33 Vaccines I

Dr. M. N. Gupta

1. Objectives

•  To understand requirement of a good vaccine

•  To understand active immunization and passive immunization.

•  To learn about various types of vaccine designs

•  To learn about merits and demerits of use of live and killed pathogens as vaccines

2. Concept Map

3. Description

Control of infectious diseases requires multipronged efforts. Animals which are carriers have to be quarantined. Improving quality of water supply, sewage discharge methods and educating people about value of personal hygiene are all important measures.

Vaccination-the induction of immunity is part of this.

Immunology started with vaccination! But design of vaccines does not have just historical importance. It is a long persued aspect of immunology which remains a frontier area even today. We now have successful vaccines today. Eradication of small pox (from all over the world) and polio (from most parts of the world) is a major triumph of vaccination.

Yet, there are many diseases for which we still do not have vaccines. Let us look at what has worked and why it has not worked with some diseases.

Jenner used „vaccinia‟ for providing immunity against small pox in humans. The word „vaccination‟ was used by him and Pasteur continued its use while developing such materials for creating protective immunity against few other infectious agents. That is how immunology started.

It is interesting to note that in 1980, WHO announced global eradication of small pox.

Vaccinia worked as it only persists briefly as a subcutaneous infection. It nevertheless stimulate immune response crossreactive with small pox virus antigens.

The broad approaches followed subsequently for developing vaccines were:

To search attenuated pathogens with reduced pathogenicity but with capacity to stimulate immune response.

To use killed organisms. These are safer as there is no risk of systemic infection especially in immunodeficient/immnuosuppersed individuals.

To use purified components which are effective immunogens.

Effective vaccination is available in the case of large number of diseases which include:

•  Measles

•  Mumps

•  Polio

•  Tetanus

•  Deptheria

•  Whooping cough

In many countries children are advised/required to be immunized against these diseases.

It is a growing list.

What is a requirement for designing a successful vaccine?

Which microbial antigen is the best immunogen?

What is the point of entry of the infectious agents? Generally, that is the site where vaccine is best injected.

What are the immune responses which needs to be manipulated? For extracellular infectious agents, antibody provides a good defence. Where intracellular infection is involved, effective CD8 T-cell response should be induced.

Each issue present some challenges.

Choice of microbial antigens

Infectious organisms which have large number of serotypes (eg. Streptococcus pyrogenes and S. pneumoniae) would require a large number of antigen to be mixed.

Often correlation between protective effect and a particular antigen or its mechanism of action is missing. In some early vaccines of pertusis, there has been occasional report of brain damage.

In such cases, mechanism of protection or such side effects are unknown.

Nevertheless, whenever the effective antigens have been identified, these are preferable to using killed microbes which have multiple antigens and may cause problem such as hypersensitivity. Also antigens can be produced more economically through recombinant methods.

These are some examples which show that the mechanism of protection by the vaccine is known. This is one example which illustrates how what is sometime described as basic research pays dividends by providing insight into medically relevant phenomena.

Local effects

Some local sites such as genitourinary tract or gut require immunization at the site of infection. For example intravaginal vaccination with Neisseria gonorrhoeae is to be preferred over systemic route. Similarly, oral vaccine for Vibriae cholera is believed to be more effective than infected form.

Passive immunization

Passive immunization consists of injection of preformed antibody intravenously or intramuscularly These antibodies are derived from animals who produce high titre of these antibodies to specific microbes.

Passive immunization is useful when a rapid protection is required such as in the case of diphtheria, rabies, some clostridium species or hepatitis B.

Passive immunization is obviously useful for immunocompromised individuals as their immune system cannot offer protection.

IgG class antibodies are generally injected every 3 weeks to keep their effective level in the body.

Horses are generally used for obtaining these antibodies. Unfortunately, repeated injection carry the risk of ag-ab complex formation and serum sickness.

Oral vaccines

The challenges in designing oral vaccines involve ensuring that:

These reach the target epithelium or lymphoid tissue and generate limited infection.

It should be a stable mutant which results in the protection and does not revert back to highly pathogenic form.

It should have only limited replication cycles so that only limited proliferation for provoking immune response is there.

