An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector

Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55(Gag) antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55(gag) replicon RNA packaged within SIN envelope glycoproteins and SIN-p55(gag) replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8(+) T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.     

A heterologous DNA prime-Venezuelan equine encephalitis virus replicon particle boost dengue vaccine regimen affords complete protection from virus challenge in cynomolgus macaques

A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA (DDD), three doses of VRP (VVV group), or a heterologous DNA prime-VRP boost regimen (DDV) using two doses of DNA vaccine and a third dose of VRP vaccine. Four weeks after the final immunization, the DDV group produced the highest dengue virus type 1-specific immunoglobulin G antibody responses and virus-neutralizing antibody titers. Moderate T-cell responses were demonstrated only in DDD- and DDV-vaccinated animals. When vaccinated animals were challenged with live virus, all vaccination regimens showed significant protection from viremia. DDV-immunized animals were completely protected from viremia (mean time of viremia = 0 days), whereas DDD- and VVV-vaccinated animals had mean times of viremia of 0.66 and 0.75 day, respectively, compared to 6.33 days for the control group of animals.     

Soluble rhesus lymphocryptovirus gp350 protects against infection and reduces viral loads in animals that become infected with virus after challenge

Epstein-Barr virus (EBV) is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350) or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]). No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV) encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a) soluble rhesus LCV gp350, (b) virus-like replicon particles (VRPs) expressing rhesus LCV gp350, (c) VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d) PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with other EBV proteins, should be considered to reduce EBV infection or virus-associated malignancies in humans.     

Transfection-independent production of alphavirus replicon particles based on poxvirus expression vectors

This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.     

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.     

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

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.     

Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.     

Characterization of human immunodeficiency virus Gag-specific gamma interferon-expressing cells following protective mucosal immunization with alphavirus replicon particles

A safe, replication-defective viral vector that can induce mucosal and systemic immune responses and confer protection against many infectious pathogens, such as human immunodeficiency virus type 1 (HIV-1), may be an ideal vaccine platform. Accordingly, we have generated and tested alphavirus replicon particles encoding HIV-1 Gag from Sindbis virus (SIN-Gag) and Venezuelan equine encephalitis virus (VEE-Gag), as well as chimeras between the two (VEE/SIN-Gag). Following intramuscular (i.m.), intranasal (i.n.), or intravaginal (IVAG) immunization with VEE/SIN-Gag and an IVAG challenge with vaccinia virus encoding HIV Gag (VV-Gag), a larger number of Gag-specific CD8+ intracellular gamma interferon-expressing cells (iIFNEC) were detected in iliac lymph nodes (ILN), which drain the vaginal/uterine mucosa (VUM), than were observed after immunizations with SIN-Gag. Moreover, a single i.n. or IVAG immunization with VEE/SIN-Gag induced a larger number of cells expressing HIV Gag in ILN, and immunizations with VEE/SIN-Gag through any route induced better protective responses than immunizations with SIN-Gag. In VUM, a larger percentage of iIFNEC expressed alpha4beta7 or alpha(Ebeta)7 integrin than expressed CD62L integrin. However, in spleens (SP), a larger percentage of iIFNEC expressed alpha4beta7 or CD62L than expressed alpha(Ebeta)7. Moreover, a larger percentage of iIFNEC expressed the chemokine receptor CCR5 in VUM and ILN than in SP. These results demonstrate a better induction of cellular and protective responses following immunizations with VEE/SIN-Gag than that following immunizations with SIN-Gag and also indicate a differential expression of homing and chemokine receptors on iIFNEC in mucosal effector and inductive sites versus systemic lymphoid tissues.     

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.     

Induction of primary virus-cross-reactive human immunodeficiency virus type 1-neutralizing antibodies in small animals by using an alphavirus-derived in vivo expression system

