Nancy L. Davis

Vaccination of Macaques against Pathogenic Simian Immunodeficiency Virus with Venezuelan Equine Encephalitis Virus Replicon Particles

ABSTRACT Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.

Severe Acute Respiratory Syndrome Coronavirus Group-Specific Open Reading Frames Encode Nonessential Functions for Replication in Cell Cultures and Mice

ABSTRACT SARS coronavirus (SARS-CoV) encodes several unique group-specific open reading frames (ORFs) relative to other known coronaviruses. To determine the significance of the SARS-CoV group-specific ORFs in virus replication in vitro and in mice, we systematically deleted five of the eight group-specific ORFs, ORF3a, OF3b, ORF6, ORF7a, and ORF7b, and characterized recombinant virus replication and gene expression in vitro. Deletion of the group-specific ORFs of SARS-CoV, either alone or in various combinations, did not dramatically influence replication efficiency in cell culture or in the levels of viral RNA synthesis. The greatest reduction in virus growth was noted following ORF3a deletion. SARS-CoV spike (S) glycoprotein does not encode a rough endoplasmic reticulum (rER)/Golgi retention signal, and it has been suggested that ORF3a interacts with and targets S glycoprotein retention in the rER/Golgi apparatus. Deletion of ORF3a did not alter subcellular localization of the S glycoprotein from distinct punctuate localization in the rER/Golgi apparatus. These data suggest that ORF3a plays little role in the targeting of S localization in the rER/Golgi apparatus. In addition, insertion of the 29-bp deletion fusing ORF8a/b into the single ORF8, noted in early-stage SARS-CoV human and civet cat isolates, had little if any impact on in vitro growth or RNA synthesis. All recombinant viruses replicated to wild-type levels in the murine model, suggesting that either the group-specific ORFs play little role in in vivo replication efficiency or that the mouse model is not of sufficient quality for discerning the role of the group-specific ORFs in disease origin and development.

Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: Construction of single and multiple mutants in a full-length cDNA clone

Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.

Role of Alpha/Beta Interferon in Venezuelan Equine Encephalitis Virus Pathogenesis: Effect of an Attenuating Mutation in the 5′ Untranslated Region

ABSTRACT Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5′ untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-α/β) activity in VEE pathogenesis. In IFN-α/βR−/− mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-α/βR−/− mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-α/β pathway. In cells derived from IFN-α/βR−/− mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-α/β plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-α/β between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-α/β in two separate in vitro assays, suggesting that the increased sensitivity to IFN-α/β plays a major role in the in vivo attenuation of V3043.

Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses

Replicon particles derived from a vaccine strain of Venezuelan equine encephalitis (VEE) virus were used as vectors for expression in vivo of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). The immunogenicity of the different replicons was evaluated in horses, as was their ability to protectively immunize horses against intranasal and intrauterine challenge with a virulent strain of EAV (EAV KY84). Horses immunized with replicons that express both the G(L) and M proteins in heterodimer form developed neutralizing antibodies to EAV, shed little or no virus, and developed only mild or inapparent signs of equine viral arteritis (EVA) after challenge with EAV KY84. In contrast, unvaccinated horses and those immunized with replicons expressing individual EAV envelope proteins (M or G(L)) shed virus for 6-10 days in their nasal secretions and developed severe signs of EVA after challenge. These data confirm that replicons that co-express the G(L) and M envelope proteins effectively, induce EAV neutralizing antibodies and protective immunity in horses.

Systemic, Mucosal, and Heterotypic Immune Induction in Mice Inoculated with Venezuelan Equine Encephalitis Replicons Expressing Norwalk Virus-Like Particles

ABSTRACT Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.

Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants.

Background In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge. Methods and Findings Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N), we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S) encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV–challenged mice. VRP-N–induced pathology presented at day 4, peaked around day 7, and persisted through day 14, and was likely mediated by cellular immune responses. Conclusions This study identifies gaps and challenges in vaccine design for controlling future SARS-CoV zoonosis, especially in vulnerable elderly populations. The availability of a SARS-CoV virus bearing heterologous S glycoproteins provides a robust challenge inoculum for evaluating vaccine efficacy against zoonotic strains, the most likely source of future outbreaks.

Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons

ABSTRACT The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.

Alphavirus Replicon Particles as Candidate HIV Vaccines

Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self‐replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix‐capsid portion of Gag, the full‐length envelope gp160, or the secreted gp140 of cloned SIVsm H‐4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H‐4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.

Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis.

We describe experiments with two monoclonal antibodies to the vesicular stomatitis virus (VSV) nucleocapsid protein N with strikingly different characteristics. Antibody 1 binds to nucleocapsids and probably the pool of free (unbound) N protein; it inhibits transcription in vitro, and when microinjected into cells, protects the cells against VSV. Antibody 2 binds poorly to nucleocapsids, does not inhibit transcription, but when microinjected into cells, binds selectively to the free N and delays the appearance of progeny virus. We have confirmed these results by analyzing the effect of these antibodies on in vitro genomic RNA synthesis. The results of both the in vivo and in vitro experiments show that the replication of the VSV genome is controlled by the availability of the nucleocapsid protein, even when the polymerase has access to the host factors and multiple phosphorylated forms of the NS protein thought to be involved in genomic RNA synthesis.