this study. While the clinical outcome of elite controllers is superior to that of elite neutralizers, knowledge of a mechanistic correlate of protection is a critical component of vaccine design. Previous vaccines failed because they were based on faulty correlates that did not actually protect individuals. For that reason, we believe that bNabs present the most promising avenues towards future vaccine design. Furthermore, our modeling indicates that a prophylactic vaccine is an important cornerstone of a global HIV/AIDS strategy. As a result, we hope to make progress towards a vaccine that can induce a broad and potent neutralizing antibody response that can prevent infection rather than a T cell response that maintains low levels of viremia or prevents disease progression. In our study, we compared the numerous HIV specific bNabs that have been characterized and developed an objective function to rank them on the basis of an optimal combination of breadth, potency, and germ line similarity. We found that the V3 loop in the outer domain of gp120 presented the most attractive epitope to instruct vaccine design due to a broad and potent class of antibodies with relatively low mutation from the germ line that have been directed towards that epitope. These antibodies were isolated from a donor that belonged to the group of “elite neutralizers". The goal of our experiment was to determine the epitope specificity for a potent member of this class, PGT121 and its clonal variants PGT123 and PGT123. We found that PGT122 utilized two glycans on the V3 loop of gp120 to neutralize virus. Interestingly, these glycans were also implicated in neutralization by PGT128, the most potent member of the PGT class of HIV-specific bNabs. Therefore, we proposed a model of antibody neutralization at the V3 loop that was dependent on the glycans at position 301 and

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332 and posited a conserved model of neutralization of glycan epitopes in general (Chapter 4) 108 . Here, we demonstrate both the ability of individual bNabs to elucidate conserved and immunogenic epitopes on HIV. Through a meta-analysis of bNabs, we can determine conserved epitope specificities. These epitopes may constitute a vulnerability of the virus. The difficulty ahead lies in turning this vulnerability into a vaccine. If we do so, we will answer a question whose impact goes far beyond HIV. Can we reverse engineer vaccines from an immunological response? In the discussion that follows, we will explore a possible path from an epitope of interest to a vaccine, the questionable assumptions of rational vaccine design, and the general distinction between the immune system’s ability to recognize pathogens and its ability to remove them. The pace at which immunogen design has burgeoned in recent years is breathtaking. While it remains a nascent and untested science, the results have been promising. The key elements of immunogen design going forward are, an improved understanding of the viral structure, the ability to determine the germ line precursors of desired bNabs, and the engineering of synthetic compounds that are both antigenic and immunogenic in hosts. First, there is a need to determine the structure of Env to determine how epitope specificities are configured on the native trimer. Much of our structural understanding of gp120 and gp41 comes from crystal structures of monomers that have been significantly modified from their original state. For example, in the first gp120 structure solved by Kwong and colleagues in 1998, both the N and C termini were removed, the protein was deglycosylated, and the variable loops were replaced with Glycine-Alanine-Glycine linker peptides. Furthermore, the gp120 construct was bound to a soluble CD4 peptide and the antigen binding fragment (Fab) of a neutralizing antibody 17b that bound the co-receptor site 34 . In this work, we have demonstrated the importance of glycans in antibody mediated neutralization. Furthermore,

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we know that gp120 binding of CD4 results in extensive conformational changes. As a result, we are unable to observe the viral envelope “seen" by the immune system of newly infected individuals. A better understanding of the Env structure will likely help us direct neutralizing responses that are effective in natural infection. Fortunately, progress has been made on this front. Recently, Sodroski and colleagues used single-particle cryo-electron tomography to reconstruct native Env at a resolution of 11 Ă… . While this resolution is not quite as high as that of a crystal structure, the Sodroski structure has provided the most detailed understanding of trimeric Env to date. The structure confirmed the requirement of a significant conformational change for CD4 binding to occur. CD4bs-specific bNabs bind gp120 in a CD4 like manner using long CDR3 loops that bind a recessed pocket between the inner and outer domains; thus, an understanding of the conformational change that occurs in gp120 will be instructive for vaccine design. Also, the structure challenged current assumptions about the disorganized nature of variable loops. This understanding is based on an analysis of monomeric gp120. Instead, the variable loops fold into stabilized states on trimeric Env 33 . This could bolster the likelihood of a variable loop related vaccine by providing a more constant target epitope than previously imagined . Indeed, additional work will need to be done to solve the structure of Env at an even higher resolution and resolve discrepancies between cryo-EM and crystalline data, and the results are sure to have broad implications for vaccine development. An inherent problem to using bNabs directly is that they are long term effects of the immune response to HIV, whereas a vaccine must stimulate the immediate causes of that response. Thus, the utilization of bNabs in vaccine design requires us to retrace the evolutionary steps and turn on the first immunological switches that led to their development. Even when the correct initial responses are primed, it is believed that bNabs have complex and prolonged affinity maturation pathways that are dependent upon persistent exposure to diverse viral