Yet another important factor is that vaccination with oral routes has a higher frequency of resulting in tolerance.

Types of vaccines

Toxoids:

Obtained by modifying toxins chemically such that it retains its immunogenicity but abolishes its toxicity completely. The extensive literature on chemical modification of proteins and bioconjugation chemistry is of considerable help. Earlier immunochemists were excellent protein chemists and that has led to early successes.

Attenuated live vaccines: These are bacterial or viral preparations modified to eliminate their pathogenic property. In general, these provide better protection. However, as these may proliferate, immunocompromised people will have a problem.

Subunit vaccines: An antigenic component isolated from the infectious organism. This is protein in nature and hence can be obtained by the recombinant route.

Vector vaccines: Antigen genes inserted into a non pathogenic viral vector such as vaccinia or adenovirus.

Conjugate vaccines: Weak antigens coupled to a toxoid. The toxoid stimulates T-cells which help B-cells producing antibody against the weak antigens. An example is 3 polysaccharide antigens (meningitis C organism) coupled to diphtheria toxoid.

DNA vaccines: So far none has been approved for use in humans. Here the DNA responsible for producing suitable antigen is taken up by the cells.

Combined vaccines: Many vaccines are given together to infants. The trivalent DPT vaccine for diphtheria, pertusis, tetanus is a well known example. UNICEF has recommended pentavalent vaccine which in addition has vaccines for hepatitis B and haemophilus influenza B.

In addition to adjuvants, vaccines may contain stabilizers and preservatives. In developing countries like India, absence of a “cold chain” (keeping the biological sample at cold temperature to prevent loss of its biological activity/property from the production point to the administration point) right upto remote nonurban areas is a big handicap. Stabilizers and preservators solve this problem in a very limited way and are not substitutes for the cold chain.

Administration of vaccines: Most vaccines are administered in the form of subcutaneous or intradermal injection. Some oral vaccines are available. Polio (Sabin) vaccine and rotavirus vaccines are examples of oral vaccines. Nasal route has been used for flu vaccines.

Nanoparticle vaccines show promise for being delivered through mucosal and systemic route.

Adjuvants:

We had earlier discussed adjuvants as materials which when administered along with an antigen enhance immune response.

Some bacterial products which are used as part of vaccine designs have the following composition:

BCG (Bacillus Calmette Guerin) is a non virulent strain of M. bovis. MDP (Muramyl dipeptide) is the cell wall component which has adjuvant action.

LPS (endotoxins/lipopolysaccharides), part of gram negative bacterial cell walls helps in B-cell proliferation and causes macrophage activation.

PTx (Bordetella partusis toxoid) binds T-cells and facilitates their proliferation.

In the context of vaccines, nanoparticles in nanoparticles vaccines actually function as adjuvants.

So, in nanoparticle vaccines, vaccine is adsorbed on nanoparticles. While current accepted definition of nanoparticles include particle size upto 100 nm, in the present case particles of 10-1000 nm have been described for such applications.

The materials used for preparing such nanoparticles include:

•  Poly (lactic acid) and poly (glycolic acid)

•  Poly (lactide-co-glycolide) (PLGA)

Nanoparticles coated with manan target mannose receptors on APC.

mAb have been used to coat nanoparticles as homing devices for specific target on dendritic cells.

Apart from nanoparticles, liposomes, muramyl dipeptide, immune stimulating complexs (ISCOMs) such as containing cholesterol and phospholipids and bacterial toxoids show high promise for being approved for human use. Right now only, aluminium and calcium salts are approved as adjuvants for vaccine preparations.

Other adjuvants being studied include IL-1, IL-2, IFNγ (cytokines), immune complexes and Freund‟s adjuvant.

Inflammation caused by the injection brings in macrophages and dendritic cells to local site.

Consequent cytokine production leads to recruitment of T-cells.

Ideally, an adjuvant should be able to stimulate both Tc and Th cells.

As has been mentioned before, vaccines containing live organism do not need adjuvants, A broader term is biological response modifiers (BRM) which mostly enhance immune response and include adjuvants. Toll like receptors and cytokines can be termed BRMs.

