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    Schematic illustration of two-step (en passant) Red mutagenesis applied in the construction of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV). A, EHV-1 strain RacH infectious bacterial artifical chromososme (BAC) genome termed pRacH. B, Unique short region of pRacH with the introduced mini-F sequences targeted for the mutagenesis. C, First recombination event resulted in incorporation of the I-Ceu I fragment containing the VEEV structural genes and the kanamycin resistance gene, aphA1, in lieu of egfp sequences. D, Second step of the en passant protocol in which the aphA1 gene was removed and the final arrangement of the mini-F region including the VEEV structural sequences is given. E, Restriction fragment length polymorphisms (RFLPs) that confirm correct insertion of VEEV structural genes into pRacH. BAC DNA was digested with Eco RI and separated by electrophoresis on a 0.8% agarose gel. Staining of the gel with ethidium bromide shows changes in RFLP patterns obtained after insertion of the I-Ceu I fragment including the VEEV structural genes and aphA1 and upon removal of the aphA1 gene (indicated by circles). DNA fragments were subsequently transferred to positively charged nylon membranes and hybridized with a digoxigenin-labeled probe specific for VEEV structural genes. kbp = kilobasepairs.

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    Immunofluorescence staining of RK13 cells infected with equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV) or parental HΔgp2 virus. RK13 cells were infected with rH_VEEV (A, B, and C) or HΔgp2 (D, E, and F) at a multiplicity of infection (MOI) of 0.0001 and plaques were stained with different primary antibodies. Plaques in panels A and D were stained with monoclonal antibody (MAb) 1A3B-7 to VEEV E2 glycoprotein. Plaques in panels B and E were stained with MAb A8 to EHV-1 glycoprotein M. Plaques in panels C and F were stained with MAb 15C5TCS to bovine viral diarrhea virus (BVDV) E2 glycoprotein, which was used as a negative control. Bound antibodies were detected with anti-mouse IgG Alexa488 (Molecular Probes, Leipzig, Germany) and visualized with an inverted fluorescent microscope (Axiovert; Zeiss, Wetzlar, Germany). Scale bar = 100 μm. G, Western blot analysis of RK13 cells infected with rH_VEEV or HΔgp2. RK13 cells were infected with rH_VEEV or HΔgp2 at an MOI of 0.1. Cell lysates were prepared at 48 hours post-infection and treated or not treated with PNGase F. Cell lysates were adjusted to equal protein concentrations and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% gel. Proteins were transferred to a nitrocellulose membrane that was incubated with anti-VEEV hyperimmune ascitic fluid detected with a secondary anti-mouse IgG antibody coupled with peroxidase. Reactive bands were visualized by enhanced chemoluminescence.

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    Comparison of in vitro growth properties of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (rH_VEEV) with those of parental HΔgp2 virus. For measuring relative sizes of plaque induced by rH_VEEV and HΔgp2, RK13 cells were infected at a multiplicity of infection (MOI) of 0.0001. At 48 hours post-infection, cells were fixed and indirect immunofluorescence staining was performed using monoclonal antibody (MAb) A8 to EHV-1 glycoprotein M and anti-mouse IgG Alexa488 (Molecular Probes, Leipzig, Germany). Fifty plaques each were measured and analyzed using the ImageJ software (http://rsb.info.nih.gov/ij/). A, For single-step growth kinetics, RK13 cells were infected at an MOI of 3 and cell culture supernatant (extracellular) and cellular fractions (cell-associated) were collected at the indicated time points. Viral titers were determined by plaque assay. Error bars show standard deviations. B, Results were computed from three independent experiments.

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    Experimental challenge of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV)–immunized mice showing complete protection against a neurovirulent strain of VEEV. A, Five groups of mice were immunized twice in 14-day intervals with the indicated recombinant viruses. All animals were challenged 14 days after the second immunization with 1,000 50% lethal doses of neurovirulent VEEV strain ZPC738. Survival was assessed daily after challenge infection. In a second experiment (B and C), animals were immunized with rH_VEEV or mock-immunized (control) with phosphate-buffered saline (PBS). In addition to survival analysis, animals were monitored for weight loss (B) and body temperature (C) on the days immediately before and after challenge infection. Groups were compared up until day 6 because only one mouse was alive on day 7 and all mice succumbed to infection in the PBS group on day 8. P values were adjusted for multiple testing (Bootstrap method) and computed with the SAS version 9.1 software (SAS Institute, Cary, NC) using the Kruskal-Wallis test for not normally distributed values.

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    Induction of IgG responses by immunization with equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV). Mice were immunized twice in a 14-day interval with 1 × 105 plaque-forming units (PFU) of rH_VEEV (I), 1 × 104 PFU of rH_VEEV (II), or 1 × 105 PFU of HΔgp2 (III). Sera from immunized mice was pooled, diluted, and incubated with purified Sin83 virus particles, which were used as the antigen in a standard indirect enzyme-linked immunosorbent assay. Binding of IgG to the VEEV antigen was detected using mouse anti-IgG1 or mouse anti-IgG (heavy plus light chain) peroxidase-conjugated antibodies. Shown are the results obtained for sera collected on day 28 (day 14 after the second vaccination). No significant IgG2a levels were observed in either group. O.D. = optical density.

