Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun 10:8:674850.
doi: 10.3389/fvets.2021.674850. eCollection 2021.

A Live-Attenuated Equine Influenza Vaccine Stimulates Innate Immunity in Equine Respiratory Epithelial Cell Cultures That Could Provide Protection From Equine Herpesvirus 1

Lila M Zarski  1 , Wendy E Vaala  2 , D Craig Barnett  2 , Fairfield T Bain  2 , Gisela Soboll Hussey  1
Affiliations

A Live-Attenuated Equine Influenza Vaccine Stimulates Innate Immunity in Equine Respiratory Epithelial Cell Cultures That Could Provide Protection From Equine Herpesvirus 1

Lila M Zarski et al. Front Vet Sci. .

Abstract

Equine herpesvirus 1 (EHV-1) ubiquitously infects horses worldwide and causes respiratory disease, abortion, and equine herpesvirus myeloencephalopathy. Protection against EHV-1 disease is elusive due to establishment of latency and immune-modulatory features of the virus. These include the modulation of interferons, cytokines, chemokines, antigen presentation, and cellular immunity. Because the modulation of immunity likely occurs at the site of first infection-the respiratory epithelium, we hypothesized that the mucosal influenza vaccine Flu Avert® I.N. (Flu Avert), which is known to stimulate strong antiviral responses, will enhance antiviral innate immunity, and that these responses would also provide protection from EHV-1 infection. To test our hypothesis, primary equine respiratory epithelial cells (ERECs) were treated with Flu Avert, and innate immunity was evaluated for 10 days following treatment. The timing of Flu Avert treatment was also evaluated for optimal effectiveness to reduce EHV-1 replication by modulating early immune responses to EHV-1. The induction of interferons, cytokine and chemokine mRNA expression, and protein secretion was evaluated by high-throughput qPCR and multiplex protein analysis. Intracellular and extracellular EHV-1 titers were determined by qPCR. Flu Avert treatment resulted in the modulation of IL-8, CCL2, and CXCL9 starting at days 5 and 6 post-treatment. Coinciding with the timing of optimal chemokine induction, our data also suggested the same timing for reduction of EHV-1 replication. In combination, our results suggest that Flu Avert may be effective at counteracting some of the immune-modulatory properties of EHV-1 at the airway epithelium and the peak for this response occurs 5-8 days post-Flu Avert treatment. Future in vivo studies are needed to investigate Flu Avert as a prophylactic in situations where EHV-1 exposure may occur.

Keywords: EHV-1; epithelial cell; equine influenza vaccine; horse; mucosal immunity.

