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Medical Microbiology PAMB 650/720

Human Immunodeficiency Virus

Lectures: 74 (part) and 75

DR RICHARD C. HUNT Expanded notes on this subject can be found the Department of Pathology and Microbiology web site.

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OBJECTIVES: To discuss the molecular structure of HIV, the involvement of this virus in AIDS pathogenesis and current treatments for AIDS. INTRODUCTION Human immunodeficiency virus (HIV) is found in all cases of ARC (AIDS related complex - a term now rarely used) and in AIDS (acquired immunodeficiency disease syndrome). It can be detected by the presence of anti-HIV antibodies or by the presence of the virus itself (using polymerase chain reaction which is very sensitive and can show HIV in situations in which it is not detectable immunologically). HIV is a retrovirus and integrates into the genome in the same manner as other retroviruses. Unlike other retroviruses, which typically bud from the infected cell for a long period of time, HIV can lyze the cell or lie dormant for many years as proviral DNA, especially in resting T4 lymphocytes. After the dormant period, recrudescence of viral production occurs that ultimately destroys the cell. However, it should be noted that while HIV may disappear from the cells of the circulation, viral replication and budding continues to occur in other tissues. The discovery of HIV depended on the ability to grow the virus in vitro. HIV AND AIDS, THE DISEASE In 1981, clusters of Kaposi’s sarcoma patients were observed in San Francisco and New York. All were young male homosexuals. Other diseases associated with immunocompromisation also occurred in the same population: e.g. Pneumocystis pneumonia, lymphadenopathy (diffuse, undifferentiated non-Hodgkins lymphoma). The number of sex partners appeared to be important in that the disease was particularly prevalent among promiscuous individuals. Later a similar immunodeficiency was found in intravenous drug users, persons who received transfusions and hemophiliacs; the sex partners of these people also got the disease. In view of this, it was obvious that an infectious agent was involved. The cell picture - the selective loss of T4 helper cells - suggests a virus. Thus, AIDS is a sexuallytransmitted viral disease. The causative agent was difficult to grow as it kills the cell that it infects. The image above shows lesions on the stomach of a patient with Kaposi’s sarcoma The virus was finally isolated by Luc Montanier and Françoise Barré-Sinoussi (Pasteur Institute, Paris – Nobel Prize, 2008) and Robert Gallo (NIH). STATISTICS AIDS is currently defined as the presence of one of 25 conditions indicative of severe immunosuppression (see web site) or HIV infection in an individual with a CD4+ cell count of <200 cells per cubic mm of blood. AIDS is the end point of an infection that is continuous, progressive and pathogenic. With the prevalence of HIV in the developing world, HIV and its complications will be with us for generations. Approximately 14000 new HIV infections occur daily around the world and over 90% of these are in developing countries. Seventeen hundred are in children less than 15 years of age. Of adult infections, about half are in women and 15% in individuals of 15 - 25 years. Perinatal infection resulted in a large number of children being born with HIV. 30-50% of mother to child transmission of HIV results from breast 2


feeding and about a quarter of babies born to HIV-infected mothers in developing countries are themselves infected. As of December 2006, 1,106,400 Americans had been reported with HIV/AIDS (up from 641,086 in 1996). Adult and adolescent AIDS cases total about 828,000 in males and 278,400 in females. Through the same time period, about 10,000 AIDS cases were reported in children under age 13. In early years of the epidemic, AIDS incidence increased by 65 - 95% each year but, partly as a result of prevention efforts targeting those at highest risk, the rate of increase fell to less than 5% per year by the mid 1990's. This was prior to the introduction of combination therapies for HIV. Deaths among people with AIDS declined for the first time in 1996, dropping 25%. This was due to advances in chemotherapy. Note: As more and more people survive with an HIV infection because of successful chemotherapeutic intervention, the number of infectious people in the population is rising even though fewer people are dying of AIDS. Thus, if declines in AIDS deaths continue, there will also be an increase in HIV prevalence, pointing to an increased need for both prevention and treatment services. The rise in HIV infections has leveled off in the west and the wave of infections threatening to affect western heterosexuals has not materialized. However, this is not the case elsewhere and there have been huge increases in southern Asia and southern Africa. In Africa (mostly sub Saharan), there are more than 20 million people with HIV infection and more than 2 million new infections per year. AIDS is responsible for a decrease in life expectancy and increase in child mortality. Child mortality rates in East Africa will double by 2010 and adult life expectancy has already declined in that region. Altogether, there are now 16 African countries in which more than one-tenth of the adult population aged 15 - 49 is infected with HIV. Several countries in sub-Saharan Africa report infection rates of over 30% in urban areas. The prevalence rate in most West African countries remains below 3%. THE COURSE OF AIDS DISEASE HIV usually enters the body in infected macrophages and is most frequently acquired via sexual intercourse. Infection takes place via the mucosa of the rectum, penis or vagina. When an organism, such a virus, invades mucosal tissues, a primary line of defense is binding of the invading organism by antigenpresenting dendritic cells. In an HIV infected person, dendritic cells accumulate the virus particles on their surfaces but usually do not usually internalize them. Interaction of dendritic cells with HIV occurs via binding of HIV gp120 to a protein called DC-SIGN, which has a higher affinity for gp120 than CD4 antigen (see below). The dendritic cells carry the virus to the lymph nodes resulting in efficient infection of CD4+ T cells. a) Acute infection Initially, in the period immediately after infection, virus titer rises and the patient sometimes experiences some mononucleosis-like symptoms (fever, rash, swollen lymph glands). The result is an initial fall in the 3


