Vaccines & Immunomodulation

1. Dr Alok Tripathi Department of Biotechnology aquaimmuno@yahoo.co.in Vaccines & Immunomodulation

2. Objectives • The role of immunological memory in protection against infectious diseases • The main differences between memory and naïve T and B cells • How vaccines can be used to manipulate the immune response to induce immunological memory • Examples of attenuated vaccines are and how they are produced. • Examples of killed vaccines are and how they are produced. • Examples of recombinant vaccines are and how they are produced • Current experimental approaches to design vaccines and their associated problems : • Recombinant plant vaccines • Live recombinant vaccines • Genetically attenuated vaccines • Peptide vaccines • DNA vaccines

3. What vaccine adjuvants are and their functions in vaccine formulations • Which adjuvants are used in human vaccines • The types of responses induced by adjuvants • The current status of research into HIV, Malaria and TB

4. Definition • Vaccines are a manipulation of the adaptive immune response whereby the host is induced to generate a protective immune response to a pathogenic organism without actually experiencing the full infection. • Vaccination involves deliberate exposure to antigen under conditions where disease should not result.

5. Role of Memory in Vaccine induced protection to infection • During this primary challenge, the immune response responds by producing not only effector cells but also memory cells Compared with naïve T cells, memory T cells: Are Long Lived • Have Increased frequency • Proliferate more rapidly • Have reduced co-stimulatory requirements (signal 2) • Compared with naïve B cells, memory B cells : Are Long Lived • Have Increased frequency • Proliferate more rapidly • Produce more Ab • Produce higher affinity Ab • Produce antibodies with better effector functions (IgG & IgA)

6. secondary antibody response to antigen. • Memory T and B cells induced during vaccination facilitate more rapid control of subsequent infections with pathogens. This can be seen most clearly in the secondary antibody response to antigen. Secondary antibody responses are T cell dependent. During the primary response, expansion of T cells specific for protein antigens is sufficient for the host to mount a secondary antibody response on subsequent exposure to that antigen, plus any other B cell antigen which is physically associated with it (e.g. a hapten). This is known as the carrier effect - it means that the secondary antibody response to a vaccine can be induced by conjugating the B cell antigen of choice to a T cell antigen to which people have already been exposed (see the Hib vaccine below)

7. History of Vaccination • Jenner performed the definitive experiments to demonstrate effective protection against smallpox (causative agent, Variola) by prior inoculation or ‘vaccination’ with cowpox (containing the closely related Vaccinia virus) in 1796. The ‘golden age of microbiology’ brought about the rapid isolation of the pathogenic agents of many diseases and subsequent attempts to produce vaccines against them. In the 1880’s Pasteur generated attenuated versions of polio virus and anthrax by in vitro culture. Subsequent methods of producing vaccines included heat or chemical inactivation of pathogens. After improvements in living conditions, vaccines are the most effective public health measure introduced. In Glasgow in the 1900’s, 1 in 5 deaths were due to smallpox infection. In May 1980, the WHO officially announced the global eradication of the smallpox virus.

8. Current statusThe following vaccines are recommended for routine vaccination in the UK

9. Producing Vaccines • The whole point of vaccination is to induce a protective immune response as would be elicited by infection, with minimal disease associated with the infection. This can be achieved by producing attenuated strains of bacteria or viruses, for example: host range mutants where bacteria or viruses (e.g. BCG or Sabin polio virus) are selected for in vitro growth in non-human cells • cold attenuated mutants where viruses are grown at temperatures of 32-34ûC, well below body temperature e.g. Measles.

