Recombinant vaccinia virus/Venezuelan equine encephalitis (VEE) virus protects mice from peripheral VEE virus challenge.

Mice immunized with recombinant vaccinia virus (VACC) expressing Venezuelan equine encephalitis (VEE) virus capsid protein and glycoproteins E1 and E2 or with attenuated VEE TC-83 virus vaccine developed VEE-specific neutralizing antibody and survived intraperitoneal challenge with virulent VEE virus strains including Trinidad donkey (subtype 1AB), P676 (subtype 1C), 3880 (subtype 1D), and Everglades (subtype 2). However, unlike immunization with TC-83 virus, immunization with the recombinant VACC/VEE virus did not protect mice from intranasal challenge with VEE Trinidad donkey virus. These results suggest that recombinant VACC/VEE virus is a vaccine candidate for equines and humans at risk of mosquito-transmitted VEE disease but not for laboratory workers at risk of accidental exposure to aerosol infection with VEE virus.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Gamma-Irradiated Venezuelan Equine Encephalitis Vaccines

The efficacy of Formalin-inactivated Venezuelan equine encephalitis (VEE) vaccine has been reported to be low for man. Although a live VEE vaccine has been shown to be highly effective for the protection of laboratory workers, local and systemic reactions have occurred in approximately 20% of inoculated individuals. Therefore, studies were initiated in an attempt to produce an inactivated vaccine of high potency with low toxicity. Inactivated VEE vaccines were prepared by exposing virus suspensions to 8 x 10(6) or 10 x 10(6) r of gamma radiation. Irradiated VEE vaccines prepared from virus suspensions produced in Maitland-type chick embryo (MTCE) cell cultures and in monolayer cultures of human diploid strain WI-38 cells were highly immunogenic for mice and guinea pigs. Guinea pigs vaccinated with a series of three inoculations of vaccine (MTCE) survived challenge with at least 10(8.4) mouse intracerebral 50% lethal doses of VEE virus. Irradiated vaccines induced high levels of serum-neutralizing and hemagglutinin-inhibiting antibodies in guinea pigs and rabbits. These findings suggest that ionizing radiation may be effective in the preparation of an inactivated VEE vaccine.     

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.     

Comparison of the Immune Responses Induced by Chimeric Alphavirus-Vectored and Formalin-Inactivated Alum-Precipitated Measles Vaccines in Mice

A variety of vaccine platforms are under study for development of new vaccines for measles. Problems with past measles vaccines are incompletely understood and underscore the need to understand the types of immune responses induced by different types of vaccines. Detailed immune response evaluation is most easily performed in mice. Although mice are not susceptible to infection with wild type or vaccine strains of measles virus, they can be used for comparative evaluation of the immune responses to measles vaccines of other types. In this study we compared the immune responses in mice to a new protective alphavirus replicon particle vaccine expressing the measles virus hemagglutinin (VEE/SIN-H) with a non-protective formalin-inactivated, alum-precipitated measles vaccine (FI-MV). MV-specific IgG levels were similar, but VEE/SIN-H antibody was high avidity IgG2a with neutralizing activity while FI-MV antibody was low-avidity IgG1 without neutralizing activity. FI-MV antibody was primarily against the nucleoprotein with no priming to H. Germinal centers appeared, peaked and resolved later for FI-MV. Lymph node MV antibody-secreting cells were more numerous after FI-MV than VEE/SIN-H, but were similar in the bone marrow. VEE/SIN-H-induced T cells produced IFN-γ and IL-4 both spontaneously ex vivo and after stimulation, while FI-MV-induced T cells produced IL-4 only after stimulation. In summary, VEE/SIN-H induced a balanced T cell response and high avidity neutralizing IgG2a while FI-MV induced a type 2 T cell response, abundant plasmablasts, late germinal centers and low avidity non-neutralizing IgG1 against the nucleoprotein.     

Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.

