Vaccine and Vaccination Department of Microbiology Introduction Vaccines

Vaccine and Vaccination Department of Microbiology

Introduction Vaccines may be defined as biological formulations composed of an infectious agent or its component (s), the administration of which results in generation of protective immune response.

Vaccines • Immunization is considered to be the most significant development in the field of health care that has saved lives of millions. • After the use of cow pox material for immunization against small pox, a large number of biological products have been shown to induce Immunity. • Presently, we have various types of biological material in use for the purpose of Immunization in human or animals. • There are two broad categories of vaccines i. e. , a) whole agent vaccine and b) sub unit vaccine. • Whole agent vaccines include vaccines that use live and inactivated organisms whereas sub-unit vaccines include vaccines which only contain the components of organism not the whole organism.

Live vaccines • A large number of live organisms are used as vaccines. Live vaccines have several advantages: • The immunity induced by live vaccines is often solid and gives long term protection. • Live organisms in the vaccine grow in the host and thus mimic the natural infection. Therefore, a more exacting type of response is mounted. • In addition to this, live vaccines generally do not require adjuvants and booster immunization.

Live vaccines However, live vaccines suffer from various disadvantages: • In live attenuated vaccines there are chances of reversal of virulence (attenuated organism again becoming virulent). • Moreover, live vaccines require cold chain maintenance to remain efficacious. • Because of the chance of killing of live organism during storage usually the shelf life of live vaccines are less and depends largely on the storage conditions.

Types of Live vaccines • This group includes four types of organism; a) Attenuated live organism, b) Naturally existing non pathogenic strains, c) Pathogenic organism in heterologous host, and d) Pathogenic organism in homologous host but through unnatural route.

a) Attenuated live organisms: • Attenuation: Attenuation is a process of reducing virulence. • Traditionally, process of attenuation involves adapting organism to grow in heterologous host or under unusual condition so that they lost their adaptation to their natural host. • Bacillus Calmette Gurein (BCG) strain of Mycobacterium bovis was rendered avirulent by being grown for 13 years on bile saturated medium. • Louis Pasteur attenuated Anthrax bacilli by growing it at 420 C. • Fowl Cholera organism got attenuated by the process of aging. • Brucella abortus strain 19 was grown under conditions in which there was a shortage of nutrients

a) Attenuated live organisms: • Viruses have traditionally been attenuated by growth in cells or species to which they are not naturally adapted. • For example RP virus, which is normally a pathogen of cattle, was first attenuated by growth in rabbits. • Eventually, a tissue culture adapted RP vaccine was developed. • Alternatively, mammalian viruses may be attenuated by growth in eggs. Flury strain of rabies was attenuated by prolonged passage in egg and lost its virulence for normal cats and dogs. • The most commonly used method of virus attenuation has been prolonged tissue culture.

b) Natural non–pathogenic strains: • Some viruses have naturally occurring non pathogenic strains that can be used for the purpose of vaccination. • Lentogenic or mesogenic strains of Newcastle disease virus is used for the purpose of vaccination whereas velogenic strain is highly virulent. • Lentogenic : F , B 1, Lasota • Mesogenic: R 2 B • Herpes Virus of Turkey (HVT) is used for immunization against Marek’s disease.

c) Pathogenic organism in heterologous host • However, sometimes even antigenically related pathogenic organism can act like a vaccine when inoculated in heterologous hosts. • For example: - Measles virus has been used to protect against Canine distemper, and - BVD virus can protect swine against hog cholera.

d) Pathogenic organism in homologous host through unnatural route. • Sometimes fully virulent virus can be used for the purpose of immunization in the homologous host by inoculating through non natural route. • Contagious Ecthyma (ORF) is a viral disease of lambs that cause massive scab formation around the mouth. • Lambs are vaccinated by rubbing dried infected scab material into scratches made in the inner aspect of thigh. • ILT virus given through cloacal route gives protection from infectious Laryngotracheitis infection in poultry.

Killed or Inactivated vaccines: • Killed or Inactivated vaccines are usually safe (as the chance of reversal of virulence is negligible) and can be easily stored. • Agents like - Formaldehyde, - Ethylene oxide, - Ethyeneimine and - β-propiolactone (BPL) are commonly used as inactivating agents. • These agents do not cause much alteration to the protein structure and thus maintains antigenicity to a larger extent.

Killed or Inactivated vaccines: • But, generally killed vaccines are of low antigenicity and thus induce less efficacious immunity. • To overcome this limitation, adjuvants are added to enhance immune response to killed vaccines. • Killed vaccines also induce short term immunity and thus multiple injections (boosters) are required to ensure long term protection.

