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MEDICAL MICROBIOLOGY MBIM 650/720 LECTURE 63

Vaccines: Past Successes, Future Prospects

Polio Virus Edward Jenner More information on this material with links may be found at http://www.med.sc.edu/micro

FACULTY: Dr Richard C. Hunt OBJECTIVES: To learn something of the past successes and latest advances in vaccine development.

Reading: Murray: Chapter 17 Introduction Vaccines are harmless agents, perceived as enemies. Immunity to a viral infection depends on the development of an immune response to antigens present on a virally infected cell or on the surface of the virion itself. Immune responses to internal antigens play little role in immunity. Thus in influenza pandemics, a novel surface glycoprotein acquired as a result of antigenic shift characterizes the new virus. Often it is the glycoprotein to which the population has not been exposed and thus, against which the population has no immunity. Surface glycoproteins are often referred to as protective antigens . To make a successful vaccine, it is likely that the identity of these antigens will have to be known unless the empirical approach of yesteryear is to be followed. There may be more than one surface glycoprotein on a virus and one may be more important in the immune response than the other. For example, influenza virus has a neuraminidase and a hemagglutin on the virus surface. The hemagglutinin is the surface protein that provokes neutralizing immunity, so this must be identified for a logical vaccine. Neutralizing antibody that blocks infectivity is required of a successful vaccine. This usually results from antibody combining with surface protein that would otherwise bind to

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surface receptor of cell. Complement can also lyse enveloped virions.

Major sites of viral infection: 1) Disease at mucosal surfaces of respiratory tract and GI tract. Rhino; myxo; corona; parainfluenza; respiratory syncytial; rota 2) Infection at mucosal surfaces followed by spread systemically via blood and/or neurones to target organs: picorna; measles; mumps; HSV; varicella; hepatitis A and B 3)Infection via needle or bites and then spread to target organs: hepatitis B; alpha; flavi; bunya Local immunity via IgA very important in 1 and 2.There is little point in having a good neutralizing humoral antibody in the circulation when the virus replicates, for example, in the upper respiratory tract. Clearly, here secreted antibodies are important. We need to know: a) Viral antigen for neutralizing antibody b) Cell surface antigen for neutralizing antibody c) The site of replication of the virus

Problems in vaccine development: 1) Different viruses may cause similar disease--e.g. common cold 2) Antigenic drift and shift -- especially true of RNA viruses and those with segmented genomes. 3) Large animal reservoirs. Reinfection may occur 4) Integration of viral DNA. Vaccines will not work on latent virions unless they express antigens on cell surface. In addition, if vaccine virus integrates it may cause problems (see later). 5) Transmission from cell to cell via syncytia--problem for potential AIDS vaccine 6) Recombination of the virulent strain (see 2 above) or of the vaccine virus. Top Left: Infant with smallpox Bottom left: Smallpox virus Bottom right: The mummified head of Ramses V (died 1157 BCE) with rash that is probably the result of smallpox

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Left: Dr Jenner about to vaccinate a child Middle: Blossom, the cow Right: Powdered smallpox scabs were used inhaled to protect against smallpox in Chinese medicine

Smallpox Fatality rate up to 25%. First control attempts in 10th century: VARIOLATION (small pox virus is Variola). If person did not die, there was lifelong immunity. Fatality rate in variolation: 1-2%. Variolation was widespread in England in 1700s and used in Pakistan, Ethiopia and Afghanistan till 1970. 1796: Jenner discovered VACCINATION using vaccinia virus, the agent of cowpox, since people who got cowpox were known to have protective immunity against the much more virulent smallpox. Vaccinated Mr Phipps and own son with cowpox from Blossom and then challenged with virulent smallpox. The original virus is not the vaccinia that was used in smallpox vaccinations until recently. Vaccinia may have arisen as recombinant from Cowpox or horse pox. Last case of natural smallpox in U.K. in 1930s; last in U.S.A. 1940s. Last natural case in world (Somalia) in October 1977. Reasons for success of smallpox vaccination: 1) No animal reservoir 2) Lifelong immunity 3) Subclinical cases rare 4) Infectivity does not precede overt symptoms 5) One Variola serotype 6) Effective vaccine 7) Major commitment by governments 3


Polio In western countries no longer a major problem. Still a major problem in less developed countries though some have had very major successes e.g. Cuba, Nicaragua. The wild type (non-vaccine) strain of polio has recently been declared to be wiped out from the Western Hemisphere.

