IMMUNOLOGICAL PRINCIPLES OF VACCINATION

General Points

1. B cells antigen receptors predominantly sees 3-Dimensional conformations.

2. T cells sees processed antigen in association with MHC molecules again as a 3-D conformation.

3. In an Antigen Presenting Cell, such as a macrophage, non-infectious proteins were endocytosed and degraded in lysosomes to peptides (endosomal pathway), some of which bound specifically to MHC II molecules. In contrast, if an agent such as a virus infected the macrophage, some newly synthesized viral antigens are likewise degraded in the cytoplasm to peptides (the cytoplasmic pathway), but these are associated with class I MHC molecules.

The different components of the immune response to infection

Antibody has 3 important functions; -

1. It is the only means to prevent an infection by neutralization of viral infectivity. The generation of protective Ab has usually been the only response measured by vaccine developers. Generally neutralizing Ab reacts with just a few epitopes on one or two surface antigens of the infecting agent. It is ineffective if the protective epitopes are subject to pronounced antigenic drift.

2. Infected cells which express viral antigen on their cell surface may be lysed by 2 antibody-dependent mechanisms - (a) complement pathway, or (b) antibody-dependent cell cytotoxicity.

3. Antibody may facilitate the removal of debris (a scavenging mechanism)

The main function of effector T cells, in particular Tc cells, is to clear an infection. Although antibody can contribute to recovery from infection, T cells are the main mechanism for achieving this. The most important feature of CMI, in direct contrast to Ab, is that it responds to many peptides from an agent. Some come from proteins that are not subject to antigenic variation. This broad T cell response is an important mechanism for overcoming antigenic variation of an agent and the genetic variability of the host population. Although such an agent may bypass the protection afforded by a preformed Ab after vaccination, the cell-mediated immune response allows most people to recover from an acute infection. Infectious agents causing chronic persistent infections have found a way of escaping a cell-mediated immune response. The mechanisms include;

1. Generation of cells that escape a cell-mediated immune response.

2. Down regulation of MHC production in infected cells so that they are not recognized and destroyed by T cells.

3. Infection of cells in immunoprivileged sites such as the brain.  

The typical events that occurs after an acute virus infection eg. murine influenza is as follows ;-

1. Virus replicates in the lungs, reaches maximum titres in 4 - 5 days,and decreases thereafter. About 12 days virus can no longer be recovered.

2. Effector Tc cell activity reaches a maximum activity after 6 - 8 days, becomes undetectable after 14 days. Tc memory cells reaches their highest level 2 - 6 weeks after infection and remain constant or decrease only slowly.

3. The number of Ab producing cells,producing first IgM then IgG and IgA peak at about 6 weeks and then steadily decline. Maximum B cell memory is found 10 - 15 weeks after infection but decreases thereafter but some are present at 18 months.

Both Ab-secreting and memory B cells are present after infection. In contrast Tc activity is generated only whilst infectious virus is present. Memory T cells persist but require further exposure to infectious virus for reactivation.

Requirements of a vaccine

To be effective a vaccine should be capable of eliciting the following ;-

1. Activation of Antigen-Presenting Cells to initiate antigen processing and producing interleukins.

2. Activation of both T and B cells to give a high a high yield of memory cells.

3. Generation of Th and Tc cells to several epitopes, to overcome the variation in the immune response in the population due to MHC polymorphism.

4. Persistence of antigen, probably on dendritic follicular cells in lymphoid tissue, where B memory cells are recruited to form antibody-secreting cells that will continue to produce antibody.

Live vaccines fulfill these criteria par excellence. Neutralizing Abs are very important. Subunit vaccines induce poor immune responses and several doses with adjuvant are required to get an adequate response. The two main functions of the adjuvant antigen are to keep the antigen at or near the injection site and to activate antigen-presenting cells to achieve effective antigen processing and interleukin production. Vaccines composed simply of one T-cell epitope and one B- cell epitope is unlikely to be effective. This is exemplified by attempts to develop a malaria vaccine.