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Protection of Mice by Equine Herpesvirus Type 1–Based Experimental Vaccine against Lethal Venezuelan Equine Encephalitis Virus Infection in the Absence of Neutralizing Antibodies

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  • 1 Department of Microbiology and Immunology, Cornell University, Ithaca, New York; Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, Texas

A vectored vaccine based on equine herpesvirus type 1 (EHV-1) was generated as an alternative for safe and efficient prophylaxis against Venezuelan equine encephalitis virus (VEEV) infection. Two-step (en passant) Red mutagenesis was used to insert VEEV structural genes into an infectious clone of EHV-1 vaccine strain RacH. The recombinant virus, rH_VEEV, efficiently and stably expressed VEEV structural proteins as detected by various antibodies, including a conformation-dependent monoclonal antibody to envelope glycoprotein E2. In addition, rH_VEEV was indistinguishable from parental bacterial artificial chromosome–derived virus with respect to growth properties in cultured cells. Immunization of mice with the vectored vaccine conferred full protection against lethal challenge infection using VEEV strain ZPC738 in the absence of neutralizing antibodies and in a dose-dependent manner. Analyses of IgG responses demonstrated production of VEEV-specific IgG1 and total IgG antibodies after vaccination, indicating that protection was dependent on either cytotoxic T cell responses or antibody-mediated protection unrelated to neutralizing activity.

INTRODUCTION

Venezuelan equine encephalitis virus (VEEV) is an enveloped, arthropod-borne virus with a non-segmented, positive-sense RNA genome of approximately 11.4 kb. The virus belongs to the genus Alphavirus in the family Togaviridae. The 5′ two-thirds of the genome encode four nonstructural proteins (nsP1 to nsP4) that form an enzyme complex required for replication.13 The viral genome is released into the cytoplasm and the nonstructural polyprotein is translated directly from this RNA and used in the production of a full-length, negative-sense, replicative RNA intermediate.3 The full-length RNA then serves as a template for the synthesis of positive-sense genomic RNA and for transcription of sub-genomic 26S RNA.2 The subgenomic RNA corresponds to the 3′ one-third of the viral genome and is translated into a structural polyprotein that is proteolytically cleaved into the capsid protein and the envelope glycoproteins E1 and E2.4 The capsid protein (240 copies) encloses the genomic viral RNA to form an icosahedral nucleocapsid, and mature virions bud from the plasma membrane, acquiring a lipid envelope with embedded protein spikes formed by E1/E2 heterodimers.5,6

VEEV is a zoonotic pathogen and six distinct antigenic subtypes are recognized.7,8 Subtypes IAB and IC were previously associated with major epidemics and equine epizootics. In VEEV epizootics, equine mortality caused by encephalitis can reach 83%. In 1995, a major outbreak occurred in Venezuela and Colombia that was associated with the VEEV subtype IC. This epidemic resulted in approximately 100,000 human cases, with an estimate of more than 300 cases of fatal encephalitis.9 In humans, although the overall mortality rate is low (< 1%), neurologic disease, including disorientation, ataxia, mental depression, and convulsions can be detected in up to 14% of infected individuals, especially children.10 Neurologic sequelae in humans are also common.11 The pathologic findings in fatal human VEE cases include 1) edema, congestion, hemorrhages, vasculitis, meningitis, and encephalitis in the central nervous system (CNS); 2) interstitial pneumonia, alveolar hemorrhage, congestion, and edema in the lungs; 3) follicular necrosis and lymphocyte depletion in lymphoid tissue; and 4) diffuse hepatocellular degeneration in the liver.1214

A murine model for VEEV-induced encephalitis and lymphotropism is well established.1518 Subcutaneous infection of mice leads to biphasic disease with initial replication in lymphoid tissues, followed by viremia and penetration into and infection of the CNS,19 where the virus replicates until death of the infected animal.2023 The infection of CNS results in an acute meningoencephalitis that ultimately leads to the destruction of large numbers of neuronal cells and 100% lethality in mice.17,18

The live-attenuated VEEV TC83 vaccine strain (TC83) was developed in 1961 and more than 8,000 individuals have received TC83 in the past four decades.2426 Previous observations indicate that 20–40% of vaccinees develop a disease with some symptoms typical of natural VEEV infection, including febrile, systemic illness, and other adverse effects.24 More recent studies in mice validated these observations, where TC83 was uniformly lethal for the C3H/HeN strain after intracerebral inoculation and produced clinical illness in BALB/c mice for almost 14 days after subcutaneous inoculation.27 A formalin-inactivated version of the TC83 vaccine, C-84, is used for individuals who fail to seroconvert after vaccination with the standard formulation of TC83.26 A more promising candidate vaccine, V3256, was described recently, in which attenuation of the TRD strain was achieved by introducing lethal mutations into the PE2 furin cleavage site of an infectious cDNA clone, followed by selection of a second-site suppressor mutation in the E1 glycoprotein gene.15,28 This virus is highly attenuated in laboratory rodents. Although this engineering minimizes the possibility of direct reversion to virulence, the potential for reversion to wild-type virulence via compensatory mutations remains unknown.

The risk of neurologic disease in horses and humans caused by VEEV infections and the threat of VEEV as a biologic weapon advocates for the development of safer vaccines. Equine herpesvirus type 1 (EHV-1) is an Alphaherpesvirus within the genus Varicellovirus. In nature, EHV-1 predominantly infects equids, although infection of other species, such as llamas and alpacas has been reported.29 Conversely, in cultured cells, EHV-1 was shown to have a broad host tropism, and can infect many cell types, including murine and human cell lines.30 In addition, the virus does not appear to be neutralized by human sera containing antibodies against other alphaherpesviruses. The lack of anti-vector immunity, the broad tissue tropism including antigen-presenting dendritic cells, and the ability to stably accept large amounts of exogenous DNA30 suggest that EHV-1 could be a candidate vectored vaccine against human infections. In addition, in light of the widespread use of both VEEV and EHV-1 vaccines in the horse population, the development of a modified-live EHV-1 that simultaneously delivers VEEV antigens to horses may present a valuable alternative to commonly used inactivated preparations, which have to be produced under conditions of high biosecurity and whose efficacy is questioned.

