Tilahun Yilma

Vaccinia Virus Vectors with an Inactivated Gamma Interferon Receptor Homolog Gene (B8R) Are Attenuated In Vivo without a Concomitant Reduction in Immunogenicity

ABSTRACT The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-γ) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-γ, including human and rat IFN-γ; it does not, however, bind significantly to murine IFN-γ. 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-γ 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.

Induction of Potent Humoral and Cell-Mediated Immune Responses by Attenuated Vaccinia Virus Vectors with Deleted Serpin Genes

ABSTRACT Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vΔB13R and vΔB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50ΔB13R and v50ΔB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F1 mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy.

Protection of goats against peste des petits ruminants with a vaccinia virus double recombinant expressing the F and H genes of rinderpest virus

Peste des petits ruminants (PPR) is a viral disease of goats and sheep characterized by necrotizing and erosive stomatitis, enteritis and pneumonia. The causative agent, PPRV, is a member of the family Paramyxoviridae and the genus Morbillivirus. Other members of the genus include rinderpest (RPV), measles, canine distemper and phocid distemper viruses. PPR has a very high rate of morbidity and mortality, and effective control of this disease is of economic importance in Africa, Asia and the Middle East. Currently, there is no safe and effective vaccine available against the disease. The tissue culture rinderpest vaccine (TCRV) protects small ruminants against severe disease; there are, however, clinical problems associated with vaccination. This laboratory has recently developed several effective vaccinia virus recombinant vaccines for rinderpest. These vaccines are easy to administer, inexpensive to produce and heat-stable. Goats were vaccinated with a vaccinia virus double recombinant expressing the haemagglutinin and fusion genes of RPV. Although vaccinated animals developed antibodies (neutralizing and ELISA) to RPV, and not to PPRV, they were completely protected against challenge inoculation with virulent PPRV. This would indicate that protection is most probably due to cell-mediated immunity. Use of the rinderpest double recombinant vaccinia virus in areas of the world where PPRV is endemic would aid in the control and eradication of PPR.

Recombinant Rift Valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice

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.

Analysis of the polypeptides synthesized in rinderpest virus-infected cells

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.

Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing GL, M and N proteins expressed from recombinant baculoviruses

Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody response to EAV. The responses of individual animals varied and ELISAs that utilized individual EAV structural proteins were not reliable for detecting antibodies in all sera that contained neutralizing antibodies to EAV. An ELISA based on a cocktail of all three EAV structural proteins, however, was used successfully to detect antibodies in most equine sera that were positive in the standard serum neutralization assay following natural or experimental EAV infection (100% specificity, 92.3% sensitivity). In contrast, this ELISA did not reliably detect antibodies in the sera of vaccinated horses. EAV frequently causes a persistent infection in stallions and all sera from carrier stallions evaluated in this study had obvious reactivity with the N protein, whereas seropositive non-carrier stallions, mares and geldings did not respond consistently to the N protein.

Long-term sterilizing immunity to rinderpest in cattle vaccinated with a recombinant vaccinia virus expressing high levels of the fusion and hemagglutinin glycoproteins

ABSTRACT Rinderpest is an acute and highly contagious viral disease of ruminants, often resulting in greater than 90% mortality. We have constructed a recombinant vaccinia virus vaccine (v2RVFH) that expresses both the fusion (F) and hemagglutinin (H) genes of rinderpest virus (RPV) under strong synthetic vaccinia virus promoters. v2RVFH-infected cells express high levels of the F and H glycoproteins and show extensive syncytium formation. Cattle vaccinated intramuscularly with as little as 103 PFU of v2RVFH and challenged 1 month later with a lethal dose of RPV were completely protected from clinical disease; the 50% protective dose was determined to be 102 PFU. Animals vaccinated with v2RVFH did not develop pock lesions and did not transmit the recombinant vaccinia virus to contact animals. Intramuscular vaccination of cattle with 108 PFU of v2RVFH provided long-term sterilizing immunity against rinderpest. In addition to being highly safe and efficacious, v2RVFH is a heat-stable, inexpensive, and easily administered vaccine that allows the serological differentiation between vaccinated and naturally infected animals. Consequently, mass vaccination of cattle with v2RVFH could eradicate rinderpest.