PubMed Disclaimer

Conflict of interest statement

WV, DB, and FB are employed by MSD Animal Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cell viability and chemokine mRNA expression following Flu Avert treatment in ERECs. (A) Microscopic analysis of EREC cultures. Mean grades from cells ± SEM of three horses following Flu Avert treatment at different MOIs. Grading of cell viability from 0 to 3 is described in Table 1. (B) Cell viability analysis. Mean percent positive cells ± SEM of three horses at each MOI as determined with propidium iodide staining. Black circle is MOI = 0. Black square is MOI = 0.1. Black upright triangle is MOI = 1. Black upside-down triangle is MOI = 5. (C) IL-8 mRNA expression. (D) CXCL9 mRNAs expression. (E) CCL2 mRNA expression. Values are mean log fold change (–ddCq) ± SEM. Black square represents untreated (media) treated ERECs. Black circle represents Flu Avert-treated ERECs. *p ≤ 0.05 and **p < 0.01, respectively, between the media and Flu Avert treatment groups.
Figure 2
Figure 2
Mean ± SEM difference in intracellular EHV-1 copy number between Flu Avert-treated and media-treated ERECs from five horses. (A) Intracellular copy number in ERECs treated with Flu Avert or media on day 1 prior to EHV-1 inoculation. (B) Intracellular copy number in ERECs treated with Flu Avert or media on day 2 prior to EHV-1 inoculation. (C) Intracellular copy number in ERECs treated with Flu Avert or media on day 5 prior to EHV-1 inoculation. (D) Intracellular copy number in ERECs treated with Flu Avert or media on day 7 prior to EHV-1 inoculation.
Figure 3
Figure 3
Mean ± SEM difference in extracellular EHV-1 copy number between Flu Avert-treated and media-treated ERECs from five horses. (A) Extracellular copy number in cells treated with Flu Avert or media on day 1 prior to EHV-1 inoculation. (B) Extracellular copy number in cells treated with Flu Avert or media on day 2 prior to EHV-1 inoculation. (C) Extracellular copy number in cells treated with Flu Avert or media on day 5 prior to EHV-1 inoculation. (D) Extracellular copy number in cells treated with Flu Avert or media on day 7 prior to EHV-1 inoculation.
Figure 4
Figure 4
Effect of Flu Avert treatment on chemokine mRNA expression in ERECs collected from five horses 24 h following EHV-1 inoculation. (A) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (B) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. (C) CXCL10 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (D) CXCL10 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. The mean log fold change (–ddCq) is represented by a bar (*p < 0.05).
Figure 5
Figure 5
Effect of Flu Avert treatment on chemokine mRNA expression in ERECs collected from five horses 48 h following EHV-1 inoculation. (A) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 1 prior to EHV-1 inoculation. (B) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 2 prior to EHV-1 inoculation. (C) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (D) IL-8 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. (E) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 1 prior to EHV-1 inoculation. (F) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 2 prior to EHV-1 inoculation. (G) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (H) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. The mean log fold change (–ddCq) is represented by a bar (**p < 0.01 and *p < 0.05).
Figure 6
Figure 6
Effect of Flu Avert treatment on chemokine mRNA expression in ERECs collected from five horses 72 h following EHV-1 inoculation. (A) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 2 prior to EHV-1 inoculation. (B) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (C) CCL2 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. (D) CXCL9 mRNA expression in ERECs that were treated with Flu Avert on day 2 prior to EHV-1 inoculation. (E) CXCL9 mRNA expression in ERECs that were treated with Flu Avert on day 5 prior to EHV-1 inoculation. (F) CXCL9 mRNA expression in ERECs that were treated with Flu Avert on day 7 prior to EHV-1 inoculation. The mean log fold change (–ddCq) is represented by a bar (*p < 0.05).
Figure 7
Figure 7
IL-10 protein expression in EREC supernatants collected from five horses. ERECs were treated with Flu Avert (or left untreated) on day 7 prior to EHV-1 inoculation, and supernatants of cultures were collected 72 h post-EHV-1 infection. The mean concentration (pg/ml) is represented by a bar (*p < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Gilkerson JR, Whalley JM, Drummer HE, Studdert MJ, Love DN. Epidemiological studies of equine herpesvirus 1 (EHV-1) in thoroughbred foals: a review of studies conducted in the hunter valley of New South Wales between 1995 and 1997. Vet Microbiol. (1999) 68:15–25. 10.1016/S0378-1135(99)00057-7 - DOI - PubMed
    1. Gilkerson JR, Whalley JM, Drummer HE, Studdert MJ, Love DN. Epidemiology of EHV-1 and EHV-4 in the mare and foal populations on a hunter valley stud farm: are mares the source of EHV-1 for unweaned foals. Vet Microbiol. (1999) 68:27–34. 10.1016/S0378-1135(99)00058-9 - DOI - PubMed
    1. Foote CE, Love DN, Gilkerson JR, Whaley JM. Detection of EHV-1 and EHV-4 DNA in unweaned thoroughbred foals from vaccinated mares on a large stud farm. Equine Vet J. (2010) 36:341–5. 10.2746/0425164044890634 - DOI - PubMed
    1. Mumford JA, Rossdale PD, Jessett DM, Gann SJ, Ousey J. Serological and virological investigations of an equid herpesvirus 1 (EHV-1) abortion storm on a stud farm in 1985. J Reprod Fertil Suppl. (1987) 35:509–18. - PubMed
    1. Pusterla N, Mapes S, Wilson WD. Prevalence of equine herpesvirus type 1 in trigeminal ganglia and submandibular lymph nodes of equids examined postmortem. Vet Rec. (2010) 167:376–8. 10.1136/vr.c3748 - DOI - PubMed