A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. The VEE-MA/CA vector replicated rapidly in the cytoplasm of baby hamster kidney (BHK) cells and expressed large quantities of antigenically identifiable MA/CA protein. When injected subcutaneously into BALB/c mice, the vector invaded and replicated in the draining lymphoid tissues, expressing HIV-1 MA/CA at a site of potent immune activity. Anti-MA/CA immunoglobulin G (IgG) and IgA antibodies were present in serum of all immunized mice, and titers increased after a second booster inoculation. IgA antibodies specific for MA/CA were detected in vaginal washes of mice that received two subcutaneous immunizations. Cytotoxic T-lymphocyte responses specific for MA/CA were detected following immunization with the MA/CA-expressing VEE vector. These findings demonstrate the ability of a VEE-based vaccine vector system to stimulate a comprehensive humoral and cellular immune response. The multifaceted nature of this response makes VEE an attractive vaccine for immunization against virus infections such as HIV-1, for which the correlates of protective immunity remain unclear, but may include multiple components of the immune system.     

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.     

Early Protection in Hamsters Immunized with Attenuated Venezuelean Equine Encephalomyelitis Vaccine

The rapid onset and persistence of homologous and heterologous protection induced by attenuated Venezuelan equine encephalomyelitis (VEE) vaccine (TC-83) were studied in the hamster, by using challenge response as the index of protection. At 8 hr postvaccination with 10³ median immunizing doses of TC-83 vaccine, 15 to 20% of animals were protected against challenge with VEE virus as well as Eastern and Western equine encephalomyelitis viruses. The percentage of protection increased with time postvaccination until 80 to 90% homologous and heterologous protection was achieved by 18 hr postvaccination. Temporal studies indicated that early protection (days 1 to 6) correlated with vaccine viremia, and that the percentage of protection against heterologous challenge decreased with the cessation of viremia. Data are presented to indicate that the early protection phenomenon is one of interference, since little or no replication of a challenge virus occurred when it was administered during the vaccine viremia stage.     

Alphavirus replicon particles as candidate HIV vaccines

Replicon particles based on Venezuelan equine encephalitis virus (VEE) contain a self-replicating RNA encoding the VEE replicase proteins and expressing a gene of interest in place of the viral structural protein genes. Structural proteins for packaging of replicon RNA into VEE replicon particles (VRPs) are expressed from separate helper RNAs. Aspects of the biology of VEE that are exploited in VRP vaccines include 1) expression of very high levels of immunogen, 2) expression of immunizing proteins in cells in the draining lymph node, and 3) the ability to induce mucosal immunity from a parental inoculation. Results of experiments with VRPs expressing green fluorescent protein or influenza virus hemagglutinin (HA) demonstrated that specific mutations in the VRP envelope glycoproteins affect both targeting in the draining lymph node and efficiency of the immune response in mice. VRPs expressing either the matrix-capsid portion of Gag, the full-length envelope gp160, or the secreted gp140 of cloned SIVsm H-4i were mixed in a cocktail and used to immunize macaques at 0, 1, and 4 months. Neutralizing antibodies against SIVsm H-4 were induced in 6 of 6 vaccinates and CTL in 4 of 6. An intrarectal challenge with the highly pathogenic SIVsm E660 was given at 5 months. A vaccine effect was seen in reduced peak virus loads, reduced virus loads both at set point and at 41 weeks postchallenge, and preserved or increased CD4 counts compared to controls. A candidate VRP HIV vaccine expressing Clade C Gag contains a sequence that is very close to the South African Clade C consensus and was selected from a recent seroconverter in the Durban cohort to represent currently circulating genotypes in South Africa. A GMP lot of this vaccine has been manufactured and tested for a phase I trial in the first months of 2002.     

TC-83 Venezuelan equine encephalitis vaccine exposure during pregnancy

A human pregnancy exposed to TC-83 live attenuated Venezuelan equine encephalitis (VEE) virus vaccine resulted in hydrops fetalis and fetal demise. Maternal seroconversion and the finding of a diffuse mononuclear cell infiltrate on postmortem examination are suggestive of a causative role for TC-83 vaccine.     

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.     

Specific in vitro lymphocyte transformation with Venezuelan equine encephalitis virus.

