34 Vaccines II

Dr. M. N. Gupta

1. Objectives

•  To learn about the various methods of attenuation and vaccines based upon attenuated preparations of pathogens.

•  To learn about principles of various vaccine designs based upon synthetic peptides, viruses as vectors and DNA Vaccines

•  To understand the concept of anti-idiotype vaccines

•  To understand the inherent challenges in immunotherapy of tumours

2. Concept Map

3. Description

We discussed few forms of vaccines in which the pathogen was not active or its subcellular components were used as antigenic molecules.

The salk vaccine, when introduced did save many individuals from polio. The success was somewhat tainted around 1960-61 when few people died of polio. The cause for this was later on identified as poor antigenicity of the killed virus of one of the three different strains.

In this module, we now will discuss how attenuated preparations of pathogens can be used as vaccines. We will also discuss some more vaccine designs and challenges inherent in this area.

Till 1940, diphtheria was a serious illness. Introduction of diphtheria vaccine around 1941 led to a gradual drop over the next decade in UK.

Similarly (as mentioned earlier) introduction of salk vaccine in the late 1950‟s controlled the number of incidences of paralytic poliomyelitis. Later, Sabin‟s vaccine eradicated polio from UK (and elsewhere!). India has been polio free for the last 03 years. Polio, in fact, is eradicated from almost all parts of the world. After small pox eradication earlier, this has been another major triumph of the vaccination approach.

In the earlier module we had briefly mentioned that attenuated organisms have few advantages over the killed pathogens as vaccines.

Parasitic worms and protozoa (to some extent) pose challenges in scaling up their production. So, using them in killed form for use as vaccines is impractical.

Hence, many such considerations have led scientists to try attenuation of the organism. Attenuation is a modification of the organism which preserves all its other properties except pathogenicity.

The organism is alive and multiplies in the host animal and that is similar to sustained dose of antigen.

In the case of salk vaccine administered parenterally, its intranasal administration produced a better response in terms of nasopharyngeal IgA response. This showed that locally administered vaccine was more successful at protecting the site and inducing the response in terms of secretory protective antibody. Nevertheless this protection was over after ~2 months.

The attenuated virus vaccine, orally administered however resulted in a more sustained IgA level.

Currently, most of the antiviral vaccines in use are of either killed/inactivated viruses or live-attenuated viruses. The latter are more potent for many reasons.

Methods of attenuating organisms

One obvious approach to reach the objective will be to choose the organism which produces very mild form of the disease. That was the ancient Chinese practice.

Another is to choose strains which are virulent in other animals but avirulent in humans. That was Jenner‟s approach of using the cowpox virus.

It was Pasteur who discovered that altering the growth condition can make organisms avirulent. He succeeded in doing it for chicken cholera bacillus and anthrax by using high temperature and anaerobic conditions.

In 1908, Calmette and Guerin at Institute Pasteur incorporated bile in the growth medium of Mycobacterium with the aim of dispersion of the growing bacteria. What resulted was the valuable chance discovery of an attenuated strain of Mycobacterium bovis. The culture became attenuated when kept for 13 years in the bile containing medium. This strain BCG (Bacilla, Calmette, Guerin) is what we know of as BCG vaccine.

Cold adaptation of influenza and other respiratory disease causing viruses has been reasonably successful for obtaining attenuated forms. The viruses can replicate in the upper respiratory tract with temperature of 32-34 °C but slightly higher temperature of 37 °C in lower respiratory tract does not let them proliferate. This prevents clinical manifestation of the disease.

A similar example is that of vaccine for bovine rhinotracheitis. The temperature sensitive mutant of the herpes virus given intranasally replicates in lower temperatures of the nasal mucosa but cannot spread to the rest of the body to cause disease as the temperature elsewhere is higher.

Culturing viruses in cells which are not normally infected is known to reduce virulence. The attenuated polio virus vaccine was obtained from viruses grown in duck embryo cells.

In some cases, viruses have been attenuated by growing in eggs. The examples of this include 17D strain of yellow fever and influenza virus strains.

One may wonder if attenuated preparations of the pathogens surpass other vaccine designs, why that has not been universally adapted design in all the cases. Are there better or equally good other approaches to vaccine design?

Also, are there any constraints and disadvantages to use of vaccines based upon attenuated preparations of the pathogens.

Let us look at both the issues further.

What attenuation does to a pathogen is obviously mutation of its genes. These are likely to be multiple mutations in different genes coding for different proteins. It is possible that with time, or inside the animal/humans/birds, the pathogenicity will re emerge by more mutations.