number of CD4+ cells and a rise in CD8+ cells but the numbers quickly return to normal. Macrophages are also infected; indeed, if acquired sexually, HIV at this stage if usually macrophage-tropic.

b) A strong anti-HIV immune defense Cytotoxic B and T lymphocytes mount a strong defense and virus is greatly reduced in the circulation. During this period, more than 10 billion new HIV particles are produced each day but they are rapidly cleared. There can be up to 102 to 107 virus particles per ml of blood. Most of this virus is coming from recently infected proliferating CD4+ cells. The infected cells that are producing this virus are destroyed either by the immune system or by the virus. However, the rate of production of CD4+ cells can compensate for the loss of cells. Most CD4+ cells at this stage are uninfected. This is the most infectious phase of the disease. Seroconversion occurs between one and four weeks after infection. c) A latent reservoir Although mainly cleared from the blood, the virus persists elsewhere such as in the lymph nodes, especially in association with dendritic cells. A small fraction of the productively infected CD4+ cells may survive long enough to revert back to the resting memory state (as do non-infected CD4+ memory cells). These carry a copy of the HIV genome, which remains latent until the cells are reactivated by antigen. These memory cells have a great potential for stability and constitute a reservoir that may be very important in drug-based therapy. Note: Although the number of HIV particles in the bloodstream falls during clinical latency, the virus is detectable. After the initial peak of virus, the virus reaches a "set point" during latency. This set point predicts the time of onset of clinical disease. With less than 1000 copies/ml of blood, disease will probably occur with a latency period of more than 10 years. With fewer than 200 copies/ml, disease does not appear to occur at all. Most patients with more than 100,000 copies per ml, lose their CD4+ cells more rapidly and progress to AIDS before 10 years. Most patients have between 10,000 and 100,000 copies per ml in the clinical latency phase (unless treated chemotherapeutically). d) Loss of CD4+ cells and loss of the immune response The major reason that the immune system fails to control HIV infection is that the CD4+ T helper cells are the target of the virus. Also dendritic cells present antigen to CD4+ cells and may bring the virus into contact with these cells at the time that they are stimulated to proliferate by antigen. There is a relentless decline of CD4+ cells with especially a loss of those specific to HIV, which occurs from the very beginning of infection and is permanent. Near the end stage of AIDS CD8+ cells also decline precipitously. 4