We have studied the induction of neutralizing antibodies by in vivo expression of the human immunodeficiency virus type 1 (HIV-1) envelope by using a Venezuelan equine encephalitis virus (VEE) replicon system with mice and rabbits. The HIV-1 envelope, clone R2, has broad sensitivity to cross-reactive neutralization and was obtained from a donor with broadly cross-reactive, primary virus-neutralizing antibodies (donor of reference serum, HIV-1-neutralizing serum 2 [HNS2]). It was expressed as gp160, as secreted gp140, and as gp160deltaCT with the cytoplasmic tail deleted. gp140 was expressed in vitro at a high level and was predominantly uncleaved oligomer. gp160deltaCT was released by cells in the form of membrane-bound vesicles. gp160deltaCT induced stronger neutralizing responses than the other forms. Use of a helper plasmid for replicon particle packaging, in which the VEE envelope gene comprised a wild-type rather than a host range-adapted sequence, also enhanced immunogenicity. Neutralizing activity fractionated with immunoglobulin G. This activity was cross-reactive among a panel of five nonhomologous primary clade B strains and a Chinese clade C strain and minimally reactive against a Chinese clade E (circulating recombinant form 1) strain. The comparative neutralization of these strains by immune mouse sera was similar to the relative neutralizing effects of HNS2, and responses induced in rabbits were similar to those induced in mice. Together, these results demonstrate that neutralizing antibody responses can be induced in mice within 2 to 3 months that are similar in potency and cross-reactivity to those found in the chronically infected, long-term nonprogressive donor of HNS2. These findings support the expectation that induction of highly cross-reactive HIV-1 primary virus-neutralizing activity by vaccination may be realized.     

Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels

Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIV(KU2) (simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.     

Venezuelan equine encephalitis virus replicon particles encoding respiratory syncytial virus surface glycoproteins induce protective mucosal responses in mice and cotton rats

Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K^d-restricted CD8⁺ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.     

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats

Background

The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo.

Methodology/Principal Findings

The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers.

Conclusions/Significance

We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.     

Determination of the protective epitopes on the glycoproteins of Venezuelan equine encephalomyelitis virus by passive transfer of monoclonal antibodies

We have previously characterized seven unique antigenic epitopes on the two envelope glycoproteins of the Venezuelan equine encephalomyelitis (VEE) virus vaccine strain, TC-83, by using monoclonal antibodies. The in vitro function of virus neutralization was primarily associated with one epitope on the gp56 (gp56c). To determine which epitopes were important in protecting animals from VEE infection, purified monoclonal antibodies were inoculated i.v. into 3-wk-old Swiss mice. Twenty-four hours later these animals were challenged i.p. with 100 IPLD50 of virulent VEE virus (Trinidad donkey). High-avidity anti-gp56c, anti-gp50b, anti-gp50c, and anti-gp50d monoclonal antibodies protected animals from virus challenge. Rabbit antisera to the gp56 and the gp50 glycoproteins were also effective in protecting mice from challenge with virulent VEE virus. Less antibody was needed to protect animals if the antibody was directed against the critical neutralization site. Less avid antibodies to the gp56c and gp50b epitopes demonstrated little or no protection in vivo. Protection, therefore, appeared to be a function of the passive antibody's specificity, avidity, and ability to bind to virion antigenic determinants topologically proximal to the critical neutralization site.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector

A replication-competent rhabdovirus-based vector expressing human immunodeficiency virus type 1 (HIV-1) Gag protein was characterized on human cell lines and analyzed for the induction of a cellular immune response in mice. We previously described a rabies virus (RV) vaccine strain-based vector expressing HIV-1 gp160. The recombinant RV was able to induce strong humoral and cellular immune responses against the HIV-1 envelope protein in mice (M. J. Schnell et al., Proc. Natl. Acad. Sci. USA 97:3544-3549, 2000; J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001). Recent research suggests that the HIV-1 Gag protein is another important target for cell-mediated host immune defense. Here we show that HIV-1 Gag can efficiently be expressed by RV on both human and nonhuman cell lines. Infection of HeLa cells with recombinant RV expressing HIV-1 Gag resulted in efficient expression of HIV-1 precursor protein p55 as indicated by both immunostaining and Western blotting. Moreover, HIV-1 p24 antigen capture enzyme-linked immunosorbent assay and electron microscopy showed efficient release of HIV-1 virus-like particles in addition to bullet-shaped RV particles in the supernatants of the infected cells. To initially screen the immunogenicity of this new vaccine vector, BALB/c mice received a single vaccination with the recombinant RV expressing HIV-1 Gag. Immunized mice developed a vigorous CD8(+) cytotoxic T-lymphocyte response against HIV-1 Gag. In addition, 26.8% of CD8(+) T cells from mice immunized with RV expressing HIV-1 Gag produced gamma interferon after challenge with a recombinant vaccinia virus expressing HIV-1 Gag. These results further confirm and extend the potency of RV-based vectors as a potential HIV-1 vaccine.     

A novel self-replicating chimeric lentivirus-like particle

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.     

A novel viral system for generating antigen-specific T cells

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with approximately 50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-alpha treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-alpha, and IFN-alpha. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-alpha-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.