The debate about pros and cons of attenuated pathogens Vs killed pathogens has often been a bitter one. Polio vaccine exemplifies it best. Let us first understand what are these two types of vaccines. Jonas Salk produced killed virus vaccine whereas Sabin‟s polio vaccine consists of attenuated “live-virus”. The debate continues, both types of vaccine have positive and negative features.

In the first module on the history of immunology, we mentioned the ancient Chinese practice of using scabs of mild cases of smallpox for vaccination. In this approach, there is an inherent hazard of disease breaking out if the organism starts multiplying inside the body at a rapid rate.

Chemical modification of proteins is a useful technique which has proved useful in large number of areas in life sciences. In those ancient days, this toolbox was not developed.

The killed organism vaccines are prepared by reacting the organism with chemicals which modify proteins and other antigens.

Earlier approach of using heat to kill organism was found unsatisfactory as antigenicity of the microorganism has to be preserved. Formaldehyde is generally used for inactivating. This reacts with amino group of proteins, purines and pyrimidines of nucleic acids). The result is introduction of conformational rigidity which blocks biological function by forming cross links.

Other examples are of alkylating agents for nucleic acids such as ethylene oxide, ethyleneimine, acetylethyleneimine, and β-propiolactone.

Vaccines based upon this approach are only able to produce humoral immunity Salk polio vaccine is given intramuscularly and uses formaldehyde for inactivation.

Such vaccine preparations can be easily mixed with other vaccine preparations. Polio viruses has three major strains. Type 1 virus is more dangerous than type 2 and 3 and seems to be far more frequent cause of paralysis. However, there is cross reactivity of antibodies. So, infection with type 2 prevents paralysis chances upon infection with type 1.

Immunity by killed microbes has shorter period as compared to attenuated vaccines. One belief among immunologist has been that “there is no immunity like convalescent immunity”. This relates to the observation of Jenner‟s days that those which survive the dreaded smallpox never got the disease again.

It is true of polio. The viral infection gives life long immunity. One has to however remember that convalescent immunity is not absolute or permanent. Sometime, a different strain may crop up without enough crossreactivity. In some cases, revaccination is required. A good example is flu vaccine. So, lasting immunity is not ensured in every disease.

The real advantage is in the case of immunodeficient people with virulence totally eliminated, the killed microbe vaccine can be safely given in such cases.

The killed polio virus vaccine does not prevent colonization of intestine by the wild polio virus. In general, whenever cell mediated immunity is important, in those cases the vaccines based upon killed microbes concept may be inadequate (Table 1).

Thus, if the natural defence mechanism against some microbes require other mechanism to operate simple antiobody production by such vaccines may be a handicap. However, as above examples show, enough protection is available the large number of diseases. Production of antibody will elicit some B-cell Th cell cooperation.

Salk polio vaccine does not lead to detection of anti-polio IgA is nasal and duodenal secretions. This has the disadvantage of not being able to fight the virus locally. Serum IgG may play some protective role, however, at epithelial surfaces.

Toxoids

Tetanus, diphtheria, gas gangrenes and cholera represent diseases which are caused by toxins produced by the pathogens. Bacteria produces these toxins to damage host cells.

which allows invasion. Toxoids prepared by reactions of formaldehyde with exotoxins of diphtheria and tetanus bacteria are examples of the success of this approach.

Toxoids are given along with adjuvants. In many cases, adsorption on aluminium hydroxide works well. Metal hydroxides are known to adsorb many proteins very well and in fact forms one of the method of enzyme immobilization.

In case of cholera vaccine, the cholera toxin subunit B is conjugated to killed vibrio and is better as it facilitates gut mucosa to form antibodies.

Purified antigen vaccine and polysaccharide vaccines

Given current state of the art in protein production, isolating a pure protein antigen is a viable proposition.

One advantage is that this avoids antigen overload from other antigens (present on the pathogens) which may lead to hypersensitivity reactions.

With advent of rDNA technology, many protein antigens can be easily produced in appropriate expression hosts.

In such cases, the antigen must have an epitope which stimulates B-cell but it is also crucial to have an epitope which is able to recruit CD4+ TH cells.

The approach benefits from the fact that extensive work on epitopes responsible for pathogenesis has been carried out in many cases. Currently, epitope identification has been adapted to the high throughput mode and can even be outsourced to some specialized companies.