e) Onset of AIDS Eventually, the virus can no longer be controlled as the virus and cytotoxic T (CD8+) cells destroy helper (T4) cells. T4 cells are also lost by apoptosis. As the T4 cells fall below 200 per cu mm, virus titers rise rapidly and immune activity falls off. It is the loss of immune competence that enables normally benign parasites such as fungi or protozoa to cause disease. Once AIDS develops, patients rarely survive more than two years without chemotherapeutic intervention. There is considerable variability at this stage. Some patients with clinical AIDS do survive for several years while others who appear relatively healthy can suddenly succumb to a major opportunistic infection. The patients die from opportunistic infections. It is the onset of HIV-associated neoplasms and opportunistic infections that defines the disease of AIDS. CO-FACTORS IN AIDS--THE ENIGMA OF KAPOSI’S SARCOMA Kaposi’s sarcoma is often a corollary of HIV infection in gay men (over 20% get it and HIV-positive patients are at 20,000 fold increased risk of Kaposi’s compared to general population). Clinically, most non-AIDS Kaposi’s is so indolent that many affected individuals die of other unrelated causes; however, the AIDSassociated form is more progressive involving many sites (skin, lymph nodes, lungs, intestine). This form of Kaposi’s sarcoma, so typical of the progression of AIDS in gay men, is not found in some other populations that are HIV-positive. It has long been suspected, therefore, that Kaposi’s is not the direct result of HIV but some co-factor that is activated in the presence of HIV. This now seems to be the case. In 1994, a Kaposi’s sarcoma-associated herpes virus (KSHV or human herpes virus-8) was identified. In AIDS patients, antibodies against KSHV are common only in those that have Kaposi’s or those that will eventually get it (greater than 80% of this population). In contrast, blood samples from infected hemophiliacs usually show no anti-KSHV antibodies. It is not known why the virus has established itself in primarily the gay population when most herpes viruses are widely disseminated throughout the population. HIV SUBTYPES Two types of HIV can be distinguished genetically and antigenically. HIV-1 is the cause of the current pandemic while HIV-2 is found in West Africa but rarely elsewhere. The HIV-2 type is more closely related to simian immunodeficiency virus (SIV) found in West Africa. There are at least 10 different HIV-1 subtypes. The major one in North America and Europe is type B. In some countries, mosaics between different subtypes have been found

HUMAN IMMUNODEFICIENCY VIRUS THE COMPONENTS OF HIV HIV is a retrovirus

VIRAL MEMBRANE This is host-derived as a result of budding from the cell surface. Some host proteins become incorporated into the viral membrane. SURFACE GLYCOPROTEIN Gp160 is encoded by the env gene. Gp160 is cleaved after translation by host enzymes in the Golgi body to form Gp120 and Gp41. Gp 41 is embedded in the membrane, Gp120 is not but is held to Gp41 by noncovalent interactions. It is readily shed from the virus particle. There are many sugar chains on gp120 (Problem for vaccine). Gp120 is the protein that interacts with a receptor on the cell to be infected. Gp41 is 5


the fusogen that is exposed after Gp120 has bound to the cell.

INTERNAL PROTEINS Internal structural proteins All are encoded in gag gene. P17 lines the inner surface of viral membrane to which it is attached by covalently bound myristic acid. Other proteins associated with the nucleocapsid include p24, p9. The group-specific antigen is made as a polyprotein and cut by virally-encoded protease encoded by the pol gene after budding of the virus. Other internal proteins All are encoded by pol gene. These are the enzymes that participate in integration and replication: reverse transcriptase, integrase, protease. GENOME Diploid positive-sense RNA

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THE GENOME OF HIV The HIV genome consists of 9749 nucleotides-- about the same size as any other retrovirus. But the genome of HIV is more complex than simple retroviruses such as RSV since it has extra open reading frames that clearly code for small proteins. Some of these are protein synthesis-controlling proteins and most do not end up in the virus particle. The HIV genome has nine open reading frames but 15 proteins are made in all (because some are cleaved) LIFE HISTORY OF HIV Cells that are infected by HIV CD4+ (T4) helper lymphocytes are the most studied but monocytes/macrophages are also infected and may provide an important reservoir. HIV also infects oligodendrocytes, astrocytes, neurones, glial cells. The virus destroys T4 cells specifically, causing profound immuno-suppression. Other cells tend to harbor the virus without lysis. Entry into cell: pH-independent fusion with plasma membrane Binding of gp120 to CD4 antigen results in a conformational change in gp41 allowing it to act as a fusion protein. No pH-dependent conformational change in the gp41 protein is necessary for fusion between the viral membrane and the membrane of the cell to be infected. Thus, no entry into endosomes or lysosomes is required. This sort of fusion of virus with the plasma membrane is associated with fusions of infected cells to form syncytia and also of infected cells with uninfected cells. Syncytium formation is a characteristic of HIV infection. This has profound significance for spread of infection between cells without any free virus. This means that virus may spread from cell to cell so that immune system circulatory antibodies cannot have any effect (problem for vaccine). Not only will a vaccine have to be able to destroy the virus, it will also have to be able to destroy infected cells. Syncytia are most often seen in the brain. CD4 antigen is the HIV receptor The apparent specificity of CD4+ cell infection observed initially, together with the observation that these cells are depleted in disease (indeed, the course of disease in the patient is followed by CD4+ cell levels), suggested that CD4 antigen might be the receptor for the virus. This was demonstrated by transfecting CD4 antigen gene into CD4- human cells and showing that they acquired the property of being able to be infected by HIV. A co-receptor for infection by HIV However, something more appears to be necessary as this experiment only works with human cells; for example, mouse cells transfected with CD4 antigen gene are not infected by HIV (although the virus does bind). It was also discovered that some strains of HIV (those adapted for life in transformed T cells) could replicate in activated human T cells but not in monocytes or macrophages. Conversely, those adapted for life in macrophages could not replicate in transformed T cells. Yet both macrophages and T4 cells possess CD4 antigen.

