Jonathan F. Smith

Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus.

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.

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.

Marburg virus vaccines: comparing classical and new approaches.

An effort to develop a safe and effective vaccine for Marburg virus (MBGV), one of the filoviruses known to cause high mortality rates in humans, led us to compare directly some of the merits of modern versus classical vaccine approaches for this agent. Prior work had established the MBGV-glycoprotein (GP), the only known virion surface antigen, as a candidate for inclusion in a vaccine. In this study, we vaccinated groups of Hartley guinea pigs with killed MBGV, live attenuated MBGV, soluble MBGV-GP expressed by baculovirus recombinants, MBGV-GP delivered as a DNA vaccine, or MBGV-GP delivered via an alphavirus RNA replicon. Serological responses were evaluated, and animals were challenged with a lethal dose of MBGV given either subcutaneously or via aerosol. Killed MBGV and replicon-delivered MBGV-GP were notably immunogenic and protective against MBGV, but results did not exclude any approach and suggested a role for DNA vaccines in immunological priming.

Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine.

A live-attenuated vaccine for Rift Valley fever virus (RVFV), MP-12, has been developed recently by undirected, serial mutagenesis of a RVFV strain (ZH548) isolated during the 1977 epidemic in Egypt. In the present study, the mutations responsible for attenuation of this virus have been examined by analysis of reassortant viruses generated between the vaccine strain and a wild RVFV strain isolated in Senegal. Reassortant viruses were generated efficiently in multiply infected Vero cells, and were readily isolated without application of selective pressures. The origin of the S and M genomic RNA segments in each cloned reassortant virus was determined with monoclonal antibodies capable of differentiating the nucleocapsid protein (S segment marker) or G1 glycoprotein (M segment marker) of the parental strains. The L segment of the vaccine strain was found to contain a temperature-sensitive (ts) mutation, and the origin of the L segment in most reassortants could be inferred by analysis of their ts phenotype. Analysis of the virulence properties of selected reassortant viruses in mice demonstrated that virulence characteristics were under polygenic control, and that at least one mutation capable of independently attenuating the virus existed on each genome segment. The L and M RNA segments were also found to contain ts mutations. These findings suggest that reversion to virulence is unlikely, and further indicate that genetic reassortment with wild-type viruses during a vaccination programme in endemic areas would also be expected to yield attenuated variants.

Improved mucosal protection against Venezuelan equine encephalitis virus is induced by the molecularly defined, live- attenuated V3526 vaccine candidate.

The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.

Candidate vaccine against botulinum neurotoxin serotype A derived from a venezuelan equine encephalitis virus vector system

ABSTRACT A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes andcis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (HC) of the BoNT/A heavy chain was cloned into the replicon vector (HC-replicon). Cotransfection of BHK cells in vitro with the HC-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, HC VEE replicon particles (HC-VRP). Cells infected with HC-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of HC-VRP, mice were vaccinated with various doses of HC-VRP at different intervals. Mice inoculated subcutaneously with HC-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus.

The complete sequence of Punta Toro virus (Phlebovirus, Bunyaviridae) middle size (M), RNA has been determined. The RNA is 4330 nucleotides long (mol wt 1.46 X 10(6), base composition: 26.7% A, 33.6% U, 18.5% G, 21.2% C) and has 3- and 5-terminal sequences that, depending on the arrangement, are complementary for some 15 residues. The viral RNA codes in its viral-complementary sequence for a single primary gene product (the viral glycoprotein precursor) that is comprised of 1313 amino acids (146,376 Da) and is abundant in cysteine residues but has few potential asparagine-linked glycosylation sites. The 5-noncoding region of the Punta Toro M viral-complementary RNA is short (16 nucleotides); the 3-noncoding sequence is much longer (372 nucleotides). The latter is rich in short stretches of adenylate residues, like the 3-noncoding regions of the Punta Toro S mRNA species (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306). No other large open reading frame has been identified in either the viral, or viral-complementary, M RNA sequences. Limited amino-terminal sequence analyses of the two viral glycoproteins have indicated the gene order and potential cleavage sites in the glycoprotein precursor. The data suggest the existence of a 30 X 10(3)-Da polypeptide (designated NSM) in the glycoprotein precursor that precedes the G1 protein (i.e., gene product order: NSM-G1-G2). Examination of the sequence of the Punta Toro M gene product reveals the presence of multiple hydrophobic sequences including a 19-amino acid, carboxy-proximal, hydrophobic region (G2). This hydrophobic sequence is followed by a 13-amino acid-terminal sequence rich in charged amino acids. The size and constitution of the carboxy-terminal region is consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. Other regions of the glycoprotein precursor contain sequences of amino acids with a predominantly hydrophobic character (23, 50, and 20 amino acids in length). Their functions are unknown. The amino terminus of the G1 protein is located near the end of the 23-amino acid-long hydrophobic sequence of the presumptive precursor, the hydrophobic 50-amino acid sequence lies within G1, and the amino terminus of G2 is located in the middle of the 20-amino acid-long hydrophobic sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoelectron microscopy of Rift Valley fever viral morphogenesis in primary rat hepatocytes.

