An equine herpesvirus type 1 (EHV-1) Ab4 open reading frame (ORF)2 deletion mutant provides immunity and protection from EHV-1 infection and disease.

Equine Herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n=8 each) were infected intranasally with Ab4ΔORF2, the parent Ab4 virus, or kept as non-infected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously non-infected horses (Control/Ab4) displayed clinical signs, shed high amounts of virus, and developed cell-associated viremia. In contrast, 5/8 and 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection and intranasal antibodies increased rapidly post challenge. Intranasal inflammatory markers were not detectable in protected horses, but quickly increased in Control/Ab4 horses during the first week after infection. Overall, our data suggest that pre-existing nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate. Importance Nasal EHV-1 shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination, emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to pre-existing local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies, and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.