Equid herpesvirus-1 (EHV-1) outbreaks continue despite widely used vaccination. We demonstrated previously that an ORF1/ORF71 gene deletion mutant of the EHV-1 strain Ab4 (Ab4ΔORF1/71) is less virulent than its parent Ab4 virus. Here, we describe the Ab4 challenge infection evaluating protection induced by the Ab4ΔORF1/71 vaccine candidate. Susceptible control horses developed respiratory disease, fever, nasal shedding, and viremia. Full protection after challenge infection was observed in 5/5 previously Ab4 infected horses and 3/5 Ab4ΔORF1/71 horses. Two Ab4ΔORF1/71 horses developed short-lasting viremia and/or virus shedding. Protective immunity in the respiratory tract was characterized by pre-existing EHV-1-specific IgG4/7 antibodies, the absence of IFN-α secretion and rapidly increasing IgG4/7 upon challenge infection. Pre-existing systemic EHV-1-specific IgG4/7 highly correlated with protection. T-cell immunity was overall low. In conclusion, protective immunity against EHV-1 infection including prevention of viremia was associated with robust systemic and intranasal IgG4/7 antibodies suggesting immediate virus neutralization at the local site.

Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses’ clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10–12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5–10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract – Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (r_sp = 0.726), while anti-RBD correlation was moderate (r_sp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.

Human IgE-binding monocytes are identified as allergic disease mediators, but it is unknown whether IgE-binding monocytes promote or prevent an allergic response. We identified IgE-binding monocytes in equine peripheral blood as IgE+/MHCII^high/CD14^low cells that bind IgE through an FcεRI αɣ variant. IgE-binding monocytes were analyzed monthly in Culicoides hypersensitive horses and nonallergic horses living together with natural exposure to Culicoides midges. The phenotype and frequency of IgE-binding monocytes remained consistent in all horses regardless of Culicoides exposure. All horses upregulated IgE-binding monocyte CD16 expression following initial Culicoides exposure. Serum total IgE concentration and monocyte surface IgE densities were positively correlated in all horses. We also demonstrated that IgE-binding monocytes produce IL-10, but not IL-4, IL-17A, or IFN-γ, following IgE crosslinking. In conclusion, we have characterized horse IgE-binding monocytes for the first time and further studies of these cells may provide important connections between regulation and cellular mechanisms of IgE-mediated diseases.

IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14⁺/MHC class II⁺/CD16⁻ monocytes, including a subpopulation of IgE-binding monocytes, following stimulation with LPS. In addition, IgE cross-linking induced IL-8 production by both peripheral blood basophils and IgE-binding monocytes. IL-8 production was compared between healthy horses and those with a naturally occurring IgE-mediated skin allergy, Culicoides hypersensitivity. Allergic horses had significantly higher percentages of IL-8⁺ IgE-binding monocytes after IgE cross-linking. In contrast, frequencies of IL-8⁺ basophils after IgE cross-linking were similar in all horses, regardless of allergic disease, highlighting IgE-binding monocytes as a novel source of IL-8 during allergy. We concluded that IgE-binding monocytes from allergic individuals have an increased capacity for IL-8 production and likely contribute to the recruitment of innate immune cells during IgE-mediated allergy and promotion of inflammation during repeated allergen contact. Key Points A novel mAb identifies a role for IL-8 in IgE-mediated allergy. IgE cross-linking induces IL-8 production in IgE-binding monocytes and basophils. Allergic horses have higher IL-8⁺/IgE⁺ monocyte percentages after IgE cross-linking.