VP22 enhances antibody responses from DNA vaccines but not by intercellular spread.

In some species DNA vaccines elicit potent humoral and cellular immune responses. However, their performance in humans and non-human primates is less impressive. There are suggestions in the literature that an increase in the intercellular distribution of protein expressed from a DNA vaccine may enhance immunogenicity. We incorporated the Herpes Simplex Virus type 1 (HSV) VP22 gene, which encodes a protein that has been described as promoting intercellular spread, into a DNA vector in which it was fused to enhanced green fluorescent protein (EGFP). Following transfection of the plasmid DNA into mammalian cells, distribution of the fusion protein VP22-EGFP was not increased compared to EGFP alone. Furthermore, we found no evidence to suggest that VP22 was capable of mediating intercellular spread. However, when these constructs were used as DNA vaccines to immunise mice, antibody levels specific to EGFP were significantly enhanced when EGFP was fused to VP22. These data suggest that amplification of the immune response may occur via mechanisms other than VP22-mediated intercellular spread of antigen.

Characterization and manipulation of the human adenovirus 4 genome.

Human adenovirus 4 (HAdV-4), the only serotype of the species HAdV-E to be isolated from man, was first identified by its association with outbreaks of acute respiratory disease in military recruits. To combat such outbreaks, a live, oral HAdV-4 vaccine that is delivered via an enteric-coated capsule was developed. This vaccine has been used for nearly 40 years and has been shown to be safe and efficacious. In this study, the complete DNA sequence (35 994 bp) of the vaccine strain is described and its genetic content is analysed. Phylogenetic comparisons confirmed that the closest sequenced relative of HAdV-4 is another serotype of HAdV-E that infects chimpanzees (SAdV-25) and that the great majority of genes in HAdV-E are related most closely to HAdV-B genes. By using the sequence data, a system was constructed to facilitate production of replication-competent HAdV-4 recombinants.

A Salmonella enterica serovar Typhi vaccine expressing Yersinia pestis F1 antigen on its surface provides protection against plague in mice.

A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.

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.

Cloning and expression of the complement receptor glycoprotein C from Herpesvirus simiae (herpes B virus): protection from complement-mediated cell lysis.

Simian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species MACACA: Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gC(SHBV)) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46.9 %), HSV-1 (44.5 %) and pseudorabies virus (21.2 %). Highly conserved cysteine residues were also noted. Similar to gC(HSV-2), gC(SHBV) is less glycosylated than gC(HSV-1), resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gC(SHBV) on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gC(SHBV) isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gC(HSV-2) and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gC(SHBV) and gC(HSV-2). Binding of recombinant gC(SHBV) to polysulphates was observed. This, together with the heparin-sensitivity of the gC(SHBV)-C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.

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 Peyer's 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.

Role for mucosal immune responses and cell-mediated immune functions in protection from airborne challenge with Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) replicates in lymphoid tissues following peripheral inoculation and a high titre viraemia develops. Encephalitis develops after the virus enters the central nervous system from the blood, with the earliest neuronal involvement being via the olfactory nerve. Following aerosol challenge with virulent VEEV, the virus is thought to replicate in the nasal mucosa and there could be direct entry into the olfactory nerve via infected neuroepithelial cells. Protection from VEEV infection is believed to be primarily mediated by virus specific antibody. The correlation between protection and neutralising serum antibody titres is, however, inconsistent when the virulent virus is administered by the airborne route. This study demonstrates a link between antibody in serum and the nasal mucosa and protection by means of passive immunisation studies. Intra-nasal administration of antibody increased protection against airborne virus in Balb/c mice. Vaccination of mu MT strain mice that do not have functional B cells and cannot produce antibody revealed normal proliferation of spleen cells in vitro and robust cytokine production. Aerosol challenge of mu MT mice demonstrated that complete protection was only achieved when passive immunisation with antibody was supplemented with active immunisation with the TC-83 vaccine strain of the virus. This implies that cell-mediated immune functions are required for protection against airborne challenge with virulent VEEV.