Neonatal Immunization with a Single IL-4/Antigen Dose Induces Increased Antibody Responses after Challenge Infection with Equine Herpesvirus Type 1 (EHV-1) at Weanling Age

Neonatal foals respond poorly to conventional vaccines. These vaccines typically target T-helper (Th) cell dependent B-cell activation. However, Th2-cell immunity is impaired in foals during the first three months of life. In contrast, neonatal basophils are potent interleukin-4 (IL-4) producers. The purpose of this study was to develop a novel vaccine triggering the natural capacity of neonatal basophils to secrete IL-4 and to evaluate if vaccination resulted in B-cell activation and antibody production against EHV-1 glycoprotein C (gC). Neonatal vaccination was performed by oral biotinylated IgE (IgE-bio) treatment at birth followed by intramuscular injection of a single dose of streptavidin-conjugated gC/IL-4 fusion protein (Sav-gC/IL-4) for crosslinking of receptor-bound IgE-bio (group 1). Neonates in group 2 received the intramuscular Sav-gC/IL-4 vaccine only. Group 3 remained non-vaccinated at birth. After vaccination, gC antibody production was not detectable. The ability of the vaccine to induce protection was evaluated by an EHV-1 challenge infection after weaning at 7 months of age. Groups 1 and 2 responded to EHV-1 infection with an earlier onset and overall significantly increased anti-gC serum antibody responses compared to control group 3. In addition, group 1 weanlings had a decreased initial fever peak after infection indicating partial protection from EHV-1 infection. This suggested that the neonatal vaccination induced a memory B-cell response at birth that was recalled at weanling age after EHV-1 challenge. In conclusion, early stimulation of neonatal immunity via the innate arm of the immune system can induce partial protection and increased antibody responses against EHV-1.Funding for this project was provided by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University ‘A Novel Strategy to Boost Antibody Production to EHV-1 in Neonates’ (http://vet.cornell.edu/research/Zweig/). Monoclonal antibody development for horse cell surface markers and cytokines was supported by USDA grant #2005-01812 ‘The US Veterinary Immune Reagent Network’ and #2015-67015-23072 ‘Equine Immune Reagents: Development of monoclonal antibodies to improve the analysis of immunity in horses’ (https://nifa.usda.gov/).Peer Reviewe

Cytokine secretion from PBMC of weanlings after experimental EHV-1 infection <i>in vivo</i> (arrow).

<p>Weanlings in group 1 received IgE and EHV-1 gC antigen as neonates and weanlings in group 2 received EHV-1 gC antigen. Weanlings in the control group 3 did not receive any neonatal. PBMC were isolated up to 5 months post EHV-1 infection. Cells were re-stimulated <i>ex vivo</i> with EHV-1 for 48 hour to provoke cytokine production. PBMC in medium served as non-stimulated control. Cytokines in cell culture supernatants were determined by cytokine multiplex analysis. The cytokine secretion values in graphs A-D were corrected by the non-stimulated control values of each weanling and day. The graphs show means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3; c = groups 1 and 2.</p

Antibodies to gC and gD in serum of weanlings after experimental EHV-1 infection (day 0; arrow).

<p>The weanling groups correspond to IgE-bio and/or EHV-1 gC antigen treatments during the neonatal period. At weanling age all three groups of weanlings were infected with the same dose of EHV-1 strain NY03. Antibodies in serum were determined by a EHV-1 Multiplex assay. The graphs show: (A) total serum anti-EHV-1 gC antibodies and (B) total serum anti-EHV-1 gD antibodies. The graphs represent means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3.</p

Body temperatures, clinical signs, nasal shedding and viremia after experimental infection of weanlings with the EHV-1 strain NY03.

<p>The groups (n = 5 per group) were established based on IgE-bio and/or EHV-1 gC antigen treatments of the foals at birth: group 1 received IgE-bio and Sav-gC/IL-4 antigen; group 2 received Sav-gC/IL-4 antigen; control group 3 no treatment at birth. EHV-1 challenge infection occurred at seven months of age (day 0; arrow) and was performed under identical conditions for all three groups. On day 0, measurements and samples were taken before infection. (A) Body temperatures; the dotted horizontal line shows the cut-off value for fever (101.5°F); (B) clinical scores; (C) nasal shedding expressed as viral copy numbers of the EHV-1 gene gB per ml nasal secretion sample and (D) viremia (gB Ct-value) per 5 x 10⁶ PBMC. Nasal shedding and viremia were evaluated by real-time PCR. The dotted horizontal line shows the positive PCR Ct-value cut-off value. All negative PCR values were set to this value. All graphs show means and standard errors per group. Significant differences between groups: a = groups 1 and 3; c = groups 1 and 2.</p

Anti-gC isotypes in the serum of weanlings after experimental EHV-1 infection (arrow).

<p>Weanlings in group 1 received IgE and EHV-1 gC antigen at birth. Weanlings in group 2 received EHV-1 gC antigen at birth. Weanlings in the control group 3 did not receive any treatment at birth. Antibody isotypes to gC were measured in a EHV-1 Multiplex assay. (A) anti-IgM; (B) anti-IgG6; (C) anti-IgG1; (D) anti-IgG1/3; (E) anti-IgG4/7; (F) anti-gC IgG3/5. The graphs show means and standard errors by group. Significant differences between groups: a = groups 1 and 3; b = groups 2 and 3.</p