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Journal of Virology logoLink to Journal of Virology
. 2014 Nov 19;89(3):1652–1659. doi: 10.1128/JVI.02449-14

A Live Attenuated Equine H3N8 Influenza Vaccine Is Highly Immunogenic and Efficacious in Mice and Ferrets

Mariana Baz a, Myeisha Paskel a, Yumiko Matsuoka a, James Zengel b, Xing Cheng b, John J Treanor c, Hong Jin b, Kanta Subbarao a,✉
Editor: S Perlman
PMCID: PMC4300743  PMID: 25410860

ABSTRACT

Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of respiratory disease in horses. Although natural infections of humans with EIV have not been reported, experimental inoculation of humans with these viruses can lead to a productive infection and elicit a neutralizing antibody response. Moreover, EIV have crossed the species barrier to infect dogs, pigs, and camels and therefore may also pose a threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a live attenuated candidate vaccine by reverse genetics with the hemagglutinin and neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 (H2N2) vaccine donor virus, which is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus induced neutralizing antibodies and conferred complete protection from homologous wt virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also induced neutralizing antibodies and conferred complete protection in mice and nearly complete protection in ferrets upon heterologous challenge with the H3N8 (eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus to humans.

IMPORTANCE Equine influenza viruses have crossed the species barrier to infect other mammals such as dogs, pigs, and camels and therefore may also pose a threat to humans. We believe that it is important to develop vaccines against equine influenza viruses in the event that an EIV evolves, adapts, and spreads in humans, causing disease. We generated a live attenuated H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and protected mice and ferrets against homologous and heterologous EIV.

INTRODUCTION

Equine influenza viruses (EIV) have been responsible for rapidly spreading outbreaks of respiratory disease in horses for centuries. Influenza A viruses contain a single-stranded, negative-sense RNA genome consisting of 8 gene segments and are further classified into subtypes on the basis of the antigenicity of the two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA) (1). Two subtypes of EIV have been isolated from horses: H7N7 and H3N8 viruses. The prototype equine H7N7 virus (A/equine/Prague/56) virus emerged in 1956 (2) but has not been isolated since the late 1970s (3), although serologic evidence suggests that this virus subtype circulated among horses in Europe and the Americas before 1956 (4, 5); its circulation in unvaccinated horses was recorded in the 1980s in India (6) and in the beginning of the 1990s in Europe and the United States (7, 8). Equine H3N8 viruses were first isolated during a major epidemic in Miami in 1963 (A/eq/Miami/1/63) (9) and since then have circulated enzootically in horses, causing significant disease and economic burdens worldwide (10). These viruses have continued to evolve and have diverged into two antigenically and genetically distinct American and European lineages since 1986. The American lineage further evolved into Kentucky, South American, and Florida sublineages. Subsequent evolution within the Florida sublineage has resulted in the emergence of two distinct clades (clades 1 and 2) (11).

Influenza A viruses can be transmitted between species, and this characteristic allows the emergence of reassortant influenza viruses (12). The H3N8 EIV has crossed the species barrier and was transmitted to racing greyhounds that shared a racing track with horses in Florida in January 2004 (13), although retrospective serologic analysis suggests that H3N8 influenza viruses were circulating in racing greyhounds since 1999 (14). Subsequently, canine H3N8 influenza viruses spread to pet dogs and became enzootic in the United States (15). Canine H3N8 infections have also been reported in the United Kingdom, Australia, and Algeria (16–19). Studies on the distribution of the sialoreceptors in the respiratory tract of horses and dogs have shown that both horses and dogs have a predominance of sialic acid alpha-2,3-galactose (SAα2,3-gal) receptors (13, 18, 20). Pecoraro et al. have recently shown by binding assays that canine and equine influenza isolates have a higher affinity for SAα2,3-gal than for SAα2,6-gal receptors (20). These data may explain the natural transmission of equine influenza virus to dogs.

