The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-␥) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-␥, including human and rat IFN-␥; it does not, however, bind significantly to murine IFN-␥. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-␥ and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.

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Rinderpest is a highly contagious ruminant viral disease manifested by a rapid course and greater than 90% mortality. Infectious vaccinia virus recombinants were constructed that express either the hemagglutinin or the fusion gene of rinderpest virus. All cattle vaccinated with either recombinant or with the combined recombinants produced neutralizing antibodies against rinderpest virus and were protected against the disease when challenged with more than 1000 times the lethal dose of the virus.

In a continuing effort to develop safe and efficacious vaccine and immunotherapeutic vectors, we constructed recombinant vaccinia virus (rVV) vaccines lacking either the B13R (SPI-2) or the B22R (SPI-1) immune-modulating gene and coexpressing IFN-␥. B13R and B22R are nonessential VV immune-modulating genes that have antiapoptotic and antiinflammatory properties with sequence homology to serine protease inhibitors (serpins). IFN-␥ is a cytokine with potent immunoregulatory, antineoplastic, and antiviral properties. We observed that these rVVs with a deletion in a serpin gene and expressing IFN-␥ replicated to high titers in tissue culture yet were avirulent in both immunocompromised and immunocompetent mice with no detectable viral replication in these animals. A single immunization elicited potent humoral, T helper, and cytotoxic T cell immune responses in mice despite the absence of any detectable virus replication in vivo. IFN-␥ coexpression and the inactivation of one or more VV immune-modulating genes provide an optimized method for increasing the safety while maintaining the efficacy of rVV vaccines. This strategy provides a method for developing highly safe and efficacious vaccines for smallpox and other diseases and immunotherapeutic vectors.vaccines ͉ safety ͉ efficacy ͉ immune-modulating genes ͉ smallpox V accinia virus (VV), the orthopoxvirus used in the global eradication of smallpox, also has served as an effective vector for eukaryotic protein expression, vaccine development, and immunotherapeutic treatments for cancer (1). The emerging threat of smallpox bioterrorism has once again brought VV vaccines to the forefront. Currently, vaccination of military and emergency response personnel with VV is recommended, and vaccination is being considered for the general public (2). Although VV has not been directly associated with any specific disease, complications have been observed in immunocompromised and immunosuppressed individuals (3). In light of the current threat of bioterrorism and the ongoing AIDS epidemic, the complications of VV infection in immunocompetent and especially in immunocompromised populations must be thoroughly addressed to circumvent any possible pathogenic effects of mass vaccination. Therefore, increasing safety while maintaining proven efficacy is one of the foremost considerations for the widespread use of VV vaccines.VV has been used extensively as a vector for the development of recombinant live vaccines (1). There are currently two well established effective recombinant VV (rVV) vaccines, one for rabies (4) and the other for rinderpest (5)(6)(7)8).We and others have demonstrated that the cytokine IFN-␥ can be used as both an adjuvant and an attenuating agent for the development of safe and efficacious live VV vaccines (9-11). IFN-␥ is a cytokine that plays an essential role in the regulation of the immune system and host defense against pathogens (12). The effects of IFN-␥ on the immune system are profound. IFN-␥ modulates macrophage tumoricidal and microbicidal activity, na...

We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for y interferon (IFN-y) and structural proteins of the human immunodeficiency virus type 1 (HIV-1).In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-y with glycoprotein gpl20, gag, and a fragment of gp4l. Afl fusion proteins retained the antigenic characteristics of both IFN-y and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-y could be demonstrated only for the IUFN-,y-gag fusion protein.In contrast, the attenuating activity of IFN-yfor nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-y was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed.

Rift Valley fever (RVF) is a zoonotic disease endemic in Africa and the Arabian Peninsula caused by the highly infectious Rift Valley fever virus (RVFV) that can be lethal to humans and animals and results in major losses in the livestock industry. RVF is exotic to the United States; however, mosquito species native to this region can serve as biological vectors for the virus. Thus, accidental or malicious introduction of this virus could result in RVFV becoming endemic in North America. Such an event would likely lead to significant morbidity and mortality in humans, and devastating economic effects on the livestock industry. Currently, there are no licensed vaccines for RVF that are both safe and efficacious. To address this issue, we developed two recombinant RVFV vaccines using vaccinia virus (VACV) as a vector for use in livestock. The first vaccine, vCOGnGc, was attenuated by the deletion of a VACV gene encoding an IFN-γ binding protein, insertional inactivation of the thymidine kinase gene, and expression of RVFV glycoproteins, Gn and Gc. The second vaccine, vCOGnGcγ, is identical to the first and also expresses the human IFN-γ gene to enhance safety. Both vaccines are extremely safe; neither resulted in weight loss nor death in severe combined immunodeficient mice, and pock lesions were smaller in baboons compared with the controls. Furthermore, both vaccines induced protective levels of antibody titers in vaccinated mice and baboons. Mice were protected from lethal RVFV challenge. Thus, we have developed two safe and efficacious recombinant vaccines for RVF.

Rinderpest is a highly contagious viral disease of ruminants with >95% morbidity and mortality. We have constructed an infectious vaccinia virus recombinant that expresses both the fusion (F) gene and the hemagglutinin (H) gene ofrinderpest virus. The Wyeth strain of vaccinia virus was used for the construction of the recombinant. Cattle vaccinated with the recombinant virus were 100% protected from challenge inoculation with >1000 times the lethal dose of rinderpest virus. No transmission of recombinant vaccinia virus from vaccinated animals to contact animals was observed. The Iyophilized form of vaccinia virus is thermostable and allows circumvention of the logistical problems associated with the distribution and administration of vaccines in the arid and hot regions of Asia and Africa. The insertional inactivation of both the thymidine kinase and the hemagglutinin genes of vaccinia virus led to increased attenuation of the virus; this was manifested by the lack of detectable pock lesions in vaccinated animals. This approach may have wide application in the development of safe and efficacious recombinant vaccines for humans and animals. This becomes quite relevant with the concern of the use of vaccinia virus in a population with high incidence of the human immunodeficiency virus.

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