Dr.D.RATHNAMMA Professor and Head Dept. of Veterinary Microbiology, Veterinary College, KVAFSU, Hebbal, Bengaluru-560 024 • Vaccination is the most efficient and cost effective method of controlling infectious diseases in human and animals. • Small pox and Rinderpest eradicated from the globe, many diseases have been controlled with use of effective vaccines. • Vaccination/ immunization : two types: Active and Passive • Passive immunization: Transfer of preformed antibodies to susceptible animal, maternal antibodies to fetus /newborns, provide immediate protection, short term immunity. • Passive immunization is routinely administered to individuals exposed to botulism, tetanus, diphtheria, hepatitis, measles, and rabies. • Passively administered antiserum is also used to provide protection from poisonous snake and insect bites. • Active immunization: involves administration of an antigen to an animal, animal responds to the antigen by producing immune response, no immediate protection, but long lasting immunity is produced. • E.Jenner and L.Pasteur are recognized as the pioneers of vaccination, or induction of active immunity. • Active immunization can be achieved by natural infection with a microorganism, or it can be acquired artificially by administration of a vaccine. • In active immunization, the immune system plays an active role; proliferation of antigen reactive T and B cells results in the generation of immune response and formation of memory cells. Vaccine ‘Vaccine’ – (Latin ) ‘vacca’ means ‘cow’ • Edward Jenner - 1798, smallpox vaccine using cow pox virus • Louis Pasteur coined the term vaccine honoring Edward Jenner; vaccines for fowl cholera, anthrax, and rabies. • Vaccine : “A suspension of attenuated live or killed microorganisms, or antigenic portions of them, introduced to a potential host to induce immunity and prevent disease”. Ideal vaccine should be; • Affordable worldwide • Heat stable • Effective after a single dose • Applicable to a number of diseases • Administered by a mucosal route • Suitable for administration early in life Two types of vaccines: I. Conventional vaccines: • Live, attenuated vaccine • Killed / Inactivated vaccines
II. Modern vaccines
• Subunit vaccines - only immunogenic protein / polysaccharide from pathogen (Toxoid vaccines, Conjugate vaccines). • Recombinant organisms- genetically attenuated • Recombinant vector vaccines • DNA vaccines Conventional vaccines: 1. Live, attenuated vaccines: • High immunogenicity • Modified live organisms infect host cells, processed as endogenous antigen triggering CMI response • More closely mimic an actual/natural infection. • Elicits good, strong, long lasting immune responses. • Elicits both the arms of immune response (HI and CMI) • Generally life-long immunity is produced. • 95% effective for many diseases. • Attenuation : Process of reducing the virulence of the organism. • Attenuation can often be achieved by growing a pathogenic bacterium or virus for a prolonged periods under abnormal culture conditions. • Eg: BCG vaccine : Mycobacterium bovis was rendered avirulent by growing in bile saturated growth medium for 13 years. • Brucella abortus S-19 vaccine (calf hood vaccine)- grown under nutrient deficient conditions. • Virus attenuation - by culturing in cells to which they are not adapted. • Eg: PPR vaccine - passaged in vero cell culture systems, unnatural host. • Canine distemper virus- attacks lymphoid cells, virus is cultured in canine kidney cells for vaccine purpose. • Rinderpest vaccine- tissue culture adapted vaccine • Flury strain of rabies virus –attenuated by prolonged passage in chicken embryos.
