Equine Herpesvirus Type 1 Vaccine Research Articles

Recombinant Glycoprotein B of Equine herpesvirus Type 1 Elicits Protective Immune Response against Challenge in BALB/c Mouse Model

Background: Equine herpesvirus type 1 (EHV-1) is the most important viral pathogen of equines, causing respiratory illness, abortion, neonatal foal mortality and neurologic disorders. Large numbers of commercial EHV-1 vaccines are available to protect equines from the disease, but they provide only partial protection. Despite immunization with inactivated and modified live virus vaccine, mares show abortions. Present study was aimed to investigate the immunogenicity and protective efficacy of EHV-1 recombinant glycoprotein B (rgB) and gB expressing plasmid DNA against EHV-1 infection in BALB/c mice model.Methods: About 3-4 weeks old 225 female BALB/c mice were selected for the comparative study of immunization followed by challenged with EHV-1/India/Tohana/96-2 strain virus in 5 different groups of 45 animals each.Result: Following immunization, rgB vaccinated mice showed optimal stimulation of EHV-1 gB specific cell mediated and humoral mediated immunity (HMI and CMI). The gB expressing plasmid DNA vaccinated mice developed only CMI while inactivated whole virus vaccinated mice had only HMI. Upon EHV-1 challenge, all infected mice displayed variable levels of clinical signs with changes in body weight, however, vaccinated mice showed very rapid recovery with optimal protection. Positive control group mice showed severe pulmonary lesions along with persistence virus infection till 5 days post challenge (dpc) whereas vaccinated mice had less pulmonary lesion only up to 3dpc. Minimal lung lesions and early virus clearance was observed in the rgB immunized mice in comparison to the gB plasmid DNA and inactivated EHV-1 vaccine immunized mice. It has been concluded that immunization with rgB elicits optimum protective immune response against EHV-1 infection in mice model. The rgB could be a potential vaccine candidate against EHV-1 infection in equine in the future.

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.

An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease.

Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate.IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.

The recombinant EHV-1 vector producing CDV hemagglutinin as potential vaccine against canine distemper

Canine distemper virus (CDV), is a pantropic agent of morbillivirus that causes fetal disease in dogs. Base on a broad host rang of CDV, the continued vaccines inoculation is unavoidable to pose gene recombination risk in vaccine virus and wild virus. The current study presents the construction of novel vectors, using equine herpesvirus type 1 (EHV-1) expressing the canine distemper virus (CDV). The recent field strain hemagglutinin protein and nucleoprotein were used for the construction of the viral vector vaccines. Based on the Bacterial artificial chromosome (BAC) genomes of EHV-1 RacH strain, the recombinant EHV-1 vaccine virus encoding CDV hemagglutinin protein (EHV-H) or CDV nucleoprotein (EHV-N) was constructed separately. The constructed BACs were rescued after 72 h post infection, and the expression of H or N in the recombinant viruses was confirmed by western-blotting. Furthermore, high levels of neutralizing antibodies were induced persistently following vaccination in the groups EHV-H&EHV-N and EHV-H, but the EHV-N group. The groups of vaccinated EHV-H and EHV-H&EHV-N pups were monitored for clinical signs, whereas the vaccinated EHV-N group developed moderate symptoms. The present study demonstrated that EHV-1 based recombinant virus carrying CDV H could be a promising vaccine candidate against canine distemper.

Protective effect of intranasal vaccination of horses with a modified live equine herpesvirus type 1 vaccine