In this study, we constructed a recombinant virus using a bacterial artificial chromosome (BAC) of EHV-1 strain RacH with the aid of a recently described markerless mutagenesis strategy.31 The resulting recombinant modified-live vaccine virus harbors and efficiently expresses the structural genes of VEEV strain TC-83. The recombinant virus was tested for genetic stability, safety, and efficacy in protecting mice against lethal VEEV infection with a recent 1D strain. Our results demonstrated that the recombinant virus, rH_VEEV, was genetically stable, did not cause any adverse side effects, and induced dose-dependent protection in mice against lethal challenge infection with VEEV in the absence of detectable neutralizing antibodies.

MATERIALS AND METHODS

Viruses and cells.

Rabbit kidney (RK13) cells were maintained in Earle’s minimum essential medium (EMEM) (Mediatech, Inc., Holly Hill, FL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gemini Bio-Products, West Sacramento, CA) and antibiotics (penicillin-streptomycin; Mediatech, Inc.). Recombinant virus HΔgp2 (EHV-1 strain RacH in which gene 71 was replaced by mini-F plasmid sequences)32 and rH_VEEV (recombinant EHV-1 expressing VEEV structural genes) were propagated in RK13 cells.

Bacterial strains.

Escherichia coli EL250 cells, which harbor the bacteriophage Red recombination genes in their genome (exo, bet, and gam) and contain the EHV-1 strain RacH genome as a mini-F-based plasmid, were maintained in Luria-Bertani (LB) broth or on LB agar plates containing 30 μg/mL of chloramphenicol. Escherichia coli strain DH10B harboring the shuttle plasmid (pEP_VEEV) was grown in LB broth or on LB agar plates containing 50 μg/mL of kanamycin.

Plasmids.

An EHV-1 BAC clone from vaccine strain RacH, pRacH1, was generated as described elsewhere with the notable exception that in this study we used a novel transfer construct that resulted in the inversion of the insertion cassette relative to the infectious clone described earlier to increase genomic stability.32 Briefly, plasmid pΔ71GFP-F and RacH viral DNA were co-transfected into RK13 cells by the calcium phosphate precipitation method.33 Virus-containing supernatant was harvested five days after transfection and titrated on RK13 cells. Fluorescent plaques were picked and purified, and recombinant viral DNA was purified and electroporated into competent E. coli EL250 cells. Electroporation was performed in 0.1-cm cuvettes (1,250 V, 200 Ω, 25 μF), and transformed bacteria were incubated for 1 hour at 32°C in LB medium and plated on LB agar plates containing 30 μg/mL of chloramphenicol. DNA from chloramphenicol-resistant bacterial clones was purified by standard alkaline lysis.33 Restriction enzyme patterns were analyzed using separation of DNA fragments by electrophoresis on a 0.8% agarose gel stained with ethidium bromide.33

Shuttle plasmid pEP_VEEV was generated from plasmid pEP_goi-in by introducing a synthetic, codon-optimized version of VEEV structural genes from strain TC-83 (sequence available upon request; Geneart, Inc., Regensburg, Germany). pEP_goi-in was generated as a plasmid specific for Red recombination within the mini-F locus previously introduced to generate pRacH1. pEP_goi-in is a modification of pCUCeu34 with additional flanking sequences of 50 basepairs (bp) that have homology to the sequences on either side of the egfp gene in pRacH1.31

Recombinant pRacH1 harboring the VEEV structural genes (pH_VEEV) was generated using a two-step Red (en passant) mutagenesis protocol.31 First, recombinant plasmid pEP_VEEV was digested with restriction endonuclease I-Ceu I to release a linear DNA fragment that contained the synthetic VEEV structural genes E3, E2, 6K, and E1 (E3-E1) under the transcriptional control of the human cytomegalo-virus immediate-early promoter (PHCMV) and the aphA1 gene that confers resistance to kanamycin. The linear I-Ceu I product also carried the flanks with homology to the sequences on either side of the egfp gene present in pRacH1 (Figure 1). Electroporation of the linear I-Ceu I product into competent EL250 cells harboring pRacH1 was performed as described above. Insertion of PHCMV_E3-E1_aphA1 into pRacH1 was achieved by recombination through the homologous regions, and the construct contained the VEEV sequences under the control of PHCMV, as well as the aphA1 gene in lieu of the egfp gene (Figure 1). The introduced aphA1 gene was flanked by an additional 50 bp of identical sequences located upstream and downstream, as well as a unique I-Sce I restriction site located upstream of the resistance gene. Cleavage at the restriction site was achieved by introducing plasmid pBAD_i-SceI into EL250 cells harboring the recombination intermediate. From this plasmid, the homing restriction endonuclease I-Sce I is expressed after addition of 0.1% arabinose to growing cultures.31 A second Red recombination resulted in excision of aphA1 via the flanking sequences and finalized the construction of recombinant pH_VEEV (Figure 1). The BAC DNA was isolated from kanamycin-negative colonies, digested using Eco RI, and analyzed by electrophoresis as described above. DNA fragments were transferred onto a positively charged nylon membrane and hybridized using digoxigenin-labeled VEEV E3-E1 sequences as a probe.33 Hybrids were detected with an anti-digoxigenin antibody and visualized using chemiluminescence (Roche Applied Science, Indianapolis, IN).