Avirulent organisms are used as live vaccine
• Eg: Anthrax vaccine for animals- Live spore vaccine Bacillus anthracis- Sterne strain , a non capsulated strain, aivrulent Advantages of Live Disadvantages of Live vaccines vaccines • Potential to revert to • Single dose often virulence sufficient to induce • Contraindicated in long-lasting immunosuppressed immunity patients • Strong immune • Interference by viruses response evoked or vaccines and passive • Both humoral and antibody CMI response • Poor stability – proper • Local and systemic storage immunity produced • Potential for contamination 2. Killed/Inactivated vaccine: • Inactivation - organisms are killed or inactivated by physical or chemical agents without changing their antigenicity. • Most commonly used chemical is Formaldehyde, which cross links proteins and nucleic acids. • Most of Bacterial vaccines are Formaldehyde inactivated vaccines. • Eg: BQ vaccine, HS vaccine, ET vaccine • Alkylating agents that cross link nucleic acid chains also suitable for inactivation of organisms. Eg: Ethylene oxide, ethyleneimine, acetyle ethyleneimine, Beta propriolactone. These are commonly used in veterinary vaccines. Binary ethyleneimine is used for inactivation of FMDV Beta propriolactone is used for rabies virus inactivation Advantages of Live and Inactivated vaccines Live vaccines Inactivated vaccines Few doses are required Stable on storage
Adjuvants are not required Unlikely cause diseases through
residual virulence Less chance of hypersensitivity Do not replicate in recipient
Induction of Interferon Unlikely to contain live
contaminating organisms Relatively cheap Will not spread to other animals
Can be given by natural route Safe in immunodeficient patients
Stimulate both Humoral and CMI Easy to store
response Long lasting immunity No risk of reversion Modern vaccines / New generation vaccines: 1. Subunit vaccines - immunogenic purified macromolecules from pathogens (Protein, Polysaccharide ) Eg: Toxoid vaccine, Conjugate vaccine, Purified polysaccharide capsule, recombinant antigens 2. Recombinant organisms - genetically attenuated 3. Recombinant vector vaccines 4. DNA vaccines Toxoid vaccine: • Inactivated bacterial exotoxins are referred as Toxoids • Eg: Diphtheria and Tetanus vaccines have been made by purifying the bacterial exotoxins and then inactivating it with formaldehyde to form a ‘toxoid’. • Vaccination with the toxoid induces anti toxoid antibodies, which are capable of binding to the specific toxin and neutralizing its effects. Conjugate vaccine: • Purified capsular polysaccharides conjugated with protein carrier. • Eg: Hib vaccine for Bacterial meningitis caused by Haemophilus influenzae type b (Hib) - type ‘b’ capsular polysaccharide covalently linked to a protein carrier, tetanus toxoid - more immunogenic • Purified polysaccharide bacterial capsules Eg: Pneumococcal vaccine: Streptococcus pneumoniae, which causes pneumonia in children, consists of 23 antigenically different capsular polysaccharides. The vaccine induces formation of opsonizing antibodies. Coating of the capsule with antibodies greatly increases the ability of macrophages and neutrophils to phagocytose pathogens. • Vaccine for Neisseria meningitidis, a common cause of bacterial meningitis, also consists of purified capsular polysaccharides. Subunit vaccine - recombinant antigen Proteins from the pathogens can be produced by recombinant DNA technology and used as subunit vaccine. Gene cloning can be used to produce large quantities of purified antigen. Eg: VP1 gene of FMD virus cloned in plasmid, plasmid inserted into E.coli and bacteria grown, the bacteria synthesized large quantities of VP1 protein, harvested, purified and incorporated into a vaccine. First commercially available recombinant subunit vaccine for veterinary use is Feline leukemia virus- major envelop protein of FeLV - gp70. Hepatitis B vaccine: first recombinant antigen (subunit vaccine) vaccine approved for human use. Developed by cloning the gene for the major surface antigen of hepatitis B virus (HBsAg) and expressing it in yeast cells. Recombinant yeast cells are grown in large fermenters, and HBsAg accumulates in the cells. Yeast cells are harvested and disrupted by high pressure, releasing the recombinant HBsAg, which is then purified by conventional biochemical techniques and used as subunit vaccine. Genetically attenuated organisms as vaccine Eg: Pseudorabies in swine • Thymidine kinase (TK) is required by this herpes virus to cause disease • If TK gene is removed from the virus, can infect cells, but not replicate, can not cause disease • Block cell invasion by virulent Psuedorabies virus. Recombinant vector vaccines Genes that encode major antigens of especially virulent pathogens can be introduced into attenuated viruses or bacteria.
The attenuated organism serves as a vector, replicating within the
host and expressing the gene product of the pathogen.
A number of organisms have been used for vector vaccines,
including vaccinia virus, the canarypox virus, attenuated poliovirus, adenoviruses, attenuated strains of Salmonella, the BCG strain of Mycobacterium bovis, and certain strains of Streptococcus that normally exist in the oral cavity. Vaccinia virus, the attenuated vaccine used to eradicate smallpox, has been widely employed as a vector vaccine.