Live Vaccine Vs Killed Vaccine Features Attenuated vaccines Inactivated whole cell vaccines Long Lasting Short lived Both humoral and Cellular Humoral No Yes Usually single Multiple Parenteral or mucosal Mostly parenteral Reversion to virulence Possible No Adventitious contaminants Possible No Stability Requirement of cold chain Less Cold chain is required More Cold chain not required Not recommended Can be given Quantity of antigen required Less More Cost of production Less More Duration of Immunity Types of Immune response Adjuvant required Number of Inoculation (requirement of boosters) Route of Inoculation Administration in Pregnant or immuno-compromised individuals

Toxoid: • The pathogenic effects of some bacteria are because of exotoxins produced by them. • The toxic effect of exotoxins can be reduced by treating them with 0. 5 % formaldehyde while they retain their immunogenicity and thus can be used for the purpose of Immunization. • These formaldehyde treated exotoxins are called “Toxoid”. • A number of toxoid vaccines are used in Veterinary. For eg: Tetanus toxoid vaccine • (DPT in Human: An example of Toxoid vaccine)

Sub-unit vaccines: • Sub-unit vaccines are made up of components of the virulent organism that are capable of inducing protective immune response rather than whole virulent organism. • The concept of sub unit vaccine is based on the fact that immune response generated against whole array of antigen from pathogen does not contribute equally in protection.

Sub-unit vaccines: • Some of the antigenic components plays major role in inducing protection while others do not. • Thus, it is possible to use only those components of the pathogen that induces protective immune response instead of using whole organism. • For example, antibody response generated against Haemagglutinin (H) and / or Neuraminidase (N) antigen of Influenza virus is protective. • Thus, sub unit vaccines against Influenza virus contain only H and N antigens. • Principally, toxoid vaccines are also a type of subunit vaccine which contains only detoxified exotoxin not the whole organism.

Modern Vaccine Technology

Modern Vaccine Technology • It is always a need to make vaccines more effective, cheaper and safer. • Thus scientist fraternity keeps trying to develop vaccines which are more efficacious, cheaper and with negligible safety concerns (side effects). • Modern molecular biological techniques are used for the development of a number of new generation vaccines like: a) Subunit vaccines, b)Genetically attenuated organism as vaccines, c) Live recombinant organisms as vaccines, d) DNA vaccines e) Marker vaccines f) Edible vaccines

Subunit vaccines: • It is possible to precisely cut any “gene of interest” from an organism and express that gene in another host. • For example, by the help of recombinant DNA technology, the VP 1 gene of FMD virus was cut, cloned into a plasmid and inserted into bacterium Escherichia coli. • This leads into the production of VP 1 protein in E. coli. The VP 1 protein was harvested, purified, adjuvanted and used for vaccination against FMD.

Genetically attenuated Organism: • Using molecular techniques, it is possible to delete virulence gene of the organism. • Virulence gene deleted organisms are capable of growth in the host but cannot produce disease. • These virulence gene deleted organism are good in inducing efficacious immune response with no risk of reversal of virulence. • eg: Pseudorabies virus (Herpesvirus) needs enzyme thymidine kinase for their replication in non dividing cells such as neuron. • Thymidine kinase deleted pseudorabies virus can infect nerve cells and thus induce protective immune response. • However, they cannot replicate in neuron and thus compromises with the virulence of the virus.

Live recombinant organism: • It is also possible to clone gene coding for protective antigen from one organism into another organism which is capable of growth in host. • Such recombinant organisms, carrying gene of other pathogen, will express the antigen in host along with their own genes. • A number of viruses like poxvirus, adenovirus, herpesvirus and bacteria like BCG, Salmonella were used for this purpose. • These organisms can easily be administered through oral, nasal routes or by rubbing on skin.

Live recombinant organism: • The advantages with these recombinant organisms are many. Along with easy administration, these organisms offer other advantages like: - Expression of protein antigen in the host, - its proper folding and - post translational modifications like glycosylations. • One such example is of Vaccinia virus recombinant which contains G protein of Rabies virus (antibodies against G protein neutralizes rabies virus) and thus induces protective immunity. • This vaccine has been used widely for oral vaccination of wild animals in bait.

Live recombinant organism: • Similarly, Newcastle disease virus gene is cloned in fowl pox virus. • Recombinant vaccinia or capripox virus carrying Haemagglutinin (H) or Fusion (F) gene of RP was also developed. • RP gene carrying recombinant capripoxvirus gives protection against both RP as well as lumpy skin disease.

DNA vaccine: • Cloning the “Gene of Interest” (gene coding for protective antigen) in plasmid vector under strong promoter and introduction of such plasmids directly into host tissue can generate immune response against the gene of interest. • These plasmids can be delivered into the host cells by various techniques. • The simplest one is by injecting them intramuscularly. Better delivery of these plasmids to intracellular locations can be achieved by shooting plasmids adsorbed on gold nanoparticles using “Gene Gun”. • In the host cells, the gene of interest is transcribed, translated, processed and presented to cell of immune system. • Such vaccines are called “DNA vaccine”. • DNA vaccine against West Nile virus infection has been used successfully in horses.

Marker vaccines: • Using molecular biological tools, immunogenic but non protective gene from virulent organism can be deleted. • The advantage of such vaccines is that immune response generated in vaccinated animals differs from animals naturally infected with the organism. • To differentiate vaccinated animals from naturally infected one, a test should be developed (for example: ELISA) which specifically detect immune response against the gene deleted in marker vaccine. • As expected, the test will be positive in naturally infected animals where as negative in vaccinated animals. • Marker vaccines are also called as DIVA vaccines (Differentiation of Infected from Vaccinated Animals).

