山羊关节炎脑炎病毒甘肃株全基因组克隆和序列分析

根据GenBank中发表的山羊关节炎脑炎病毒CAEV-CO株（caprine arthritis encephalitis virus-CO,CAEV-CO）的全基因组序列（序列号：NC-001463）,设计合成了7对引物,对CAEV甘肃株的全基因组进行了PCR扩增,并对扩增产物进行了克隆和测序。结果表明,CAEV-甘肃株基因组全长为9186nt。与国际标准毒株CAEV—CO株相比,在编码区有12个碱基的缺失,二者的核苷酸同源性为91．0％;在非编码区有27个核苷酸的插人,核苷酸同源性为97．0％。gag、pol、蛋白Q、tat、env基因编码的氨基酸同源性分别为94．6％、94．7％、87．9％、94．7％和91．5％。     

IRES-Containing VEEV Vaccine Protects Cynomolgus Macaques from IE Venezuelan Equine Encephalitis Virus Aerosol Challenge

Venezuelan equine encephalitis virus (VEEV) is an arbovirus endemic to the Americas that is responsible for severe, sometimes fatal, disease in humans and horses. We previously described an IRES-based VEE vaccine candidate based up the IE serotype that offers complete protection against a lethal subtype IE VEEV challenge in mice. Here we demonstrate the IRES-based vaccine’s ability to protect against febrile disease in cynomolgus macaques. Vaccination was well tolerated and elicited robust neutralizing antibody titers noticed as early as day 14. Moreover, complete protection from disease characterized by absence of viremia and characteristic fever following aerosolized IE VEEV challenge was observed in all vaccinees compared to control animals, which developed clinical disease. Together, these results highlight the safety and efficacy of IRES-based VEEV vaccine to protect against an endemic, pathogenic VEEV IE serotype.     

Acute Infection with Venezuelan Equine Encephalitis Virus Replicon Particles Catalyzes a Systemic Antiviral State and Protects from Lethal Virus Challenge