In herpes virus glycoprotein D (Gly D) results in protective CTL response. Effective delivery methods resulting in complete immune response remains a challenge in this approach.

Vaccines with purified capsular polysaccharides have been designed for infections of S. pneumonia and Neisseria meningtidis. The former was produced by merck with pneumovax 23 and by lederle labs with pneu-immune 23 as trade names. The disadvantage is that polysaccharides are poor antigens in class switching and producing B-cell memory.

To overcome this, a vaccine for H-infuenza (Hib) has been designed by linking type b polysaccharide to tetanus toxoid as a carrier. It did not produce memory T cells specific to influenza infection.

The first successful example of purified antigen approach has been hepatitis B virus surface antigen (HBsAg) produced by cloning the viral gene in yeast. Again it does not produce Tc response.

Given the advances in protein expression by cloning, this would continue to be an active area in vaccine designs.

Vaccination schedules

Many vaccines which are now commercially available and are accepted by the medical community worldwide are given to humans either routinely or just before travel and people whose working/occupational environment makes them more prone to infection with a particular microbe.

Herd immunity is a useful concept in vaccination programmes if significant % of this population is vaccinated, it creates islands of disruption in the spread of infection. This eliminates the number of hosts wherein the pathogens may multiply. Both factors protect the non-vaccinated individuals from that pathogen.

Now, it is a standard practice to immunize children against pathogens for which safe and useful vaccines are available. In USA, for example pediatric population is given vaccines for following diseases:

• Diphtheria

• Tetanus (toxoids with pertusis vaccina)

• Oral poliovirus vaccines (attenuated virus type 1, 2 and 3)

• Combined vaccine for measles, mumps and rubella

• Haemophilus b diphtheria toxoid conjugate

In India, polio oral vaccine administration program has been launched few years back and is already showing remarkable results.

BCG vaccination program are different in various countries. In USA and UK, it is based upon risk perception. Incidences of > 40 per 100,000 individuals are taken as indicator of high risk population. In some countries, BCG vaccination is mandatory.

Not all individuals respond to vaccination in a similar way. 100% population thus will never be protected by vaccination. However, because of herd immunity concept, vaccination is still effective in controlling large number of cases.

Immunodeficient individuals, individuals on immunosuppressive drugs, under extreme conditions of temperature or stress situations and malnourished individuals all show poor immune response. It should be rembered that vaccination efficacy depends upon the individuals immune response.

Epidermiological studies on a disease form the basis for designing vaccination strategies. Continuous monitoring with accurate record keeping are critical in assessing the success of any mass vaccination program.

Immigration authorities of different countries require proof of immunization against specific diseases.

Malaria infects nearly over 200 million people globally. About 10% of these are serious cases. Malaria is caused by the protozoan parasite called plasmodium falciparum which infects erythrocytes. In rodents, the malaria parasite is plasmodium yoelii.

Antibody mediated mechanism involve blocking invasion by inhibiting its attachment to a new host cell. Plasmodium species infects erythrocytes as merozoites through binding to a receptor. This binding is blocked by specific antibody. Opsonisation by ab also encourage phagocytosis of infected cells, by macrophages.

Malarial parasite evades immune response by antigenic drift.

This parasite also releases soluble antigens. These are polymorphic and have repetitive aminoacid sequences. These antigens act as decoy by multiple mechanisms. As a consequence of these properties of malarial parasites a successful vaccine for malaria poses enormous challenge.

In general, as all parasites evade immune system by different mechanisms, vaccines against them (such as Trypanosoma, Leishmania, Schistosoma mansoni) are difficult to design.

We have just discussed various types of vaccines. Many such designs need to be looked at more closely. For example, we need to understand how live pathogens can be used safely as vaccines. After all, it all started with Jenner using a live virus !

There are some new concepts like DNA vaccines. When we discussed Jerne‟s theory of network selection, we had hinted that this concept of networks is not totally abandoned. We will see its application is vaccine design.

Summary

•  Properties of a good vaccine

•  Active and passive immunization

•  Various types of vaccine designs

•  Toxoids and killed pathogens as vaccines