Mucosal immunity in the nasal cavity is considered to play an essential role in protecting horses from disease caused by equine herpesvirus type 1 (EHV-1), and immunoglobulin A (IgA) antibody in the nasal secretions is a key component of this mucosal immunity. Although intranasal administration of live or killed EHV-1 vaccines has been attempted with the aim of protecting horses from viral challenge, induction of virus-specific IgA after these vaccinations was not observed, and only slight mitigation of clinical signs was observed after viral challenge. Here, we measured immunological responses and performed a challenge experiment in horses intranasally immunized with a modified live EHV-1 vaccine (Nisseiken, Tokyo, Japan) that was originally licensed for intramuscular injection. Horses (n ¼ 3) were intranasally inoculated with the vaccine (1.3 106 plaque-forming units [pfu]/10 mL/head) twice with a 4-week interval. Control horses (n ¼ 2) were inoculated in the same way with dilution medium. Four weeks after the second dose had been given, the horses were challenged by intranasal inoculation of EHV-1 (strain 10-I-224, 4.0 106 pfu/10 mL/head). Sequential nasal wash samples were concentrated by ultrafiltration and subjected to ELISA to measure EHV-1-specific IgA. Viral nasal shedding and viremia were monitored by virus isolation and real-time PCR. The results of real-time PCR were considered negative if there were � 10 gene copies in the analyzed sample. EHV-1-specific IgA levels in the nasal wash increased in all of the vaccinated horses after two intranasal doses of vaccine, whereas those in the control horses remained stable. Serum virus-neutralizing titers also increased in the vaccinated horses. After viral challenge, pyrexia over 39.0 � C was observed at 1 day post-inoculation (dpi) in both of the control horses. In contrast, none of the vaccinated horses was pyretic (over 39.0 � C) throughout the experiment, although one horse had a temperature of 38.7 � C at 7 dpi. Viral nasal shedding in the control horses was detected at 1 to 6 dpi, with a highest mean gene copy number of 103.8. In contrast, viral nasal shedding in the vaccinated horses was detected only at 1 to 2 dpi, with a highest mean gene copy number of 102.3. Viremia was detected in all horses at 5 to 11 dpi, with individual durations of 2 to 6 days. Intranasal immunization of horses with the live EHV-1 vaccine resulted in the production of EHV-1-specific IgA in the nasal mucosa and virus-neutralizing antibody in the whole body. Virus replication in the nasal mucosa after viral challenge was lower in vaccinated horses than in control horses; virus-specific IgA induced by the intranasal vaccination might have been responsible for this. The absence of pyrexia over 39.0 � C in the vaccinated horses indicated that the immunity induced by intranasal vaccination was effective in protecting horses from EHV-1-induced pyrexia.

Mitigation of Pyrexia by a Th-1-Biased IgG Subclass Response after Infection with Equine Herpesvirus Type 1 in Horses Pre-Immunized with Inactivated Vaccine

The immunoglobulin G (IgG) subclass response was investigated in horses with or without pyrexia after natural infection with equine herpesvirus type 1 (EHV-1) in the field. All horses were kept at the training centers of the Japan Racing Association and were immunized with an inactivated EHV-1 vaccine before EHV-1 infection. An IgG subclass response dominated by IgGa and IgGb was induced in horses without pyrexia after EHV-1 infection. In contrast, horses that developed pyrexia showed increased IgGc and IgG (T) subclass production in addition to IgGa and IgGb. Although inactivated EHV-1 vaccines are considered to induce a mainly Th-2-biased response, these results indicated that the responses in horses inoculated with inactivated EHV-1 vaccine were not uniform, and that horses with a Th-1-biased response were likely to be protected from pyrexia.

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

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.

The equine herpesvirus 1 UL11 gene product localizes to the trans-golgi network and is involved in cell-to-cell spread

Experiments were conducted to identify and characterize the equine herpesvirus type 1 (EHV-1) UL11 homologous protein. At early-late times after EHV-1 infection of Rk13 cells several proteins at an M r of 8000 to 12,000 were detected using a UL11 protein-specific antiserum. Particularly, an M r of 11,000 protein was found abundantly in purified virions and could be assigned to the tegument fraction. As demonstrated by confocal laser scanning microscopy, UL11 reactivity localized predominantly to the trans-Golgi network of infected cells, but was also noted at the plasma membrane, specifically of transfected cells. Deletion of UL11 sequences in EHV-1 vaccine strain RacH (HΔ11) and in the virulent isolate RacL22 (LΔ11) resulted in viruses that were able to replicate on noncomplementing cells. It was shown in one-step growth kinetics on Rk13 cells that the reduction of intracellular and of extracellular virus titers caused by the absence of UL11 expression in either virus was somewhat variable, but approximately 10- to 20-fold. In contrast, a marked influence on the plaque phenotype was noted, as mean maximal diameters of plaques were reduced to 23.2% (RacL22) or 34.7% (RacH) of parental virus plaques and as an effect on the ability of RacH to cause syncytia upon infection was noted. It was therefore concluded that the EHV-1 UL11 product is not essential for virus replication in Rk13 cells but is involved in cell-to-cell spread.