In addition, two H3N8 influenza viruses were isolated from pigs in central China during surveillance for swine influenza in 2004 to 2006. Sequence and phylogenetic analyses of the eight gene segments revealed that the two swine isolates were of equine origin and were most closely related to European H3N8 EIV from the early 1990s (21). Recently, an EIV (H3N8) was isolated from a Bactrian camel in Mongolia, highlighting a novel interspecies transmission (22).

While natural transmission of EIV to humans has not been documented, experimental challenge studies done in the 1960s indicate that the influenza A/equi 2/Miami/1/63 virus was able to infect 64% of 33 human volunteers who received an intranasal dose of between 104.6 and 105.3 50% tissue culture infectious doses (TCID50) of virus. However, illness occurred in only 12% of the volunteers, suggesting that the virus was more virulent for horses than for humans (23–25). Human birth cohorts from the late 19th century, particularly for individuals born before 1890, demonstrated serologic reactivity with equine H3N8 viruses many decades later (26). However, a recent study reported by Burnell et al. showed sparse evidence for H3N8 infection in 100 subjects enrolled during equine events in Australia (27). In that study, only nine subjects showed serologic reactivity against EIV antigens, and although eight of the subjects reported horse exposure, antibody titers were low except in the case of one subject who had a titer of 1:80 by microneutralization (MN) assay. Another study done by Khurelbaatar et al. also showed sparse evidence for EIV infection in 439 subjects ≥18 years of age (28), though 76% of the participants reported exposure to horses.

The social and economic impact of widespread disease caused by EIV in humans could be devastating, since people in different regions of the world still rely heavily upon horses for recreation, communication, and military or general transport and EIV has already crossed the species barrier to dogs. We believe that it is important to develop vaccines against animal influenza viruses of the H3 subtype. The ongoing circulation of seasonal H3N2 viruses does not preclude the possibility of a pandemic caused by an antigenically distinct animal H3 virus, as demonstrated by the unexpected emergence of a swine origin H1N1 influenza virus as a pandemic strain in 2009 despite the ongoing circulation of seasonal H1N1 viruses.

Seasonal live attenuated influenza vaccines (LAIV) have been licensed in the United States since 2003, and they elicit both systemic and local mucosal immunity (29, 30). Our laboratories have previously generated live attenuated H1N1, H2N2, H5N1, H6N1, H7N3, H7N7, H7N9, and H9N2 viruses and found that these candidate vaccines were safe and efficacious in conferring protection against wild-type (wt) viruses in mice and ferrets (31–36); several of these vaccines have been evaluated in phase 1 clinical trials (33, 37, 38).

We analyzed the antigenic relatedness and replicative capacity of H3N8 EIV from the predivergent and American lineage (sublineage Florida clade 1 and 2) viruses using postinfection mouse and ferret sera (39). We selected A/equine/Georgia/1/1981 (eq/GA/81) for vaccine development because it induced the most broadly cross-neutralizing antibodies (NtAb) and replicated to high titers in the upper respiratory tracts of mice and ferrets (39). We used reverse genetics to generate a live attenuated cold-adapted (ca) H3N8 virus bearing wt HA and NA genes from the eq/GA/81 wt virus and the six internal protein gene segments from the ca influenza A vaccine donor strain, A/Ann Arbor/6/60 (H2N2). The immunogenicity and protective efficacy against challenge with the homologous wt eq/GA/81 and heterologous A/equine/Newmarket/5/2003 (eq/Newm/03) viruses were evaluated in mice and ferrets.

MATERIALS AND METHODS

Viruses.