To gain a perspective, type 3 Sabin polio vaccine strain had 10 different nucleotides out of 7429 nucleotides as compared to the wild strain. While the reversions to pathogenicity may be rare, it cannot be ruled out. The live viral vaccines also run the risk of viral nucleic acid getting integrated with the host.

Some complications can occur with live viruses. Measles immunization in rarest of rare cases can cause encephalitis. The natural injection by measles has the larger5 probability of 1:2000 of this occurring.

Live pathogens have to be kept under appropriate conditions of temperature. Developing countries do not have cold chain which enables vaccines to be produced, transported and stored appropriately. In many remote non-urban areas, while mobiles may be available, appropriate infrastructure with appropriate conditions and trained medical personnels are not always available. It is against this backdrop that the national polio eradication program‟s success must be appreciated.

When mammalian tissue culture is used for attenuation, the contamination is a serious possibility. In 1960 (when Sabin‟s vaccine was introduced), the oncogenic virus was found as a contaminant in the monkey kidney cells in a batch of vaccine.

Using rDNA technology for attenuation

The gene responsible for virulence is isolated and in vitro mutagenesis is used to modify it in such a way that virulence is abolished without affecting the desired antigenicity.

The virus genome is reconstituted. This attenuated strain can be used as vaccine. The gene for virulence can also be deleted if it does not affect its performance as a vaccine.

A herpes virus causes a disease called pseudorabies in pigs. The virus needs the thymidine kinase gene for replication in non dividing cells such as neurons. Removal of this gene led to a virus which could infect nerve calls but could not replicate and result in pseudorabies. It may be noted that in such cases, the vaccinated pigs were not carriers of the virulent virus any longer and spread of the infection could be halted.

The vaccine for foot-and-mouth disease virus was the first one to be produced by recombinant approach. The viral genes for the protective antigen (VPI) was known. The RNA genome of this RNA virus was duplicated as DNA by reverse transcriptase. The gene for VPI was cloned into E.coli.

The vaccine against enteropathogenic E.coli was also obtained by this approach. The enterotoxin is heat labile and has dimeric structure. The α-subunit is responsible for toxicity.

whereas β-subunit enables binding to enteric cells. The β-subunit based toxoid was found to have protective function as a vaccine. Hence, the β-subunit was cloned into a non-pathogenic E.coli strain.

The attachment pili of E.coli such as F4 or F5 are another attractive vaccine design targets as attachment of the pathogenic E.coli strain to the intestinal wall can be prevented.

The vaccine design of influenza viruses constitutes a challenging problem. Two major surface antigens are its haemagglutinin and neuraminidase. The agglutinin is involved in binding of the virus to the human cell and antibodies against it constitute protective antibody.

The problem is that the virus goes on changing its surface antigen in a limited way (antigenic drift) or to a significant level (antigenic shift).

Antigenic shifts have been tracked since early years. The influenza virus antigen nomenclature is in terms of HxNy.

The antigenic drifts and antigenic shifts render any vaccine based upon antibody against these antigens ineffective with time. Thus, it allows the virus to reinfect the host several times as the immune memory is only against earlier virus before the antigenic shift.

Hence, the killed virus vaccine is made every year based upon the prevailing virus. In principle, it should be able to develop a rDNA based approach which creates attenuated virus easily to match the wild virus strain. A number of mutants creating attenuation can be incorporated into the gene for viral polymerase PB2. The mutated viral gene can be each time substituted for the virulent variant. This may lead to faster production of the vaccine each year.

Another approach called “infection-permissive immunization” has also been developed. A recombinant virus containing the neuraminidase but a agglutinin (which is no longer relevant to the current strain) can be produced. This has shown partial immunity. It produces infection but may not permit full blown disease.

Using attenuated organisms as hosts

Vaccinia virus has a large genome which makes it easy to insert foreign genes into it. It has high level of protein expression. It has glycosylation and secretion is possible. In fact, simple dermal scratching is good enough for its administration.

The properties make vaccinia virus a valuable vehicle to carry genes for other antigens which can vaccinate against other organisms.

In fact, multiple vaccinia genes can be simultaneously introduced by this approach. While, vaccinia itself is no longer useful for small pox vaccination, as a carrier of other genes it is still considered an attractive candidate.

However, in this approach, the carrier virus cannot be used second time as a vehicle for different antigens. This restriction arises because of the phenomenon called “original antigen sin” which we had discussed earlier. To recollect it again, it implies that if an individual is exposed to one variant of a virus, infection with second variant will result in immune system generating antibodies against only those epitopes which were present in the first variant.

