Evaluation of immune responses following infection of ponies with an EHV-1 ORF1/2 deletion mutant

Abstract

Equine herpesvirus-1 (EHV-1) infection remains a significant problem despite the widespread use of vaccines. The inability to generate a protective immune response to EHV-1 vaccination or infection is thought to be due to immunomodulatory properties of the virus, and the ORF1 and ORF2 gene products have been hypothesized as potential candidates with immunoregulatory properties. A pony infection study was performed to define immune responses to EHV-1, and to determine if an EHV-1 ORF1/2 deletion mutant (ΔORF1/2) would have different disease and immunoregulatory effects compared to wild type EHV-1 (WT). Infection with either virus led to cytokine responses that coincided with the course of clinical disease, particularly the biphasic pyrexia, which correlates with respiratory disease and viremia, respectively. Similarly, both viruses caused suppression of proliferative T-cell responses on day 7 post infection (pi). The ΔORF1/ORF2 virus caused significantly shorter primary pyrexia and significantly reduced nasal shedding, and an attenuated decrease in PBMC IL-8 as well as increased Tbet responses compared to WT-infected ponies. In conclusion, our findings are (i) that infection of ponies with EHV-1 leads to modulation of immune responses, which are correlated with disease pathogenesis, and (ii) that the ORF1/2 genes are of importance for disease outcome and modulation of cytokine responses.

Figure 1

(A). Genomic organization of RacH, Ab4 wild type and the recombinant Ab4 OFR1/2 deletion mutant. Shown is the RacH and Ab4genome with a detailed organization of parts of the unique long (UL) and unique short (US) regions, along with parts of the inverted and terminal repeat regions (IR & TR, shaded in grey). In addition, the genome of the recombinant Ab4 ORF1/2 mutant is shown where the genes ORF1 and ORF2 were deleted. B. Restriction fragment length polymorphisms (RFLPs) that confirm correct deletion of the ORF1 and 2 genes in the pAb4 background. BAC DNA was digested with NotI and separated by electrophoresis on a 0.8% agarose gel. Staining of the gel with ethidium bromide (EtBr) shows changes in RFLP patters obtained, indicated by circles, after the first recombination (insertion of the aphA1 gene; pAb4 intermediate) and the second recombination (removal of aphA1 gene and the ORF1/2 genes; pAb4ΔORF1/2). (C). Western blot analysis detecting the expression of gp2 in cellysates of cells infected with reconstituted or not reconstituted Ab4ΔORF1/2. Mock-infected cells (RK13 cells) were included as controls. Cellysates were loaded and separated by SDS-12% PAGE under reducing conditions. Separated proteins were transferred to a nitrocellulose membrane and incubated with monoclonal antibodies (mAb) 3B12 (against gp2; 1:10) or the control mAb L3ab (against ETIF, 1:2000). The sizes of the PageRuler Prestained protein Ladder (Fermentas) are given in thousands.

Figure 2

Mean clinical score, body temperature, viral nasal shedding, and viremia following infection. Clinical scores (a). Body temperatures are indicated in degree Celcius and the dotted line indicates fever (defined as a T ≥ 38.6°C) (b). Viral nasal shedding is represented as mean log gB copy numbers in nasal swabs as determined by real time PCR (c) and viremia is represented as log gB copy numbers/106 copies of β-actin in PBMCs (d). Controls (n = 5): white circles, Ab4 WT infected (n = 7): grey triangles, Ab4 delta ORF1/2 infected (n = 7): black squares. Data are displayed as means + SEM.

Figure 3

EHV-1 serum neutralization titers (a) and kinetic ELISA IgG subclass anti-EHV-1 antibody responses (b-e). Controls (n = 5): white circles, Ab4 WT infected (n = 7): grey triangles, Ab4 delta ORF1/2 infected (n = 7): black squares. Data are displayed as means + SEM. Asterisks indicate statistically significant differences between infection groups, + indicates significant differences with the controls (p < 0.05).

Figure 4

EHV-1 specific cytokine mRNA responses at days 1-7 pi. Relative quantities (RQ) represent x-fold increases over average levels observed for the same cytokine on day -2 prior the experimental infections. Controls (n = 5): white bars, Ab4 WT infected (n = 7): grey bars, Ab4 delta ORF1/2 infected (n = 7): black bars. Days 1-7 pi are represented by figure a-g respectively. Data are displayed as means + SEM. A statistical difference from the control group is indicated by an * (p < 0.05) and between the two infection groups by an + (p < 0.05).

Figure 5

Lymphoproliferative responses to in vitro stimulation with PHA on day 7 post infection. Controls (n = 5): white bars, Ab4 WT infected (n = 7): grey bars, Ab4 delta ORF1/2 infected (n = 7): black bars. Data are displayed as means + SEM. A statistical difference from the control group is indicated by * (p < 0.05).