Virus Replicon Particles Research Articles

Evaluation of neurovirulence and biodistribution of Venezuelan equine encephalitis replicon particles expressing herpes simplex virus type 2 glycoprotein D

The safety of a propagation-defective Venezuelan equine encephalitis virus (VEEV) replicon particle vaccine was examined in mice. After intracranial inoculation we observed approximately 5% body weight loss, modest inflammatory changes in the brain, genome replication, and foreign gene expression. These changes were transient and significantly less severe than those caused by TC-83, a live-attenuated vaccinal strain of VEEV that has been safely used to immunize military personnel and laboratory workers. Replicon particles injected intramuscularly or intravenously were detected at limited sites 3 days post-administration, and were undetectable by day 22. There was no evidence of dissemination to spinal cord or brain after systemic administration. These results demonstrate that propagation-defective VEEV replicon particles are minimally neurovirulent and lack neuroinvasive potential.

Vaccine Efficacy in Senescent Mice Challenged with Recombinant SARS-CoV Bearing Epidemic and Zoonotic Spike Variants

BackgroundIn 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge.Methods and FindingsUsing Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N), we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S) encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV–challenged mice. VRP-N–induced pathology presented at day 4, peaked around day 7, and persisted through day 14, and was likely mediated by cellular immune responses.ConclusionsThis study identifies gaps and challenges in vaccine design for controlling future SARS-CoV zoonosis, especially in vulnerable elderly populations. The availability of a SARS-CoV virus bearing heterologous S glycoproteins provides a robust challenge inoculum for evaluating vaccine efficacy against zoonotic strains, the most likely source of future outbreaks.

Distinct Contributions of Vaccine-Induced Immunoglobulin G1 (IgG1) and IgG2a Antibodies to Protective Immunity against Influenza

Vaccination represents the most effective form of protection against influenza infection. While neutralizing antibodies are typically measured as a correlate of vaccine-induced protective immunity against influenza, nonneutralizing antibodies may contribute to protection or amelioration of disease. The goal of this study was to dissect the individual contributions of the immunoglobulin G1 (IgG1) and IgG2a antibody isotypes to vaccine-induced immunity against influenza virus. To accomplish this, we utilized an influenza vaccine regimen that selectively enhanced IgG1 or IgG2a antibodies by using either DNA or viral replicon particle (VRP) vectors expressing influenza virus hemagglutinin (HA) (HA-DNA or HA-VRP, respectively). After HA-DNA vaccination, neutralizing antibodies were detected by both in vitro (microneutralization) and in vivo (lung viral titer) methods and were associated with increased IgG1 expression by enzyme-linked immunosorbent assay (ELISA). Vaccination with HA-VRP did not strongly stimulate either neutralizing or IgG1 antibodies but did induce IgG2a antibodies. Expression of IgG2a antibodies in this context correlated with clearance of virus and increased protection against lethal influenza challenge. Increased induction of both antibody isotypes as measured by ELISA was a better correlate for vaccine efficacy than neutralization alone. This study details separate but important roles for both IgG1 and IgG2a expression in vaccination against influenza and argues for the development of vaccine regimens that stimulate and measure expression of both antibody isotypes.

Classical swine fever virus replicon particles lacking the Ernsgene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E(rns) were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E(rns) (A187delE(rns)). For packaging of A187delE(rns) into virus particles, porcine kidney cell lines constitutively expressing E(rns) of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10(7) TCID(50) of VRP A187delE(rns) elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-gamma producing cells. No anti-E(rns) antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus.

Mucosal and systemic adjuvant activity of alphavirus replicon particles

Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines.

Vaccination of Mice with Gonococcal TbpB Expressed In Vivo from Venezuelan Equine Encephalitis Viral Replicon Particles

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.

Comparison of Immune Responses to Gonococcal PorB Delivered as Outer Membrane Vesicles, Recombinant Protein, or Venezuelan Equine Encephalitis Virus Replicon Particles

