The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses

Abstract

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs

“…We assume that these viral proteins (ORF2 and ORF17) are involved in modulating several pathways in PBMC, including cell signaling, adhesion, and immune pathways, thereby ORF2 and ORF17 gene deletions resulted in reduced virus transfer to EC. Our findings clearly correlate with previous in vivo findings where ORF2, ORF1/ORF71, and ORF1/ORF2 deletion mutants showed respiratory infection, comparable viremia, but no noticeable endothelial cell infection in four different experimental studies in horses [48][49][50]76].…”

“…Further, ORF1 and ORF2 deletion mutants showed significantly reduced virus shedding, a shorter course of pyrexia, and modulated cytokine response with attenuation of virulence in comparison to Ab4-wt in in vivo studies in ponies [50]. Experimental infection with ORF1 and ORF71 deletion mutants revealed a brief period of pyrexia, low virus shedding, and decreased cytokine response (IFNα, IL-10, and soluble CD14); however, they had comparable viremia to Ab4-wt [48]. In another study, ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [49,76].…”

“…In another study, ORF2 deletion mutant was attenuated; however, it had strong immunogenicity without altering viremia in Icelandic horses [49,76]. Failure to induce T-cell response was suggested as a reason for viremia in both Ab4-wt and mutant viruses infected horses [48][49][50]. As animal experiments were performed in non-pregnant horses and no distinct neurological signs were observed following infection with Ab4-wt and mutant viruses, it is unclear that cell-associated viremia with mutant viruses can result in subsequent endothelial infection or not.…”

“…The ORF2 gene product is considered a virulence factor for EHV-1 [ 47 ]. Previous in vivo studies have revealed the involvement of ORF1 and ORF2 in modulating cytokine and chemokine responses in infected horses and subsequently minimizing the course of the disease [ 48 , 49 , 50 ].…”

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

“…IgG1 and IgG4/7 specific for EHV-1 envelope glycoproteins C and D (gC and gD, respectively) also dominated the host immune response after vaccination with an inactivated vaccine, and the glycoprotein-specific IgG4/7 antibodies correlated highly with EHV-1 SN titers (17). Experimental infection with EHV-1 strain Ab4 likewise induced serum antibody responses against gB, gC, and gD with IgG4/7 antibodies carrying the long-lasting adaptive immunity (18,19).…”