Total reported cases in Sweden and Finland (right) (1950-76) which use the killed vaccine

Reported (rate cases per 100,000 population) cases of paralytic poliomyelitis in the United States 19511992

The residual cases in countries such as the U.S. using live vaccine result from mutation of vaccine strain to virulence. About half of the few cases are in vaccinees and half in contacts of vaccinees. Paralytic polio arises in 1 in 100 cases of infection by wild type virus and 1 in 4 million vaccinations as a result of back reversion to virrulence. This was deemed acceptable as the use of the attenuated virus means that the vaccine strain of the virus still replicates in the body and gives gut immunity via IgA. Thus, wild type virus does not replicate in the gut of vaccinee, nor can it go to neural tissue where the disease manifests itself.

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The vaccinee who has received killed vaccine still allows wild type virus to replicate in his/her gastrointestinal tract. Thus, wild type virus would not die out in populations who have received only the killed vaccine since wild type polio virus would be shed in the feces. It should be noted, however, that studies in The Netherlands during a polio outbreak in 1992 (among people who had refused the vaccine) showed that immunity produced by the Salk vaccine did prevent circulation of wild type virus in the general population. The previous policy adopted in the U.S. has been reevaluated with the idea that one could give the killed vaccine first and then the attenuated vaccine. The killed vaccine would stop the revertants of the live vaccine giving trouble by moving to the nervous system. To reduce the vaccine associated cases (8 to 10 per year), the CDC Immunization Practices Advisory Committee has recommended (January 1997) a regimen of two doses of the injectable killed (inactivated: Salk) vaccine followed by two doses of the oral attenuated vaccine on a schedule of 2 months of age (inactivated), 4 months (inactivated), 12-18 months (oral) and 4-6 years (oral). Currently four doses of the oral vaccine are typically administered in the f irst two years of life. It is thought that the new schedule will eliminate most of the cases of vaccine-associated disease. This regimen has already been adopted by several European countries and some of Canada. NEW RECOMMENDATIONS: To eliminate the risk for Vaccine-Associated Paralytic Poliomyelitis, the ACIP recommended an all-inactivated poliovirus vaccine (IPV) schedule for routine childhood polio vaccination in the United States. As of January 1, 2000, all children should receive four doses of IPV at ages 2 months, 4 months, 6-18 months, and 4-6 years. Other important early attenuated vaccines: Yellow fever vaccine . Rabies developed by Pasteur. It was the first attenuated viral vaccine. Passed through nerve cords of rabbits. Current vaccine is inactivated ATTENUATION is usually achieved by passage of the virus in foreign host such as embryonated eggs or tissue culture cells. From among the many mutant viruses that exist in a population (especially so in RNA viruses), some will be selected in new host that have better ability to grow in the foreign host (higher virulence). These tend to be less virulent for the original host. In Sabin polio vaccine, attenuation was only achieved with use of high inocula and rapid passage in primary monkey kidney cells. The viruses became overgrown with a less virulent strain (for humans) that could grow well in non-nervous tissue but not in CNS. Non-virulent strains of all three polio types produced for the vaccine. Molecular basis of attenuation. We do not know why attenuated in most cases. The empirical foreign-cell passage method causes many mutations in a virus and it is difficult to determine which are the important mutations. Many attenuated viruses are temperature sensitive (grow better at 32-35 degrees than 37 degrees) or cold adapted (may grow as low as 25 degrees). In type 1 polio virus attenuated vaccine strain 57 nucleotide changes, resulting in 21 amino acid changes in the genome. One third in VP1 gene (this gene is only 12% of genome). Suggests that attenuation results from change in surface glycoproteins of the virus 5


There has recently (Fall 1997) been an announcement of the efficacy of an attenuated nasal vaccine for influenza. This contains cold-adapted vaccine strains of the influenza virus that have been grown in tissue culture at progressively lower temperatures. After a dozen or more of these passages, the virus grows well only at around 25째 and in vivo growth is restricted to the upper respiratory tract. The manufacturers used a trivalent vaccine similar to the annually formulated killed vaccine that is currently in use. Studies showed that influenza illness occurred in only 7 percent of volunteers who received the intra-nasal influenza vaccine, versus 13 percent injected with trivalent inactivated influenza vaccine and 45 percent of volunteers who were given placebo. Both vaccine comparisons with placebo were statistically significant.