H3N8 EIV isolates were provided by Richard Webby, St. Jude Children's Research Hospital, Memphis, TN (A/equine/Georgia/1/1981 [H3N8]), and Debra Elton, Animal Health Trust, Newmarket, United Kingdom (A/equine/Newmarket/5/2003 [H3N8]). The HA amino acid sequence identity between eq/GA/81 and eq/Newm/03 is 97.3% (39). eq/GA/81 belongs to the Florida clade 1 sublineage, and eq/Newm/03 belongs to the Florida clade 2 sublineage (40). We have previously reported (39) that sera from ferrets infected with eq/GA/81 showed cross-reactivity against the homologous virus (NtAb titer ≥ 1,280) as well as eq/Newm/03 (NtAb titer ≥ 640). Virus stocks were propagated in the allantoic cavities of 9- to 11-day-old embryonated specific-pathogen-free hens' eggs (Charles River Laboratories, North Franklin, CT) at 35°C. The allantoic fluid was harvested at 72 h postinfection (p.i.), tested for hemagglutinating activity using 0.5% turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA), pooled, aliquoted, and stored at −80°C until use. Virus titers were determined in Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA) and calculated using the method of Reed and Muench (41).

Generation of reassortant eq/GA/81 ca vaccine virus by reverse genetics.

The HA and NA gene segments of eq/GA/81 (H3N8) were amplified from viral RNA (vRNA) by reverse transcription-PCR (RT-PCR) using primers that are universal to the HA and NA genes, sequenced, and cloned into the plasmid vector pAD3000 (42). The 6:2 reassortant vaccine virus was generated by cotransfecting eight plasmids encoding the HA and NA of the eq/GA/81 virus and the 6 internal protein gene segments of the A/Ann Arbor/6/60 ca virus into cocultured 293T and MDCK cells. At 3 to 5 days posttransfection, the transfected cell supernatant was inoculated into the allantoic cavities of 10- to 11-day-old embryonated chicken eggs (Charles River Laboratories) and incubated at 33°C for 2 days. Virus titer was determined by immunostaining plaques using an anti-NP monoclonal antibody and expressed as log10 PFU (PFU)/ml as previously described (43). The HA and NA sequences of the rescued virus were verified by sequencing the genes amplified from viral RNA by RT-PCR.

Serologic assays.

Anti-influenza virus antibody titers in serum samples were measured by hemagglutination inhibition (HAI) according to standard protocols (44) or by microneutralization (MN) assay as previously described (45). For the HAI assay, nonspecific inhibitors were removed from serum by overnight treatment with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan). Sera were 2-fold serially diluted in 96-well V-bottom plates starting at a dilution of 1:10, and 4 HA units of virus were added. Control wells received phosphate-buffered saline (PBS) alone. Virus and sera were incubated together for 30 min at room temperature, and 50 μl of a 0.5% (vol/vol) suspension of turkey erythrocytes was added. The virus-serum mixture and erythrocytes were gently mixed, and the results were recorded after incubation for 45 min at room temperature. HAI titers were recorded as the inverse of the highest antibody dilution that inhibited hemagglutination. A cross-reactive antibody response was defined as a ≤4-fold difference between the homologous HAI titer and the titer generated against the heterologous virus. For the MN assay, serial 2-fold dilutions of heat-inactivated serum were prepared starting from a 1:20 dilution. Equal volumes of serum and virus were mixed and incubated for 60 min at room temperature. The residual infectivity of the virus-serum mixture was determined in MDCK cells in 4 replicates for each dilution of serum. The NtAb titer was defined as the reciprocal of the serum dilution that completely neutralized the infectivity of 100 TCID50 of the virus as determined by the absence of cytopathic effect on MDCK cells at day 4. A cross-reactive antibody response was defined as a ≤4-fold difference between the homologous NtAb titer and the titer generated against the heterologous virus.

Immunogenicity and protective efficacy of the H3N8 ca virus in mice.

Six- to 8-week-old female BALB/c mice (Taconic Farms, Inc., Germantown, NY) were used in all mouse experiments. Animal studies were conducted in biosafety level 2 (BSL2) laboratories at the National Institutes of Health (NIH), and protocols were approved by the National Institutes of Health Animal Care and Use Committee.

