Bombyx mori nucleopolyhedrovirus F-like protein Bm14 affects the morphogenesis and production of occlusion bodies and the embedding of ODVs.

In group I nucleopolyhedrovirus such as Bombyx mori nucleopolyhedrovirus (BmNPV), the biological functions of F-like protein (Bm14) still remain elusive. Here, we found that the deletion of Bm14 reduced the production rate of infectious budded viruses in cell culture, delayed the lethal time of infected larvae by approximately 26 h, and produced less occlusion bodies (OBs). Scanning electron microscopy demonstrated that its disruption affected OB morphogenesis, forming irregular OBs with a pitted surface and irregular profiles. Moreover, almost 45% less DNA was present in OBs produced by Bm14-null virus. This reduction in DNA content was consistent with fewer virions embedded into OBs. The titers of occlusion-derived viruses was 7.5 times less in mutant OBs. Western blot analysis revealed that Bm14 is present in the envelope of both BV and ODV. Taken together, Bm14 is a viral factor that affects OB morphogenesis and production, and the number of ODVs occluded into OBs.

Recovery of Replication-Competent and G-Gene-Deleted Rabies Viruses from cDNA.

This protocol describes the recovery of replication-competent rabies viruses (RV) such as SAD L16 or SAD L16-eGFP. It is suggested that at least three parallel transfection experiments are performed to increase the success rate (in three 3.5-cm(2) dishes or in three wells in a six-well plate). The entire protocol takes 10 d, and successful rescue can be obtained after 6 d. An additional procedure for the recovery of G-gene-deleted viruses is also included. Please note that appropriate biosafety measures are needed.

Pseudotyping of G-Gene-Deficient Rabies Virus.

G-deleted fluorescent rabies virus (RV) pseudotyped with RV G proteins, SAD ΔG eGFP (RV CVS-G), can be used as single-round vectors for efficient retrograde labeling of neurons. For these experiments, as well as for monosynaptic tracing, which involves pseudotyping in situ, the use of the CVS strain G is recommended because of its high tropism for neurons. Pseudotype virus stocks generated by transfection of pCAGGS-G (or in MG139-on cells) contain the G protein of the vaccine strain SAD L16, which is broader in its tropism, and infects astrocytes, glia, and oligodendrocytes. We also describe a procedure for pseudotyping with ASLV Env A, which uses a cell-line expressing a version of the EnvA protein that is incorporated efficiently into the RV envelope (EnvARG(RGct)).

Elite Control, Gut CD4 T Cell Sparing, and Enhanced Mucosal T Cell Responses in Macaca nemestrina Infected by a Simian Immunodeficiency Virus Lacking a gp41 Trafficking Motif.

UNLABELLED: Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4(+) T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4(+) T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4(+) T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4(+) T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8(+) cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE: The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

Widely Used Herpes Simplex Virus 1 ICP0 Deletion Mutant Strain dl1403 and Its Derivative Viruses Do Not Express Glycoprotein C Due to a Secondary Mutation in the gC Gene.

Herpes simplex virus 1 (HSV-1) ICP0 is a multi-functional phosphoprotein expressed with immediate early kinetics. An ICP0 deletion mutant, HSV-1 dl1403, has been widely used to study the roles of ICP0 in the HSV-1 replication cycle including gene expression, latency, entry and assembly. We show that HSV-1 dl1403 virions lack detectable levels of envelope protein gC, and that gC is not synthesized in infected cells. Sequencing of the gC gene from HSV-1 dl1403 revealed a single amino acid deletion that results in a frameshift mutation. The HSV-1 dl1403 gC gene is predicted to encode a polypeptide consisting of the original 62 N-terminal amino acids of the gC protein followed by 112 irrelevant, non-gC residues. The mutation was also present in a rescuant virus and in two dl1403-derived viruses, D8 and FXE, but absent from the parental 17+, suggesting that the mutation was introduced during the construction of the dl1403 virus, and not as a result of passage in culture.

Rabies virus vector transgene expression level and cytotoxicity improvement induced by deletion of glycoprotein gene.

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.

Infection-enhancing lipopeptides do not improve intranasal immunization of cotton rats with a delta-G candidate live-attenuated human respiratory syncytial virus vaccine.