Advantages of Attenuated Vaccine 1) Activates all phases of immune system. Can get humoral IgG and local IgA 2) Raises immune response to all protective antigens . Inactivation may alter antigenicity. 3) More durable immunity; more cross-reactive 4) Low cost 5) Quick immunity in majority of vaccinees 6) In case of polio and adeno vaccines, easy administration 7) Easy transport in field 8) Can lead to elimination of wild type virus from the community 6


Disadvantages of Attenuated vaccine 1) Mutation; reversion to virulence 2) Spread to contacts of vaccinee who have not consented to be vaccinated (could also be an advantage in communities where vaccination is not 100%) 3) Spread vaccine not standardized--may be mutated 4) Poor "take" in tropics 5) Problem in immunodeficiency disease.

Advantages of inactivated vaccine 1) Gives sufficient humoral immunity if boosters given 2) No mutation or reversion 3) Can be used with immuno-deficient patients 4) Sometimes better in tropics

Disadvantages of inactivated vaccines 1) Many vaccinees do not raise immunity 2) Boosters needed 3) No local immunity (important) 4) Higher cost 5) Shortage of monkeys (polio) 6) Failure in inactivation and immunization with virulent virus.

New methods of vaccine production 1) Selection for missense. Conditional lethal mutants. Temperature-sensitive mutants in influenza A and RSV made by mutation with 5-fluorouracil, then selected for temperature sensitivity. Can reassort the TS gene. Can also produce cold adapted mutants in this way. Get mis-sense in all six non-surface genes. A new attenuated influenza vaccine uses a cold-sensitive mutant that can be reassorted with any new virulent influenza strain that appears. The reassorted virus will have the genes for the internal proteins from the attenuated virus (and hence will be attenuated) but will display the surface proteins of the new virulent antigenic variant. Because this is based on a live, attenuated virus, the customization of the

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vaccine to each year's new flu variants is much more rapid than the current process of predicting what influenza strains will be important for the coming flu season and combining these in a killed vaccine. 2) Synthetic peptides. Problem with poor antigenicity. Epitope may depend on conformation of the virus as a whole. A non-viral example that has achieved some limited success is an anti-malarial vaccine. 3) Anti-idiotype vaccines. Antigen binding site is a reflection of the structure of the antigen. This unique amino acid structure is known as the idiotype. Antibodies can be raised against the idiotype (anti-ids). The anti id can bind to the same idiotope to that which the antigen binds. It therefore mimics the antigen and antibodies against it might potentially neutralize the virus. This happens: Anti-ids raised against antibodies to hepatitis B S antigen elicit anti-viral antibodies. 4) Recombinant DNA techniques. a) Attenuation of virus. Make deletion mutations which are large enough that they are unlikely to revert (though suppression a problem). Problem: In some vaccines, virus could still retain other unwanted characteristics such as oncogenicity (e.g. with adenovirus, herpes virus, HIV) b) Single gene approach. Expression of single gene (for protective antigen) in foreign host. Use expression vectors--bacteria (post-translational processing a problem); yeast (better). Has disadvantages of a killed vaccine. The current hepatitis B vaccine is this type. This has been used to make several HIV vaccines. The trouble is that they provoke little cell-mediated immunity. c) Cloning of protein into another virus--Vaccinia is a good candidate. Can make a multivalent vaccine virus strain in this way as Vaccinia will accept several foreign genes. A candidate HIV vaccine is now in trials. The use of vaccinia against smallpox has shown rare but serious complications in immunocompromised patients, alternatives have been sought. One is a recombinant canary pox virus that does not replicate in humans but does infect cells. Immunization with live recombinant canary pox vector expressing HIV env has induced HIV-1 envelope specific CTL response. Similar constructs with gag, protease, nef and parts of pol genes are in clinical trials.

DNA Vaccines "The Third Vaccine Revolution" The deliberate introduction of a DNA plasmid carrying a protein-coding gene that transfects cells in vivo at very low efficiency and expresses an antigen that causes an immune response. These are often called DNA vaccines but would better be called DNA-mediated or DNA-based immunization since it is not the purpose to raise antibodies against the DNA molecules themselves. The plasmid DNA is taken up by muscle cells after intramuscular injection. It has also be shown that DNA can be introduced into tissues by bombarding the skin with DNA-coated gold particles. It is also possible to introduce DNA into nasal tissue in nose drops. In the case of the gold bombardment method, one nanogram of DNA coated on gold produced an immune response. One microgram of DNA could potentially introduce a thousand different genes into the vaccinee. 8