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antigens 108,131 . This process is difficult to reconstitute in a prophylactic vaccine. For this reason, germ line similarity is a desirable attribute in bNabs, as extensive somatic hypermutation reduces the possibility of reelicitation de novo (Devin Sok, personal communication). Recent work has elucidated the activation of B cells, revealing the initial immunological responses that lead to the development of bNabs. B cells have the extraordinary capability to recognize foreign pathogens through antibody receptors. These pathogens are subsequently internalized and degraded, and pieces of them are presented on MHC-II peptides. Costimulation by CD4+ T cells with specificity for the same epitope results in the activation of germ line B cells, class switching, the production of secreted antibodies, and the initiation of affinity maturation 121 . Thus, a critical juncture in the development of bNabs is the activation of germ line B cells through detection of pathogens by cell-surface receptors. By following an HIV infected individual closely and tracking the evolution of viral isolates at several time points, scientists were able to study the co-evolution of both the virus and the antibody response. While an antibody that could neutralize 55% of heterologous viral isolates (CH103) was eventually formed, that antibody failed to bind the transmitter/founder virus that initiated the infection. Interestingly, the germ line precursor to CH103 was able to bind this precursor, and this recognition mediated co-evolution of viral escape and broad neutralization. Thus, the notion of activating a germ line precursor to bNabs is thought to be an effective way to prime the immune system against HIV infection. Unfortunately, the target antibody of this analysis had only modest breadth (55%) 131 . A similar analysis was performed using the far broader antibody VRC01. Scientists identified the germ line precursors to this antibody to be B cells harboring the VH1-2*02 variable heavy gene 132 . Unfortunately, while a prime of VRC01 precursors may be possible, the high degree of somatic hypermutation in the variable heavy gene precludes an easy path to boost the immune response thereafter. It would be interesting to apply this approach to V3-specific bNabs such as PGT121 and PGT128,

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whose breadth almost reaches VRC01 level but whose germ line similarity closely resembles CH103, providing the best of both worlds. Furthermore, it is interesting to note that PGT antibodies developed a conserved epitope specificity from two different germ line precursors, suggesting a V3-specific immune response may be easier to elicit with a vaccine. Even when the initial immunological triggers of bNab development are identified, the process of developing an antigen that can activate them in vivo is non-trivial. The reconstitution of bNab-specific epitopes as immunogens has thus far been unsuccessful, but progress in achieving receptor binding and B cell activation in vitro is a significant stepping stone to that end. Once the germ line precursors of VRC01 were identified, scientists attempted to construct an immunogen that would activate them. To do so, they scaffolded the epitope on an engineered outer domain of gp120, and used computational techniques to introduce selective mutations that would remove interfering residues and glycans and strengthen existing interactions. Even after optimizing binding to VRC01 germ line precursors, the compound they produced did not activate B cells. Only after expressing it on a self-assembling 60mer were they able to activate B cells in vitro, possibly due to the ability of B cell receptors to cross-link when binding the 60mer. Unfortunately, the lack of cognate VH genes in animal models precluded an analysis of B cell activation in vitro 132 . Overall, these results demonstrate a progressive understanding of the initial priming of an effective anti-HIV immune response. However, how to subsequently tailor that response to yield the broad and potent antibodies required for rapid virus neutralization and clearance remains uncertain. This methodology of turning an epitope of interest into a safe and effective vaccine treads into unchartered territory. Indeed, advances in molecular biology have allowed for an unprecedented view of viral genomes and proteomes. The goal of vaccine design is to observe the repertoire of viral antigens and use those most likely to be detected by the immune system as templates for vaccine design. It is this rationale that was applied in our study as well as