Indirect immunofluorescence analysis (IFA) and Western blotting.

For IFA, monoclonal antibody (MAb) A8 directed against EHV-1 glycoprotein M (gM) was used to detect EHV-1 infection, and MAb1A3B-7, which is directed against VEEV E2 and is reactive with a conformation-dependent epitope within the neutralization domain, was routinely used for detection of expression of VEEV structural proteins.32,35,36 The RK13 cells were seeded in six-well-plates and infected with either HΔgp2 or rH_VEEV virus at a multiplicity of infection (MOI) of 0.0001. One hour post-infection, medium was removed and infected cells were overlaid with 0.25% methylcellulose in EMEM-10% FBS. At 48 hours post-infection, cells were fixed with 90% ice-cold acetone for 10 minutes at −20°C. After re-hydration with phosphate-buffered saline (PBS), cells were blocked with PBS-0.5% bovine serum albumin for 30 minutes at room temperature. Cells were incubated with the primary antibodies at a 1:50 dilution for 30 minutes at room temperature and washed extensively with PBS before the secondary antibody (Alexa Fluor488-conjugated goat anti-mouse IgG antibody; Molecular Probes, Leipzig, Germany) was added for 30 minutes at room temperature. After thorough washing, plaques were inspected using an inverted fluorescent microscope (Axiovert 25; Zeiss, Wetzlar, Germany).

For Western blot analysis, pellets of 1 × 106 RK13 cells infected at an MOI of 1 were resuspended in radioimmuno-precipitation assay buffer (20 mM Tris, pH 7.5,150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate [SDS]) containing a protease inhibitor cocktail (Roche Applied Science). Additionally, N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) was added to the samples following the manufacturer’s recommendations. Sample buffer (0.15 M Tris, pH 6.8, 1.2% SDS, 0.3% glycerol, 0.15% β-mercaptoethanol, 0.018% [w/v] bromophenol blue) was added and protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel to separate the proteins. Either anti-VEEV hyperimmune ascitic fluid or A8 were used at 1:100 dilutions to detect proteins after transfer to nitrocellulose sheets (Schleicher & Schuell Biosciences, Inc., Keene, NH) by the semi-dry method.37 The secondary antibody was anti-mouse IgG coupled with peroxidase (Sigma, St. Louis, MO) and reactive bands were visualized by enhanced chemiluminescence (Pierce, Rockford, IL).

Plaque area measurements and single-step growth kinetics.

For plaque area measurements, RK13 cells were infected at an MOI of 0.0001 and at 1 hour post-infection infected cells were overlaid with 0.25% methylcellulose-containing EMEM-FBS. At three days post-infection, plaques were visualized by IFA using anti-EHV-1 gM MAb A8, 50 plaques were photographed, and areas were determined using the ImageJ software (http://rsb.info.nih.gov/ij/) in at least three independent experiments. Values were calculated and compared with parental HΔgp2 plaque areas, which were set to 100%. For single-step growth kinetics, RK13 cells were infected at an MOI of 3. Virus was allowed to attach for 1 hour at 4°C, followed by a penetration period of 1.5 hour at 37°C. At the indicated time points, supernatants were harvested, and viral titers were determined by conventional plaque assays. At 3 days post-infection, cells were fixed with 10% formalin in PBS, stained with 0.3% crystal violet solution, and plaques were counted. Single-step growth curves were calculated from three independent experiments and average titers were plotted.

Mouse experiments.

Six-week-old, female, NIH SWISS mice were obtained from Harlan (Indianapolis, IN) and were acclimatized in the Animal Biosafety Level-2 (BSL-2) facility for a week prior to vaccination. Challenge infection with VEEV was performed in approved BSL-3 laboratory facilities at the University of Texas Medical Branch, Galveston, TX, which are registered by the Centers for Disease Control and Prevention, Atlanta, GA for work with VEEV. Five mice were inoculated on day −28 subcutaneously with chimeric rH_VEEV virus at doses of 1 × 104, 1 × 103, or 1 × 102 plaque-forming units (PFU) or HΔgp2 virus (1 × 104) in a total volume of 0.1 mL of PBS. An additional group was immunized on day −28 by subcutaneous inoculation with 1 × 103 PFU of SINZPC, a recombinant Sindbis virus/VEEV vaccine,38 which was used as a positive control for the vaccination procedure. All animals received a booster vaccination on day −14, which was performed in the same way as the initial immunization. At day 0, immunized mice were challenge-infected with a recent VEEV subtype ID strain ZPC738 (ZPC738) by subcutaneous inoculation of 103 PFU (approximately 1,000 50% lethal doses) in 0.1 mL of PBS per animal. Mice were observed twice daily for a period of 28 days for clinical illness (anorexia and/or paralysis) and/or death. All of the animals were bled on days 0 and 28 after challenge. Samples for serology were heat-inactivated at 56°C for 30 minutes and stored at −70°C.