General Principles 

The most successful immunization programs have been those directed against viral diseases such as smallpox, poliomyelitis and measles. Before embarking on any vaccination program, the need for a vaccine in a community must be evaluated. An epidemiological assessment of the incidence and severity of an infection will determine whether it is worth preventing. No vaccine is completely safe and potential benefits from immunization should be weighed against the risk of side effects. Two ingredients are needed for a successful immunization program.

1. A safe and effective vaccine

2. An appropriate strategy with adequate vaccine coverage

The strategy required depend on whether the aim of the program is eradication, elimination or containment. Eradication is the complete extinction of the organism in question. In elimination, the disease disappears but the organism remains. Containment is the control of the disease to the point at which it no longer constitutes a public health problem.

When eradication or elimination is the aim, mass immunization in early life of both sexes is usually necessary. In practice, it is very difficult to achieve 100% coverage and the success of the program depends on the ability of the vaccine to interrupt transmission of the wild virus, thereby protecting the unvaccinated. When containment is the aim, selective immunization of those most at risk is normally sufficient. The usual indications for selective immunization are travel, occupational risk, outbreak control and for individuals at special risk of severe illness. Herd immunity plays little part, as the virus continues to circulate widely among the unvaccinated. In theory, selective immunization is less expensive than mass immunization. In practice, it is not always easy to identify and vaccinate those most at risk and mass immunization may be an easier option.

Vaccination policy

1. Varies between different countries

2. Maternal antibodies present up to 6 months after birth, which may interfere with the induction of an effective immune response against the vaccine by the infant. This should be duly taken into account when formulating a vaccination policy.

WHO expanded program for immunization (EPI)

1. Aimed at developing countries and initiated in 1974.

2. Aims to control and not eradicate 6 common disease through national health programs.

3. The six diseases are TB, DPT, polio and measles.

Different Types of Vaccine

Whole virus vaccines. either live or killed, constitute the vast majority of vaccines in use at present. However, recent advances in molecular biology had provided alternative methods for producing vaccines. Listed below are the possibilities;-  

1. Live whole virus vaccines

2. Killed whole virus vaccines

3. Subunit vaccines;- purified or recombinant viral antigen

4. Recombinant virus vaccines

5.  Anti-idiotype antibodies

6. DNA vaccines

1. Live Vaccines

Live virus vaccines are prepared from attenuated strains that are almost or completely devoid of pathogenicity but are capable of inducing a protective immune response. They multiply in the human host and provide continuous antigenic stimulation over a period of time, Primary vaccine failures are uncommon and are usually the result of inadequate storage or administration. Another possibility is interference by related viruses as is suspected in the case of oral polio vaccine in developing countries. Several methods have been used to attenuate viruses for vaccine production.

Use of a related virus from another animal - the earliest example was the use of cowpox to prevent smallpox. The origin of the vaccinia viruses used for production is uncertain.

Administration of pathogenic or partially attenuated virus by an unnatural route - the virulence of the virus is often reduced when administered by an unnatural route. This principle is used in the immunization of military recruits against adult respiratory distress syndrome using enterically coated live adenovirus type 4, 7 and (21).

Passage of the virus in an "unnatural host" or host cell - the major vaccines used in man and animals have all been derived this way. After repeated passages, the virus is administered to the natural host. The initial passages are made in healthy animals or in primary cell cultures. There are several examples of this approach: the 17D strain of yellow fever was developed by passage in mice and then in chick embryos. Polioviruses were passaged in monkey kidney cells and measles in chick embryo fibroblasts. Human diploid cells are now widely used such as the WI-38 and MRC-5. The molecular basis for host range mutation is now beginning to be understood.

Development of temperature sensitive mutants - this method may be used in conjunction with the above method.  

