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.

Fig 1. Body temperatures, clinical scores, nasal viral shedding and viremia after experimental EHV-1 infection of naïve horses.

The horses were infected with EHV-1 strain Ab4 (n = 5) or deletion mutant strain Ab4ΔORF1/71 (n = 5). The arrow points to the time of infection (d0). A non-infected control group (n = 5) was included. (A) Body temperatures. The dotted line shows the cut-off value for fever of 38.5°C; (B) total clinical score determined by the summation of several clinical variables evaluated on a numerical scale; (C) EHV-1 in nasal secretions measured by virus isolation; (D) viremia measured by virus isolation from PBMC; (E) viremia determined by real-time PCR. The dotted horizontal line shows the positive PCR Ct-value cut-off value. All graphs show means and standard errors by group. Significant differences between groups are marked: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.

Fig 2. Cytokines and total anti-EHV-1 gC antibodies in nasal secretions of naïve horses (n = 5 per group) after infection with EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF1/71.

Non-infected horses were kept as controls. The arrow marks the time of infection. Nasal secretion samples were collected before and at various times after infection. Cytokine concentrations were analyzed by a bead-based Cytokine Multiplex assay and antibodies were evaluated by EHV-1 multiplex assay: (A) IFN-α (B) sCD14, (C) IL-10, and (D) total anti-gC antibodies expressed as median fluorescence intensities (MFI). Mean and standard errors are displayed. Significant differences between groups: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.

Fig 3. Total anti-EHV-1 gC and IgG isotype responses in serum from horses after infection with the EHV-1 strain Ab4 or its deletion mutant Ab4ΔORF1/71.

EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection on d0. Serum antibodies were measured by an EHV-1 Multiplex assay. Antibody values are expressed as median fluorescence intensities (MFI) for (A) total Ig, (B) IgG1, (C) IgG1/3, and (D) IgG4/7. All graphs show means and standard errors by group over time. The dotted vertical lines mark d8 and d12pi to illustrate the time of the onset of the response for total Ig and the different IgG isotypes. Significant differences between the groups are shown: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.

Fig 4. Cytokine secretion from PBMC of EHV-1 infected horses after re-stimulation ex vivo.

EHV-1 naïve horses were infected with one of two EHV-1 strains (Ab4 or Ab4ΔORF1/71). A group of non-infected control horses was included (n = 5 per group). The arrow points to the time of infection. PBMC were harvested before and at several time points after infection and were re-stimulated with the EHV-1 strain Ab4 or were kept in cell culture medium. After 48 hours of incubation cell culture supernatants were harvested and cytokine production was analyzed by a bead-based Cytokine Multiplex assay. All EHV-1 re-stimulation values shown in the figures are corrected by the value of the respective medium control. Mean and standard errors for (A) IFN-γ, (B) IL-10, and (C) IL-4 in the cell culture supernatants are displayed. Significant differences between groups: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.

Fig 5. EHV-1 specific IFN-γ producing lymphocytes in PBMC.

EHV-1 naïve horses (n = 5 per group) were infected with the EHV-1 strain Ab4 or the deletion mutant virus Ab4ΔORF1/71. Non-infected horses were kept as controls. The arrow marks the time of infection. PBMC were isolated at different times post infection and re-stimulated with EHV-1 strain Ab4 ex vivo or were kept in cell culture medium for control. The secretion inhibitor Brefeldin A was added to the cultures for the last 24 hours. Cells were harvested at 48 hours, fixed, stained for intracellular IFN-γ production and cell surface CD4 and CD8, and measured by flow cytometry. The analyzing gate was set on small lymphocytes. Mean and standard errors for the percentages of (A) total IFN-γ⁺ lymphocytes, (B) CD8⁺/IFN-γ⁺ lymphocytes, and (C) CD4⁺/IFN-γ⁺ lymphocytes are displayed over time. All percentages for IFN-γ⁺ cells were corrected by the respective medium control values. The dotted horizontal line represents a suggested cutoff of 0.05% IFN-γ⁺ lymphocytes. Values below this cutoff were typically reached for IFN-γ⁺ cells in the non-infected control group prior to correction by medium control values. Significant differences between groups are marked: a = Ab4 vs. controls, b = Ab4ΔORF1/71 vs. controls, and c = Ab4 vs. Ab4ΔORF1/71.