Porin (PorB) is a major outer membrane protein produced by all Neisseria gonorrhoeae strains and has been a focus of intense interest as a vaccine candidate. In this study, the immunogenicity of PorB in mice was investigated after several immunization regimens. Outer membrane vesicles (OMV), recombinant renatured PorB (rrPorB), and PorB-expressing Venezuelan equine encephalitis (VEE) virus replicon particles (PorB VRP) were delivered intranasally (i.n.) or subcutaneously (s.c.) into the dorsal area or the hind footpad in three-dose schedules; the PorB VRP-immunized mice were given a single additional booster dose of rrPorB in Ribi adjuvant. Different delivery systems and administration routes induced different immune responses. Mice immunized s.c. with rrPorB in Ribi had the highest levels of PorB-specific serum immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. Surprisingly, there was an apparent Th1 bias, based on IgG1/IgG2a ratios, after immunization with rrPorB in Ribi in the footpad while the same vaccine given in the dorsal area gave a strongly Th2-biased response. PorB VRP-immunized mice produced a consistent Th1 response with a high gamma interferon response in stimulated splenic lymphocytes and very low IgG1/IgG2a ratios. Immunization by OMV delivered i.n. was the only regimen that resulted in a serum bactericidal response, and it generated an excellent mucosal IgA response. Serum from mice immunized with rrPorB preferentially recognized the surface of whole gonococci expressing a homologous PorB, whereas serum from PorB VRP-immunized mice had relatively low whole-cell binding activity but recognized both heterologous and homologous PorB equally. The data resulting from this direct comparison suggested that important aspects of the immune response can be manipulated by altering the form of the antigen and its delivery. This information coupled with an understanding of protective antigonococcal immune responses will enable the design of the optimal vaccine for N. gonorrhoeae.

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.

Modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles.

Measles remains a major worldwide problem partly because of difficulties with vaccination of young infants. New vaccine strategies need to be safe and to provide sustained protective immunity. We have developed Sindbis virus replicon particles that express the measles virus (MV) hemagglutinin (SIN-H) or fusion (SIN-F) proteins. In mice, SIN-H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination. SIN-F, or SIN-H and SIN-F combined, induced somewhat lower responses. To assess protective efficacy, juvenile macaques were vaccinated with a single dose of 10(6) or 10(8) SIN-H particles and infant macaques with two doses of 10(8) particles. A dose of 10(8) particles induced sustained levels of high-titered, MV-neutralizing antibody and IFN-gamma-producing memory T cells, and most monkeys were protected from rash when challenged with wild-type MV 18 months later. After challenge, there was a biphasic appearance of H- and F-specific IFN-gamma-secreting CD4+ and CD8+ T cells in vaccinated monkeys, with peaks approximately 1 and 3-4 months after challenge. Viremia was cleared within 14 days, but MV RNA was detectable for 4-5 months. These studies suggest that complete clearance of MV after infection is a prolonged, phased, and complex process influenced by prior vaccination.

Vaccination of macaques with SIV immunogens delivered by Venezuelan equine encephalitis virus replicon particle vectors followed by a mucosal challenge with SIVsmE660

VEE replicon particles (VRP), non-propagating vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), were engineered to express immunogens from the cloned isolate SIVsmH-4, combined in a vaccine cocktail and inoculated subcutaneously to immunize rhesus macaques. The virulent, uncloned challenge stock, SIVsmE660, represented a type of heterologous challenge and the intrarectal challenge modeled infection across a mucosal surface. Prechallenge neutralizing antibodies against SIVsmH-4 were induced in all vaccinates, and a prechallenge cellular immune response could be detected in one of six. Post-challenge, virus loads were reduced at the peak, at set point and at termination (41 weeks post-challenge), although these differences did not reach statistical significance. Significantly elevated levels of CD4 + T cells were observed post-challenge. A strong correlation was noted between a net increase in CD4 + T cell count and lowered virus load at set point.

Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV

A cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187 [Ruggli N, Tratschin JD, Mittelholzer C, Hofmann MA. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol 1996;70(6):3478–87] was used to construct two E2 deletion mutants lacking either the complete E2 gene or, alternatively, a stretch of 204 nucleotides encoding 68 amino acids located in the C-terminal region of the E2 glycoprotein. The respective in vitro synthesized mutant RNAs replicated in SK-6 cells but no infectious virus was generated. Both replicons could be packaged into virus particles in SK-6 cells constitutively expressing E2 of CSFV. For the resulting CSF virus replicon particles (CSF-VRP) A187-E2del373 and A187-E2del68 titers of 10 6 and 10 7 TCID 50/ml, respectively, were obtained. Oronasal vaccination with 10 7 TCID 50 of either of the two CSF-VRP protected pigs against a challenge with a lethal dose of CSFV strain Eystrup. In contrast, after intradermal vaccination VRP A187-E2del68 but not VRP A187-E2del373 lacking the complete E2 gene induced a protective immune response. We conclude that E2-complemented CSF-VRP have the potential to be used as live-attenuated non-transmissible oral vaccines for pigs. In addition, our data suggest that E2 of CSFV is dispensable for the induction of mucosal but not of parenteral immunity.

