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Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones., (Published by Elsevier Ltd.)

Interferon-γ (IFN-γ) is an attenuating factor for vaccinia virus (VACV), decreasing its virulence in vivo by more than a million fold. It is also a highly effective adjuvant when administered at the time of immunization with protein antigens. However, recombinant VACV (rVACV) vaccines expressing IFN-γ do not induce enhanced immune responses. It is possible that the IFN-γ expressed by rVACVs induces both an antiviral state and increased immunological clearance, thus resulting in decreased levels of antigen expression due to reduced viral replication and spread. We conjectured that delaying expression of IFN-γ would result in enhanced production of antigens by rVACVs thus resulting in increased immune responses to foreign antigens. Interleukin (IL)-18, also known as IFN-γ inducing factor, is a cytokine that induces T and NK cells to produce IFN-γ. In this study, we demonstrated that an rVACV expressing bioactive murine IL-18 replicated to low but detectable levels in vivo, unlike an rVACV expressing IFN-γ. Moreover, the rVACV expressing IL-18 was significantly attenuated in both immunocompromised and immunocompetent mice. This attenuation was dependent on IFN-γ, as IL-18 expression failed to attenuate VACV in IFN-γ knock-out mice. Cytotoxic T-cell (CTL) and anamnestic antibody responses were slightly increased in animals vaccinated with the rVACV expressing IL-18. Thus, induction of IFN-γ because of IL-18 expression resulted in an rVACV that replicated to low but detectable levels in vivo, yet elicited slightly better CTL and anamnestic humoral immune responses.

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

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.

A vaccine for human immunodeficiency virus (HIV) infection is desperately needed to control the AIDS pandemic. To address this problem, we constructed single-cycle simian immunodeficiency viruses (SIVs) pseudotyped with the glycoprotein of vesicular stomatitis virus and expressing different levels of gamma interferon (IFN-gamma) as a potential vaccine strategy. We previously showed that IFN-gamma expression by pseudotyped SIVs does not alter viral single-cycle infectivity. T cells primed with dendritic cells transduced by pseudotyped SIVs expressing high levels of IFN-gamma had stronger T-cell responses than those primed with dendritic cells transduced by constructs lacking IFN-gamma. In the present study, we tested the immunogenicities of these pseudotyped SIVs in a rat model. The construct expressing low levels of rat IFN-gamma (dSIV(LRgamma)) induced higher levels of cell-mediated and humoral immune responses than the construct lacking IFN-gamma (dSIV(R)). Rats vaccinated with dSIV(LRgamma) also had lower viral loads than those vaccinated with dSIV(R) when inoculated with a recombinant vaccinia virus expressing SIV Gag-Pol as a surrogate challenge. The construct expressing high levels of IFN-gamma (dSIV(HRgamma)) did not further enhance immunity and was less protective than dSIV(LRgamma). In conclusion, the data indicated that IFN-gamma functioned as an adjuvant to augment antigen-specific immune responses in a dose- and cell type-related manner in vivo. Thus, fine-tuning of the cytokine expression appears to be essential in designing vaccine vectors expressing adjuvant genes such as the gene for IFN-gamma. Furthermore, we provide evidence of the utility of the rat model to evaluate the immunogenicities of single-cycle HIV/SIV recombinant vaccines before initiating studies with nonhuman primate models.

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.

To increase the safety and efficacy of human immunodeficiency virus vaccines, several groups have conducted studies using the macaque model with single-cycle replicating simian immunodeficiency viruses (SIVs). However, these constructs had poor or diminished efficacy compared to live attenuated vaccines. We previously showed that immunization of macaques with live attenuated SIV with a deletion in the nef gene and expressing gamma interferon (IFN-gamma) results in significantly enhanced safety and efficacy. To further enhance safety, we constructed and characterized single-cycle SIVs, pseudotyped with the glycoprotein of vesicular stomatitis virus, expressing different levels of macaque IFN-gamma. Expression of IFN-gamma did not alter the infectivity or antigenicity of pseudotyped SIV. The transduction of dendritic cells (DCs) by IFN-gamma-expressing particles resulted in the up-regulation of costimulatory and major histocompatibility complex molecules. Furthermore, T cells primed with DCs transduced by SIV particles expressing high levels of IFN-gamma and then stimulated with SIV induced significantly higher numbers of spot-forming cells in an enzyme-linked immunospot assay than did T cells primed with DCs transduced with SIV particles lacking the cytokine. In conclusion, we demonstrated that the transduction of DCs in vitro with pseudotyped single-cycle SIVs expressing IFN-gamma increased DC activation and augmented T-cell priming activity.

The nef gene of human and simian immunodeficiency viruses (HIV and SIV) is important for pathogenicity and maintenance of high virus loads. We previously reported that recombinant vaccinia viruses (rVVs) expressing nef from attenuated SIVmac1A11 (vNef1A11) produced typical plaques on thymidine kinase-deficient 143B cells, whereas rVVs expressing nef derived from the pathogenic SIVmac239 (vNef157) formed plaques with altered morphology. Here, we show that vNef157 is attenuated in normal and nude mice, whereas the pathogenicity of vNef1A11 is similar to that of a control virus. Thus, Nef157 is an attenuating factor in the vaccinia virus (VV) system, contrasting sharply with its function in lentiviruses. We also show that Nef157 inhibits VV cell-to-cell spread, causing formation of atypical plaques regardless of thymidine kinase deficiency, neoplasticity, and species of the infected cell line. We hypothesized that Nef157 interferes with VV spread by association with actin, but no direct colocalization of Nef and the cytoskeletal actin network was detected. Instead, higher levels of Nef157 protein were observed, although mRNAs for both nef genes were produced at comparable levels. Thus, the mechanism behind such Nef157 protein accumulation and Nef157-mediated VV attenuation could be related to the process that causes an opposite effect in its native SIV system, making SIVmac239 more pathogenic than SIVmac1A11.

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-gamma. B13R and B22R are nonessential VV immune-modulating genes that have antiapoptotic and antiinflammatory properties with sequence homology to serine protease inhibitors (serpins). IFN-gamma 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-gamma 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-gamma 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.

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 (vDeltaB13R and vDeltaB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50DeltaB13R and v50DeltaB22R). 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 CB6F(1) 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.

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 10(3) 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 10(2) 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 10(8) 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.

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

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.

The gene encoding the P6-like protein of Pasteurella multocida was cloned in the baculovirus expression system. Baculovirus-expressed recombinant protein was used to parenterally immunize 6-wk-old Nicholas broad-breasted white turkeys. Turkeys developed significant antibody titers to the recombinant protein as measured by enzyme-linked immunosorbent assay. Two weeks after the last immunizing injection, vaccinated turkeys were placed in contact with turkeys infected with P. multocida strain P1059, as were nonvaccinated control birds. No differences occurred in percent mortality between the two groups. We conclude that parenterally administered recombinant P6-like protein does not protect turkeys from avian cholera.

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.

Rinderpest, a major economic problem of Africa and Asia, is a highly contagious viral disease of cattle and buffalo with greater than 90% mortality in susceptible herds. We have developed an effective vaccinia virus recombinant vaccine for rinderpest which protects cattle against challenge by more than 1000 times the lethal dose of the virus. The vaccine circumvents the major logistical problems of the Plowright tissue culture vaccine that occasioned previous failures to eradicate rinderpest by vaccination. The recombinant can easily be propagated and administered by scarification, using methods employed in the world-wide eradication of smallpox. More importantly, the lyophilized form of vaccinia virus is thermostable and does not require maintenance of the cold chain, a distinct advantage in the hot and arid regions of Africa and Asia.