The worries about rare complications of small pox vaccination are associated with vaccinia virus. The approach has been used to incorporate genes for influenza virus haemagglutinin, vescicular stomatis virus glycoprotein, HIVgp120 and herpes simplex virus glycoprotein D.

Chimpanzees were protected successfully from hepatitis B virus. Hepatitis surface antigen (HBsAg) was found to be secreted by the vaccinia infected cells as 22 nm particles.

Similarly, mice injected with the influenza haemagglutinin formed specific Tc cells and did not show any clinical symptoms of influenza.

Some alternatives to vaccinia virus have also been tried. Attenuated salmonella organism have been used as a host for genes for antigens from tetanus, Listeria monocytogenes, Bacillus anthracis, Leishmania major, Yersinia pestis and Sapitosoma mansoni. These have been oral vaccines for mice.Another interesting variation is to use plant viruses in this „piggy back‟ approach. Plant viruses are not pathogens for humans. So, while use of other viruses carry the same risk as using live viruses as vaccines, this is a safer approach. On the minus side, the carrier has no protective role.

Mice were protected against rabies virus by prior feeding with spinach leaves containing recombinant alfa alfa mosaic virus containing a rabies virus peptide. The challenge is to improve protein expression in plant systems as a host. The attractive part is the administration route.

Synthetic Peptides

Synthetic peptides are now easy to make. Considering epitopes are generally of peptide sizes in antigens, use of synthetic peptides as a “synthetic epitope” is another powerful approach with huge potential.

What makes this approach increasingly more feasible is the possibility of identifying epitopes by use of monoclonals or through computer based modelling.

Epitopes, however, many times are not just a peptide sequence. In many case, conformation of the protein antigen brings together different peptide segments from different parts of the protein molecule to make an epitope. So, one would think that this approach will be of limited application. In fact, in practice, it is not so!

The „loop‟ peptide of diphtheria toxin induced antibodies with good neutralising effect. Similarly, a short peptide from the foot and mouth virus protein (VPI) also gave encouraging results. A peptide from polio virus VPI induced an antibody with poor neutralising effect but formed a good primary injection for protection against the virus. It is believed that this unexpected beneficial effect results from such peptides selecting specific T-cells in the animal. Please recollect that T-cell recognition is for peptides derived from processed antigens.

Another approach which has been called “reverse immunogenetics” is also based upon the above fact. The T-cells recognize these peptides in association with MHC molecules of the animals.

It was found that there was a correlation between human MHC class I antigen HLA-B53 and resistance to cerebral malaria. Cerebral malaria may not be frequent but when developed, it is usually fatal. It was believed that these MHC molecules present peptides which is good in inducing participation of Tc cells.

Cells infected with the pathogen had relevant peptides bound to these MHC molecules which could be eluted. Such nona-peptides were found to have proline with highest frequency at the second position. Variants of such peptides were identified in several cases.

In the next step, the candidate nona-peptides were screened by checking whether they fitted well in the groove of HLA-B53. This could be done by checking whether HLA-B53 and the peptide form a stable cell surface heterodimer.

This approach was used to identify peptides from four proteins of Plasmodium falciparum expressed early during hepatocyte infection. One candidate liver stage antigen-1 in association with HLA-B53 was found to be recognized by Tc cells.

Similar approach has been tried with a peptide eluted from MHC Class II molecules in macrophages infected with Leishmania. The peptide could help in isolation of the gene from Leishmania which in turn was exploited to produce a protein antigen based vaccine for Leishmania for mice successfully.

The constraint is that not all individuals will necessarily have the MHCvariant used to produce the vaccine. In fact, those people because of this may be more prone to the infection and need protection more than others!

Another constraint is that peptides are not strong immunogens, they are more of haptens.

Possibly, gene segments for such peptides could be integrated into carrier virus genes.

Use of immune stimulatory complexes (ISCOMs) is another way to overcome the constraint of poor immunogenicity of the peptides. ISCOMs are lipid adjuvants with very low toxicity. Acting like carriers, these help peptides to reach cytoplasm and facilitate the MHC class I restricted T-cell responses.

DNA Vaccines

cDNA which codes for henagglutinin of influenza virus injected into muscle tissues was found to result in the production of antibody and CTL response could be increased by incorporating a plasmid for GM0CSF in the vaccine. As only single gene is involved in such DNA vaccines, it does not have the risk associated with live vaccines. DNA delivery methods developed for gene therapy etc may be useful in this approach as well. This approach is attractive as the productive cost may be much less than many other forms of vaccines.

