Alice M. Bennett

DNA vaccination protects against botulinum neurotoxin type F.

A DNA vaccine was constructed which expressed the binding domain of Clostridium botulinum neurotoxin serotype F fused to a signal peptide. Three intra-muscular doses fully protected Balb/c mice against 10(4) MLD of serotype F toxin. Priming of the immune response by DNA vaccination followed by a single booster with type F binding domain protein resulted in high levels of antibody against the binding domain. This study demonstrates the utility of DNA vaccination for protection against botulinum neurotoxin type F and indicates that a prime-boost regimen could be an efficient method of generating antibody for passive immune therapy in cases of botulism involving serotype F toxin.

Co-immunisation with a plasmid DNA cocktail primes mice against anthrax and plague.

The protective antigen (PA) of Bacillus anthracis and the V antigen of Yersinia pestis are potent immunogens and candidate vaccine sub-units. When plasmid DNA encoding either PA or V antigen was used to immunise the Balb/c mouse, a low serum IgG titre was detected (log (10)1.0 or less) which was slightly increased by boosting with plasmid DNA. However, when mice immunised with plasmid DNA were later boosted with the respective recombinant protein, a significant increase in titre (up to 100-fold) was observed. Mice primed with a combination of each plasmid and boosted with a combination of the recombinant proteins, were fully protected (6/6) against challenge with Y. pestis. This compared favourably with mice primed only with plasmid DNA encoding the V antigen and boosted with rV, which were partially protected (3/6) against homologous challenge or with mice primed and boosted with plasmid DNA encoding the V antigen which were poorly protected (1/6). Combined immunisation with the two plasmid DNA constructs followed by boosting with a combination of the encoded recombinant proteins enhanced the protective immune response to Y. pestis compared with priming only with plasmid DNA encoding the V antigen and boosting with rV. This enhancement may be due to the effect of CpG motifs known to be present in the plasmid DNA construct encoding PA.

Construction and evaluation of a eukaryotic expression plasmid for stable delivery using attenuated Salmonella

An approach to enhancing the stability of eukaryotic expression plasmids for delivery using attenuated Salmonella has been evaluated. The expression apparatus and beta-galactosidase gene from the expression plasmid, pCMVbeta, was cloned into the low copy number plasmid pLG339. The resulting construct, pLGbetaGAL, was shown to have a lower copy number than pCMVbeta in Salmonella enterica var Typhimurium aroA strain SL7207. Furthermore, beta-galactosidase-specific antibody was induced in mice following intramuscular inoculation with pLGbetaGAL as naked DNA. Following oral administration of mice with SL7207/pCMVbeta, recombinants could not be detected in tissues 3 days after inoculation. In comparison, SL7207/pLGbetaGAL recombinant bacteria could be detected in the Peyers patches and spleens indicating that the Salmonella strain was stable. However, both SL7207/pCMVbeta and SL7207/pLGbetaGAL failed to induce beta-galactosidase-specific IgG in vivo. The mechanism by which attenuated Salmonella are able to release heterologous DNA for antigen processing and presentation is not yet understood. These results suggest that the mechanism needs to be further elucidated in order to rationally improve the system.

Protection against herpes B virus infection in rabbits with a recombinant vaccinia virus expressing glycoprotein D

Herpes B virus infects naturally monkeys of the macaque genus in whom it can cause recurrent oral and genital lesions. However, when the virus infects humans it causes a neurological illness with a high case fatality rate. Successful treatment is possible but this depends on diagnosis prior to the onset of respiratory arrest, and fatalities over the last 10 years have been the result of late or no diagnostic data on which to base anti‐viral intervention. An effective vaccine would be an ideal way to combat the risk of herpes B virus disease in humans working with potentially infected monkeys or their tissues. A recombinant vaccinia virus expressing herpes B virus glycoprotein D (gD) was constructed and rabbits inoculated with the chimeric virus were tested for immunoglobulin responses to herpes B virus by virus neutralisation, ELISA and Western blot analyses. Anti‐gD humoral responses were detected in all vaccinated animals by ELISA and Western blot but neutralising antibody was not detected prior to challenge with herpes B virus. Non‐vaccinated rabbits died within 8 days of challenge while 10/11 vaccinated animals were protected against herpes B virus disease. No antibodies to herpes B virus proteins other than gD were detectable in surviving animals, suggesting minimal herpes B virus replication post challenge. Autopsies were carried out on 4/10 rabbits which had remained healthy at 31 days post challenge and the dorsal root ganglia adjacent to the inoculation site were removed. Attempts to detect herpes B virus DNA by PCR followed by hybridisation proved negative suggesting protection against latent herpes B virus infection. J. Med. Virol. 57:47–56, 1999.

Efficacy of a B virus gD DNA vaccine for induction of humoral and cellular immune responses in Japanese macaques

It is desirable to prevent dissemination of B virus (BV) in macaque colonies because transmission of BV to humans causes deadly encephalomyelitis. Vaccination of monkeys is one method that could confine spread of BV within macaque colonies. Availability of a BV DNA vaccine for use in macaques would eliminate the risk of working with infectious BV. Toward this end, we constructed a plasmid expressing the BV glycoprotein D (gD). Immunogenicity of this construct as a DNA vaccine was assessed in adult Japanese macaques by four intracutaneous injections at a dose of 500 microg per head. Results of enzyme-linked immunosorbent assay (ELISA) using a recombinant herpes simplex virus type 1 (HSV1) gD, a homologue of BV gD, showed that significant levels of antibody was induced in all vaccinated animals following each booster injection. Western blot of sera from vaccinated macaques confirmed the specific recognition of authentic BV gD. Immune sera were also demonstrated to contain neutralizing activity against infectious BV. Weak lymphoproliferative responses were also observed in vaccinated macaques using recombinant HSV1 gD as a stimulating antigen and flow cytometry analysis of one individual revealed the presence of HSV1 gD-responsive effector T cells. Thus, the BV gD DNA vaccine was demonstrated to induce both humoral and cellular immune responses in macaques which recognized BV gD.