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Practically useful vaccination procedures may require both an initial priming exposure to antigen, plus an additional boosting exposure. Diversified "prime and boost protocols" provide a means of generating elevated immune responses and extending the duration of immunity for new generation vaccines based on recombinant DNA expressed antigens (as subunit antigens, vector based or naked DNA). Enhanced responses have been observed for viral, parasite and cancer vaccines. In any vaccine study an evaluation of diversified prime and boost protocols is warranted. The immunological mechanisms responsible for the elevated responses have not been defined. A detailed investigation of the mechanisms is needed.

Cost effective delivery of new vaccines for disease control in man and animals is a major challenge for vaccine technologists and vaccine manufacturers. The most attractive vaccines are those that can be delivered as single shot multivalent vaccines and that induce long lasting immunity, but the performance of many current vaccines falls short of these ideal parameters. Successful application of recombinant DNA based vaccines will be dependent in many circumstances upon the selection of antigen delivery format and upon the timing of priming and boosting vaccinations.

Many experimental vaccines based upon rDNA expressed antigens, such as those based on vaccine vectors (bacterial or viral) or injected naked DNA, have not yet been able to achieved these ideal performance parameters when used singly for priming and boosting. However, there is a growing body of evidence that the combined use of antigens expressed in different formats e.g. subunit antigen followed by vector-expressed can lead to elevated immune responses which exceed those of either format alone. These also appear to offer increased longevity of responses. Timing of doses and optimisation of parameters of combined antigen formats have not been extensively explored nor have the underlying immunological mechanisms for the synergistic or enhance responses been defined.

Examples of Successful Prime and Boost Protocols

Iritani et.al. (1991) demonstrated that a 10 to 80 fold elevation of haemagglutination inhibiting (HI) antibody responses to Newcastle disease virus (NDV) could be achieved by priming with one vaccine format and boosting with an alternative. Chickens vaccinated at three days of age with live NDV vaccine and revaccinated 36 days later with recombinant fowlpox virus expressing the haemagglutinin-neuraminidase (HN) gene developed HI antibody titres approximately 80 fold higher than either vaccine alone. In 5-week-old chickens the combined use of inactivated NDV vaccine with FPV-HN recombinant (29 days later) generated HI antibody titres 10 to 20 fold higher than either vaccine alone.

In Phase I human vaccine trials with HIV gp160 as a vaccinia virus recombinant or recombinant protein, T cell and antibody responses were of low magnitude and transient (no neutralizing antibodies developed). In contrast, a combined vaccination regimen of priming with vaccinia-gp160 followed by boosting with baculovirus-expressed gp160, generated T cell responses 3 to 10 fold higher than responses with either vaccine alone and the responses were long lived in 75 % of vaccinees (> 18 months). Following the combined regimen of vaccination, antibody responses (titres of 1:800 to 1:102,400) to homologous HIV envelope developed in all vaccinees (7/13 developed neutralizing antibodies). (Cooney et.al., 1993).

An avipox virus recombinant expressing Japanese encephalitis virus (JEV) antigens generated protective immune responses in mice. However, a combined vaccination regimen of priming with the avipox recombinant boosted by non-infectious extracellular particles induced higher antibody levels (32 fold higher) than immunization twice with the extracellular particles alone (Konishi et.al., 1994). The levels of antibodies induced by twice vaccination with the avipox recombinant were similar to the combination regimen. Interestingly a single time point simultaneous vaccination with a mixture of recombinant avipox and extracellular particles induced neutralizing antibody titres 8 to 16 fold higher than immunization with recombinant virus or extracellular particles alone (at a single time point) and equal to titres achieved by two immunizations with recombinant virus or extracellular particles alone.

Naked DNA Plus Another Dose Format

There is also evidence that combining naked DNA vectors with recombinant expressed antigens or viral vector-expressed antigens can substantially enhance immune responses. A combined vaccination regimen using recombinant fowlpox and naked DNA expressing influenza haemagglutinin generated antibodies to haemagglutinin 30 to 120 fold higher than those obtained with naked DNA or fowlpox alone (Leong et.al., 1995 ; Ramsay et. al., 1997). The high antibody response was maintained for a long duration. Similar potentiation of immune responses was generated in combined vaccination regimens with HIV/SIV antigens expressed by naked DNA, vaccinia virus or subunit antigens. A single vaccinia virus or subunit antigen boost following primary vaccination with a naked DNA vaccine generated elevated antibody responses - higher than responses seen in animals receiving naked DNA or vaccinia virus vectors alone (Fuller et.al., 1997 ; Barnett et.al., 1997). Animals primed with vaccinia virus-expressed antigen also responded with elevated responses when boosted with naked DNA showing that, in this case, the synergistic effect appears not to be dependent upon the order of vaccine administration.