While DNA vaccines have shown considerable promise with animals, no DNA vaccine has yet been released for human vaccination program. An attractive target is anthrax vaccine which is being developed in India at JNU, New Delhi.

Anti-idiotype vaccines

It may be recollected that idiotypes are the binding sites in the antibody corresponding to antigenic epitopes. We had discussed this at some length while discussing Jerne‟s network theory of immune responses.

The antibody raised against an idiotype will have similar shape at its binding site as the original epitope. This is because both epitope and anti-idiotypic antibody both bind the antibody (against the epitope).

What will happen if we make antibody against anti-idiotype? The anti-idiotype will bind not only to anti-idiotype but also to the original epitope.

For raising vaccines against parasites, it is useful to separate determinants (epitopes) which induce Th action from Ts action. Thus, idiotypes can be exploited for designing the vaccine directed against the chosen epitope.

We have earlier in this module referred to the limitation of synthetic peptides as vaccines because many epitopes are conformational in nature. Using idiotype based approach overcomes this problem. There are two kinds of anti-idiotypes: Ab2α which corresponds to cross reactive idiotypes and Ab2β which are the internal images of the epitopes.

In cases, where an idiotype is strongly associated with specificity (such as murine T15id on anti-phosphorylcholine), Ab2α anti-idiotypes can form the basis for vaccines. However, in general, Ab2β are better stimulator for wider range of lymphocytes. Vaccines designed on this principle have shown good results with animals. For example, both Ab2α and Ab2β anti-idiotypes have shown good results for vaccination against hepatitis B in chimpanzees.

Immunotherapy against tumors

The vaccines against tumors are not available. The reasons are best understood by understanding the challenges inherent in immunotherapy (of tumours).

Some early approaches to immunotherapy of tumors were aimed at enhancing the response of the host‟s immune response to the tumors. Such approaches included attempts at increasing both non-specific immunity.

Infecting the tumor with an attenuated/killed virus, using hapten-tumor surface antigen conjugates and using biological response modifiers illustrate such approaches.

The use of antibodies in the therapy presents some challenges. F (ab)‟ fragments or engineered single domain antibodies are more suitable for penetration into large tumor mass as compared to whole antibody molecules. The most basic issue is of selectivity: the discrimination between tumor cells and normal cells.

There are many ways by which tumors escape what Burnet called “immune surveillance”. Lack of novel peptides generated during antigen processing confers low immunogenicity on tumors. Some tumors which escape immune response have selective advantage. Tumors often suppress T-cell mediated immunity by secreting immune suppressive factors such as TGF-β.

The result of these escape mechanisms is that tumors are almost never effectively attacked by T-cells. Tumors genes are also prone to mutation, so the mutants are one step ahead of the immune response. Tumor cells of colon and cervical cancers show loss of expression of MHC class I molecules, this prevents attack by CD8 T-cells.

However, such tumors can be attacked by NK cells. Please recollect that NK cells have receptors for MHC class I molecules so that tumor cells with even low expression of MHC class I molecules are attacked by NK cells.

With experimental animal systems, it is known that nude mice (which lack T-cells) have actually higher levels of NK cells.

mAbs alone or linked to toxins have given encouraging results with some tumors. About a quarter of breast cancer patients overexpress HER-2/neu, a growth factor receptor. A humanized mAb called herceptin against this blocks the receptor-factor interaction.

Rituximab is the commercial name for a mAb which binds to CD20 and has shown promise in treatment of non-Hodgkin‟s B-cell lymphoma. The binding clusters CD20 and the resulting signal results in lymphocyte apoptosis.

In fact, the first successful application of mAb for tumor treatment used anti-idiotypic antibody which target B-cell lymphomas. However, genetic instability resulted in re-appearance of tumor after initial remission.

Currently, vaccines for many diseases have been designed. While limited side effects for drugs are now considered acceptable risks, in case of vaccines, the collateral damage in case of a flawed design may have more serious consequences. Immune system has lot of internal synergy. Hence, vaccines for many pathogens and tumors have remained at experimental stage. At the time of writing this, a promising ebola vaccine has been announced.

Vaccine design is both challenging and exciting.

Summary

•  Attenuation methods and attenuated pathogens

•  rDNA technology in vaccine designs

•  Synthetic peptides as vaccines

•  DNA vaccines

•  Anti idiotypic vaccines

•  Immunotherapy for tumors