2. Inactivated whole virus vaccines

These were the easiest preparations to use. The preparation was simply inactivated. The outer virion coat should be left intact but the replicative function should be destroyed. To be effective, non-replicating virus vaccines must contain much more antigen than live vaccines that are able to replicate in the host. Preparation of killed vaccines may take the route of heat or chemicals. The chemicals used include formaldehyde or beta- propiolactone. The traditional agent for inactivation of the virus is formalin. Excessive treatment can destroy immunogenicity whereas insufficient treatment can leave infectious virus capable of causing disease. Soon after the introduction of inactivated polio vaccine, there was an outbreak of paralytic poliomyelitis in the USA use to the distribution of inadequately inactivated polio vaccine. This incident led to a review of the formalin inactivation procedure and other inactivating agents are now available, such as Beta-propiolactone. Another problem was that SV40 was occasionally found as a contaminant and there were fears of the potential oncogenic nature of the virus.
 

Live vs Dead vaccines

Feature                 Live         Dead

Dose low high

no. of doses             single         multiple

need for adjuvant         no               yes

Duration of immunity     many years        less

antibody response         IgG,          IgA IgG

CMI                       good             poor

Reversion to virulence   possible not     possible

Because live vaccines replicate inside host cells, bits of virus antigen are presented to the cell surface and recognized by cytotoxic cells

Potential safety problems

Live vaccines

1. Underattenuation

2. Mutation leading to reversion to virulence

3. Preparation instability

4. Contaminating viruses in cultured cells

5. Heat lability

6. Should not be given to immunocompromized or pregnant patients

Killed vaccines

1. Incomplete inactivation

2. Increased risk of allergic reactions due to large amounts of antigen involved

Present problems with vaccine development include

1. Failure to grow large amounts of organisms in laboratory

2. Crude antigen preparations often give poor protection. eg. Key antigen not identified, ignorance of the nature of the protective or the protective immune response.

3. Live vaccines of certain viruses can  (1). induce reactivation, (2) be oncogenic in nature  

3._Subunit Vaccines

Originally, non-replicating vaccines were derived from crude preparations of virus from animal tissues. As the technology for growing viruses to high titres in cell cultures advanced, it became practicable to purify virus and viral antigens. It is now possible to identify the peptide sites encompassing the major antigenic sites of viral antigens, from which highly purified subunit vaccines can be produced. Increasing purification may lead to loss of immunogenicity, and this may necessitate coupling to an immunogenic carrier protein or adjuvant, such as an aluminum salt. Examples of purified subunit vaccines include the HA vaccines for influenza A and B, and HBsAg derived from the plasma of carriers.  

4. Recombinant viral proteins

Virus proteins have been expressed in bacteria, yeast, mammalian cells, and viruses. E. Coli cells were first to be used for this purpose but the expressed proteins were not glycosylated, which was a major drawback since many of the immunogenic proteins of viruses such as the envelope glycoproteins, were glycosylated. Nevertheless, in many instances, it was demonstrated that the non-glycosylated protein backbone was just as immunogenic. Recombinant hepatitis B vaccine is the only recombinant vaccine licensed at present.

An alternative application of recombinant DNA technology is the production of hybrid virus vaccines. The best known example is vaccinia; the DNA sequence coding for the foreign gene is inserted into the plasmid vector along with a vaccinia virus promoter and vaccinia thymidine kinase sequences. The resultant recombination vector is then introduced into cells infected with vaccinia virus to generate a virus that expresses the foreign gene. The recombinant virus vaccine can then multiply in infected cells and produce the antigens of a wide range of viruses. The genes of several viruses can be inserted, so the potential exists for producing polyvalent live vaccines. HBsAg, rabies, HSV and other viruses have been expressed in vaccinia.

Hybrid virus vaccines are stable and stimulate both cellular and humoral immunity. They are relatively cheap and simple to produce. Being live vaccines, smaller quantities are required for immunization. As yet, there are no accepted laboratory markers of attenuation or virulence of vaccinia virus for man. Alterations in the genome of vaccinia virus during the selection of recombinant may alter the virulence of the virus. The use of vaccinia also carries the risk of adverse reactions associated with the vaccine and the virus may spread to susceptible contacts. At present, efforts are being made to attenuate vaccinia virus further and the possibility of using other recombinant vectors is being explored, such as attenuated poliovirus and adenovirus.  

