Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/ß receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics.

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo.

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge.

Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the V(H) and V(L) domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus.

In murine models of Venezuelan equine encephalitis virus (VEEV) infection, the neutralising monoclonal antibody 1A3B-7 has been shown to be effective in passive protection from challenge by the aerosol route with serogroups I, II and Mucambo virus (formally VEE complex subtype IIIA). This antibody is able to bind to all serogroups of the VEEV complex when used in ELISA and therefore is an excellent candidate for protein engineering in order to derive a humanised molecule suitable for therapeutic use in humans. A Complementarity Determining Region (CDR) grafting approach using human germline IgG frameworks was used to produce a panel of humanised variants of 1A3B-7, from which a single candidate molecule with retained binding specificity was identified. Evaluation of humanised 1A3B-7 (Hu1A3B-7) in in vitro studies indicated that Hu1A3B-7 retained both broad specificity and neutralising activity. Furthermore, in vivo experiments showed that Hu1A3B-7 successfully protected mice against lethal subcutaneous and aerosol challenges with VEEV strain TrD (serogroup I). Hu1A3B-7 is therefore a promising candidate for the future development of a broad-spectrum antiviral therapy to treat VEEV disease in humans.

A recombinant humanized monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus.

A recombinant humanized antibody to Venezuelan equine encephalitis virus (VEEV) was constructed in a monocistronic adenoviral expression vector with a foot-and-mouth-disease virus-derived 2A self-cleavage oligopeptide inserted between the antibody heavy and light chains. After expression in mammalian cells, the heavy and light chains of the humanized antibody (hu1A4A1IgG1-2A) were completely cleaved and properly dimerized. The purified hu1A4A1IgG1-2A retained VEEV binding affinity and neutralizing activity similar to its parental murine antibody. The half-life of hu1A4A1IgG1-2A in mice was approximately 2 days. Passive immunization of hu1A4A1IgG1-2A in mice (50 microg/mouse) 24 h before or after virulent VEEV challenge provided complete protection, indicating that hu1A4A1IgG1-2A has potent prophylactic and therapeutic effects against VEEV infection.

Towards a Brucella vaccine for humans.

There is currently no licensed vaccine for brucellosis in humans. Available animal vaccines may cause disease and are considered unsuitable for use in humans. However, the causative pathogen, Brucella, is among the most common causes of laboratory-acquired infections and is a Center for Disease Control category B select agent. Thus, human vaccines for brucellosis are required. This review highlights the considerations that are needed in the journey to develop a human vaccine, including animal models, and includes an assessment of the current status of novel vaccine candidates.

Improved efficacy of a gene optimised adenovirus-based vaccine for venezuelan equine encephalitis virus.

BACKGROUND: Optimisation of genes has been shown to be beneficial for expression of proteins in a range of applications. Optimisation has increased protein expression levels through improved codon usage of the genes and an increase in levels of messenger RNA. We have applied this to an adenovirus (ad)-based vaccine encoding structural proteins (E3-E2-6K) of Venezuelan equine encephalitis virus (VEEV). RESULTS: Following administration of this vaccine to Balb/c mice, an approximately ten-fold increase in antibody response was elicited and increased protective efficacy compared to an ad-based vaccine containing non-optimised genes was observed after challenge. CONCLUSION: This study, in which the utility of optimising genes encoding the structural proteins of VEEV is demonstrated for the first time, informs us that including optimised genes in gene-based vaccines for VEEV is essential to obtain maximum immunogenicity and protective efficacy.

Development and characterization of mouse models of infection with aerosolized Brucella melitensis and Brucella suis.

There is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensis and Brucella suis and demonstrated their utility for the evaluation of vaccines using the model live B. melitensis vaccine strain Rev.1.

CpG used as an adjuvant for an adenovirus-based Venezuelan equine encephalitis virus vaccine increases the immune response to the vector, but not to the transgene product.

An adenovirus-based (ad-based) vaccine delivering antigens from the Alphavirus Venezuelan equine encephalitis virus (VEEV) is a strategy that offers clinical potential. A vaccine against VEEV is desirable because of the re-emerging nature of this virus, and also the potential that it may be used as a biological weapon. This study was designed to investigate whether the co-administration of CpG oligodeoxynucleotides (ODNs) with an ad-based VEEV vaccine could enhance the protective efficacy of the vaccine. We report that the co-administration of CpG ODN was unable to increase VEEV-specific antibody responses in mice, and was unable to increase the protective efficacy of the vaccine against aerosol challenge with virulent VEEV. However, it was noted that antibody responses directed against the adenovirus vaccine vector were increased, which may be detrimental, particularly in the context of homologous boosting.

Boosting with an adenovirus-based vaccine improves protective efficacy against Venezuelan equine encephalitis virus following DNA vaccination.

There is a requirement for a vaccine that protects against the alphavirus, Venezuelan equine encephalitis virus (VEEV). Previous work has shown that DNA vaccines encoding structural proteins of VEEV can elicit immune responses and protection against VEEV though this protection is incomplete against airborne VEEV. In this study, we demonstrate that particle-mediated epidermal delivery of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein. This results in an improved Th2-type IgG response, an increase in neutralising antibody and a significant increase in protection against airborne VEEV. This indicates that prime-boost may be a suitable immunisation regimen for providing protection against airborne VEEV.

DNA vaccines for biodefence.

The advantages associated with DNA vaccines include the speed with which they may be constructed and produced at large-scale, the ability to produce a broad spectrum of immune responses, and the ability for delivery using non-invasive means. In addition, DNA vaccines may be manipulated to express multiple antigens and may be tailored for the induction of appropriate immune responses. These advantages make DNA vaccination a promising approach for the development of vaccines for biodefence. In this review, the potential of DNA vaccines for biodefence is discussed.

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax.

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.

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.

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.

The effect of recombinant plasmids on in vivo colonisation of Salmonella enterica serovar Typhimurium strains is not reflected by in vitro cellular invasion assays.

Attenuated strains of Salmonella enterica serovar Typhimurium are used as carriers of heterologous antigens as candidate oral vaccines and, more recently, as carriers of DNA vaccines. In this study, recombinant Salmonella strains that were altered in their ability to colonise murine tissues in vivo when compared to parent strains were not, however, equally altered in their ability to invade murine cells in vitro. These results suggest that in vitro invasion studies may not be a representative model for colonisation of tissues in vivo, and that in vitro studies should ideally be used in conjunction with in vivo studies for the assessment of potential Salmonella vaccines.