Comparison of Immune Responses to Gonococcal PorB Delivered as Outer Membrane Vesicles, Recombinant Protein, or Venezuelan Equine Encephalitis Virus Replicon Particles

Comparison of Immune Responses to Gonococcal PorB Delivered as Outer Membrane Vesicles, Recombinant Protein, or Venezuelan Equine Encephalitis Virus Replicon Particles

Alphavirus immunotherapy of NPM/ALK+ anaplastic large cell lymphoma in a mouse model

2574 Background: \(\underline{\mathrm{A}}\)naplastic \(\underline{\mathrm{L}}\)arge \(\underline{\mathrm{C}}\)ell \(\underline{\mathrm{L}}\)ymphoma (ALCL) is a non-Hodgkin's lymphoma. 2/3 of Kit+ (CD30) ALCL cases are associated with the expression of the fusion oncogene, NPM/ALK. Due to the effective immunogenicity of Venezuelan equine encephalitis virus replicon particles (VRP) expressing foreign antigens, we propose that VRP expressing a mutated form of NPM/ALK (NPM/ALK-VRP) will protect against tumor establishment and induce tumor regression in a murine model of ALCL. Methods: Stably transformed NPM/ALK pro-B cells inoculated intravenously into syngeneic immune competent mice resulted in tumor growth with 50–70% mortality at 2 months and a second phase of death at 4–6 months. To protect against tumor establishment, mice were inoculated in the footpad with NPM/ALK-VRP or GFP-VRP as an irrelevant antigen, boosted 3 weeks later and challenged with tumor cells. To test tumor regression, naive mice were transfused with tumor cells and 2-days later inoculated with either PBS, GFP-VRP or NPM/ALK-VRP, followed by a boost 3 weeks later. In both experiments, mice were scored for mortality (% survival) and morbidity; clinical ranking of 1–6 (6 = no symptoms and 1= death) and weight loss. Results: Immunohistological analysis confirmed the expression of NPM/ALK in tumors. Pre-treatment of mice with NPM/ALK-VRP compared to GFP-VRP significantly increased survival following tumor transfer (80%, 10%, respectively; Chi Square, P=0.0077). In addition, the NPM/ALK-VRP group had an overall higher clinical score and less weight loss compared to the GFP-VRP group. In the regression experiment, both NPM/ALK-VRP and GFP-VRP treated mice had significantly increased survival compared to the PBS group at 3 months post tumors (100%, 100% and 35%, respectively; Chi Square, P<0.0001), higher clinical scores and less weight loss. Conclusions: Pre-treatment of mice with NPM/ALK-VRP protected against tumor establishment. When administered after tumor transfer, treatment with VRP significantly reduced mortality and morbidity, regardless of the antigen expressed. No significant financial relationships to disclose.

Evidence for arbovirus dissemination conduits from the mosquito (Diptera: Culicidae) midgut.

The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.

Doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against HER 2/neu in a murine mammary carcinoma model.

IntroductionThe purpose of the present study was to determine whether cytotoxic chemotherapeutic agents administered prior to immunotherapy with gene vaccines could augment the efficacy of the vaccines.MethodsMice were injected in the mammary fat pad with an aggressive breast tumor cell line that expresses HER2/neu. The mice were treated 3 days later with a noncurative dose of either doxorubicin or paclitaxel, and the following day with a gene vaccine to HER2/neu. Two more doses of vaccine were given 14 days apart. Two types of gene vaccines were tested: a plasmid vaccine encoding a self-replicating RNA (replicon) of Sindbis virus (SINCP), in which the viral structural proteins were replaced by the gene for neu; and a viral replicon particle derived from an attenuated strain of Venezuelan equine encephalitis virus, containing a replicon RNA in which the Venezuelan equine encephalitis virus structural proteins were replaced by the gene for neu.ResultsNeither vaccination alone nor chemotherapy alone significantly reduced the growth of the mammary carcinoma. In contrast, chemotherapy followed by vaccination reduced tumor growth by a small, but significant amount. Antigen-specific CD8+ T lymphocytes were induced by the combined treatment, indicating that the control of tumor growth was most probably due to an immunological mechanism. The results demonstrated that doxorubicin and paclitaxel, commonly used chemotherapeutic agents for the treatment of breast cancer, when used at immunomodulating doses augmented the antitumor efficacy of gene vaccines directed against HER2/neu.ConclusionsThe combination of chemotherapeutic agents plus vaccine immunotherapy may induce a tumor-specific immune response that could be beneficial for the adjuvant treatment of patients with minimal residual disease. The regimen warrants further evaluation in a clinical setting.