Edible vaccine: • It is also possible to clone gene of interest in plants like tobacco, potato and corn. • The genes of Transmissible gastro-enteritis and Newcastle disease, coding for protective antigen, were cloned in plants and these plant products were used for vaccination. • In US, tobacco based Newcastle disease vaccine has been licensed.

ADJUVANTS

ADJUVANTS • Adjuvants are substance when administered along with antigen enhances the magnitude of immune response to that antigen. • In general, inactivated or killed vaccines are poorly immunogenic. • Thus, these vaccine formulations include adjuvants so that they could mount a better and efficacious immune response. • Addition of adjuvants offer several advantages such as a) lesser amount of antigen is required, b) Induces better or heightened immune response, c) reduces the number of doses required, d) Induces long term immunity.

ADJUVANTS: Mechanism of action • The mechanism through which adjuvants exert their effect is not well established. • However, better antigen presentation by activation of antigen presenting cells (APCs) and antigen depot formation are the two most plausible explanations. • The adjuvant that forms antigen depot is thought to slow down the degradation of antigen and mediates sustained release of antigen. • This led to the prolonged stimuli for the immune system and thus resulting into a heightened immune response.

Commonly used ADJUVANTS • The most commonly used adjuvants include aluminum salts such as: - Aluminum hydroxide gel, - Aluminum phosphate and - Aluminum potassium sulphate (alum) as well as - Calcium sulphate. • The main disadvantages associated with Aluminum based adjuvants are that they augment (promote) humoral immune response but have very less stimulatory effect on cell mediated immunity.

Water in oil emulsions • Water in oil emulsions are other important means of inducing better immune response. • Mixing of antigen with mineral or vegetable oil leads to formation of “water in oil emulsions” which creates antigen depot upon inoculation. • This ensures better immune response to the antigen due to depot effect. • Moreover, local tissue irritation produced by oil also activates antigen presenting cells and thus increases the antigen presentation.

Water in oil emulsions • Two most important examples of “water in oil” based adjuvants are - Freund’s complete adjuvant (FCA) and - Freud’s incomplete adjuvant (FIA). • The composition of both the adjuvants is same except for the fact that FIA does not contain the Mycobacterial component which is present in FCA. • FCA is considered to be the most potent adjuvant which is used for purpose of primary vaccination whereas FIA is used for boosters under mainly experimental circumstances. • FCA is not used in cattle and other species because its use may leads to false positive reaction on tuberculin testing. • Moreover, oil based adjuvants also spoil the quality of meat.

ADJUVANTS • Generally, in comparison to soluble antigens particulate antigens are better taken up by the cells of Immune system and thus induce better immune response. • Thus, a number of newer adjuvants formulations like Immune stimulating complexes (ISCOMS), Liposomes are also tried with success. • ISCOMs are stable complexes containing cholesterol, phospholipid, saponin (Quil A) and antigens. • Liposomes are lipid based synthetic microparticles encapsulating antigens. These biodegradable micro-particles are readily taken up by the phagocytes.

ADJUVANTS • A number of chemical compounds of microbial origin are identified which can be used as adjuvants in near future. • Most notable examples are LPS, Monophosphoryl Lipid A, flagellin, Cp. G containing DNA etc. • These microbial products are reported to act as TLR agonists and thus they exhibit their adjuvancity by activating APCs. • Some plant products have also shown immense promise for their use as adjuvants. One noteworthy example is Saponin (from bark of Quillaza saponaria).

QUALITY CONTROL OF VACCINES • As vaccination is perceived as a means of protection, it should itself not pose any threat to the animals health. • Therefore, vaccination of animals with high quality vaccines is in the forefront of any Immunization programme. • The quality of a vaccine should be tested on various parameters like: - potency, - efficacy, - safety, - sterility, - purity etc. • Quality vaccines should be potent and efficacious, safe and sterile, pure and provide durable or long term immunity.

Vaccine quality parameters • Potency: of a vaccine is its relative strength to generate protective immune response in suitable laboratory animals and the host species. • For live vaccines, bacterial or viral count, at any time point prior to expiry date, should be sufficiently greater than the live count shown to be protective (efficacious). • Efficacy: of a vaccine is the quality that confers solid protection to the host against any challenge (experimental or natural) by the pathogens.

Vaccine quality parameters • Safety: safety of a vaccine indicates the absence of associated risks in the inoculated individuals. • Purity: Purity of a vaccine indicates the lack of contaminants in the vaccine formulation i. e. , it must contain only the protective agents and no other organisms. Each batch of vaccine should be tested for presence of any extraneous viruses, bacteria, mycoplasma or fungi. • Sterility: Sterility of a vaccine means lack of residual virulence in inactivated vaccines and lack of reversion of attenuated organism to virulent forms in live vaccines. The term is often confused with “purity” of vaccine. •

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