Groups of eight mice were lightly anesthetized and inoculated intranasally (i.n.) with 50 μl containing 106 PFU of the H3N8 ca vaccine virus in one or two doses. Mock-inoculated controls received Leibovitz-15 (L15) medium alone. Neutralizing antibody responses to homologous (eq/GA/81) and heterologous (eq/Newm/03) H3N8 wt viruses were determined from sera collected prior to inoculation (prebleed) and at 38 days after the first or second immunization by MN assay (45).

On day 38 after the first or second dose of vaccine, groups of eight mice were challenged i.n. with 105 TCID50 of the H3N8 equine wt viruses, eq/GA/81 or eq/Newm/03. Four mice per challenge virus were sacrificed on days 2 and 4 postchallenge (p.c.), and lungs and nasal turbinates (NTs) were harvested and stored at −80°C. We chose these time points based on previous observations in our laboratory that equine wt viruses replicate to high titers in the NTs and lungs of mice from days 2 to 4 postinfection (39). Organs were weighed and homogenized in L15 medium containing 2× antibiotic-antimycotic (penicillin, streptomycin, and amphotericin B) (Invitrogen-GIBCO) to make 10% and 5% (wt/vol) homogenates of lung and NT tissues, respectively. Tissue homogenates were clarified by centrifugation at 1,500 rpm for 10 min, and virus titers were determined in 24- and 96-well tissue culture plates containing MDCK cell monolayers. The virus titer for each organ was determined by the method of Reed and Muench (41) and was expressed as log10 TCID50/g of tissue.

Replication of H3N8 equine wt and ca viruses in the respiratory tract of ferrets.

Ten- to 12-week-old ferrets (Triple F Farms, Sayre, PA) were used in these experiments. Animals were seronegative for antibodies to circulating human H3N2, H1N1, and B influenza viruses. Ferret studies were conducted in BSL2 facilities at MedImmune and NIH, and protocols were approved by the MedImmune and NIH Animal Care and Use Committees.

Groups of ferrets were lightly anesthetized with isoflurane and inoculated i.n. with 500 μl containing 107 PFU of wt or ca virus. At 3 and 5 days p.i., ferrets were euthanized, and the right middle and the caudal portion of the left cranial lobe of the lungs and the NTs were harvested and stored at −80°C. Organs were thawed, weighed, and homogenized in L15 medium as described above to make a 10% (wt/vol) suspension, and titers were determined by plaque assay on MDCK cells using an anti-NP monoclonal antibody and expressed as log10 PFU/g of tissue.

Immunogenicity and protective efficacy of the H3N8 ca virus in ferrets.

Groups of ferrets were inoculated i.n. with one or two doses 28 days apart of 500 μl containing 107 PFU of eq/GA/81 ca or L15 medium (mock immunized), and serum samples were collected on day 0 (preimmunization), 28, or 56 p.i. Antibody titers in pre- and postinfection ferret sera were determined by MN and HAI assays, as described above.

On day 28 or 56 p.i., ferrets were challenged i.n. with 107 PFU of each of the H3N8 equine wt viruses, eq/GA/81 or eq/Newm/03. Three ferrets per challenge virus were euthanized on days 3 and 5 p.c., and lungs and NTs were harvested and stored at −80°C. We chose these time points based on previous observations in our laboratory that wt EIV are detected at high titers in the NTs of ferrets from days 1 to 5 p.i. (39). Challenge virus titers were determined in MDCK cells and expressed as log10TCID50 per gram of NT or lung tissue as described above.

Human sera.

Sera collected during a study in 2009 from healthy adult men and nonpregnant women before vaccination with the monovalent inactivated 2009 pH1N1 vaccine were provided by John Treanor (University of Rochester) (46). Subjects were enrolled in 3 age cohorts: 18 to 32 years (n = 19), 60 to 69 years (n = 19), and ≥70 years (n = 18). The study was conducted under a protocol approved by the University of Rochester Research Subjects Review Board. Informed written consent was obtained from each participant.

RESULTS

Immunogenicity of the eq/GA/81ca virus in mice.