13. Viruses • • Vaccinia: the first recombinant virus was produced and tested in 1982. • Since then it has been extensively used as a recombinant carrier for over 100 different antigens. • The extremely large genome of the poxviruses has facilitated the insertion of larger parasite and bacterial genes into Vaccinia. • NYVAC, a highly attenuated form of Vaccinia, has been used as a recombinant carrier for HIV vaccine and clinical trials are underway. • Constructs have also been made incorporating cytokine genes (ALVAC). (see Letvin, Science (1998) 280 (Jun 19) 1875-80)

14. Adenovirus • have also been used as a carriers for HIV vaccines, but with limited success. • This is probably due to the highly cytolytic nature of adenovirus. Letvin, Science (1998) 280 (Jun 19) 1875-80)

15. Bacteria • BCG : Used in vaccines against: Malaria (Merozoite surface protein -1), HIV-1 (Letvin, Science (1998) 280 (Jun 19) 1875-80), Borreliaburgdorferi and Streptococcus pneumoniae• Salmonella : The EvansTy21a attenuated strain used in vaccines was produced by chemical mutagenesis. Recently, aro mutants, which lack enzymes involved in the production of aromatic amino acids have extensively studied as genetically attenuated alternative and as carrier for other vaccines (reviewed Chatfield et al.Vaccine (1989) 7, 495-498). • Cholera : The Cholera toxin A (CTA) subunit has ADP ribosylating activity and is responsible for Cholera induced diarrhoea. The CTB subunit is highly immunogenic, especially when given orally. Attempts to produce a vaccine have therefore concentrated on attenuating the A subunit (clinical trials with CVD103Hg-R are underway) • Bordetellapertussis: intranasal administration of a recombinant vaccine encoding the 28-kDa glutathione S-transferase antigen from Schistosomamansonii has been demonstrated to mediate protection to infection.

16. Problems associated with attenuated vaccines:• undefined genetic lesions (not the case with genetically attenuated vaccines)• possibly of reversion to virulence/toxicity (e.g. in HIV/SIV vaccines: Johnson, Nature Medicine (1999), 5(2), 154-155)• cannot be used in immunocompromised hosts• possibility of zoonosis• loss of replication due to interference by other infections• public acceptance of genetically modified vaccines

17. Subunit vaccinesA number of subunit vaccines are already licensed for use (See Section 2) and interest in producing new subunit vaccines continues, mainly due to safety concerns. For example, the immunogenicity and toxicity of Bordetellapertussis vaccine, an inactivated whole cell vaccine is greatly affected by culture conditions. Therefore development of acellular whooping cough vaccines, containing pertussis toxin and fimbral HA, are of interest.

18. Recombinant subunit vaccines • To avoid the problems involved in bulk culture of pathogens and to increase the yield of protective antigens, recombinant vaccines have been introduced. • Hepatitis B was the first recombinant vaccine licensed for human use. • The surface antigen (HBsAg) was expressed in yeast, the antigen thus produced spontaneously forms multimeric particles similar in appearance to the non-infectious Dane particles produced during hepatitis infection.

19. Recombinant plant vaccines • Hepatitis B surface Ag expressed in potato/tobacco is immunogenic when fed to mice. • This has also been the case when Cholera toxin B subunit and E.colienterotoxin B subunit (LT-B) were expressed in plants. • Furthermore, a clinical trial of LT-B expressed in potato (Tacket et al.Nature Medicine 1998, 4 (May), 607-9), demonstrated that administration of 3 doses of 50g of potato (4 - 15 ug of LT-B) produced :

21. Peptide Vaccines • Whole proteins only contain a handful of protective epitopes, these can be synthesised and produced in large scale by peptide synthesis techniques. • Information from the primary sequence of proteins can be used to predict which sequences may be T cell epitopes. • Relative merits of peptide vaccines

23. Adjuvants • One approach to increase the immunogenicity of a protein is to formulate it with a vaccine adjuvant. Adjuvants are described in the Dictionary of Immunology as "Agents which act non-specifically to increase the specific immune response or responses to an antigen" or alternatively "the immunologists dirty little secret" by Charles Janeway. • Essentially, adjuvants appear be able to provide signal 2 to T cells, a feature absent in purified proteins. The effects of adjuvants are mainly mediated indirectly via antigen presenting cells. • The only adjuvant currently licenced for use in humans in the UK are the Aluminium compounds. Their adjuvant activity was first described in 1926 and they form a component of vaccines against Hepatitis A, Hepatitis B, Anthrax and DTP (Diphtheria, Tetanus, Pertussis). • However one of the major limitations of Aluminium compounds is that they only stimulate the induction of Th2 responses. In contrast some experimental adjuvants(such as Freund's Complete Adjuvant (FCA)) can stimulate Th1 responses, but are too toxic to be used clinically.