5. Synthetic Peptides

The development of synthetic peptides that might be useful as vaccines depends on the identification of immunogenic sites. Several methods have been used. The best known example is foot and mouth disease, where protection was achieved by immunizing animals with a linear sequence of 20 aminoacids. Synthetic peptide vaccines would have many advantages. Their antigens are precisely defined and free from unnecessary components which may be associated with side effects. They are stable and relatively cheap to manufacture. Furthermore, less quality assurance is required. Changes due to natural variation of the virus can be readily accommodated, which would be a great advantage for unstable viruses such as influenza.

Synthetic peptides do not readily stimulate T cells. It was generally assumed that, because of their small size, peptides would behave like haptens and would therefore require coupling to a protein carrier which is recognized by T-cells. It is now known that synthetic peptides can be highly immunogenic in their free form provided they contain, in addition to the B cell epitope, T- cell epitopes recognized by T-helper cells. Such T-cell epitopes can be provided by carrier protein molecules, foreign antigens. or within the synthetic peptide molecule itself.

Synthetic peptides are not applicable to all viruses. This approach did not work in the case of polioviruses because the important antigenic sites were made up of 2 or more different viral capsid proteins so that it was in a concise 3-D conformation.  

Advantages of defined viral antigens or peptides include:

1. Production and quality control simpler

2. No NA or other viral or external proteins, therefore less toxic.

3. Safer in cases where viruses are oncogenic or establish a persistent infection

4. Feasible even if virus cannot be cultivated

Disadvantages:

1. May be less immunogenic than conventional inactivated whole-virus vaccines

2. Requires adjuvant

3. Requires primary course of injections followed by boosters

4. Fails to elicit CMI.  

6. Anti-idiotype antibodies

The ability of anti-idiotype antibodies to mimic foreign antigens has led to their development as vaccines to induce immunity against viruses, bacteria and protozoa in experimental animals. Anti-idiotypes have many potential uses as viral vaccines, particularly when the antigen is difficult to grow or hazardous. They have been used to induce immunity against a wide range of viruses, including HBV, rabies, Newcastle disease virus and FeLV, reoviruses and polioviruses.

7. DNA vaccines

Recently, encouraging results were reported for DNA vaccines whereby DNA coding for the foreign antigen is directly injected into the animal so that the foreign antigen is directly produced by the host cells. In theory these vaccines would be extremely safe and devoid of side effects since the foreign antigens would be directly produced by the host animal. In addition, DNA is relatively inexpensive and easier to produce than conventional vaccines and thus this technology may one day increase the availability of vaccines to developing countries. Moreover, the time for development is relatively short which may enable timely immunization against emerging infectious diseases. In addition,  DNA vaccines can theoretically result in more long-term production of an antigenic protein when introduced into a relatively nondividing tissue, such as muscle.

Indeed some observers have already dubbed the new technology the "third revolution" in vaccine development—on par with Pasteur's ground-breaking work with whole organisms and the development of subunit vaccines. The first clinical trials using injections of DNA to stimulate an  immune response against a foreign protein began for HIV in 1995. Four other clinical trials using DNA vaccines against  influenza, herpes simplex virus, T-cell lymphoma, and an additional trial for HIV were started in 1996.

The technique that is being tested in humans involves the direct injection of plasmids - loops of DNA that contain genes for proteins produced by the organism being targeted for immunity. Once injected into the host's muscle tissue, the DNA is taken up by host cells, which then start expressing the foreign protein. The protein serves as an antigen that stimulate an immune responses and protective immunological memory.