Advantages of DNA vaccines 1) Plasmids are easily manufactured in large amounts 2) DNA is very stable 3) DNA resists temperature extremes so storage and transport are straight forward 4) DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the infectious agent 5) By using the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than, for example, using a recombinant plasmid to produce an antigen in yeast (e.g. the HBV vaccine), purifying that protein and using it as an immunogen. 6) Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced 7) The plasmid does not replicate and encodes only the proteins of interest 8) No protein component so there will be no immune response against the vector itself 9) Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the pathogen. A CTL response also offers protection against diseases cause by certain obligate intracellular pathogens (e.g. Mycobacterium tuberculosis) All of the above means that DNA vaccines are cheap and therefore likely to be developed against pathogens of lesser economic importance (at least to drug companies) Possible Problems 1) Potential integration of plasmid into host genome leading to insertional mutagenesis 2) Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies) 3) Induction of immunologic tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that antigen)

Initial studies Most work has been done on DNA vaccines against viruses since DNA-based plasmid immunization actually resembles virus infection. When they have been well-characterized, the immune responses are broad-based and mimic the situation seen in a normal infection by the homologous virus. The immune response can be remarkably long-lasting and even more so after one booster injection of plasmid. CTL responses are also well produced as might be expected since the immune system is seeing what is a model of an infected cell. One important demonstration using a DNA vaccine has been the induction of cytotoxic cellular immunity to a conserved protein of influenza A to determine if it might be possible to overcome the seasonal variation of the virus. CTLs were derived in mice against the conserved flu nucleoprotein and this was 9


effective at protecting the mice against disease even when they were challenged with a lethal dose of a virulent heterologous virus with a different surface hemagglutinin. Because transfer of antinucleoprotein antibodies to untreated mice does not protect them from disease, the protective effect of the vaccine must have been cell-mediated. The current influenza vaccine is an inactivated preparation containing antigens from the >flu strains that are predicted to infect during the next >flu season. If such a prediction goes awry, the vaccine is of little use. It is the surface antigens that change as a result of reassortment of the virus in the animal (duck) reservoir. The vaccine is injected intramuscularly and elicits an IgG response (humoral antibody in the circulation). The vaccine is protective because enough of the IgG gets across the mucosa of the lungs where it can bind and neutralize incoming virus by binding to surface antigens. If a plasmid-based vaccine is used, both humoral and cytotoxic T lymphocytes are produced, which recognize antigens presented by plasmid-infected cells. The CTLs are produced because the infected muscle cells present >flu antigens in association with MHC class I molecules. If the antigen presented is the nucleocapsid protein (which is a conserved protein) this overcomes the problem of antigenic variation. Such an approach could revolutionize the influenza vaccine. Other studies have used a mix of plasmids encoding both nucleoprotein and surface antigens. Protection by DNA vaccines has also been demonstrated with rabies, mycoplasma and Plasmodium yoelii. Human trials with the flu DNA vaccine are now in progress. Anti-HIV vaccines are also being tested. In the HIV lectures I alluded to the fact that progress on AIDS vaccines has been stymied by the fact that present vaccines only elicit humoral antibodies while the use of whole virus vaccines (which might elicit CTLs) has been rejected because of other potential problems. Plasmid-based vaccines may overcome these problems; indeed, the currently experimental anti-HIV plasmid-based vaccine elicits CTLs.

Common currently used anti-viral vaccines Polio (Salk) Polio (Sabin) Rabies Mumps Measles Rubella Influenza Hepatitis B Varicella Hepatitis A Rotavirus

Inactivated Attenuated Inactivated Attenuated Attenuated Attenuated Inactivated Subunit Attenuated Attenuated Attenuated (use suspended because of higher than normal incidence of intusseption - may not be statistically significant)

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TABLE 1. The maximum number of cases of specified vaccine-preventable diseases ever reported for a calendar year compared with the number of cases of disease and vaccine adverse events reported for 1995 C United States

Category

Maximum no. reported cases during prevaccine era

Year(s). maximum no cases reported

Reported no. cases during 1995

*Percentage change in morbidity

Disease Congenital rubella syndrome 20,000 H Diphtheria 206,939 Invasive Haemophilus influenzae 20,000 H Measles 894,134 Mumps 152,209 Pertussis 265,269 Poliomyelitis (wild) 21,269 Rubella 57,686 Tetanus 601

1964B65 1921

0

7 (B99.99)

1984 1941 1968 1934 1952 1969 1948

1,164 309 840 4,315 0 146 34

(B94.18) (B99.97) (B99.45) (B98.37) (B99.99) (B99.75) (B97.82)

Vaccine adverse events '

(B99.96)

10,594

*Provisional totals. H Estimated

because national reporting did not exist in the prevaccine era. ' Total number reported to the Vaccine Adverse Events Reporting System (VAERS).

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Vaccins for students2000