Toll‐Like Receptor and Cytokine Expression Patterns of CD56+T Cells Are Similar to Natural Killer Cells in Response to Infection with Venezuelan Equine Encephalitis Virus Replicons

Using the natural killer (NK) cell-surface marker CD56 to study NK T cells in peripheral blood, we found that their frequency in mononuclear cells among healthy individuals was 1%-20% (average, 7.3%) and sporadically increased 4-5-fold within individuals over the course of 8 months. Infection of mononuclear cells in vitro with Venezuelan equine encephalitis virus replicon particles (VRPs) resulted in a significant increase in CD56(+) T cells and in the expression of interferon-alpha, tumor necrosis factor (TNF)-alpha, and interferon-gamma by CD56(+) but not CD56(-) T cells. NK and CD56(+) T cells expressed higher levels of Toll-like receptor (TLR)-3 and TLR4 after infection with VRPs, whereas only NK cells expressed inducible TNF-alpha and TLR2. Most of these effects were duplicated by activating mononuclear cells with double-stranded RNA. These expression patterns indicate that T cells coexpressing NK markers respond like NK cells to viral infection or double-stranded RNA, potentially fulfilling innate and adaptive immune functions.

Boosting with recombinant vaccinia increases HPV-16 E7-Specific T cell precursor frequencies and antitumor effects of HPV-16 E7-Expressing sindbis virus replicon particles

Immunotherapy using the heterologous prime–boost regimen has emerged as an attractive approach for generating antigen-specific T-cell-mediated immune responses against tumors and infectious diseases. We have previously linked the Mycobacterium tuberculosis heat-shock protein 70 (HSP70) to the HPV-16 E7 antigen creating a chimera, E7/HSP70. We found that nucleic acid vaccines encoding E7/HSP70 can generate strong antitumor immunity. Recently, replication-defective Sindbis virus replicon particle vaccines have been considered as an important vector system for vaccine development. In this study, we assessed whether the combination of E7/HSP70 Sindbis virus replicon particles (SINrep5-E7/HSP70) and E7/HSP70 vaccinia (Vac-E7/HSP70) can further enhance E7-specific immune responses using sequential vaccination. We found that priming with SINrep5-E7/HSP70 and boosting with Vac-E7/HSP70 generated the highest number of E7-specific CD8+ T cells and best antitumor effect compared to other combinations. Moreover, our data showed that at the dosage and route of immunization used in this study, mice treated with the Sindbis virus replicon particle prime–vaccinia boost regimen generated stronger antitumor responses compared to mice treated with the DNA prime–vaccinia boost vaccine regimen. Our results encourage the use of the Sindbis virus replicon particle prime–vaccinia boost regimen in future clinical trials.

DNA Immunization of mice with a plasmid encoding Neisseria gonorrhea PorB protein by intramuscular injection and epidermal particle bombardment

Immunogenicity of a DNA vaccine encoding PorB from Neisseria gonorrhoeae strain FA1090 was analyzed in BALB/C mice immunized by intramuscular needle injection or epidermal gene gun bombardment. Both delivery routes generated measurable specific antibodies although the gene gun response was slower. Antibody isotypes were indicative of Th2 activation following gene gun immunization and of Th1 activation following intramuscular injection. In both immunization protocols, boosting with either renatured recombinant (rr) PorB protein or PorB expressed from Venezuelan equine encephalitis virus replicon particles (VRPs) significantly increased anti-PorB antibody levels. Boosting with rrPorB protein had little effect on antibody isotypes, while boosting with VRPs expressing PorB-enhanced a Th1 type response. Whole cell binding experiments showed that a portion of the antibodies recognized the surface of the homologous N. gonorrhoeae strain. Serum from groups with high antibody levels showed some opsonization of the homologous strain using human neutrophils. These results showed the potential of DNA vaccination for the purpose of priming an antibody response against PorB of N. gonorrhoeae. When combined with a protein or VRP boost, DNA priming resulted in high-titer and long-lasting responses. Based on different prime-boost protocols, we could polarize immune responses to predominantly Th1 or Th2, which should enable future studies of the types of immune responses that are protective in mouse models of gonorrhea.

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7 49–57 epitope and generated comparable antitumor responses in several HPV16 E6 +E7 + tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7 49–57 epitope was observed in 90% of human leukocyte antigen (HLA)-A ∗0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.

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.