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many other studies for vaccines to various diseases. However, the known successes of rational vaccine design are quite limited. The successful development of a vaccine for serogroup B Neisseria meningitis, the pathogen responsible for the majority of meningitis cases in the developed world, appeared to validate the paradigm of rational vaccine design. However, despite the development of this vaccine, the assumptions made regarding which antigens to pursue can be problematic when applied to other diseases. Specifically, rational vaccine design assumes that the epitopes that confer vaccine-mediated immunity are detected by the immune system, and more specifically are limited to surface antigens. The former part of the theory is sound. Our ability to sequence the genomes of viruses has given us the key to discovering putative antigens for vaccine design and analyzing them in high throughput studies. However, the restriction of these antigens to those expressed on pathogen surfaces and detected by immune responses may not hold for highly mutable pathogens with immune suppressing activity 133 . In other words, the advent of the era of genomics has provided us with tools to look into the haystack. However, finding the needle remains a difficult task. A major assumption we have made in studying broadly neutralizing antibodies is that protection is dependent on the recognition of surface antigen. In essence, we believe that the binding of HIV Env by bNabs prevents CD4 receptor binding, fusion, and entry, and furthermore, that this is the mechanism of vaccine derived immunity. This is still an issue of contention and not supported thoroughly by previous vaccines. In fact, the component of the pathogen that confers protection is only known for two vaccines, and in one it is a surface protein (hepatitis B) while in the other it is a capsid (human papillomavirus) (Adel Mahmoud, personal communication). The poorly understood mechanisms of vaccine mediated protection prevent us from knowing whether surface antigens are the exception, the rule, or somewhere in between. In some sense, the very term “broadly neutralizing antibody" may be a misnomer, as a protective vaccine will probably need to activate several immune

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mechanisms in addition to serum antibody mediated neutralization. Therefore, more research is required to fully understand how vaccines work, and advance our limited understanding of correlates of immunity into a broad understanding of mechanisms of immunity. In some sense, we are flipping switches in one room and observing flickers of light in another, without much understanding of the circuitry in between 134 . Beyond assuming that protective responses are mounted against surface antigens, we make the broader assumption that the critical viral vulnerabilities that can be exploited to confer protection can be identified by the immune system in the first place. Our reliance on antibodies, and immune responses in general, to discover vulnerable viral epitopes, occludes the study of several epitopes the immune system does not recognize during natural infection. We already know that natural infection is a poor model for vaccine-derived immunity in HIV. We also know that HIV severely diminishes the capability of the adaptive immunity to mount an effective response against itself and other pathogens. One of the known correlates of bNab development is a reduction in the pool of CD4+ T cells, suggesting that immunological responses to HIV are produced by a system not at its best (Devin Sok, personal communication). Even though HIV affects CD4+ T cells, the activation of these cells is required for B cell activation, class switching, and affinity maturation, which are required for the development broadly neutralizing antibodies. Thus, it is questionable whether the most effective pathway to vaccination is being elucidated by assessing weak and incomplete immune responses 134 . Fundamentally, we do not know what distinguishes the immune system’s ability to detect pathogens, which is universal, and its ability to control them, which is not. We know that B cells can recognize virtually any structure in nature. On the other hands, T cells have more limited recognition capability; they can only detect peptide epitopes on MHC molecules. While this may be one source of the disparity, even infections such as HIV which produce a limited T cell response cannot be cleared by the adaptive immunity. Tearing down the wall

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between virology and immunology will be required to understand the role that neutralization plays, if any, in protecting individuals from infection. Nevertheless, a remarkable degree of progress has been made thus far towards an HIV vaccine. While it is easy to be skeptical about the possibility of rational vaccine design, it is hard to not be optimistic. Our understanding of the mechanisms of protective immunity is far from complete, but the resources we have to understand it and the progress of new discovery is unprecedented. Just imagine, when Jenner happened upon the protective effects of pus from cowpox infection, he did not know what a virus or neutralizing antibody was; yet today, we are able to observe both of these entities at an atomic resolution. Centuries after Pasteur discovered the process of attenuation, we now have the capability to manipulate the genomic and proteomic elements of pathogens at will and harness their rapid evolutionary power under the selective pressures of our choosing. Where we must remain wary, however, is to believe that rational vaccine design, if it succeeds, signifies a conclusive victory against pathogens. Just two years after HIV was found to be the etiologic agent of AIDS, then Secretary of Health and Human Services Margaret Heckler boldly declared that an HIV vaccine would be ready for testing in two years 135 . While it is easy to smile a superior smile at the inaccuracy of this prediction, it is more revealing to reflect on just how little any of us knew about the virus then. No matter how the chapter on HIV ends, it will not be the last in the book of infectious diseases. Even as HIV has caused so much destruction and tragedy throughout the world, it should also humble us into remembering that we will never possess the replicative and rapid evolutionary advantages of the pathogens. But all is not lost, because we too have been endowed with advantageous gifts that the pathogens do not possess: the ability to think and to collaborate through science. No matter how wily the pathogen is, it battles alone. We, on the other hand, can battle it together. For that is our human gift. We owe it to our fellow human beings to use these gifts of observation, experimentation, and analysis to approach the biggest challenges of our time.

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And it is the hope of this author, that this thesis, and several after it, can advance the cause of an HIV vaccine so that some day in the future, researchers can look back on the history of this pandemic, having learnt from our struggles, and yes, even our setbacks, on the road to triumph.

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