In a second animal experiment we analyzed the quality of the protection observed in challenged animals after vaccination with rH_VEEV virus. Two groups of five mice each were subcutaneously inoculated with 0.1 mL of PBS (control) or 1 × 104 PFU/animal of rH_VEEV on days −28 and −14 as indicated above. At day 0, all mice were challenged-infected as described above. Measurement of body weight and telemetric monitoring of body temperature was performed on days −3, 0, 1, 4, and 8 after infection. For measurement of body temperature, animals were anaesthetized with 5% isoflurane and implanted subcutaneously with BMDS IPTT-300 transponders (Bio Medic Data Systems, Inc., Seaford, DE) using a trocar needle assembly. Animals were monitored for signs of infection or migration of transponder prior to temperature recording. Chips were scanned using a DAS-6007 transponder reader (Bio Medic Data Systems, Inc.). Downloading of digital temperature data was performed in accordance with the manufacturer’s protocol. As described above, mice were observed twice daily for a period of 28 days for clinical illness (anorexia and/or paralysis) and/or death.

Antibody assays.

For anti-VEEV-specific antibodies, plaque reduction neutralization tests were performed on baby hamster kidney-21 cell monolayers. A stock of TC83 virus was incubated for 1 hour at 37°C with serial dilutions of sera from individual mice. Subsequently, cell monolayers were incubated with virus/serum mixtures for 1 hour at 37°C, overlaid with 0.5% agarose, maintained for 36 hours at 37°C, and stained with crystal violet. The serum dilution corresponding to an endpoint of 80% plaque reduction was determined.

For enzyme-linked immunosorbent assay (ELISA), Nunc MaxiSorp plates (eBioscience, San Diego, CA) were coated with purified Sin83 virus particles38 overnight at 4°C. Plates were blocked with 1% gelatin in PBS containing 0.03% Tween 20 (PBST) for 3 hours, followed by an incubation with serially diluted mouse sera (50 μL of pooled sera from individual mouse groups) for 1.5 hours. Antibody responses were detected with peroxidase-conjugated antibodies against IgG1, IgG2a, or whole IgG (heavy plus light chain), diluted in PBST for 1 hour. Anti-VEEV hyperimmune ascitic fluid was used as a positive control for the assay. The ELISA plates were washed 10 times after each incubation period with PBST and developed with o-phenylenediamine and H2O2 as a substrate. Absorbance was measured at 450 nm and relative optical densities were normalized with sera from mock-vaccinated animals.

RESULTS

Construction of recombinant pH_VEEV.

Two-step en passant mutagenesis was used to achieve insertion of the VEEV E3-E1 sequences and subsequent excision of aphA1 gene31 (Figure 1A–D). In the first recombination step, insertion of the VEEV E3-E1 region and the aphA1 gene, which results in transfer of kanamycin resistance into pRacH1 and formation of an intermediate recombinant DNA that differed from parental pRacH1 DNA in the Eco RI restriction pattern such that a 7.5 kilobasepair (kbp) fragment was increased in size to 10.8 kbp (Figure 1E). In a second recombination step, the aphA1 was removed from the intermediate construct, which resulted in the final recombinant EHV-1 BAC clone that was termed pH_VEEV. The pH_VEEV construct could be differentiated from the intermediate construct by the generation of a 9.8-kbp fragment that resulted from the deletion of aphA1 gene (1 kbp) from the 10.8 kbp fragment present in the intermediate (Figure 1E). In a Southern blot analysis using a probe for VEEV E3-E1 sequences, a specific reaction of the probe was obtained with the 10.8-kbp and 9.8-kbp Eco RI fragments in the intermediate or final construct, respectively, thereby confirming the successful insertion of E3-E1 sequences (Figure 1E).

Transgene expression and in vitro growth properties of recombinant H_VEEV virus.

The pRacH1 or pH_VEEV DNA was transfected into RK13 cells. Three days after transfection, progeny virus was harvested and subsequently characterized by IFA and Western blotting using VEEV E1- and E2-specific and EHV-1 anti-gM MAbs. In RK13 cells infected with rH_VEEV, virus plaques were reactive with the conformation-dependent VEEV E2-specific antibody (MAb1A3B-7) used, and those induced by parental virus, HΔgp2, were not (Figure 2A–F). In addition, VEEV E1 and E2 were also detected in lysates prepared from rH_VEEV-infected cells using VEEV-specific hyperimmune ascitic fluid, but, as expected, these proteins were absent in lysates derived from mock- or HΔgp2-infected cells Furthermore, addition of PNGase F to cell lysates from cells infected with rH_VEEV resulted in deglycosylation of E1 and E2, an indication of the correct processing of the inserted VEEV proteins (Figure 2G). The relative expression of VEEV structural proteins and rH_VEEV virus titers remained stable over at least 10 virus passages as tested by continuous propagation of rH_VEEV in RK13 cells and subsequent IFA. On the basis of these results, we concluded that rH_VEEV stably expressed the structural proteins of VEEV in a properly processed and folded form.

After determination of transgene expression, viral growth properties in cultured cells were analyzed. First, the ability of rH_VEEV to spread from cell to cell was investigated by measuring plaque areas and comparison to those formed by the parental HΔgp2 virus. Also, to determine whether the VEEV recombinant virus had any growth defects, virus replication of the parental and the rH_VEEV virus was compared by performing single-step growth kinetics. No significant difference was observed between the two viruses with respect to plaque areas measured at three days post-infection (Figure 3A). At earlier times post-infection, intracellular titers of rH_VEEV were reduced by approximately 10-fold compared with that of parental HΔgp2. Similarly, extracellular titers of rH_VEEV at 12 hours post-infection were reduced by 25-fold relative to those of HΔgp2. At later times post-infection (24 and 28 hours), however, intracellular and extracellular virus titers were comparable between the parental and rH_VEEV virus (Figure 3B). On the basis of these results, we concluded that in vitro growth of rH_VEEV was delayed with respect to onset of infectious virus production but that viral end titers were virtually indistinguishable from those of parental HΔgp2.