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Chemokine receptors seem to be the key to the gateway of the cell -- a family of proteins on the surface of immune cells CCR5 - Several laboratories identified an essential co-receptor for those HIV strains involved in critical early stages of infection (these are the macrophage-tropic strains). All of these studies found CCR5 as the partner of CD4 in allowing entry into macrophages. CXCR4 (also known as fusin) is also a co-receptor for HIV in otherwise non-infectable CD4+ cells. The amount of fusin on the cell surface may explain differences in tropism. People who are infected but do not exhibit disease: Long-term non-progressors Ever since the AIDS epidemic began there have been people who are clearly exposed to the virus who seem to exhibit no symptoms and normal CD4+ T cell counts. These are called long term non-progressors, i.e. people who have been infected with HIV for more than 7 years that have stable CD4+ T cell counts above 600 per cu mm and have no history of symptoms and have not been taking anti-retroviral drugs. The CD4+ T lymphocytes of these patients fall after primary infection and seroconversion but remain at normal levels thereafter. This seems to be a heterogeneous group of people whose long-term nonprogressive disease results from a robust immune response against HIV or a poorly replicative virus. Co-receptors and disease severity As noted above, a chemokine receptor on the surface of macrophages and T4 CD4+ cells is a co-receptor for HIV. The nature of this co-receptor may be one explanation of the people who are exposed repeatedly to HIV but remain uninfected. It has been found that the T-cells of some people are very resistant to HIV infection because they have mutant chemokine receptors. If both copies of the gene for CCR5 are mutated, it is virtually impossible for virus to enter the cell and exposed patients are immune from HIV infection. Approximately 1 in 100 Caucasians have this double mutation. Interestingly, the same CCR5 mutation (called â&#x20AC;&#x153;delta 32â&#x20AC;?) is thought to be the mutation that rendered some people immune to the plague in the middle ages. Rare HLA antigens and disease There are other reasons why some people are exposed, yet uninfected. A study of Nairobi prostitutes, repeatedly exposed to HIV (25% or more of their clients are HIV positive), has shown that many of these women have been free of disease for many years. They seem to be completely resistant to infection. They may present epitopes that are highly conserved between different HIV-1 variant strains. For example, one epitope, to which there is a strong CTL response in these women, is found to be located in a highly conserved part of HIV p24 protein. It appears to be conserved because it is very important in the assembly of the virus. Another important epitope is in the protease. The protease may not be able to bear much mutation in this region without losing enzymic activity. HIV can infect some CD4-negative cells via a galactocerebroside It was originally thought that only cells that have CD4 antigen are infected. It is now known, however, that some non-CD4+ cells, such as those in brain and intestine, can be infected via a galactocerebroside receptor. Other cells can be infected in a different way; for example, in macrophages an Fc or complement receptor may be used. In these cases, the HIV must be bound by anti-HIV antibodies (opsonized) that interact with receptors on the cell surface. Thus, anything that can up-regulate Fc receptors on macrophages will augment infection.

