Increase in Virus-Specific Mucosal Antibodies in the Upper Respiratory Tract Following Intramuscular Vaccination of Previously Exposed Horses Against Equine Herpesvirus Type-1/4

Abstract

Background/objectives: Equine herpesvirus type-1 (EHV-1) enters through the upper respiratory tract (URT) and causes respiratory disease, abortions, and myeloencephalopathy in equids. Pre-existing immunity at the viral entry site, especially mucosal IgG4/7 antibodies, has recently been shown to correlate with protection from disease and incomplete viral replication at the URT. Here, we tested whether intramuscular (i.m.) vaccination with a commercial inactivated EHV-1/4 vaccine can induce mucosal antibodies (mucAbs) at the URT.

Methods: Adult horses with complete EHV-1 vaccination and/or exposure histories were vaccinated i.m. six times within eight months. Before and after each vaccination, blood and nasal swab samples were obtained. Serum and mucAbs were measured in fluorescent bead-based EHV-1 assays.

Results: All horses still had existing EHV-1 specific serum and mucAbs prior to vaccination, which were mainly composed of IgG4/7 antibody isotypes. Serum IgG4/7 significantly increased after the first vaccination and stayed high until the end of the study. An additional short-lasting serum IgG1 response was only induced by the first vaccine application. At the URT, mucAbs increased after five out of six i.m. vaccine injections. Like the systemic antibody response, mucAbs were dominated by IgG4/7 and a small IgG1 increase after the first vaccination.

Conclusions: Our data emphasize that robust EHV-1 specific mucAb levels are obtained after i.m. vaccination with the inactivated EHV-1/4 vaccine used here. The findings have important implications for evaluating EHV-1/4 vaccines for their ability to induce and maintain protective mucosal IgG4/7 antibodies.

Keywords: IgA; IgG1; IgG4/7; antibody; equine herpesvirus; frequent vaccination; inactivated vaccine; mucosal immunity; upper respiratory tract.

Figure 1

Study timeline of frequent EHV-1/4 vaccination and sampling. Horses with existing immunity from previous vaccination against EHV-1/4 (n = 6) or experimental infection with EHV-1 (n = 8) were repeatedly vaccinated with Calvenza EHV^® (V1–V6). The vaccine was given i.m. into the pectoral muscles. Serum and nasal swab samples for antibody measurement were taken immediately before each vaccination and at several additional time points for this study. The red arrows show all sampling times. A similar frequent vaccination approach with the same vaccine was previously performed in pregnant mares and resulted in decreasing serum antibodies after some of the vaccine administrations. In this study, we aimed to analyze if adult non-pregnant mares and/or geldings would respond similarly to repeated vaccination.

Figure 2

Anti-EHV-1 gC serum antibody decline in 14 adult horses in a 22-month period without EHV-1/4 exposure. The graph starts after the last EHV-1/4 exposure (arrow) and ends before the first vaccination (V1, arrow)) of the frequent EHV-1/4 vaccination approach described in this article. All horses had a complete EHV-1/4 vaccination and infection history. The last EHV-1/4 exposure of these horses was 22 or 21 months prior to the start of the frequent vaccination. Samples were taken at 21 or 20, 15 or 14, 11, 4, and 2 months, and at 0 month directly before V1. Serum antibodies against EHV-1 glycoprotein C (gC) were quantified in a fluorescent bead-based assay (EHV-1 Risk Evaluation assay). EHV-1 gC-specific total Ig and IgG4/7 antibodies were measured, and values are shown as median fluorescent intensity (MFI). The black dotted horizontal line shows the anti-gC total Ig cut-off value at 3000 MFI and the green dotted line at 400 MFI is the anti-gC IgG4/7 cut-off value. Antibody values above the cut-off value are indicative of a low infection risk if horses are exposed to EHV-1. p-values (*** p < 0.001, **** p < 0.0001) mark serum antibody decreases compared to antibodies at 21/20 months before V1. The color of the asterisks represents serum total Ig (black) or IgG4/7 (green). Brackets span identical p-value ranges.

Figure 3

Serum antibody responses after frequent i.m. vaccination of horses with an inactivated EHV-1/4 vaccine. Adult horses (n = 14) with pre-existing immunity against EHV-1 were repeatedly vaccinated (V1–V6). Serum antibodies against EHV-1 glycoprotein C (gC) were quantified in a fluorescent bead-based assay (EHV-1 Risk Evaluation assay) at a serum dilution of 1:400. (A) EHV-1-specific total Ig and (B) IgG1, IgG4/7 and IgG3/5 isotypes in serum were measured as median fluorescent intensity (MFI). The gray dashed vertical lines show the times of vaccination after the serum sample were taken on the respective days. The dotted horizontal line in the upper graph shows the anti-gC total Ig cut-off value at 3000 MFI and antibody values above the cut-off value are indicative of a low infection risk if horses are exposed to EHV-1. p-values (* p < 0.05, *** p < 0.001, **** p < 0.0001) mark serum antibody increases compared to pre-V1 antibodies. The color of the asterisks represents the respective serum total Ig or isotypes. Brackets span identical p-value ranges.

Figure 4

Nasal mucosal antibodies (mucAbs) after frequent EHV-1/4 vaccination. Horses with pre-existing immunity against EHV-1 (n = 14) were repeatedly vaccinated i.m. with an inactivated EHV-1/4 vaccine (Calvenza EHV^®, V1–V6). MucAbs against EHV-1 gC were measured in undiluted nasal secretion samples using a fluorescent bead-based multiplex assay. (A) MucAb total Ig levels and (B) mucosal IgG1(blue), IgG4/7 (green), and IgA (sky blue) isotypes are shown as median fluorescent intensity (MFI). The dotted horizontal line in (A) represents the anti-gC total Ig cut-off value for serum. The dashed vertical lines mark the times of vaccine administration. p-values (* p < 0.05, ** p < 0.01, *** p < 0.001) show mucAb increases in comparison to mucAbs pre-V1 vaccination. The color of the asterisks represents the respective mucAb isotypes. The bracket spans IgG4/7 results with identical p-values.