Complete protection by rH_VEEV against a neurovirulent strain of VEEV in mice.

To determine whether the constructed recombinant virus was capable of inducing protective immunity, three groups of mice (n = 5) were immunized twice in a 14-day interval by subcutaneous injection with three different doses (1 × 104, 1 × 103, or 1 × 102 PFU/animal). Control groups were immunized accordingly; mice in the negative control group were immunized with 1 × 104 PFU of parental HΔgp2 virus per animal, and mice in the positive control group were inoculated with 1 × 103 PFU/animal of SINZPC.38 Fourteen days after the last immunization, all animals were challenged with 1,000 50% lethal doses of the neurovirulent VEEV strain ZPC738. Serum neutralization assays showed that none of the mice immunized with the rH_VEEV construct exhibited any detectable VEEV-specific neutralizing antibodies at any time point tested. However, five of five animals immunized with the highest dose of rH_VEEV (1 × 104 PFU) and four of five animals vaccinated with 1 × 103 PFU of rH_VEEV survived challenge infection with ZPC738. In contrast, all negative control animals and the mice immunized with the lowest dose of 1 × 102 PFU of rH_VEEV died at days 7 or 8 after challenge infection (Figure 4A). On the basis of these results, we concluded that complete protection against VEEV challenge infection could be induced by the EHV-1 recombinant and that this protection was dose-dependent.

In addition, the observed immunity was independent of detectable levels of serum neutralizing antibodies prior to challenge. A second animal experiment was designed to validate the quality of the protection observed in the first experiment. Two groups (n = 5) of mice were vaccinated according to the schedule of the first experiment with rH_VEEV (1 × 104 PFU/animal) or mock-vaccinated with 0.1 mL of PBS. All animals were challenged with ZPC738 on day 0 and monitored twice a day for 28 days. Weight loss and changes in body temperature, as well as clinical illness, were recorded on days 0, 1, 4, 6, and 8 after challenge infection. In agreement with the above results, all mock-immunized animals died by day 8 after challenge infection (4 on day 7 and 1 on day 8), and all rH_VEEV-immunized animals survived without signs of clinical illness. From day 4 post-infection, a significant weight loss and a dramatic pre-exitus decrease in body temperature were observed in mock-immunized animals compared with rH_VEEV-immunized animals, whose weight and temperature remained unchanged throughout the experiment (Figure 4B and C). These results further corroborated the complete protection against VEEV challenge infection induced as a result of vaccination with the EHV-1 recombinant.

Induction of IgG responses in mice by vaccination with rH_VEEV.

To determine if any antibody responses were induced in vaccinated mice, three groups of mice (n = 5) were immunized twice in a 14-day interval by subcutaneous injection with two different doses (1 × 104 or 1 × 105 PFU/animal) or HΔgp2 (1 × 105 PFU/animal) after the regimen that resulted in complete protection of mice against subsequent challenge infection. Serum samples were collected and pooled at the time of the second vaccination and 14 days after the last vaccination. Samples were analyzed by ELISA for anti-VEEV specific IgG1, IgG2a, and whole IgG. Significant levels of IgG2a were not detected in any of the rH_VEEV-vaccinated groups compared with HΔgp2 vaccinated animals at any time point. However, low levels of VEEV-specific IgG1 and whole IgG were detected on the day the animals received the second vaccination. Booster injection increased VEEV-specific IgG1 and whole IgG titers in rH_VEEV vaccinated animals, and VEEV-specific IgG2a antibody titers remained undetectable in both groups (Figure 5).

DISCUSSION

In this study, we describe the generation and characterization of a recombinant EHV-1 modified-live virus vaccine harboring synthetic genes that encode for structural proteins E3, E2, 6K, and E1 of VEEV. We show that the recombinant herpesvirus generated exhibited growth properties that were comparable to those of its parental virus and that immunization of mice with the recombinant results in complete protection from lethal challenge with a neurovirulent strain of VEEV (ZPC738).

Our first goal was to streamline the recently described markerless mutagenesis method for quick and reliable generation of vectored EHV-1 constructs based on the manipulation of the RacH BAC.31 One of the important findings of this study was the genetic stability of the vectored construct as seen by the reliable, high-level expression of the introduced VEEV genes assayed by IFA and Western blot analysis for more than 10 passages of the recombinant virus in the RK13 production cell line. Herpesviruses are attractive candidates for vaccine vectors because they retain their ability to replicate to high titers, even after the introduction of foreign DNA of substantial length.39 Consistent with earlier findings, the results reported here demonstrate that rH_VEEV was not impaired in its ability to spread directly from cell to cell despite the introduction of a large foreign DNA construct encoding four VEEV genes. However, rH_VEEV, replicated slightly slower at earlier time points as demonstrated by single-step growth kinetics, an impairment that is overcome at later times in infection. One possible explanation for this unexpected behavior of rH_VEEV is that herpes viral, as well as alphaviral membrane (glyco)proteins, are synthesized at the endoplasmic reticulum and have to be transported through the Golgi and trans-Golgi network along the secretory pathway to ultimately arrive at cytoplasmic vesicles to be incorporated into nascent EHV-1 virions. An accumulation of large amounts of viral (glyco)proteins in the endoplasmic reticulum Golgi, and/or vesicles into which EHV-1 has to bud, especially of the VEEV structural proteins at early times of infection through PHCMV control, may cause stress in the endoplasmic reticulum and decelerate the process of secondary envelopment and virion formation, thus limiting production of infectious virus. A second explanation for the reduced growth at early times after infection may be that the entry process of rH_VEEV is slowed by the presence of VEEV glycoproteins because VEEV glycoproteins are incorporated into the envelope of recombinant virions.