A single dose of eq/GA/81ca virus induced a robust neutralizing antibody response against the homologous virus, with a geometric mean titer (GMT) of 418 (range, 57 to 1,016) (Table 1). The titers of NtAb against the heterologous virus, eq/Newm/03, were similar (GMT of 490; range, 160 to 1,613). When two doses of the eq/GA/81 ca vaccine were administered to mice, the GMT achieved was 264 (range, 57 to 905) following the first dose, with a further increase (GMT of 2,426; range, 640 to 7,241) 38 days after the second dose (Table 1). A similar profile against the heterologous virus was observed (Table 1). As expected, mock-immunized mice did not develop detectable NtAb antibodies. These results demonstrate that the eq/GA/81 ca vaccine candidate is highly immunogenic in mice, and serum antibodies cross-reacted well with the heterologous eq/Newm/03 wt virus.

TABLE 1.

Serum neutralizing antibody response to the eq/GA/81 ca vaccine in micea

Test antigen GMT of serum NtAb achieved at indicated days postimmunization in miceb
1 dose (day 38) 2 doses (days 28 and 66)
wt eq/GA/81 418 264/2,426
wt eq/Newm/03 490 225/1,540
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a

Groups of eight mice were inoculated i.n. with 106 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after the first immunization. Homologous antibody titers are in bold.

b

Mice were bled on day 38 after the first and second immunization for technical reasons.

Efficacy of the eq/GA/81 ca virus in mice.

To determine whether immunization with the eq/GA/81 ca virus induced protection, we inoculated mice with either a single dose or two doses of the vaccine candidate. Thirty-eight days after the final vaccination, mice were challenged with 106 PFU/50 μl of the homologous (eq/GA/81) or the heterologous (eq/Newm/03) wt virus. In mock-immunized mice, the mean titers on days 2 and 4 p.c. in the NTs after challenge with the eq/GA/81 wt virus were 105.5 and 105.25 TCID50/g, respectively, and the mean titers in the lungs were 106.1 and 104.6 TCID50/g, respectively. In mock-immunized mice, the mean titers in the NTs on days 2 and 4 after challenge with the heterologous eq/Newm/03 wt virus were 106.8 and 104.9 TCID50/g, respectively, and the mean titers in the lungs were 106.4 and 104.95 TCID50/g on days 2 and 4 p.c., respectively (Fig. 1). A single dose of the eq/GA/81 ca virus provided complete protection against challenge with homologous and heterologous wt viruses in both the upper and lower respiratory tracts (Fig. 1). A similar pattern was observed for mice vaccinated with two doses of the eq/GA/81 ca virus (Fig. 1). We had previously observed that the eq/GA/81 and eq/Newm/03 wt viruses did not cause weight loss or mortality in mice, so we did not assess protection from clinical illness in mice (39). Thus, the eq/GA/81 ca vaccine candidate offered complete protection against homologous and heterologous H3N8 wt virus challenge in mice.

FIG 1.

FIG 1

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Protection conferred by the eq/GA/81 ca vaccine against homologous and heterologous challenge in the upper (A) and lower (B) respiratory tracts of mice. Mice were intranasally inoculated with either L15 medium (mock) or 1 or 2 doses of 106 PFU/mouse of eq/GA/81 ca vaccine and challenged 38 days following the last vaccine dose with 106 PFU/mouse of the indicated challenge virus. Virus titers were determined on days 2 and 4 postchallenge. The dotted horizontal line represents the lower limit of detection.

Level of replication of the eq/GA/81 wt and ca viruses in ferrets.