Enthusiasm for DNA vaccination in humans is tempered by the fact that delivery of the DNA to cells is still not optimal, particularly in larger animals. Another concern is the possibility, which exists with all gene therapy, that the vaccine's DNA will be integrated into host chromosomes and will turn on oncogenes or turn off tumor suppressor genes. Another potential downside is that extended immunostimulation by the foreign antigen could in theory provoke chronic inflammation or autoantibody production  

Presentation of immunogenic proteins and peptides

Proteins separated from virus particles are generally much less immunogenic than the intact particles. This difference in activity is usually attributed to the change in configuration of a protein when it is released from the structural requirements of the virus particle. Many attempts have been made to enhance the immunogenic activity of separated proteins.  

Adjuvants

Used to potentiate the immune response

1. Functions to localize and slowly release antigen at or near the site of administration.

2. Functions to activate APCs to achieve effective antigen processing or presentation

Materials that have been used include;-

1. Aluminum salts

2. Mineral oils

3. Mycobacterial products, eg. Freud's adjuvants

Immunostimulating complexes (ISCOMS)

1. An alternative vaccine vehicle

2. The antigen is presented in an accessible, multimeric, physically well defined complex

3. Composed of adjuvant (Quil A) and antigen held in a cage like structure

4. Adjuvant is held to the antigen by lipids

5. Can stimulate CMI

6. Mean diameter 35nm

In the most successful procedure, a mixture of the plant glycoside saponin, cholesterol and phosphatidylcholine provides a vehicle for presentation of several copies of the protein on a cage-like structure. Such a multimeric presentation mimics the natural situation of antigens on microorganisms. These immunostimulating complexes have activities equivalent to those of the virus particles from which the proteins are derived, thus holding out great promise for the presentation of genetically engineered proteins.

Similar considerations apply to the presentation of peptides. It has been shown that by building the peptide into a framework of lysine residues so that 8 copies instead of 1 copy are present, the immune response induced was of a much greater magnitude. A novel approach involves the presentation of the peptide in a polymeric form combined with T cell epitopes. The sequence coding for the foot and mouth disease virus peptide was expressed as part of a fusion protein with the gene coding for the Hepatitis B core protein. The hybrid protein, which forms spherical particles 22nm in diameter, elicited levels of neutralizing antibodies against foot and mouth disease virus that were at least a hundred times greater than those produced by the monomeric peptide.  

Immunization and Herd Immunity  

The following questions should be asked when a vaccination policy against a particular virus is being developed.

1. What proportion of the population should be immunized to achieve eradication.

2. What is the best age to immunize?

3. How is this affected by birth rates and other factors

4. How does immunization affect the age distribution of susceptible individuals, particularly those in age-classes most at risk of serious disease?

5. How significant are genetic, social, or spatial heterogeneities in susceptibility to infection?

6. Hoe does this affect herd immunity?

A basic concept is that of the basic rate of the infection R₀. for most viral infections, R₀ is the average number of secondary cases produced by a primary case in a wholly susceptible population. Clearly, an infection cannot maintain itself or spread if R₀ is less than 1. R₀ can be estimated from as B/(A-D);B = life expectancy, A = average age at which infection is acquired, D = the characteristic duration of maternal antibodies.

The larger the value of R₀, the harder it is to eradicate the infection from the community in question. A rough estimate of the level of immunization coverage required can be estimated in the following manner: eradication will be achieved if the proportion immunized exceeds a critical value pinc = 1-1/R₀. Thus the larger the R₀, the higher the coverage is required to eliminate the infection. Thus the global eradication of measles, with its R₀ of 10 to 20 or more, is almost sure to be more difficult to eradicate than smallpox, with its estimated R₀ of 2 to 4. Another example is rubella and measles immunization in the US. Rubella (A = 9 years) has an Ro roughly half that of measles (A = 5 years) and indeed rubella has been effectively eradicated in the US while the incidence of measles have declined more slowly.

Why do we not require 100% coverage to eradicate an infection? Immunization has both a direct and indirect effect. The susceptible host population is reduced by mass immunization so that the transmission of infection has become correspondingly less efficient and eventually, the infection will be unable to maintain itself.