Venezuelan equine encephalitis virus complex-specific monoclonal antibody provides broad protection, in murine models, against airborne challenge with viruses from serogroups I, II and III.

The alphavirus Venezuelan equine encephalitis virus (VEEV) is highly infectious by the airborne route. It is a hazard to laboratory workers, has been developed as a biological weapon and is a potential bioterrorist agent. A suitable vaccine appears in an advanced stage of development but there remains a need for antiviral drugs, effective in prophylaxis of disease prior to or a short time after exposure to airborne virus. Using a murine model to study monoclonal antibody (MAB) a VEEV complex-specific, glycoprotein E2-binding MAB was identified, able to protect against disease induced by exposure to aerosolised VEEV from serogroups I, II and IIIA (mouse-virulent strains). There was no synergy in protection between anti-E1 and anti-E2 MAB. Assays of MAB virus neutralising activity in a homologous (mouse fibroblast) cell line suggested that neutralisation played a significant role in protection in addition to the previously reported mechanism of Fc receptor-binding [Mathews et al., 1985. J. Virol. 55, 594-600]. Development of an analogous human MAB with identical VEEV epitope specificity may be informed and monitored by reference to these properties.

Intranasal immunisation with defective adenovirus serotype 5 expressing the Venezuelan equine encephalitis virus E2 glycoprotein protects against airborne challenge with virulent virus.

There is no vaccine licensed for human use to protect laboratory or field workers against infection with Venezuelan equine encephalitis virus (VEEV). Infection of these groups is most likely to occur via the airborne route and there is evidence to suggest that protection against airborne infection may require high antibody levels and the presence of antibody on the mucosal surface of the respiratory tract. Recombinant defective type 5 adenoviruses, expressing the E3E26K structural genes of VEEV were examined for their ability to protect mice against airborne challenge with virulent virus. After intranasal administration, good protection was achieved against the homologous serogroup 1A/B challenge virus (strain Trinidad donkey). There was less protection against enzootic serogroup II and III viruses, indicating that inclusion of more than one E3E26K sequence in a putative vaccine may be necessary. These studies confirm the potential of recombinant adenoviruses as vaccine vectors for VEEV and will inform the development of a live replicating adenovirus-based VEEV vaccine, deliverable by a mucosal route and suitable for use in humans.

Antibody and interleukin-12 treatment in murine models of encephalitogenic flavivirus (St. Louis encephalitis, tick-borne encephalitis) and alphavirus (Venezuelan equine encephalitis) infection.

Early and sustained treatment with interleukin-12 (IL-12) ameliorated disease in a mouse model of infection with the encephalitogenic flavivirus, St. Louis encephalitis virus (SLEV, Japanese encephalitis serogroup). However, this effect was not reproduced in murine infections with either the flavivirus tick-bore encephalitis virus (TBEV) or the alphavirus Venezuelan equine encephalitis virus (VEEV). IL-12 exacerbated TBEV disease when used in conjunction with monoclonal antibody (mAb), suggesting an enhancement of immunopathology, and was without clinical effects in VEEV infection. These data confirm the need to fully understand the pathogenesis of viral infection before cytokine intervention may be employed as a broad-spectrum antiviral therapy.

Cytotoxic T-cell activity is not detectable in Venezuelan equine encephalitis virus-infected mice.

Previously published research has established that the immune response to the Venezuelan equine encephalitis virus (VEEV) vaccine strain TC-83 is Th 1-mediated, with local activation of both CD4+ and CD8+ T cells. This suggests that cytotoxic lymphocytes CTL may play a role in protection against virulent VEEV. Studies involving a variety of immunisation schedules with either TC-83 or strain CAAR 508 (serogroup 5) of VEEV, and six different haplotypes of mice, failed to reveal functional CTL activity against VEEV-infected targets in secondary antigen-stimulated lymphocyte cultures from either the draining lymph nodes (LN) or spleen. Nor were VEEV-specific CTL detected after immunisation of mice (three haplotypes) with recombinant vaccinia viruses (VV) expressing either the non-structural (nsP1-4) or the structural (C-E3-E2-6K-E1) genes of TC-83. Reciprocal experiments in which mice were immunised with TC-83, and their lymphocytes tested against VV recombinant-infected targets also failed to detect CTL activity. These data suggest that VEEV infection of mice does not elicit detectable CTL activity, and that CTL are unlikely to play a role in protection against virulent VEEV.