Development of live-attenuated human respiratory syncytial virus (HRSV) vaccines has proven to be difficult. Several vaccine candidates were found to be over-attenuated and displayed limited immunogenicity. Recently, we identified three synthetic cationic lipopeptides that enhanced paramyxovirus infections in vitro. The infection enhancement proved to be mediated by enhanced virus binding to target cells. We hypothesized that these lipopeptides can be used as adjuvants to promote immune responses induced by live-attenuated paramyxovirus vaccines. This hypothesis was tested in a vaccination and challenge model in cotton rats, using a previously described recombinant live-attenuated candidate HRSV vaccine lacking the gene encoding the G glycoprotein (rHRSVΔG). Surprisingly, intranasal vaccination of cotton rats with rHRSVΔG formulated in infection-enhancing lipopeptides resulted in reduced virus loads in nasopharyngeal lavages, reduced seroconversion levels and reduced protection from wild-type HRSV challenge. In conclusion, we were unable to demonstrate the feasibility of lipopeptides as adjuvants for a candidate live-attenuated HRSV vaccine in the cotton rat model.

Complementation of the function of glycoprotein H of human herpesvirus 6 variant A by glycoprotein H of variant B in the virus life cycle.

Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle.

Elevated tumor-associated antigen expression suppresses variant peptide vaccine responses.

Variant peptide vaccines are used clinically to expand T cells that cross-react with tumor-associated Ags (TAA). To investigate the effects of elevated endogenous TAA expression on variant peptide-induced responses, we used the GP70 TAA model. Although young BALB/c mice display T cell tolerance to the TAA GP70(423-431) (AH1), expression of GP70 and suppression of AH1-specific responses increases with age. We hypothesized that as TAA expression increases, the AH1 cross-reactivity of variant peptide-elicited T cell responses diminishes. Controlling for immunosenescence, we showed that elevated GP70 expression suppressed AH1 cross-reactive responses elicited by two AH1 peptide variants. A variant that elicited almost exclusively AH1 cross-reactive T cells in young mice elicited few or no T cells in aging mice with Ab-detectable GP70 expression. In contrast, a variant that elicited a less AH1 cross-reactive T cell response in young mice successfully expanded AH1 cross-reactive T cells in all aging mice tested. However, these T cells bound the AH1/MHC complex with a relatively short half-life and responded poorly to ex vivo stimulation with the AH1 peptide. Variant peptide vaccine responses were also suppressed when AH1 peptide is administered tolerogenically to young mice before vaccination. Analyses of variant-specific precursor T cells from naive mice with Ab-detectable GP70 expression determined that these T cells expressed PD-1 and had downregulated IL-7Rα expression, suggesting they were anergic or undergoing deletion. Although variant peptide vaccines were less effective as TAA expression increases, data presented in this article also suggest that complementary immunotherapies may induce the expansion of T cells with functional TAA recognition.

Horizontal transmission dynamics of a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus and the effect of vaccination on transmission of virulent virus.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R0, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R0 was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs.

The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication.

Refined methods for propagating vesicular stomatitis virus vectors that are defective for G protein expression.

Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-DeltaG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing non-propagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-DeltaG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1 x 10(7) to 1 x 10(8) infectious units (I.U.s) of vaccine vector per milliliter. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-DeltaG.

Herpes simplex virus type 2 glycoprotein G is targeted by the sulfated oligo- and polysaccharide inhibitors of virus attachment to cells.

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans.

Herpesvirus chemokine-binding glycoprotein G (gG) efficiently inhibits neutrophil chemotaxis in vitro and in vivo.

Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernatants of EHV-1-infected cells significantly inhibited IL-8-induced chemotaxis of equine neutrophils. Identification of gG as the responsible vCKBP was achieved by repeating similar experiments with supernatants from cells infected with a gG-negative mutant, which were unable to alter IL-8-induced equine neutrophil migration. Furthermore, rEHV-1 gG was able to significantly reduce neutrophil migration, establishing gG as a bona fide vCKBP. Second, and importantly, in vivo analyses in a murine model of EHV-1 infection showed that neutrophil migration in the target organ lung was significantly reduced in the presence of gG. In summary, we demonstrate for the first time that EHV-1 gG not only binds to chemokines but is also capable of inhibiting their chemotactic function both in vitro and in vivo, thereby contributing to viral pathogenesis and virulence.

Glycoprotein G deficient infectious laryngotracheitis virus is a candidate attenuated vaccine.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is currently controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations because of residual pathogenicity and reversion to virulence, suggesting that a novel vaccine strain that lacks virulence gene(s) may enhance disease control. Glycoprotein G (gG) has recently been identified as a virulence factor in ILTV. In this study the immunogenicity and relative pathogenicity of gG deficient ILTV was investigated in SPF chickens. Birds vaccinated with gG deficient ILTV were protected against clinical signs of disease following challenge with virulent ILTV and gG deficient ILTV was also shown to be less pathogenic than currently available commercial vaccine strains. Thus gG deficient ILTV appears to have potential as a vaccine candidate.