To determine whether the eq/GA/81 ca vaccine virus was attenuated in ferrets, the levels of replication in the NTs and lungs 3 and 5 days following i.n. administration were compared with those of the eq/GA/81 wt virus. The mean titers on days 3 and 5 in the NTs of ferrets inoculated with the eq/GA/81 wt virus were 107.2 and 106.9 PFU/g, respectively, and those in ferrets inoculated with the eq/GA/81 ca virus were 104.8 and 105.7 PFU/g. Thus, the replication of the eq/GA/81 ca virus was 16- to 250-fold lower than that of the corresponding wt virus in the upper respiratory tract of ferrets. The eq/GA/81 wt virus did not replicate well in the lower respiratory tract of ferrets, as previously reported (39), and replication of the vaccine candidate was not detected in the lungs of ferrets (Fig. 2). No notable signs of disease were observed in ferrets infected with the equine ca or wt viruses. These data indicate that the eq/GA/81 ca virus was attenuated in ferrets.

FIG 2.

FIG 2

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Level of replication of the eq/GA/81 ca vaccine virus compared with the corresponding wt virus in the upper (A) and lower (B) respiratory tract of ferrets. Lightly anesthetized ferrets were inoculated intranasally with 107 PFU/ferret, and tissues were harvested on days 3 and 5 postinfection. The dotted horizontal line represents the lower limit of detection.

Immunogenicity of the eq/GA/81 ca virus in ferrets.

Ferrets that received a single dose of eq/GA/81 ca virus developed neutralizing and HAI antibodies to the homologous wt virus at titers that ranged from 320 to 905 (GMT = 538) and from 40 to 160 (GMT = 85), respectively (Table 2). One dose of the eq/GA/81 ca virus elicited cross-reactive neutralizing and HAI antibodies to the heterologous eq/Newm/03 wt virus at titers that ranged from 113 to 453 (GMT = 196) and from 160 to 640 (GMT = 226), respectively. In animals that received two doses of the vaccine, the first dose of the eq/GA/81 ca vaccine induced homologous NtAb and HAI responses at titers that ranged from 226 to 1,280 (GMT = 559) and 40 to 160 (GMT = 90), respectively. Titers increased after the second dose and ranged from 453 to 3,620 (GMT = 932) and 80 to 1,280 (GMT = 302), respectively. One dose of the eq/GA/81 ca virus elicited cross-reactive neutralizing and HAI antibodies to the heterologous wt virus at titers that ranged from 320 to 1,280 (GMT = 512) and 80 to 640 (GMT = 151), respectively. Again, titers increased after the second dose and ranged from 320 to 5,120 (GMT = 1243) and 160 to 2,560 (GMT = 678), respectively (Table 2). Consistent with findings from the study with mice, these data indicate that a single dose of the eq/GA/81 ca virus was immunogenic in ferrets and that serum antibodies cross-reacted with a heterologous H3N8 virus. Sera from vaccinated ferrets failed to neutralize an older (A/Port Chalmers/1973) and a recent (A/Texas/50/2012) human H3N2 virus (data not shown).

TABLE 2.

Serum antibody responses to the eq/GA/81 ca vaccine in ferretsa

Test antigen Assay GMT of HAI or neutralizing Ab achieved at indicated days postimmunization in ferrets
1 dose (day 28) 2 doses (days 28 and 56)
wt eq/GA/81 MN 538 559/932
HAI 85 90/302
wt eq/Newm/03 MN 196 512/1,243
HAI 226 151/678
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a

Groups of 12 ferrets were inoculated i.n. with 107 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after immunization. Homologous antibody titers are in bold.

Efficacy of the eq/GA/81 ca virus in ferrets.