The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.Venezuelan equine encephalitis virus (VEE) is an arthropod-borne, single-stranded, message-sense RNA virus belonging to the Alphavirus genus and Togaviridae family. Associated with periodic epidemics and equine epizootics in the Western Hemisphere, VEE also serves as a leading model for the study of alphavirus pathogenesis in vivo. In the murine model, which closely mimics infection of horses in nature, VEE causes a two-phase disease: an initial, acute lymphotropic phase characterized by a high serum viremia, followed by invasion of the central nervous system during a neurotropic phase that leads to fatal encephalitis (22, 27). Using the infectious molecular clone of VEE and an extensive panel of mutants blocked at various stages of infection, the course of infection and disease in the mouse model has been well characterized (3, 14, 15, 17, 27).Studies examining the molecular aspects of VEE pathogenesis have underscored the critical role of virus genetics and the subsequent host response in dictating the course and outcome of infection (6, 12, 23, 27, 35, 60, 64, 73). However, many details of the earliest host-pathogen interactions during VEE infection remain largely unknown. A tool paramount to studying early events in infection are VEE replicon particles (VRP). VRP are propagation-defective particles that undergo only one round of infection, as the structural genes which normally drive the assembly of progeny virions are deleted from the replicon genome (51). Infection of cells by VRP results in amplification of replicon viral RNA, but there is no packaging of new progeny and thus no further spread to other cells. As such, VRP infection is limited to the first round of targeted cells, allowing examination of the earliest interactions between virus and host.VRP infection of mice facilitated the identification of the draining lymph node (DLN) as the initial site of VEE viral amplification in vivo (44). Following footpad inoculation of mice with VRP, resident dendritic cells in the skin serve as the cellular target for infection. These infected dendritic cells then rapidly migrate from the site of inoculation in the skin to the local DLN (44). In the case of VRP infection, while no new viral progeny are packaged or released, the replicon genome continues to be replicated within these initially infected skin dendritic cells that have migrated and reside in the DLN. However, during infection with VEE virus, new viral progeny are eventually released into the DLN environment and infection spreads to adjacent cells.Based on these observations, we hypothesize that the earliest host-pathogen interactions within the DLN set the stage for the specific course of events that define VEE-induced pathogenesis. The innate immune response, including interferon (IFN) signaling, has been extensively documented as a critical component of controlling viral infection and spread (45, 47, 62, 66). In fact, utilizing a VRP-based mRNP-tagging system in vivo, we recently reported the robust activation of the host innate antiviral response directly within the infected cells of the DLN, as well as in surrounding uninfected bystander cells, at early times postinoculation (39). A consequence of this early, robust innate immune response at the initial site of replication is likely a contemporaneous induction of an antiviral state in tissues distal to the primary infection.We postulated that if early viral replication in the DLN induces the production of soluble immune mediators, such as IFN-α/β, then the induction of innate immune responses may be rapidly transmitted downstream from this primary site to distal tissues. Utilizing VRP to limit viral spread, we examined the host antiviral response within the DLN and tissues remote from the site of replication at early times following infection. In the liver and brain, the robust expression of a panel of IFN-stimulated genes, a hallmark of the antiviral state, was detected by 1 to 3 h following VRP footpad inoculation and peaked at expression levels >100-fold over mock animals. These results suggest that the early innate response to VRP infection is capable of rapidly inducing a systemically active antiviral state within the entire infected animal. Moreover, we found that mice pretreated by footpad inoculation with VRP for 6, 12, or 24 h were protected from an otherwise lethal VEE footpad or intranasal challenge, and the average survival time of mice challenged intracranially with VEE was significantly extended.Protection from VEE infection has typically been associated with the presence of neutralizing antibody (11, 24, 29, 49, 55). However, nonspecific protection against VEE has been suggested, including the involvement of the innate immune response (10, 26, 28, 33, 61, 73). In one instance, mice “vaccinated” with an attenuated clone of VEE were protected against lethal VEE challenge administered just 24 h after vaccination (26). In separate studies, the complete attenuation of a VEE mutant harboring a single noncoding nucleotide change was attributed to a heightened sensitivity of the virus to the host antiviral state (73). Additionally, mice with severe combined immunodeficiency survive longer than immunocompetent mice (9 days as opposed to 6 days) following infection with virulent VEE (12). These findings firmly indicate that the nonspecific host response to VEE is a critical component of controlling the earliest stages of infection.While IFN and the IFN-induced antiviral state are undoubtedly key mediators of the initial response to VRP infection in vivo, they may not solely be responsible for a rapidly induced protective state. In the challenge model presented here, VRP pretreatment was unable to protect mice from death following heterologous challenge with another IFN-sensitive virus, vesicular stomatitis virus (VSV). However, VRP pretreatment successfully protected mice from lethal challenge with influenza virus. Collectively, our results raise at least three important implications. First, the innate host response is rapidly mobilized following infection with VRP/VEE, at areas both proximal and distal to the site of active replication. Second, there exist components of the innate immune response to VEE that remain uncharacterized. Third, viruses are specifically and differentially sensitive to unique innate immune response profiles. These data provide new insight into the rapid mobilization of the host response to viral infection and present an effective pretreatment/challenge model to further investigate specific components of the innate response critical to protection against infectious pathogens.     

Immunization of Pigs with a Particle-Mediated DNA Vaccine to Influenza A Virus Protects against Challenge with Homologous Virus

Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.     

Proviral DNA of caprine arthritis encephalitis virus (CAEV) is detected in cumulus oophorus cells but not in oocytes from naturally infected goats