24. The ability to modulate Th1 or Th2 responses, has been assigned to a third signal in T cells, therefore adjuvants can clearly influence the provision of signal 3. This is a major problem as induction of Th1 responses are thought to be required for vaccine induced protection against the big 3 pathogens (HIV, Tuberculosis and Malaria). Therefore a number of new adjuvants are under development by various companies and institutes to try and induce strong antigen specific Th1 responses to vaccines. • Particles • Liposomes (Swiss Serum and Vaccine Institute)These are composed of lipid bilayers (like plasma membranes) which can have antigens entrapped inside • ISCOMsThese contain a glycoside extract (Quil A) prepared from tree bark. When mixed with virus spike proteins (surface proteins) they form micelles • Bacterially derived adjuvants • Monophosphoryl lipid A (SKB)Is a relatively de-toxified derivative of lethally toxic endotoxin from gram negative bacteria (LPS) • Natural/Synthetic surfactants • QS21 (SKB)A more purified version of Quil A, but not micellar like ISCOMs • Oil/Water emulsions • MF-59 (Chiron) • These adjuvants are highly diverse

25. Previous studies have used molecular biology to express genes encoding protective antigens in expression vectors. These antigens, such as the Hepatitis B vaccine expressed in yeast cells, are then isolated and used as vaccines. More recent approaches have used naked plasmid DNA containing genes encoding the protective antigen to actually transfect the host. A typical plasmid vector such as pcDNA, contains strong promoters to induce transcription of the protective antigen DNA. Host cells then express the protein antigen in situ, and host immune responses are generated to the foreign protein. Methods used to introduce DNA include : • Intramuscular injection • Intradermal injection • Gene Gun • DNA bound to gold particles and shot under gas pressure at high speed into epidermis • Jet injection • Using even higher pressure and speed, it is possible to shoot DNA (or proteins) into epidermal cells without the requirement for gold particles

26. DNA vaccines have been applied to numerous infectious agents and has been frequently successful in the mouse. This is partly due to plasmid DNA having its own built-in adjuvant. Prokaryotic DNA contains CpG motifs which are largely absent in mammalian DNA, however, CpGs are recognised as foreign by the mammalian immune system and directly activate it.  

28. With our expanded understanding of how the immune system works, experimental approaches to vaccine development are aimed at developing vaccines that target protective immune responses. One area of interest is the development of better vaccines to stimulate mucosal immunity, since most pathogens enter the body through mucosal membranes. The oral polio vaccine is an example of a vaccine that enters by the pathogen's normal route and stimulates protective neutralizing antibody. Difficulties with oral vaccine administration include antigen destruction in the stomach or intestines and risk of inducing tolerance.

29. Another area of vigorous research is targeting antigens to APC. Antigens have been covered with mannose to bind macrophage mannose receptor and made into immune complexes to stimulate uptake by FcR+ cells. Pathogen DNA has been complexed with CTLA-4 to promote its uptake and expression by B7+ APC. ISCOMs target antigen to Class I MHC, while antigen coupled to a particular signal peptide can be used to move antigen into endosomes for processing and presentation on Class II MHC. The outer membrane protein of Salmonella typhimurium binds M cells and may be useful for targeting antigen to the mucosal immune system.

30. Finally, the ability of vaccination or cytokine administration to control ongoing infection is being studied. Chronic infections occur with Herpes simplex viruses, hepatitis B and C viruses, Mycobacterium tuberculosis and M. leprae, and the parasites Leishmania, Plasmodium, and Schistosoma. Persisting infections lead to prolonged infectivity, tissue damage from immune hypersensitivity, and tumor development. Established immune responses are very difficult to modify or eliminate; but there is hope that with a properly-targeted vaccine boost the immune system may be able to completely eliminate pathogen.