To determine whether immunization with the eq/GA/81 ca virus induced protection in ferrets, we inoculated animals intranasally with either a single dose or two doses of the vaccine candidate and challenged them 28 days later with 107 PFU of the homologous (eq/GA/81) or the heterologous (eq/Newm/03) wt virus. In mock-immunized ferrets, the titers of eq/GA/81 wt challenge virus on days 3 and 5 p.c. in the NTs were 107.1 and 105.0 TCID50/g, respectively, and the mean virus titers in the lungs were 102.3 and 102.5 TCID50/g on days 3 and 5 p.c. In mock-immunized ferrets, the mean titers of the heterologous eq/Newm/03 wt virus on days 3 and 5 p.c. in the NTs were 108.1 and 107.2 TCID50/g, respectively, and the mean virus titers in the lungs were 102.5 and 101.7 TCID50/g on days 3 and 5 p.c. (Fig. 3). A single dose of the eq/GA/81 ca virus provided complete protection against challenge (no detectable replication) with homologous wt virus in the upper respiratory tract of the ferrets and restricted replication and early clearance of the heterologous wt challenge virus (only one ferret out of 3 had 102 TCID50/g on day 5 p.c.) (Fig. 3). Similar results were obtained for ferrets that received a second dose of the vaccine. Because the eq/GA/81 and the eq/Newm/03 wt viruses did not replicate well in the lower respiratory tract of ferrets, the protection conferred by the eq/GA/81 ca virus in the lungs could not be evaluated (Fig. 3).

FIG 3.

FIG 3

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Protection conferred by the eq/GA/81 ca vaccines against homologous and heterologous challenge in ferrets. Animals were intranasally inoculated with either L15 medium (mock) or 1 or 2 doses of 107 PFU/ferret of eq/GA/81 ca vaccine and challenged 28 days following the last vaccine administration with 107 PFU/ferret of the indicated challenge virus. Virus titers were determined on days 3 and 5 postchallenge. Shown are levels of replication of the indicated challenge viruses in the upper (A) and lower (B) respiratory tracts of ferrets that were challenged following 1 dose of the ca vaccine. The dotted horizontal line represents the lower limit of detection.

Testing for the presence of cross-reactive antibodies in human sera representing three age cohorts.

Because human H3N2 viruses have circulated since 1968, we sought to determine whether prior exposure to seasonal H3N2 viruses induced cross-reactive Ab against EIV.

We assessed the presence of antibodies that cross-reacted with eq/GA/81 (H3N8) virus in human sera collected in 2009. As a control, we assayed the levels of antibodies against the seasonal influenza virus A/Wisconsin/67/2005 (H3N2) that was circulating at the time the sera were collected. Subjects from three age groups, 18 to 32 years old (n = 19), 60 to 69 years old (n = 19), and 70 years or older (n = 18), were enrolled in a clinical trial of a monovalent 2009 H1N1pdm vaccine that has been reported previously (46).

The 18- to 32-year-old subjects failed to show detectable NtAb against the equine H3N8 virus (Fig. 4A). Interestingly, subjects from the other two cohorts, 60 to 69 and ≥70 years old, had GMTs of 22 (range, 10 to 57) and 20 (range, 10 to 113), respectively. Among subjects older than 60 years of age (n = 37), seven had an NtAb titer of 40, one had a titer of 50, three had a titer of 57, and one had a titer of 113. Nine subjects showed lower levels of NtAb (between 20 and 28). In total, 22 out of 37 individuals had titers of NtAB against the equine H3N8 virus of ≥20. Unfortunately, we did not enroll subjects who were between 33 and 59 years of age in the study, so we cannot comment on the level of cross-reactive antibody in this age group. However, the study was conducted in 2009 to 2010, and therefore, subjects born after 1968, when H3N2 viruses emerged and became established in humans, would have been 42 years of age or younger. Therefore, we would expect that people over 42 years of age would have been exposed to H3N2 viruses and could have some cross-reactive antibody. The GMTs in the 18- to 32-year-old, 60- to 69-year-old, and ≥70-year-old subjects against the A/Wisconsin/67/2005 (H3N2) virus were 248 (range, 10 to 3,620), 232 (range, 10 to 3,620), and 134.5 (range, 10 to 1,613), respectively (Fig. 4A).

FIG 4.

FIG 4

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Serum neutralizing antibody (A) and hemagglutination inhibiting antibody (B) titers in individuals of different age groups. The titers of serum antibody against eq/GA/81 (circles) and A/WI/67/05 (triangles) are shown for individual subjects. Bars identify geometric mean titer of the group. The dotted horizontal line represents the lower limit of detection.

