Haemagglutinin and nucleoprotein replicon particle vaccination of swine protects against the pandemic H1N1 2009 virus.

The recent emergence of the pandemic H1N1 (pH1N1) and H3N2 variant influenza A viruses (IAV) in 2009 and 2011-2012, respectively, highlight the zoonotic potential of influenza viruses and the need for vaccines capable of eliciting heterosubtypic protection. In these studies, single-cycle, propagation-defective replicon particle (RP) vaccines expressing IAV haemagglutinin (HA) and nucleoprotein (NP) genes were constructed and efficacy was evaluated in homologous and heterologous pig challenge studies with the pH1N1 2009 influenza virus (A/California/04/2009). Homologous HA RP vaccination eliminated virus shedding and decreased pulmonary pathology in pigs following pH1N1 2009 challenge. An RP vaccine expressing an H3N2-derived NP gene was able to decrease nasal shedding and viral load following heterosubtypic pH1N1 2009 challenge in pigs. These studies indicate that although homologous vaccination of swine remains the most effective means of preventing IAV infection, other vaccine alternatives do offer a level of heterosubtypic protection, and should continue to be evaluated for their ability to provide broader protection.

Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle.

Alphavirus-based replicon systems are frequently used as preclinical vectors and as antigen discovery tools, and they have recently been assessed in clinical vaccine trials. Typically, alphavirus replicon RNAs are delivered within virus-like replicon particles (VRP) that are produced following transfection of replicon RNA and two helper RNAs into permissive cells in vitro. The non-structural proteins expressed from the replicon RNA amplify the replicon RNA in cis and the helper RNAs in trans, the latter providing the viral structural proteins necessary to package the replicon RNA into VRP. Current helper RNA designs incorporate the alphavirus 26S promoter to direct the transcription of high levels of structural gene mRNAs. We demonstrate here that the 26S promoter is not required on helper RNAs to produce VRP and propose that such promoterless helper RNAs, by design, reduce the probability of generating replication-competent virus that may otherwise result from RNA recombination.

Alphavirus replicon particle vaccines developed for use in humans induce high levels of antibodies to influenza virus hemagglutinin in swine: proof of concept.

A propagation-defective, single-cycle, alphavirus replicon particle (RP) system was used to produce two vaccines against human influenza virus A/Wyoming/03/2003 (H3N2). One vaccine was prepared from Venezeulan equine encephalitis virus (VEEV) strain 3014 and the other from VEEV strain TC-83. Both vaccines induced high antibody titers to the influenza hemagglutinin (HA) protein and illustrated the potential of using alphavirus RP influenza vaccines in swine.

Alphavirus replicon approach to promoterless analysis of IRES elements.

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced >95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters.

Seoul virus (SEOV) is a member of the Hantavirus genus (family Bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. The medium (M) and small (S) gene segments of SEOV encode the viral envelope glycoproteins and nucleocapsid protein, respectively. We compared the immunogenicity and protective efficacy of naked DNA (pWRG7077), DNA-based Sindbis replicon (pSIN2.5), and packaged Sindbis replicon vectors (pSINrep5), containing either the M or S gene segment of SEOV in Syrian hamsters. All of the vectors elicited an anti-SEOV immune response to the expressed SEOV gene products. Vaccinated hamsters were challenged with SEOV and monitored for evidence of infection. Protection from infection was strongly associated with M-gene vaccination. A small number of S-gene-vaccinated animals also were protected. Hamsters vaccinated with the pWRG7077 vector expressing the M gene demonstrated the most consistent protection from SEOV infection and also were protected from heterologous hantavirus (Hantaan virus) infection.

Murine leukemia virus pseudotypes of La Crosse and Hantaan Bunyaviruses: a system for analysis of cell tropism.

We have developed a system for the preparation of La Crosse virus (LAC) and Hantaan virus (HTN) pseudotypes using a murine leukemia virus vector. After concentration, the pseudotypes were present in quantities sufficient to analyze cell tropism and neutralization. Cells resistant to LAC could not be infected with the MLV (LAC) pseudotypes, and the pseudotypes were sensitive to neutralizing monoclonal antibodies prepared against LAC glycoproteins, as well as to inhibition by a soluble form of the virus cell-attachment protein, G1. Perhaps because of lower expression of the HTN glycoproteins at the cell surface, MLV (HTN) pseudotypes were present at lower titers. However, they were also sensitive to appropriate neutralizing antibodies. This pseudotype system will be useful for analysis of the entry process of the Bunyaviridae, and for neutralization studies with some Bunyaviruses whose high virulence normally requires specialized containment facilities.

DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection.

Seoul virus (SEOV) is one of four known hantaviruses causing hemorrhagic fever with renal syndrome (HFRS). Candidate naked DNA vaccines for HFRS were constructed by subcloning cDNA representing the medium (M; encoding the G1 and G2 glycoproteins) or small (S; encoding the nucleocapsid protein) genome segment of SEOV into the DNA expression vector pWRG7077. We vaccinated BALB/c mice with three doses of the M or S DNA vaccine at 4-week intervals by either gene gun inoculation of the epidermis or needle inoculation into the gastrocnemius muscle. Both routes of vaccination resulted in antibody responses as measured by ELISA; however, gene gun inoculation elicited a higher frequency of seroconversion and higher levels of antibodies in individual mice. We vaccinated Syrian hamsters with the M or S construct using the gene gun and found hantavirus-specific antibodies in five of five and four of five hamsters, respectively. Animals vaccinated with the M construct developed a neutralizing antibody response that was greatly enhanced in the presence of guinea pig complement. Immunized hamsters were challenged with SEOV and, after 28 days, were monitored for evidence of infection. Hamsters vaccinated with M were protected from infection, but hamsters vaccinated with S were not protected.