Molecular cloning of the rinderpest virus matrix gene: Comparative sequence analysis with other paramyxoviruses

The nucleotide sequence of the gene encoding the matrix or membrane (M) protein of the virulent (Kabete-O) strain of rinderpest virus (RPV) has been determined. The M gene is 1457 nucleotides long with a single, large open reading frame. The derived polypeptide has 335 amino acids, corresponding to a calculated molecular weight of 38,289 and contains both small hydrophobic regions and many basic residues. The predicted amino acid sequence was compared to the M proteins of paramyxoviruses. Sequence comparison and hydropathy profiles among the morbilliviruses revealed that the M protein of RPV exhibits features similar to those of the M protein of MV and CDV. There is 78.2% homology at the amino acid level between the M protein of RPV and MV, and 77.6% between RPV and CDV. This indicates that a high degree of homology exists among the members of the genus Morbillivirus. In contrast, there is only 37.3 and 18% homology between RPV and bovine parainfluenza type 3 (BPV3), and RPV and Newcastle disease virus (NDV) M proteins, respectively. Thus the M proteins of the morbilliviruses are highly conserved whereas the M proteins of the genus Paramyxovirus show more divergence.

Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV

Abstract: Analysis of immune responses generated by live‐attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV‐specific T‐helper responses in macaques immunized with the live‐attenuated SIV strains SIVmac239Δnef and SIVmac239Δ3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV‐specific proliferative responses, with peak stimulation indices of up to 84. SIV‐specific proliferative responses were mediated by CD4 + T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV‐specific T‐helper precursor frequencies of up to 96 per 106 peripheral blood mononuclear cells (PBMC). Intracellular flow‐cytometric analysis demonstrated the production of interleukin (IL)‐2, interferon (IFN)‐γ, RANTES and macrophage inhibitory protein‐1α (MIP‐1α) by T lymphocytes from SIVmac239Δnef‐vaccinated animals following SIV p55 stimulation. Induction of strong SIV‐specific T‐helper responses by live‐attenuated SIV vaccines may play a role in their ability to induce protective immunity.

Incorporation of CD40 Ligand into the Envelope of Pseudotyped Single-Cycle Simian Immunodeficiency Viruses Enhances Immunogenicity

ABSTRACT A vaccine for the prevention of human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we developed vesicular stomatitis virus glycoprotein-pseudotyped replication-defective simian immunodeficiency viruses (dSIVs) as an AIDS vaccine strategy. The dSIVs retain characteristics of a live attenuated virus without the drawbacks of potential virulence caused by replicating virus. To improve vaccine immunogenicity, we incorporated CD40 ligand (CD40L) into the dSIV envelope. CD40L is one of the most potent stimuli for dendritic cell (DC) maturation and activation. Binding of CD40L to its receptor upregulates expression of major histocompatibility complex class I, class II, and costimulatory molecules on DCs and increases production of proinflammatory cytokines and chemokines, especially interleukin 12 (IL-12). This cytokine polarizes CD4+ T cells to Th1-type immune responses. DC activation and mixed lymphocyte reaction (MLR) studies were performed to evaluate the immunogenicity of CD40L-dSIV in vitro. Expression levels of CD80, CD86, HLA-DR, and CD54 on DCs transduced with the dSIV incorporating CD40L (CD40L-dSIV) were significantly higher than on those transduced with dSIV. Moreover, CD40L-dSIV-transduced DCs expressed up to 10-fold more IL-12 than dSIV-transduced DCs. CD40L-dSIV-transduced DCs enhanced proliferation and gamma interferon secretion by naive T cells in an MLR. In addition, CD40L-dSIV-immunized mice exhibited stronger humoral and cell-mediated immune responses than dSIV-vaccinated animals. The results show that incorporating CD40L into the dSIV envelope significantly enhances immunogenicity. As a result, CD40L-dSIVs can be strong candidates for development of a safe and highly immunogenic AIDS vaccine.