The Equine Herpesvirus 1 IR6 Protein Influences Virus Growth at Elevated Temperature and Is a Major Determinant of Virulence

The diploid IR6 gene (ORF 67) of equine herpesvirus type 1 (EHV-1) is absent in the modified live EHV-1 vaccine strain RacH and is present in a mutated form in the avirulent EHV-1 strains RacM24 and RacM36, such that the IR6 protein fails to form the typical rod-like structures observed for wild-type EHV-1 RacL11. To assess the role of the IR6 protein in EHV-1 replication and virulence, two recombinant RacH viruses, HIR6-1 and HIR6-2, that harbor a single copy of the wild-type IR6 gene were engineered and characterized. It was shown that: (i) HIR6-1 or HIR6-2 virus encoded for an IR6 protein that was capable of forming the rod-like structures typical of cells infected with the wild-type virulent virus strain RacL11. (ii) Whereas the avirulent EHV-1 strains RacH and RacM36 are temperature-sensitive (in that virus replication at 40° versus that at 37° was reduced by as much as 7,500-fold), the HIR6-1 and HIR6-2 viruses, like RacL11 virus, were capable of significant replication at the elevated temperature. (iii) Electron microscopic analyses revealed that cells infected with the HIR6-1 or HIR6-2 virus, like those infected with virulent RacL11 virus, produced and released comparable numbers of virus particles at both 37 and 40°. In cells infected with the RacH virus at 40°, however, release of extracellular particles was inhibited by greater than 90% and relatively few of the particles were enveloped. (iv) Infections of BALB/cA mice revealed that both the HIR6-1 and HIR6-2 viruses, unlike the parent RacH virus, were as virulent as the wild-type RacL11 strain as judged by the criteria of body weight loss, development of clinical signs of EHV-1 infection, virus titers in the lung, and ability to cause viremia. These findings and those of our recent studies indicate that the IR6 protein is a major determinant of EHV-1 virulence and that the IR6 protein may play a role in virus maturation and/or egress.

In-situ hybridization for demonstration of equine herpesvirus type 1 DNA in paraffin wax-embedded tissues and its use in horses with disseminated necrotizing myeloencephalitis

The detection of equine herpesvirus type 1 (EHV-1) in infected cell cultures, and in tissues taken at necropsy, by the in-situ hybridization technique is described. A 4.9 kb Bam HI fragment of EHV-1 vaccine strain RacH was used as a probe after labelling with [alpha-32P] thymidine 5'-triphosphate ([32P]TTP) or digoxigenin-deoxyuridine 5'-triphosphate (dUTP). Both probes specifically detected EHV-1 DNA in either cytospin or paraffin wax-embedded preparations of infected cells. The digoxigenin-labelled probe was further used to examine tissue sections of equine fetuses which had been aborted due to EHV-1 infection. In all cases positive hybridization signals were mainly associated with the nuclei. Positive results were confirmed by immunostaining of EHV-1 antigen in adjacent sections. However, both methods failed to detect EHV-1 in spinal cord sections of six horses suffering from disseminated necrotizing myeloencephalitis (DNM). These results support the hypothesis that DNM is not caused by a productive viral infection of parenchyma of the nervous system but is immunologically mediated.

Equine herpesvirus type 1 (EHV-1) infection and vaccination with an inactivated EHV-1 vaccine

Abstract Extract Madam:- We have been investigating episodes of severe respiratory disease in foals during their first year of life in the Waikato district of New Zealand. Most foals are affected at six to ten weeks of age and again at weaning time (4) Horner, G.W., Hunter, R., O'Flaherty, J.D. and Dickinson, L.G. 1976. Isolation of equine herpesviruses from horses with respiratory disease. N.Z. vet. J., 24: 171–176. [Taylor & Francis Online] , [Google Scholar] and the problem has been especially severe on one stud, ten foals having died of respiratory disease there in the past four years. Preliminary investigations on this stud have indicated that equine herpesviruse type 1 (EHV-1) and type 2 (EHV-2) were frequently isolated and might be the initiators of the respiratory disease observed. (5) Jolly, P.D. 1983. The development of an enzyme linked immunosorbent assayfor the detection of antibodies to equid herpesvirus type 1 in horsesera, Palemerston North: Massey University. B. Phil, thesis [Google Scholar] As part of an attempt to control this problem, an inactivated vaccine * *Pneumabort-K, Fort Dodge Laboratories, Iowa, U.S.A. derived from EHV-1 abortifacient strain (1) Bryans, J.T. 1978. Immunizaton of pregnant mares with an inactivated equine herpesvirus 1 vaccine. Proc 4th. Int. Conf. Equine Infec. Dis., Lyon, 1976, : 83–92. [Google Scholar] had been used since 1981. However, neither the epidemiology of these herpesviruses nor the efficacy of the vaccine has been thoroughly examined. In order to more clearly define the problem, an investigation was carried out to establish the patterns of EHV-1 and EHV-2 infections and to determine the effectiveness of the vaccination regime in preventing respiratory disease in foals.