Specific lymphocyte transformation to viral antigen was detected in individuals vaccinated with live, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain TC-83. Suspensions of purified, inactivated virus were used an antigen in 250-μ1 lymphocyte cultures, and under optimal conditions the assay demonstrated 10-fold greater incorporation of ¹⁴C-thymidine by lymphocytes from immune than from nonimmune people. The rise in lymphocyte transformation response occurred 1 week after vaccination. The magnitude and range of the lymphocyte transformation response to the VEE viral antigen were similar to the responses seen using antigens derived from five other microbial sources: Francisella tularensis, Coccidioides immitis, Mycobacterium tuberculosis, Streptococcus, and parainfluenza virus. Autologous plasma containing antibody exerts an inhibitory effect on cultures from immune individuals. The onset, magnitude of response, and specificity of this in vitro assay are correlated with the clinical and pathological events of VEE virus infection.     

Resistance to Alpha/Beta Interferons Correlates with the Epizootic and Virulence Potential of Venezuelan Equine Encephalitis Viruses and Is Determined by the 5′ Noncoding Region and Glycoproteins

We compared the alpha/beta interferon (IFN-α/β) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-α/β resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5′ noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.     

Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies.

We have previously characterized with monoclonal antibodies (MAbs) seven unique epitopes on the two envelope glycoproteins of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83. The epitopes important in protection from VEE virus infection were determined in passive antibody transfer studies, with virulent VEE (Trinidad donkey) virus as the challenge virus. Selected high-avidity MAbs to the three major protective epitopes (E2c, E1b, and E1d) were assayed for in vitro complement activity. All three fixed murine complement to high titer. Limited pepsin digestion of the anti-E2c in the presence of cysteine resulted in a rapid decrease and complete loss of complement-fixing ability by 2 h, but the majority of mice, except at the lowest dilution of MAb, were protected until the Fc termini were cleaved at 3 h. Anti-E2c F(ab')2 would neutralize VEE (Trinidad donkey) virus more efficiently than either Fab' or Fab; none of the fragments would fix complement or was effective in passive protection. C5-deficient mice and mice depleted of C3 with cobra venom factor were still protected from VEE (Trinidad donkey) virus challenge after passive transfer of either anti-E2c or anti-E1b MAb. The results show that the anti-E2c MAb mediates neutralization through bivalent binding at a critical site on the virion and that Fc effector functions, other than complement, are necessary for protection. Although the ability of the anti-E2c MAb to fix complement was associated with its ability to protect in vivo, no direct cause-and-effect relationship was found. Since the epitope defined by the anti-E1d antibody is found on the cell membrane, but is not expressed on the infectious virion, protection in mice was most likely mediated at the cellular level, possibly by inhibition of the final stages of virion maturation.     

Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.     

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.     

Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein.

The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.     

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.     

Melatonin Decreases Nitric oxide Production, Inducible Nitric oxide Synthase Expression and Lipid Peroxidation Induced by Venezuelan Encephalitis Equine Virus in Neuroblastoma Cell Cultures

Increased expression of inducible nitric oxide synthase has been shown in murine Venezuelan equine encephalitis (VEE) virus infection. In this experimental model, melatonin (MTL) treatment has shown to be beneficial. The aim of this study was to determine the effect of VEE virus on the nitric oxide (NO) production and lipid peroxidation in neuroblastoma cell cultures, and to investigate the role of MTL during cell-virus interaction. Neuroblastoma cells were co-cultured with VEE virus and treated with MTL at doses ranging from 0 to 1.8 mM, for 6, 12, 24 and 48 h. NO and lipid peroxidation were measured in culture supernatants and in the cellular content by nitrite concentration and thiobarbituric acid assay, respectively. Expression of inducible nitric oxide synthase (iNOS) was determined by indirect immunofluorescence. Increased production of NO and lipid peroxidation products were found in supernatants and cellular contents of VEE virus treated cultures. Both NO and lipid peroxidation were decreased by MTL treatment in a time dependent manner. Increased iNOS expression was observed in VEE virus infected cultures that was reduced by MTL treatment. These results could be related to the beneficial role of MTL in the VEE experimental disease and address the possible therapeutic potential of the hormone in human VEE virus infection.