Monoclonal antibody protects mice against infection and disease when given either before or up to 24 h after airborne challenge with virulent Venezuelan equine encephalitis virus.

Airborne infection with Venezuelan equine encephalitis virus (VEEV) is a significant hazard for laboratory workers, who may not be immunised against VEEV infection as there is no vaccine currently available suitable for human use. We describe a potential alternative strategy that could protect workers exposed to VEEV or similar viruses. VEEV-specific murine monoclonal antibodies (MAB), given by intraperitoneal (i.p.) injection to mice as a single dose of 100 microg, have a half-life of 6-10 days in serum and spread by transudation to respiratory secretions. Administration of MAB (approximately 4 mg/kg) to mice 24h before challenge with approximately 100LD50 of virulent VEEV protected up to 100% animals. The same dose of MAB delivered up to 24h after challenge protected approximately 50%. Two MAB that were synergistic in vitro in plaque reduction neutralisation tests were not synergistic in vivo in protection assays. An examination of virus multiplication, in the blood and internal organs (brain, spleen, lung) of MAB-treated mice infected by the airborne route with VEEV, suggested that therapeutic activity depended both upon the prevention of virus infection of the brain, and the rapid clearance of virus from the periphery. Antiviral therapy with VEEV-specific human or "humanised" MAB, providing that they are administered early, may offer an alternative means of specific medical intervention for those with a known exposure to VEEV.

An immunological profile of Balb/c mice protected from airborne challenge following vaccination with a live attenuated Venezuelan equine encephalitis virus vaccine.

The live attenuated vaccine strain of Venezuelan equine encephalitis virus (VEEV), TC-83, protects mice against challenge (subcutaneous and aerosol) with virulent VEEV but is not suitable for widescale human use. Elucidation of the immune response profile of protected mice should assist in the development of an improved vaccine. We determined the optimum dose of TC-83 required to consistently protect Balb/c mice from airborne challenge with the virulent Trinidad Donkey strain of VEEV and studied the development of humoral and cellular immune responses in protected mice between 6 h and 21 days post-vaccination. The most dramatic immune responses occurred in draining lymph nodes 24 h following vaccination with increased levels of activated B cells and T cells of both CD4(+) and CD8(+) subtypes. Activated monocyte/macrophages and natural killer cells were also seen between 6 h and 7 days post-vaccination. Serum contained detectable VEEV-specific IgG on day 5 post-vaccination with titres continuing to rise on days 7, 14 and 21. Isotypes of IgG measured on days 7 and 21 were predominantly of the IgG2a subclass, indicating that the immune response was Th1-mediated. Cytokine mRNA was quantified by RT-PCR and revealed production of the Th1 cytokine IFN-gamma and the inflammatory cytokine TNF-alpha, whereas the Th2 cytokine IL4 was not detected above control levels at any of the time points studied. This data describes key cellular immune responses at early times post-vaccination and is consistent with previous data demonstrating protection against aerosol challenge with VEEV in the absence of detectable levels of specific IgG or IgA antibody.

Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.

The immune response to vaccinia virus is significantly reduced after scarification with TK- recombinants as compared to wild-type virus.