The aim of this study was to determine whether oocytes taken from ovarian follicles in 123 naturally infected goats were carrying the proviral CAEV genome. Examination of DNA isolated from 190 batches of oocytes with intact cumulus cells and 190 batches of oocytes whose cumulus cells had been removed, taken from follicles of the same ovaries, demonstrated that 42/190 batches of oocytes with intact cumulus cells had the proviral CAEV genome, whereas none of the 190 batches of oocytes without cumulus cells were positive for the provirus. To confirm that the proviral genome was present in the cumulus cells and not in the oocyte cells, 586 oocytes from 56 different ovaries, were separated from their cumulus cells. The DNA was then extracted from each fraction and examined. The purity of the oocyte fraction was verified by searching for granulosa cell-specific mRNA, using RT-PCR; this was negative in all the batches of oocytes in which the cumulus cells had been removed. PCR analysis demonstrated that none of the oocytes without cumulus cells were positive, whereas 22/56 of the batches with cumulus cells were found to be positive. This study clearly demonstrates that despite being surrounded by infected cumulus cells, the oocytes are not infected, and that the enzymatic and mechanical technique for removing the cells surrounding the oocyte, as used in this study, is effective, thus enabling CAEV-free oocytes to be obtained from infected goats.     

Limited Protection from a Pathogenic Chimeric Simian-Human Immunodeficiency Virus Challenge following Immunization with Attenuated Simian Immunodeficiency Virus

Two live attenuated single-deletion mutant simian immunodeficiency virus (SIV) constructs, SIV_239Δnef and SIV_PBj6.6Δnef, were tested for their abilities to stimulate protective immunity in macaques. During the immunization period the animals were examined for specific immune responses and virus growth. Each construct generated high levels of specific immunity in all of the immunized animals. The SIV_239Δnef construct was found to grow to high levels in all immunized animals, with some animals remaining positive for virus isolation and plasma RNA throughout the immunization period. The SIV_PBj6.6Δnef was effectively controlled by all of the immunized animals, with virus mostly isolated only during the first few months following immunization and plasma RNA never detected. Following an extended period of immunization of over 80 weeks, the animals were challenged with a pathogenic simian-human immunodeficiency virus (SHIV) isolate, SIV_89.6PD, by intravenous injection. All of the SIV_239Δnef-immunized animals became infected with the SHIV isolate; two of five animals eventually controlled the challenge and three of five animals, which failed to check the immunizing virus, progressed to disease state before the unvaccinated controls. One of five animals immunized with SIV_PBj6.6Δnef totally resisted infection by the challenge virus, while three others limited its growth and the remaining animal became persistently infected and eventually died of a pulmonary thrombus. These data indicate that vaccination with attenuated SIV can protect macaques from disease and in some cases from infection by a divergent SHIV. However, if animals are unable to control the immunizing virus, potential damage that can accelerate the disease course of a pathogenic challenge virus may occur.     

Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8⁺ and CD4⁺ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4⁺ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

Profile of the Caprine arthritis-encephalitis virus (CAEV) in blood, semen from bucks naturally and experimentally infected in the semi-arid region of Brazil

The aim of this study was to report the chronology of Caprine arthritis-encephalitis virus elimination and compare the blood and semen viral profiles of animals naturally and experimentally infected by SRLV raised in the semi-arid region of Brazil. The experiment was carried out at the Brazilian Center for Goat Research (Embrapa). Nine bucks were selected, four naturally infected by CAEV and five animals proven negative that were inoculated with the goat lentivirus (CAEV-Cork strain). Every week the animals were submitted to semen collection using an artificial vagina. The blood was collected by puncturing the jugular vein with tubes containing EDTA, 7 days after inoculation (experimentally infected group) or at the start of the experiment (naturally infected group) and then at every 30 days. The genomic viral DNA was extracted from semen and blood and then Nested-PCR was applied. An agar gel microimmunodiffusion was performed to detect anti-CAEV antibodies. The results were described in percentage and analyzed by the Chi square test (P < 0.05). The presence of anti-CAEV antibodies was detected in the 16th week after inoculation that characterized the seroconversion from four of the five naturally infected goat bucks (80%). The fifth reproducer presented late seroconversion, totaling 32 weeks post-inoculation. A quantity was observed in the total of samples collected of 12.50 and 17.14% positive results in the blood and 10.98 and 11.25% positive results in the semen of the naturally and experimentally infected animals, respectively, and there was no statistical difference. No statistically significant differences were observed regarding the presence of proviral DNA in the blood and semen of the naturally and experimentally infected animals. A viral elimination pattern was not identified during the assessment period, but the presence of proviral DNA was shown at shorter intervals after the 18th week and the 22nd week, for the experimentally and naturally infected bucks, respectively. Therefore, recently infected goats in the period prior to seroconversion eliminated small ruminant lentivirus proviral DNA in the semen and are important sources of infection that should be considered in a control program of this lentivirus, and the Nested-PCR technique is a relevant tool to select virus-free ejaculates.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous,but Not Heterologous,Challenge