The morphogenesis of the hepatotropic phlebovirus Rift Valley fever virus (RVFV) has been examined by immuno-electron microscopy in primary hepatocyte cultures derived from genetically susceptible and resistant rat strains. RVFV replicates in both cell types with growth kinetics comparable with those seen in other permissive cells. However, in contrast to that has been observed in other cell types, RVFV replication in hepatocytes is associated with maturation at cellular surface membranes in addition to the smooth internal membranes of the Golgi and endoplasmic reticulum. Envelope acquisition at surface membranes occurred primarily on basolateral membranes. The events occurring in RVFV morphogenesis were indistinguishable in hepatocytes from resistant and susceptible animals; however, hepatocytes from susceptible animals produced significantly higher titers of virus.

Immune Protection against Staphylococcal Enterotoxin-Induced Toxic Shock by Vaccination with a Venezuelan Equine Encephalitis Virus Replicon

A candidate vaccine against staphylococcal enterotoxin B (SEB) was developed using a Venezuelan equine encephalitis (VEE) virus vector. This vaccine is composed of a self-replicating RNA, termed replicon, containing the VEE nonstructural genes and cis-acting elements and a gene encoding mutagenized SEB (mSEB). Cotransfection of baby hamster kidney cells with the mSEB replicon and 2 helper RNA molecules resulted in the release of propagation-deficient mSEB-VEE replicon particles (mSEB-VRPs). Mice inoculated subcutaneously with mSEB-VRPs were protected (15 of 20 mice) from a challenge with 5 median lethal dose units of wild-type (wt) SEB. T cells from mice vaccinated with mSEB-VRP responded normally both in vitro to wt SEB and in recall response to the inactivated mSEB polypeptide. The profile of cytokines measured after challenge with wt SEB suggested that the mode of protection was predominantly Th1 dependent. Our results suggest that the VEE replicon is a practical and convenient model system for evaluating efficacy of vaccines for the control of bacterial diseases.

Selection for accelerated penetration in cell culture coselects for attenuated mutants of Venezuelan equine encephalitis virus.

Previous studies with Sindbis virus (SB) suggested that a single point mutation in glycoprotein E2 (serine 114 to arginine 114) conferred three phenotypic alterations: attenuation in neonatal mice, accelerated penetration of cultured cells, and efficient neutralization by two E2-specific monoclonal antibodies (Davis, Fuller, Dougherty, Olmsted, and Johnston (1986) Proc. Natl. Acad. Sci. USA 83, 6771-6775). Moreover, selection for rapidly penetrating mutants of SB coselected for attenuation in vivo, indicating that a domain of SB E2 which influences penetration in culture overlaps an E2 domain which influences pathogenesis (Olmsted, Meyer, and Johnston (1986) Virology 148, 245-254). To test the possibility that overlapping penetration and pathogenesis domains exist in other alphaviruses, the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus (TRD-VEE) was serially passed in baby hamster kidney (BHK) cells under a stringent selective pressure for accelerated penetration. Isolates were biologically cloned from the first through the fourth passages and were characterized as to penetration time course in BHK cells and virulence in adult mice following intraperitoneal inoculation. Twenty-two of the 27 isolates segregated into two major categories: slowly penetrating and virulent (like the TRD-VEE parent) and rapidly penetrating and avirulent. Mice which received the avirulent mutants were positive for anti-VEE neutralizing antibody and were refractory to challenge with TRD-VEE. Of the seven mouse avirulent mutants, two also were attenuated in hamsters, indicating the presence of at least two genetic loci at which mutations may influence both pathogenesis and penetration.