Although it is unlikely that large-scale vaccination against smallpox will ever be required again, it is conceivable that the need may arise to vaccinate against a human orthopoxvirus infection. A possible example could be the emergence of monkey poxvirus (MPV) as a significant human disease in Africa. Vaccinia virus (VV) recombinants, genetically modified to carry the immunogenic proteins of other pathogenic organisms, have potential use as vaccines against other diseases present in this region. The immune response to parental wild-type (wt) or recombinant VV was examined by binding and functional assays, relevant to protection: total IgG, IgG subclass profile, B5R gene product (gp42)-specific IgG, neutralizing antibodies and class 1-mediated cytotoxic lymphocyte activity. There was a substantial reduction in the immune response to VV after scarification with about 10(8) PFU of recombinant as compared to wt virus. These data suggest that to achieve the levels of immunity associated with protection against human orthopoxvirus infection, and to control a possible future outbreak of orthopoxvirus disease, the use of wt VV would be necessary.

Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus.

Plasmids expressing the V antigen of Yersinia pestis or the E2 glycoprotein of Venezuelan Equine Encephalitis (VEE) virus were used to vaccinate mice by intra-dermal or intra-muscular injection, or by particle-mediated bombardment using the Helios gene gun. After two immunizations, groups of mice which had received 4 microg doses of plasmid DNA using the gene gun had IgG levels which were higher than in other groups manually immunised with 12-fold more plasmid DNA. The immunoglobulin isotype profile was predominantly IgG1 following inoculation with either plasmid. Our results indicate that gene gun mediated vaccination can be used to increase the magnitude of the immune response to both bacterial and viral antigens expressed by plasmid DNA.

Recombinant vaccinia viruses protect against Clostridium perfringens alpha-toxin.

Recombinant vaccinia viruses that expressed the nontoxic C-domain of Clostridium perfringens alpha-toxin were constructed. The J2R (thymidine kinase [TK] gene) and B13R (serpin 2 [SPI-2] gene) loci were used as insertion sites for the clostridial DNA, and expression of the foreign protein was measured in each case. A double recombinant that encoded the alpha-toxin truncate at the B13R locus and the protective antigen of Bacillus anthracis at the J2R locus was also constructed. Although differences in expression of the alpha-toxin C-domain were recorded, all of the vaccinia recombinants protected mice against a lethal challenge with alpha-toxin demonstrating that a recombinant vaccinia virus can be used to provide protection against a toxin challenge that is known to be solely antibody mediated.

Immunity to airborne challenge with Venezuelan equine encephalitis virus develops rapidly after immunization with the attenuated vaccine strain TC-83.

Mice vaccinated subcutaneously with the attenuated vaccine strain of Venezuelan equine encephalitis virus (VEEV) rapidly develop immunity to subcutaneous or airborne challenge with virulent VEEV. The specificity of this immune response was demonstrated by challenge with a heterologous virus (St. Louis encephalitis virus). Examination of the levels of VEEV-specific antibody classes in serum and respiratory secretions suggested that the rapid development of immunity was coincident with the appearance of specific IgM and IgG (but not IgA) in the respiratory tract. In order to confirm the role of respiratory tract antibody, mice were passively immunised either intraperitoneally or intranasally with polyclonal VEEV-specific IgG. Intranasal administration of specific IgG significantly enhanced protection against airborne challenge. These results confirm the need to emphasise local antibody production in the development of improved VEEV vaccines.

Interferon-alpha protects mice against lethal infection with St Louis encephalitis virus delivered by the aerosol and subcutaneous routes.

In common with other flaviviruses, there is no specific therapy for St Louis encephalitis (SLE) virus infections. A number of cases have occurred where infection may have been acquired by the aerosol route in laboratory accidents. The recombinant human interferon hybrids IFN-alpha A/D (Roche Laboratories) and IFN-alpha B/D (Ciba-Geigy) have activity in murine models. Given for several days around the time of exposure to the virus or shortly after, these compounds reduce the mortality from SLE virus administered to mice subcutaneously by up to 70%. In an aerosol model of SLE disease, the mortality was reduced to 30-50% compared to 100% in controls, depending on the challenge level of virus. These results suggest that interferon-alpha could be used to reduce the mortality from SLE infection after known exposure to the virus.

