A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

No abstract

Marburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE replicons (packaged into VEE-like particles), inoculated with MBGV, and evaluated for viremia and survival. Results indicated that either GP or NP were protective antigens while VP35 afforded incomplete protection. As a more definitive test of vaccine efficacy, nonhuman primates (cynomolgus macaques) were inoculated with VEE replicons expressing MBGV GP and/or NP. Three monkeys received packaged control replicons (influenza HA); these died 9 or 10 days after challenge, with typical MBGV disease. MBGV NP afforded incomplete protection, sufficient to prevent death but not disease in two of three macaques. Three monkeys vaccinated with replicons which expressed MBGV GP, and three others vaccinated with both replicons that expressed GP or NP, remained aviremic and were completely protected from disease.

Ebola virus (EBOV) causes acute hemorrhagic fever that is fatal in up to 90% of cases in both humans and nonhuman primates. No vaccines or treatments are available for human use. We evaluated the effects in nonhuman primates of vaccine strategies that had protected mice or guinea pigs from lethal EBOV infection. The following immunogens were used: RNA replicon particles derived from an attenuated strain of Venezuelan equine encephalitis virus (VEEV) expressing EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus expressing EBOV glycoprotein; liposomes containing lipid A and inactivated EBOV; and a concentrated, inactivated whole-virion preparation. None of these strategies successfully protected nonhuman primates from robust challenge with EBOV. The disease observed in primates differed from that in rodents, suggesting that rodent models of EBOV may not predict the efficacy of candidate vaccines in primates and that protection of primates may require different mechanisms.

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins GN and GC. To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both GN and GC. Using these MAbs, we found that GN predominantly colocalized with a Golgi marker when expressed alone or with GC, while GC was transported to the Golgi apparatus only in the presence of GN. Both proteins remained endo-β-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the GN ectodomain, because a soluble version of GN lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of GN and GC also resulted in localization of soluble GC to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the GN precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of GN when it was expressed alone but were dispensable when GC was coexpressed. In neutralization assays on SW-13 cells, MAbs to GC, but not to GN, prevented CCHFV infection. However, only a subset of GC MAbs protected mice in passive-immunization experiments, while some nonneutralizing GN MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to GC