This large, complex virus, with a genome of about 200 genes, can be engineered to carry several dozen foreign genes without impairing its capacity to infect host cells and replicate. Recombinant vector vaccine Vaccinia virus : A good candidate for a live recombinant viral vector vaccine Large stable genome express high levels of new antigens. The gene that encodes the desired antigen from pathogen is inserted into a plasmid vector adjacent to a vaccinia promoter and flanked on either side by the vaccinia thymidine kinase (TK) gene. When tissue culture cells are incubated simultaneously with vaccinia virus and the recombinant plasmid, the antigen gene and promoter are inserted into the vaccinia virus genome by homologous recombination at the site of the nonessential TK gene, resulting in a TK- recombinant virus. Cells containing the recombinant vaccinia virus are selected by addition of bromodeoxyuridine (BUdr), which kills TK + cells. Eg: Vaccinia vectored rabies vaccine Gene for rabies virus envelop G protein inserted into Vaccinia virus. Used as oral bait vaccine for wild carnivores (Fox, Raccoons, Coyotes) against rabies.
Fowlpox vectored NDV-
approved in USA; H and F genes of NDV inserted into fowlpox virus, confer immunity against both diseases DNA vaccine “Plasmid DNA encoding antigenic protein from a pathogen is injected directly into the muscle of the recipient”. • Muscle cells take up the DNA and the encoded protein antigen is expressed, leading to both humoral antibody response and CMI response. • Injected DNA is taken up and expressed by the muscle cells with much greater efficiency than in tissue culture. • Easy to prepare, safe, stable, no need of cold chain. • Administered intramuscularly • DNA coding for the specific immunodominant epitopes are used. • Naked DNA/DNA coated gold nanoparticles are injected/ inoculated into body as DNA vaccine DNA vaccine induces both humoral and cellular immunity: antigenic proteins are expressed and processed as endogenous and exogenous antigens. Commercially available DNA Vaccine for Veterinary use DNA vaccine for West Nile virus Combined vaccines/ Multiple antigen vaccines • Two (or more) different viruses are grown separately in same cell lines, Separately attenuated and titrated then added in a single base and given at ones to the animal • Eg: PPR and sheep/goat pox • PPRV grown in vero cell line attenuated by giving 70 passages , titrated to have 10 log 3 TCID 50 virus per dose (0.5 ml) • Sheep/goat pox virus grown in vero cell line attenuated by giving62 passages , titrated to have 10 log 3 TCID 50 virus per dose (0.5 ml) • Made into a single container of 1ml, lyophilized. • Incoulated to animals by s/c, 1ml. Advantages • Avoids multiple visit to the animal • Gives the same immunity as given separately • Drastic reduction in cost of production • Other examples • Dogs: CD, Parvo, ICH , Parainfluenza, Rabies, Leptospira bacterin • Cattle: FMD and HS vaccine • Cattle : BHV-1, BVD, Parainfluenza 3 Other Biologicals : Diagnostic antigens • Brucella Diagnostic antigens: Agglutination test 1) RBPT(Rose Bengal Plate Test) antigen 2) Brucella abortus plain antigen for Standard tube agglutination(STT) 3) ABR (Abortus Bang Ring ) antigen for Milk ring test • Salmonella Pullorum coloured antigen – Whole blood agglutination test for screening of poultry flock for Salmonella • Salmonella Gallinarum – Plain antigen - Serum agglutination test for Fowl typhoid • Bacterial strain used : Brucella abortus S-99 • Colored antigen: It contains 11% B.abortus cell suspension diluted in formal saline. Cells are stained with crystal violet or malachite green or Rose Bengal dye. Most commonly used antigen is Rose Bengal plate antigen, the test is called Rose Bengal plate test(RBPT) which is a rapid plate test. • Plain antigen: It contains 1% B. abortus cell suspension diluted in phenol saline for inactivation. This antigen is used for standard tube agglutination test (STT). • Abortus Bang Ring antigen: It contains 8% of cell suspension stained with haematoxylin & tetrazolium, used for milk ring test for herd diagnosis of Brucellosis in animals. • Raw milk from animal has to be tested. ?????
