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. 2012 Jun 6;30(26):3965-74.
doi: 10.1016/j.vaccine.2012.03.026. Epub 2012 Mar 23.

Immunogenicity and clinical protection against equine influenza by DNA vaccination of ponies

Alida Ault  1 , Alyse M Zajac, Wing-Pui Kong, J Patrick Gorres, Michael Royals, Chih-Jen Wei, Saran Bao, Zhi-yong Yang, Stephanie E Reedy, Tracy L Sturgill, Allen E Page, Jennifer Donofrio-Newman, Amanda A Adams, Udeni B R Balasuriya, David W Horohov, Thomas M Chambers, Gary J Nabel, Srinivas S Rao
Affiliations

Immunogenicity and clinical protection against equine influenza by DNA vaccination of ponies

Alida Ault et al. Vaccine. .

Abstract

Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza.

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Conflict of interest statement

To fully disclose any potential conflicts of interest, authors Gary J. Nabel, Srinivas S. Rao, Wing-Pui Kong, and Chih-Jen Wei are each listed on a patent filing for our DNA vaccine technology, entitled “U.S. Continuation in Part Patent Application No. 12/838,292”, which is an adjunct to an existing patent entitled “Influenza DNA Vaccination and Methods of Use thereof”, Serial #61/023,341.

Figures

Figure 1
Figure 1. Experimental schema
Ponies were immunized at weeks 0, 4 and 8 with blood draws in between vaccinations. All animals were challenged at week 15 with Ohio/03 virus. Pre- and post-challenge rectal temperatures, clinical scores, blood, and nasal swabs were collected for assays.
Figure 2
Figure 2. Microneutralization titers against H3N8 viruses from mouse studies
Serum endpoint dilutions calculated to completely neutralize 200 TCID50 units of Ohio/03 (striped bar), Newmarket/2/93 (solid bar) and canine/KY/06 (open bar) viruses in 50% of wells are shown. The x-axis indicates the different vaccination groups. B = Bari/05, O = Ohio/03, N = Newmarket/93. Horse convalescent serum challenged with Ohio/03 was used as the positive control.
Figure 3
Figure 3. HI antibody responses following vaccination and challenge
Mean HI titers to Ohio/03 (a), Aboyne/05 (b), and Richmond/07 (c) antigen. For graphing purposes, a titer of <10 was assumed to be 5 and is denoted as a value of log10(5), which also serves as the y origin. Error bars represent Standard Error of the Mean (SEM). Ponies received three doses of vaccine (V1, V2 and V3) and were challenged with live equine influenza virus (Ch). Monovalent = Ohio/03 HA, Trivalent = Ohio/03 HA + Aboyne/05 HA + Bari/05 HA, Control = empty CMV/R vector, NS = needle and syringe, NF = needle-free device.
Figure 4
Figure 4. SRH antibody responses following vaccination and challenge
Mean SRH antibody in vaccinated horses measured against (a) Ohio/03, (b) Aboyne/05, and (c) Richmond/07. Error bars represent SEM. Ponies received three doses of vaccine (V1, V2 and V3) and were challenged with live equine influenza virus (Ch). Monovalent = Ohio/03 HA, Trivalent = Ohio/03 HA + Aboyne/05 HA + Bari/05 HA, Control = empty CMV/R vector, NS = needle and syringe, NF = needle-free device.
Figure 5
Figure 5. Mean clinical scores post-challenge
(a) Mean daily clinical scores for 8 days post-challenge. (b) Mean summed clinical scores for days 1 through 8 broken down by clinical sign. Error bars represent SEM. See Table 1 for scoring index. (Resp = respiration, Dem = demeanor, N Dis = nasal discharge, Cough = coughing) Ponies received three doses of vaccine (V1, V2 and V3) and were challenged with live equine influenza virus (Ch). Monovalent = Ohio/03 HA, Trivalent = Ohio/03 HA + Aboyne/05 HA + Bari/05 HA, Control = empty CMV/R vector, NS = needle and syringe, NF = needle-free device. One pony in the monovalent NF group was euthanized at wk 10 due to unrelated cecocolic intusseption with secondary peritonitis. Necropsy investigation revealed that this was not a vaccine-related event.
Figure 6
Figure 6. Mean daily rectal temperature post-challenge
The mean rectal temperatures for the first three days following challenge. After day +3, all temperatures returned to normal range. Error bars represent SEM. Ponies received three doses of vaccine (V1, V2 and V3) and were challenged with live equine influenza virus (Ch). Monovalent = Ohio/03 HA, Trivalent = Ohio/03 HA + Aboyne/05 HA + Bari/05 HA, Control = empty CMV/R vector, NS = needle and syringe, NF = needle-free device.
Figure 7
Figure 7. Pro-inflammatory cytokine mRNA induction post-challenge
Interleukin 6 (IL-6) (a), interleukin 1 (IL-1) (b), tumor necrosis factor-alpha (TNFα) (c), and interferon-gamma (IFNγ) (d) expression in whole blood collected in PAXgene tubes was determined by quantitative RT-PCR. RQ is the ratio of each daily sample’s normalized measurement to that of the Day -1 average (calibrator). Error bars represent SEM. Ponies received three doses of vaccine (V1, V2 and V3) and were challenged with live equine influenza virus (Ch). Monovalent = Ohio/03 HA, Trivalent = Ohio/03 HA + Aboyne/05 HA + Bari/05 HA, Control = empty CMV/R vector, NS = needle and syringe, NF = needle-free device.
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