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Reverse transcription and integration This is similar to other retroviruses. HIV uses reverse transcriptase imported during infection. RNA synthesis, translation and assembly This is also similar to other retroviruses Note: After the virus has budded from the cell, the protease cuts itself free and cuts up the rest of the proteins in gag or gag/pol, releasing the various structural proteins and reverse transcriptase. This specific protease is vital as the proteins are not functional unless separated. This specific HIV protease is a good candidate for the site of action of an anti-HIV drug (see below and chemotherapy lecture notes). Note gp160 is cut to gp120 and gp41 by a host enzyme in the Golgi apparatus since it gets to the cell membrane via the exocytic pathway. THE LATENCY OF HIV Cellular latency The virus rapidly kills most infected, activated CD4 cells but these are initially replaced. CD4+ lymphocytes replicate HIV only when they are activated by contact with an antigen when they produce prodigious amounts of virus that leads to cell lysis and cell death. Thus, a variety of viral and bacterial infections (and unfortunately vaccinations) can markedly increase HIV load in plasma. When activated, most CD4+ cells are ultimately removed by apoptosis in a normal immune response but some revert to the resting state resulting in immunologic memory. These memory T4 cells cannot replicate virus (if they happen to be infected) but harbor the virus in a proviral (DNA) form that can reinitiate replication when the memory cell is itself reactivated. WHY IS THERE A PROGRESSIVE, INEVITABLE LOSS OF THE CD4+ HELPER T-CELLS? WHY DO CD8+ KILLER T CELLS DISAPPEAR LATE IN DISEASE? Why, when only 1 in 10,000 (early) or 1 in 40 (later) cells show productive infection, do all of the T4 cells disappear? It is still unclear why so many of the CD4+ cells disappear but there are several possibilities: a) Membranes are punctured as virus buds (for this, the cell needs to be infected) b) Syncytia formation (resulting in spread to uninfected cells) c) Infected cells look like virus since they are gp120 positive (and destroyed by cytotoxic T cells) d) Shed gp120/gp41 binds to uninfected cells via CD4 antigen. As a result, they are seen as infected and destroyed e) HIV initiates apoptosis in all T4 cells (this is normal in some T4 cells to overcome autoimmunity). This is currently thought to be the most important factor in the loss of CD4 cells Some of the above may explain why only a minority of T4 cells appear to be infected at a given time yet almost all disappear in the later stages of the disease. It could also be that the virus switches from one T4 cell population to another as it switches its co-receptor (see above). CD8+ cells are not easily infected by HIV (since they have only low levels of CD4 antigen) and their numbers remain high during the course of the disease for many years. And then, until recently inexplicably, they rapidly die off. It appears that some of the HIV subtypes that occur late in infection prompt a mass apoptosis of CD8 cells. Although CD8 killer cells are CD4-, they do have CXCR4 co-receptor and HIV can bind to this. Since little CD4 antigen is present there is no infection but binding to CXCR4 sends a signal to the cell, the signal for apoptosis and there is mass CD8+ cell suicide.

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MACROPHAGES Although CD4+ T4 cells are usually considered to be the most important cells in the course of AIDS and it is their loss that leads to immune suppression, other cells get infected. Macrophages are very important as they form a reservoir outside the blood and carry the virus into tissues. Non-proliferating mature macrophages can support HIV production for a long time without being killed. Cytokine production by the infected macrophages is also aberrant leading to a variety of secondary effects. The slim disease that is characteristic of HIV infections in Africa may result from macrophage cytokine disruption. This wasting is very reminiscent of Visna in sheep and Visna infections involve the macrophages. Macrophages are infected via CD4 antigen. Also since the virus raises good antibodies in the host, cells that express Fc or complement receptors will take up the virus. POPULATION POLYMORPHISM AND HIV VARIANTS Population polymorphism results from the high error rate of reverse transcriptase and host RNA polymerase II which are used in genome replication: 1 in 10,000. This, together with the high rate of CD4+ cell production and infection, means that every possible single point mutation in the viral genome arises daily and almost 1% of all possible double mutations occur each day. This means that the virus isolated from an AIDS patient is very different from infecting virus. Distinct sub-strains differ in cell tropism. Some form syncytia, some do not. The non-syncytium-inducing macrophage-tropic type is probably the initial infectious form. The major variable protein is gp120, although there are some conserved sites where mutations are presumably non-viable (e.g. CD4 binding site). Glycosylation often masks these conserved sites (Masking and polymorphism are problems for a vaccine). Gp41 is not so glycosylated and the fusion site needs to be conserved (making this protein a possible vaccine site).

STRATEGIES TO COMBAT VIRUS Chemotherapy Most anti-HIV drugs are toxic. In addition, anti-HIV chemotherapy will not stop infection and is unlikely to cure the infected host (see chemotherapy lecture notes). The most we can hope for is suppression of virion production making AIDS a more tractable disease. Great strides towards this goal have been made using triple drug therapy (Highly active anti-retroviral therapies (HAART) â&#x20AC;&#x201C; see chemotherapy notes. Education HIV is (fortunately) not highly infectious. It can be avoided by taking the correct precautions. Vaccine The best way to protect against an original infection is to develop a vaccine. But HIV is a retrovirus and this poses enormous problems for vaccine development.

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