The importance of neutralizing antibodies (NAbs) in protection against hepatitis C virus (HCV) remains controversial. We infused a chimpanzee with H06 immunoglobulin from a genotype 1a HCV-infected patient and challenged with genotype strains efficiently neutralized by H06 in vitro. Genotype 1a NAbs afforded no protection against genotype 4a or 5a. Protection against homologous 1a lasted 18 weeks, but infection emerged when NAb titers waned. However, 6a infection was prevented. The differential in vivo neutralization patterns have implications for HCV vaccine development.     

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.     

Novel SNPs of the Caprine Growth Hormone Secretagogue Receptor (GHSR) Gene and Their Association with Growth Traits in Goats

Growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor that binds ghrelin, plays an important role in the central regulation of pituitary growth hormone secretion, food intake, and energy homeostasis. This study analyzed polymorphism of the caprine GHSR gene as a genetic marker candidate for growth traits in goats. Two single nucleotide polymorphisms (GU014697:g.165G→A and GU014697:g.548T→C) were identified in exon 2 of the caprine GHSR gene by PCR-single-strand conformation polymorphism and DNA sequencing methods. Their associations with growth traits were analyzed in 313 Xuhuai goats. The results indicated that GU014697:g.548T→C had significant effects on growth traits. Body length and body length index were significantly higher in individuals with genotype TT than CC and CT in (P < 0.05). TT individuals also tended to have better performance in other traits, such as body height and chest circumference, although there were no statistical differences (P > 0.05). This suggests that GHSR is a strong candidate gene that affects growth traits in goats.     

Rapid Appearance of Secondary Immune Responses and Protection from Acute CD4 Depletion after a Highly Pathogenic Immunodeficiency Virus Challenge in Macaques Vaccinated with a DNA Prime/Sendai Virus Vector Boost Regimen

Heterologous prime/boost regimens are AIDS vaccine candidates because of their potential for inducing cellular immune responses. Here, we have developed a prime/boost regimen leading to rapid control of highly pathogenic immunodeficiency virus infection in macaques. The strategy, priming by an env and nef deletion-containing simian-human immunodeficiency virus (SHIV) proviral DNA followed by a single booster with a Gag-expressing Sendai virus (SeV-Gag), efficiently induced virus-specific T cells, which were maintained for more than 3 months until challenge. While all naive control macaques showed acute CD4(+) T-cell depletion at week 2 after an intravenous SHIV89.6PD challenge, all the macaques vaccinated with the prime/boost regimen were protected from depletion and showed greatly reduced peak viral loads compared with controls. Vaccination with the DNA alone or SeV-Gag alone was not enough to confer the consistent protection from the depletion, although it led to efficient secondary CD8(+) T-cell responses at week 2 after challenge. At week 1, a difference in the secondary responses between the protected and the unprotected macaques was clear; rapid augmentation of virus-specific CD8(+) T cells was detected in the former but not in the latter. Thus, our results indicate the importance of rapid secondary responses for reduction in the peak viral loads and protection from acute CD4(+) T-cell depletion.     

Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus

Background

Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.

Methodology/Principal Findings

We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.

Conclusion/Significance

These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.     

BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD₁₀₆ΔTK, BoHV-4-A-EF1α-M1RgD₁₀₆ΔTK and BoHV-4-A-EF1α-B6RgD₁₀₆ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD₁₀₆ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD₁₀₆ΔTK and BoHV-4-A-EF1α-B6RgD₁₀₆ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1^(-/-) mice against mortality and morbidity. This work demonstrated the efficacy of BoHV-4 based vectors and the use of BoHV-4 as a vaccine-vector platform.