With respect to the immune responses induced by rH_VEEV, we observed a surprising result. Complete protection against lethal VEEV challenge infection in mice was achieved in the absence of detectable neutralizing antibody responses. Although the possible role of cytotoxic T lymphocyte (CTL) responses in protection against alphavirus infection has been addressed, humoral responses (i.e., neutralizing antibodies) are considered essential for virus clearance and thus protection.6 In the experimental challenge with VEEV described here, mice were fully protected in the absence of detectable neutralizing antibodies, which strongly suggests the involvement of CTL or antibody-dependent cytotoxicity in the protection of mice against lethal VEEV infection.

Previously, it was shown that the passive transfer of non-neutralizing MAb into animals infected with neuroadapted Sindbis virus can prolong survival or even completely protect mice against challenge infection.40 The efficacy of such treatment as described by Schmaljohn and others40 strongly correlated with the attenuation of CNS infection that was reflected by reduced viral titers in the brains of infected mice receiving antibodies against VEEV compared with untreated mice. Similar to results from experimental work with Sindbis virus, Hunt and others have demonstrated the protective potential of non-neutralizing antibody to the amino terminus of E2 of Trinidad Donkey strain of VEEV (TC-83), which limited virus replication in brains of infected mice and resulted in reduction of viral load in the brain.41,42 In our evaluation of protection against VEEV infection, viral loads were not measured because of limited capability to use BSL-3 laboratory space. Nonetheless, we measured IgG1, IgG2a, and whole IgG induction in vaccinated animals. The rH_VEEV-vaccinated animals had increased levels of VEEV-specific IgG1 and total IgG antibodies in the sera after two injections. On the basis of these data, we concluded that non-neutralizing antibodies of the IgG subclass are produced as a result of vaccination and may potentially play a role in the protection induced by the EHV-1-VEEV recombinant, conceivably by antibody-dependent cytotoxicity.

It is tempting to speculate, however, that vaccination with rH_VEEV could confer a predominantly CTL-mediated protection against VEEV because it is well known that live attenuated (herpesvirus) vaccines are potent inducers of cell-mediated responses, although, at first glance, the absence of VEEV-specific IgG2a antibodies, which would be indicative of a Th1-biased immune response, seems to not fully support that scenario. Upon delivery by the vector, antigens can be classically processed and presented through the proteasome-major histocombatibility class I pathway. In the light of recent data that indicate an important role for γδ T cells in VEEV clearance from the brain (Paessler S and others, unpublished data), we currently favor a model where induction of CTLs is the protective mechanism against lethal challenge infection with VEEV after immunization with recombinant EHV-1. The question of the protective correlate after vaccination with rH_VEEV will be addressed by experiments in mice that involve the adoptive transfer of T lymphocyte subsets from vaccinated to non-vaccinated animals and subsequent challenge infections, as well as the use of various knockout mice that are unable to produce certain B or T cell lineages.

Because synthetic genes have a higher degree of nucleotide sequence disparity with naturally circulating VEEV strains, it is predicted that these genes offer a high degree of vaccine safety by reducing the possibility of recombination between vaccinal preparations and VEEV strains in the field. We believe that use of synthetic genes in a vector system, as presented in this study, would result in high environmental safety and allow immunization of horses in areas with endemic virus activity without the risk of genetic recombination between the live-attenuated VEEV vaccine and the enzootic VEEV strains. It is also noteworthy that in contrast to the only presently available live-attenuated VEEV vaccine TC-83, residual virulence was not observed in any of the immunized animals. Additionally, generation of rH_VEEV does not rely on handling VEEV, a BSL3 pathogen. Therefore, rH_VEEV could be a safe, convenient, and efficient vaccine against VEEV, which can also be applied intranasally and therefore be used to induce a local immunity (Rosas CT and others, unpublished data).

Overall, our results show that recombinant EHV-1 virus expressing VEEV E3-E1 is stable and protects against lethal infection with the neurovirulent VEEV strain ZPC738. Future studies will address the exact mechanism of protection against lethal challenge in mice and vaccine safety and efficacy in horses.

Figure 1.
Figure 1.

Schematic illustration of two-step (en passant) Red mutagenesis applied in the construction of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV). A, EHV-1 strain RacH infectious bacterial artifical chromososme (BAC) genome termed pRacH. B, Unique short region of pRacH with the introduced mini-F sequences targeted for the mutagenesis. C, First recombination event resulted in incorporation of the I-Ceu I fragment containing the VEEV structural genes and the kanamycin resistance gene, aphA1, in lieu of egfp sequences. D, Second step of the en passant protocol in which the aphA1 gene was removed and the final arrangement of the mini-F region including the VEEV structural sequences is given. E, Restriction fragment length polymorphisms (RFLPs) that confirm correct insertion of VEEV structural genes into pRacH. BAC DNA was digested with Eco RI and separated by electrophoresis on a 0.8% agarose gel. Staining of the gel with ethidium bromide shows changes in RFLP patterns obtained after insertion of the I-Ceu I fragment including the VEEV structural genes and aphA1 and upon removal of the aphA1 gene (indicated by circles). DNA fragments were subsequently transferred to positively charged nylon membranes and hybridized with a digoxigenin-labeled probe specific for VEEV structural genes. kbp = kilobasepairs.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.83

Figure 2.
Figure 2.