Enhanced immune responses have also been observed with parasite and cancer antigens. Prime-boost vaccination strategies involving the use of naked DNA and adenovirus recombinants expressing the Taenia ovis 45W antigen generated responses comparable to those induced by Quil A adjuvanted antigen. This was observed even though two vaccinations with the naked DNA or adenovirus alone induced barely detectable antibody responses. There was also a marked difference in the IgG subtype induced by the prime-boost vaccination with the naked DNA and adenovirus. In this instance the order of vaccination affected the response. Using a model cancer antigen (beta-galactosidase) Irvine et.al. 1997 observed that heterologous boosting with antigen expressed by poxvirus vectors or naked DNA resulted in longer mouse survival than homologous boosting. It was suggested that with the poxvirus vectors the poor efficacy of homologous boosting resulted from the induction of strong antibody responses to the vector virus thus inhibiting the efficacy of the homologous boosting. Priming and boosting with heterologous vectors generated strong antigen-specific cytotoxic T cell responses believed to be responsible for the longer survival times.

In summary, these experiments suggest that the combined use of different antigen formats (including naked DNA, viral vectors and rDNA expressed antigens) may provide a means of generating elevated responses and extend the term of immunity. In the case of Japanese encephalits virus, one experiment suggested that elevated responses might be achievable by delivery of different antigen formats at a single time of vaccination. In any vaccine study using recombinant DNA expressed antigens an evaluation of diversified prime-boost strategies is warranted as they may well lead to better responses - higher and potentially more efficacious than the use of a single antigen format. None of the studies have examined the immunological mechanisms responsible for the elevated responses - this warrants detailed investigation.

References

Barnett, S.W., Rajasekar, S., Legg, H., Doe, B., Fuller, D., Haynes, J., Walker, C.M. and Steimer, K.S. (1997) Vaccination with HIV DNA induces immune responses that are boosted by a recombinant gp120 protein subunit. Vaccine 15 : 869-873.

Cooney, E.L., McElrath, M.J., Corey, L., Hu, S-L., Collier, A.C., Arditti, D., Hoffman, M., Coombs, R.W., Smith, G.E. and Greenberg, P.D. (1993). Enhanced immunity to human immunodeficiency virus (HIV) envelope elicited by a combined vaccine regimen consisting of priming with a vaccinia recombinant expressing HIV envelope and boosting with gp160 protein. Proc. Natl. Acad. Sci. USA 90 : 1882-1886.

Fuller, D.H, Simpson, L., Cole, K.S., Clements, J.E., Panicali, D.L., Montelaro, R.C. , Murphey-Corb, M. and Haynes, J.R. (1997) Gene gun-based nucleic acid immunization alone or in combination with recombinant vaccinia vectors suppresses virus burden in rhesus macaques challenged with a heterologous SIV. Immunol. Cell Biol. 75 : 389-396.

Iritani, Y., Aoyama, S., Takigami, S., Hayashi, Y., Ogawa, R., Yanagida, N., Saeki, S. and Kamogawa, K. (1991). Antibody responses to Newcastle Disease Virus (NDV) of recombinant fowlpox virus (FPV) expressing a hemagglutinin-neuraminidase of NDV into chickens in the presence of antibody to NDV or FPV. Avian Diseases 35 : 659-661.

Irvine, K.R., Chamberlain, R.S., Shulman, E.P., Surman, D.R., Rosenberg, S.A. and Restifo, N.P. (1997). Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors. J.Natl. Cancer Inst. 89 : 1595 _1601.

Konishi, E., Pincus, S., Paoletti, E., Shope, R.E. and Mason, P.W. (1994). Avipox virus-vectored Japanese encephalitis virus vaccines : use as vaccine candidates in combination with purified subunit immunogens. Vaccine. 12 : 633-638.

Leong, K.H., Ramsay, A.J., Ramshaw, I.A., Morin, M.J., Robinson, H.L. and Boyle, D.B. (1995). Generation of enhanced immune responses by consecutive immunization with DNA and recombinant fowl pox vectors. Vaccines 95. Cold Spring Harbor Laboratory Press 1995 pages 327-331.

Ramsay, A.J., Leong, K.H. and Ramshaw, I.A. (1997). DNA vaccination against virus infection and enhancement of antiviral immunity following consecutive immunization with DNA and viral vectors. Immunol. Cell. Biol. 75 : 382-388.

Rothel, J.S., Boyle, D.B., Both, G.W., Pye, A.D., Waterkeyn, J.G., Wood, P.R. and Lightowlers, M.W. (1997). Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep. Parasite Immunology. 19 : 221-227.

Copyright 1998 Australian Biotechnology Association Ltd.