B5-deficient vaccinia virus as a vaccine vector for the expression of a foreign antigen in vaccinia immune animals.

Recombinant vaccinia viruses have shown promise as vaccine vectors. However, their effectiveness is markedly reduced by pre-existing anti-vaccinia immunity. The possibility of new vaccinia immunizations in the event of a bioterror-related smallpox release poses an additional negative impact on the utility of vaccinia-based vectors. Thus, we aimed to design a vaccinia vector that would enhance the immune response to an expressed foreign protein in a pre-immune animal model. To do this, we made use of the finding that most neutralizing antibodies against the extracellular form of vaccinia virus are directed against the B5 protein. We found that mice immunized with vaccinia, primed with Gag plasmid DNA, and boosted with a recombinant vaccinia virus lacking the majority of the B5 ectodomain expressing a test antigen, HIV Gag, had stronger anti-Gag immune responses than mice that were boosted with a wild-type virus-expressing Gag. These findings are particularly striking given the more attenuated phenotype of this virus, as compared to its wild-type counterpart. Importantly, we found that vaccination with a B5R deletion virus, followed by boosting with the Gag-expressing virus lacking the majority of the B5 ectodomain, resulted in poorer anti-Gag immune responses. Thus, recombinant vaccinia viruses lacking the B5 ectodomain may serve as vaccine vectors in DNA prime-vaccinia boost vaccinations of individuals with pre-existing immunity against vaccinia. These data open the possibility of extending the potential benefit of replication competent recombinant vaccinia virus vectors to a larger population.

Alpha-herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites.

Alpha-herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine alpha-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of alpha-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus.

In the absence of glycoprotein I (gI), gE determines bovine herpesvirus type 5 neuroinvasiveness and neurovirulence.

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus that causes fatal encephalitis in calves. Envelope glycoproteins E (gE) and gI of alphaherpesviruses are important for the pathogenesis in vivo. Previously the authors determined that BHV-5 gE is important for BHV-5 neurovirulence. To determine the role of gI in BHV-5 neurovirulence, the authors have constructed gI-deleted and gI-revertant BHV-5 and analyzed their neuropathogenic properties in a rabbit seizure model. Following intranasal infection, 40% of the rabbits infected with the gI-deleted virus showed severe neurological signs. gI-deleted BHV-5 invaded all the central nervous system (CNS) structures invaded by the gI-revertant BHV-5; however, the number of neurons infected by the gI-deleted virus was similar or slightly reduced (two to four fold). Thus, the gI-deleted virus retained significant neurovirulence and/or neuroinvasive properties when compared with the gE-deleted BHV-5. Pulse-chase analysis revealed that the gE of gI-deleted virus was processed to a larger and a diffused 94- to 100-kDa protein (instead of 94 kDa). The 94- to 100-kDa protein was processed in the Golgi with delayed kinetics but it was endoglycosidase H (EndoH) resistant. In cells infected with gI-deleted virus, there was a reduction in cell-surface gE expression compared to wild-type, which correlated to reduced amount of gE processed in the Golgi. The authors believe that in the absence of gI, BHV-5 gE is sufficient for BHV-5 neurovirulence.

Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus.

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of non-aggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

Adjuvanticity of an IL-12 fusion protein expressed by recombinant deltaG-vesicular stomatitis virus.

The remarkable immunomodulatory and adjuvant properties of rIL-12 have been well described. Many early studies documenting the adjuvanticity of IL-12 were performed using the murine model of Listeria monocytogenes infection. In this report, we describe the construction of an attenuated recombinant vesicular stomatitis virus (VSV-deltaG) that encodes a single-chain IL-12 fusion protein (IL-12F), and the use of this virus as an expression vector to produce large quantities of IL-12F. VSV-expressed IL-12F (vIL-12F) was then co-administered to mice along with a poorly immunogenic listerial antigen preparation as a vaccine regimen and the resulting immune responses were monitored. The vIL-12F was found to have adjuvant properties similar to those observed for rIL-12. Co-administration of vIL-12F and listerial antigen elicited powerful cell-mediated immune responses that conferred long-lived protective listerial immunity. These studies demonstrated that VSVdeltaG-IL12F-infected cells secrete bioactive single-chain IL-12, and laid the foundation for studies using VSVdeltaG-IL12F as a vector for delivery of IL-12F in vivo.