Use of the Sindbis replicon system for expression of LaCrosse virus envelope proteins in mosquito cells.

The Sindbis replicon expression system was used to express La Crosse (LAC) virus envelope glycoprotein genes in both mammalian and mosquito cell culture. Replicon expressed LAC proteins had correct molecular mass (Mr) and were antigenically similar to wild type LAC envelope proteins. In addition, LAC G1 and G2 proteins colocalized when expressed from separate constructs in both mammalian and mosquito cells suggesting that they were trafficked through the cell similarly to wild type LAC proteins. A truncated form of the G1 protein was secreted from mosquito cells when expressed alone. The truncated G1 protein was also secreted from mosquito cells when expressed with the G2 protein, but to a lesser extent than when expressed alone, suggesting that the G2 protein sequestered G1 protein intracellularly. The Sindbis replicon system is a powerful tool for the study of LAC virus protein maturation within mosquito cells and mosquitoes.

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Genetically engineered resistance to dengue-2 virus transmission in mosquitoes.

The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus.

Molecularly engineered resistance to California serogroup virus replication in mosquito cells and mosquitoes.

Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.

Replication and expression of a recombinant Sindbis virus in mosquitoes.

A recombinant Sindbis virus, TE/3'2J/ANTI-S, containing LaCrosse virus small segment cDNA in antisense orientation, was inoculated into Aedes triseriatus mosquitoes. Virus replication and LAC-ANTI-S RNA expression were analysed temporally and spatially. TE/3'2J/ANTI-S virus titre peaked at 5.0 log10 TCID50 in heads 6-9 days post infection (p.i.) and decreased to 3.4 log10 TCID50 by 37 days p.i. Salivary glands contained 4.4 log10 TCID50 of virus 6 days p.i.; titres were lower in other organs. LAC-ANTI-S RNA levels paralleled virus titre. SIN E1 antigen was detected in many mosquito organ systems, but in specific cells and tissues of some organs. TE/3'2J/ANTI-S virus exhibited different cellular tropisms in salivary glands of Aedes and Culex mosquitoes.

The expression of chloramphenicol acetyltransferase in mosquitoes and mosquito cells using a packaged Sindbis replicon system.

Sindbis (SIN) replicon virus was used to express chloramphenicol acetyltransferase (CAT) in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes. RNA transcribed in vitro from a SIN replicon plasmid (pSINrep5/CAT) and from SIN virus helper constructs (pDH-EB or pDH(26S)5'SIN) was coelectroporated into BHK-21 cells to generate replicon viruses, designated rep5/CAT/EB and rep5/CAT/26S. C6/36 cells infected with rep5/CAT/EB or rep5/CAT/26S virus at a multiplicity of infection of 3, expressed 3.8 x 10(6) and 6.0 x 10(6) CAT trimers per cell, respectively, at 2 days postinfection (pi). Both viruses attained peak titers by Day 2 pi. Adult female A. triseriatus mosquitoes were intrathoracically inoculated with 7 x 10(4) IFU rep5/CAT/EB or 1 x 10(5) IFU rep5/CAT/26S virus. Virus titers remained at approximately 10(5) IFU/ml through Day 2 pi and decreased roughly 1 log by Day 10 pi. CAT enzyme activity was detected 2 days pi (rep5/CAT/EB, 1.49 x 10(-4) units CAT/10 micrograms protein; rep5/CAT/26S, 2.03 x 10(-5) units CAT/10 micrograms protein) and remained near these levels through Day 10 pi. CAT was detected in the head, salivary glands, midgut, and ovaries of inoculated mosquitoes by indirect immunofluorescence or CAT activity assays. These results suggest that packaged replicon viruses can be useful for expression of heterologous genes in mosquito cells and whole mosquitoes.

Expression strategy of the M genome segment of Hantaan virus.

The medium (M) genome segment of Hantaan virus encodes the envelope glycoproteins, G1 and G2, in a continuous open reading frame with a gene order of 5'-G1-G2-3' with respect to the virus-complementary sense RNA. Because potential translation initiation codons and amino acids constituting typical signal sequences precede both the G1 and G2 genes, we sought to determine if G1 and G2 can be expressed independently. To investigate translational requirements for G1 and G2, we constructed M segment genes in which portions of the coding information were mutated or deleted, and transiently expressed these genes in eukaryotic cells by using a vaccinia virus/T7 RNA polymerase system. We found that G2 expression can occur by ribosomal access to the translation initiation codon preceding the G2 signal sequence (nucleotides 1934-1936), but that other upstream AUG codons cannot be used as efficiently. The presence of this codon, however, was not required for G2 expression because changing nucleotides 1934-1936 to CUG, GCG or AUG did not abrogate expression of G2. We also found that leaky ribosomal scanning, rather than internal initiation of translation was the most likely explanation for the observed independent translational initiation of G2, but that not all upstream, in-frame AUGs could serve as initiator codons. To assess the requirement for a continuous open reading frame for G1 and G2 expression, we expressed a gene which had G1 and G2 coding information in different reading frames. Although G1 was expressed at apparently normal levels, little or no G2 was expressed. In contrast, only G2 was expressed from a gene in which the carboxy-terminal G1 coding information was deleted and the remaining, truncated G1 was placed out of frame with respect to G2. These data suggest that reinitiation of translation may occur under some, but not all, circumstances when the polyprotein coding information is perturbed. Our results are consistent with biogenesis of G1 and G2 primarily or entirely according to the ribosomal scanning model.