TC-83 vaccine protects against airborne or subcutaneous challenge with heterologous mouse-virulent strains of Venezuelan equine encephalitis virus.

Vaccination with TC-83 virus produced solid protection against subcutaneous challenge with Venezuelan equine encephalitis (VEEV) viruses from homologous and heterologous serogroups, but breakthrough infection and disease occurred after airborne challenge. Breakthrough occurred more often with time after vaccination, and was more frequent with epizootic, homologous serogroup 1A/B viruses than with enzootic, heterologous serogroup viruses. A decrease in VEEV-specific IgA levels in the respiratory tract of vaccinated mice may explain the increased frequency of breakthrough with time after vaccination. However increased breakthrough with the highly virulent homologous serogroup 1A/B viruses (compared to less virulent viruses from heterologous serogroups) may be a consequence of their greater ability to invade the brain via the olfactory neuroepithelium and olfactory nerve.

Humane endpoints are an objective measure of morbidity in Venezuelan encephalomyelitis virus infection of mice.

The clinical signs are described of Venezuelan encephalomyelitis virus (VEEV) infection in mice after both airborne and subcutaneous (s.c.) challenge. Group clinical scores reflected the known pathogenesis of infection by both s.c. and airborne challenge, and with epizootic and enzootic strains of VEEV. This observation confirms the specific relationship of the observed clinical signs to VEEV infection. Within an experiment, those who are assessing the animals for clinical signs must have a common understanding of their appearance, including severity, and should be unaware of the allocation of treatments. If these conditions are met, the progress of clinical signs may be used to determine objectively the time of culling for humane endpoints.

Improved protection against Venezuelan equine encephalitis by genetic engineering of a recombinant vaccinia virus.

An improved vaccine is needed against Venezuelan equine encephalitis (VEE) virus because the existing live attenuated vaccine, TC-83, causes a high incidence of adverse effects, and the Formalin-inactivated vaccine, C-84, does not protect against airborne infection. A recombinant vaccine had previously been constructed in which the VEE structural proteins were expressed by vaccinia virus. Although protection against subcutaneous challenge with VEE was achieved, the vaccine had limited efficacy against aerosolized virus. We made a similar construct (WR100) and compared its performance with that of a recombinant vaccinia virus which had been altered in two ways (WR103) in order to improve its performance as a vaccine: a synthetic promoter was inserted upstream of the VEE coding sequence to increase the amount of VEE proteins produced, and a single nucleotide in the E2 glycoprotein gene was altered to enhance immunogenicity. The WR103 virus expressed greater amounts of VEE proteins on the surface of infected cells than did WR100, and this difference was found to correspond to a 3.5-fold increase in VEE protein production. Sera from mice immunized with WR103 contained elevated levels of antibody to VEE, and enhanced protection against subcutaneous challenge with the pathogenic Trinidad donkey strain was achieved. This altered construct could form the basis for a better vaccine against VEE.

Improved protection against Venezuelan equine encephalitis by genetic engineering of a recombinant vaccinia virus

An improved vaccine is needed against Venezuelan equine encephalitis (VEE) virus because the existing live attenuated vaccine, TC-83, causes a high incidence of adverse effects, and the Formalin-inactivated vaccine, C-84, does not protect against airborne infection. A recombinant vaccine had previously been constructed in which the VEE structural proteins were expressed by vaccinia virus. Although protection against subcutaneous challenge with VEE was achieved, the vaccine had limited efficacy against aerosolized virus. We made a similar construct (WR100) and compared its performance as a vaccine: a synthetic promoter was inserted upstream of the VEE coding sequence to increase the amount of VEE proteins produced, and a single nucleotide in the E2 glycoprotein gene was altered to enhance immunogenicity. The WR103 virus expressed greater amounts of VEE proteins on the surface of infected cells than did WR100, and this difference was production. Sera from mice immunized with WR103 contained elevated levels of antibody to VEE, and enhanced protection against subcutaneous challenge with the pathogenic Trinidad donkey strain was achieved. This altered construct could form the basis for a better vaccine against VEE.(Au)

A simple device for the exposure of animals to infectious microorganisms by the airborne route.