Immunofluorescence staining of RK13 cells infected with equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV) or parental HΔgp2 virus. RK13 cells were infected with rH_VEEV (A, B, and C) or HΔgp2 (D, E, and F) at a multiplicity of infection (MOI) of 0.0001 and plaques were stained with different primary antibodies. Plaques in panels A and D were stained with monoclonal antibody (MAb) 1A3B-7 to VEEV E2 glycoprotein. Plaques in panels B and E were stained with MAb A8 to EHV-1 glycoprotein M. Plaques in panels C and F were stained with MAb 15C5TCS to bovine viral diarrhea virus (BVDV) E2 glycoprotein, which was used as a negative control. Bound antibodies were detected with anti-mouse IgG Alexa488 (Molecular Probes, Leipzig, Germany) and visualized with an inverted fluorescent microscope (Axiovert; Zeiss, Wetzlar, Germany). Scale bar = 100 μm. G, Western blot analysis of RK13 cells infected with rH_VEEV or HΔgp2. RK13 cells were infected with rH_VEEV or HΔgp2 at an MOI of 0.1. Cell lysates were prepared at 48 hours post-infection and treated or not treated with PNGase F. Cell lysates were adjusted to equal protein concentrations and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% gel. Proteins were transferred to a nitrocellulose membrane that was incubated with anti-VEEV hyperimmune ascitic fluid detected with a secondary anti-mouse IgG antibody coupled with peroxidase. Reactive bands were visualized by enhanced chemoluminescence.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.83

Figure 3.
Figure 3.

Comparison of in vitro growth properties of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (rH_VEEV) with those of parental HΔgp2 virus. For measuring relative sizes of plaque induced by rH_VEEV and HΔgp2, RK13 cells were infected at a multiplicity of infection (MOI) of 0.0001. At 48 hours post-infection, cells were fixed and indirect immunofluorescence staining was performed using monoclonal antibody (MAb) A8 to EHV-1 glycoprotein M and anti-mouse IgG Alexa488 (Molecular Probes, Leipzig, Germany). Fifty plaques each were measured and analyzed using the ImageJ software (http://rsb.info.nih.gov/ij/). A, For single-step growth kinetics, RK13 cells were infected at an MOI of 3 and cell culture supernatant (extracellular) and cellular fractions (cell-associated) were collected at the indicated time points. Viral titers were determined by plaque assay. Error bars show standard deviations. B, Results were computed from three independent experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.83

Figure 4.
Figure 4.

Experimental challenge of equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV)–immunized mice showing complete protection against a neurovirulent strain of VEEV. A, Five groups of mice were immunized twice in 14-day intervals with the indicated recombinant viruses. All animals were challenged 14 days after the second immunization with 1,000 50% lethal doses of neurovirulent VEEV strain ZPC738. Survival was assessed daily after challenge infection. In a second experiment (B and C), animals were immunized with rH_VEEV or mock-immunized (control) with phosphate-buffered saline (PBS). In addition to survival analysis, animals were monitored for weight loss (B) and body temperature (C) on the days immediately before and after challenge infection. Groups were compared up until day 6 because only one mouse was alive on day 7 and all mice succumbed to infection in the PBS group on day 8. P values were adjusted for multiple testing (Bootstrap method) and computed with the SAS version 9.1 software (SAS Institute, Cary, NC) using the Kruskal-Wallis test for not normally distributed values.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.83

Figure 5.
Figure 5.

Induction of IgG responses by immunization with equine herpesvirus type 1 (EHV-1)–Venezuelan equine encephalitis virus (VEEV) recombinant virus (pH_VEEV). Mice were immunized twice in a 14-day interval with 1 × 105 plaque-forming units (PFU) of rH_VEEV (I), 1 × 104 PFU of rH_VEEV (II), or 1 × 105 PFU of HΔgp2 (III). Sera from immunized mice was pooled, diluted, and incubated with purified Sin83 virus particles, which were used as the antigen in a standard indirect enzyme-linked immunosorbent assay. Binding of IgG to the VEEV antigen was detected using mouse anti-IgG1 or mouse anti-IgG (heavy plus light chain) peroxidase-conjugated antibodies. Shown are the results obtained for sera collected on day 28 (day 14 after the second vaccination). No significant IgG2a levels were observed in either group. O.D. = optical density.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 1; 10.4269/ajtmh.2008.78.83

*

Address correspondence to Nikolaus Osterrieder, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail: no34@cornell.edu

Authors’ addresses: Cristina Rosas, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, Telephone: 607-253-4014, Fax: 607-253-3384, E-mail: ctr8@cornell.edu. Slobodan Paessler, Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, Telephone: 409-747-0764, Fax: 409-747-0762, E-mail: slpaessl@utmb.edu. Haolin Ni, Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609, Telephone: 409-747-2489, Fax: 409-747-0762, E-mail: shni@utmb.edu. Nikolaus Osterrieder, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, E-mail: no34@cornell.edu.

Acknowledgments: We would like to thank Jennifer Smith for technical assistance and Kerstin Osterrieder for statistical analysis.

Financial support: This study work was supported by National Institutes of Health (NIH) grant AI061412 and the Harry M. Zweig Memorial Fund for Equine Research (to Nikolaus Osterrieder). Slaoboaan Paessler was supported by NIH K08 grant AI059491.

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