In order to evaluate prophylaxis and therapy for individuals infected with pathogens by the airborne route, we have designed and built a simple apparatus in which small laboratory animals may be exposed to aerosols of infectious microorganisms. Animals are kept in a chamber closed by a HEPA filter and exposed to the pathogen aerosolized using a Collison nebulizer. Air in the exposure chamber may be sampled to show that the infectious agent is present but the dose of agent must be expressed as 50% effective doses determined by titration. An effective dose may be defined by whatever criteria are chosen to judge disease. Using this apparatus we have shown that St Louis encephalitis (SLE) virus is infectious for mice by the airborne route. These data support the idea that there may be significant hazard to personnel exposed to aerosols of infectious SLE after a laboratory accident.

Production of virus-specific antisera using synthetic peptides corresponding to sequences in the yellow fever E protein: brief report.

Synthetic peptides have gained widespread acceptance for use in epitope mapping and as immunogens for monoclonal antibody and polyclonal serum production. Putative antigenic peptides homologous to regions in the primary sequence of the envelope protein (E) of yellow fever virus (YF17D) were synthesized and evaluated for their ability to produce polyclonal antisera specific for the parent protein and for their reactivity with a panel of E-specific mAb. Antipeptide sera were reacted with native virus in ELISA, Western blot, neutralization, hemagglutination-inhibition, and immunofluorescence tests. Reactive sera were in most cases specific for the original peptide. However, despite the diversity of peptide selection processes, we were unable to identify any antipeptide serum that reacted specifically with authentic YF E protein.

Evaluation of monoclonal antibodies for generic detection of flaviviruses by ELISA.

Three monoclonal antibodies (Mabs) specific for the envelope (E) protein of flaviviruses were evaluated for use in an antigen capture ELISA. Three combinations of Mabs and a combination of polyclonal antibodies (Pabs) were evaluated in antigen capture ELISAs for their ability to detect 18 flaviviruses. The Mab ELISAs detected 50% of flavivirus antigens with a sensitivity between 1 and 9 x 10(4)/ng viral protein/ml, however, none of the ELISAs evaluated proved to be useful for generic detection of flaviviruses, being unable to detect tick-borne flaviviruses and some mosquito-borne flaviviruses. The inability of the ELISAs to detect tick-borne flaviviruses is thought to be due to the conformation of surface epitopes, which the Mabs were unable to recognise. This was again observed using recombinant TBE virus prM/E protein as antigen in direct and antigen capture ELISAs. The Mabs reacted with the prM/E protein when it was denatured by binding directly onto the solid phase, but the antibodies were unable to detect the native protein in antigen capture ELISAs. The antigen capture ELISAs evaluated in this study were considered to be unsuitable for the generic detection of flaviviruses, but may provide a sensitive diagnostic assay for specific flavivirus infection.

Immunisation with DNA polynucleotides protects mice against lethal challenge with St. Louis encephalitis virus.

In vivo transfection by intramuscular injection with plasmids expressing the immunogenic proteins of microbial pathogens has considerable potential as a vaccination strategy against many pathogens of both man and animals. Here we report that weanling mice given a single intramuscular injection of 50 micrograms of a plasmid, pSLE1 expressing the St. Louis encephalitis virus (SLE) prM/E protein under the control of the cytomegalovirus immediate early protein promoter produced SLE-specific antibody and were protected against lethal challenge with the virulent virus. Polynucleotide vaccine technology provides a unique opportunity to produce vaccines against flavivirus diseases of low incidence cheaply and rapidly, and to produce multivalent vaccines such as would be required for immunisation against dengue virus disease.