Interestingly, none of the subjects had detectable HAI antibody against the equine H3N8 virus (Fig. 4B). The GMTs of HAI antibodies in the 18- to 32-year-old, 60- to 69-year-old, and ≥70-year-old subjects against the A/Wisconsin/67/2005 (H3N2) virus were 46 (range, 10 to 1,280), 67 (range, 10 to 640), and 27 (range 10 to 640), respectively (Fig. 4B). The detection of cross-reactive NtAb in the absence of HAI antibodies in 59% of subjects over 60 years of age suggests that the antibody was induced by prior or repeated exposure by infection or vaccination with older seasonal H3N2 viruses and that the Abs could be directed at the HA stalk.

DISCUSSION

Although direct transmission of EIV to humans has not been reported, experimental infection of humans with EIV can lead to a productive infection and elicit a significant NtAb response (24, 25). The facts that EIV can infect humans and these viruses have crossed the species barrier and infected dogs (13, 16, 18), pigs (21), and camels (21) underscore the potential threat posed to human health by viruses of this subtype. The emergence and pandemic spread of the swine origin H1N1 influenza virus in 2009, despite the ongoing circulation of human H1N1 viruses, suggests that an antigenically distant animal origin H3 virus may pose a pandemic threat despite the circulation of H3N2 viruses in humans since 1968.

The purpose of our study was to generate and evaluate a vaccine candidate to be used in humans in the event that an EIV evolves, adapts, and spreads in humans, causing disease. To this end, we previously evaluated three H3N8 equine influenza viruses from different lineages and selected the eq/GA/81 virus for vaccine development because it elicited cross-reactive antibodies against heterologous EIV (39). We generated an eq/GA/81 candidate LAIV by plasmid-based reverse genetics on the backbone of the A/Ann Arbor/6/60 ca donor virus that is used to produce the licensed seasonal live attenuated influenza vaccine.

In mice, a single dose of the eq/GA/81 ca vaccine virus induced robust neutralizing antibody titers against the homologous and heterologous wt challenge viruses and conferred full protection against homologous and heterologous virus challenge in both the upper and lower respiratory tracts. In ferrets, as in mice, one dose of the vaccine was highly immunogenic and conferred complete protection against homologous challenge virus and near complete protection against the heterologous challenge virus. We observed a direct correlation between serum antibody response and protection against challenge in mice and ferrets that received one dose of the eq/GA/81 ca vaccine virus, although contributions from other arms of the immune system such as the cellular or mucosal immune response cannot be excluded.

In 2009, when the novel H1N1pdm virus emerged, it was assumed that two doses of vaccine would be needed to immunize the human population against the pandemic virus because studies in 1977 had demonstrated the need for two doses of vaccine in a naive population; preclinical evaluation of 2009 H1N1pdm vaccines in influenza-naive animal models supported this conclusion. However, when clinical trials of the inactivated 2009 H1N1pdm vaccine were undertaken, a single dose of vaccine was sufficient in all except children younger than 3 years of age, indicating that most of the population had been primed by prior exposure or vaccination with seasonal H1N1 viruses. We evaluated this phenomenon in a mouse model and demonstrated that priming was achieved by infection with seasonal H1N1 influenza virus or seasonal LAIV but not by seasonal inactivated influenza vaccine (47). In the present study, we evaluated sera from 56 subjects from three age groups, 18 to 32 years old, 60 to 69 years old, and 70 years and older, who were enrolled in a previous study for cross-reactive H3 antibodies. Most subjects in each cohort reported receiving the 2009-2010 seasonal trivalent influenza vaccine 2 to 4 months before their blood samples were collected (46). We do not know if the participants had been exposed to horses. We observed reactivity against the eq/GA/81 virus in individuals over 60 years of age and speculate that the detectable cross-reactive NtAb titers may be explained by cross-reactivity due to previously circulating human influenza A H3N2 viruses. In the event of a pandemic caused by a related virus, a large portion of the human population may be immunologically primed because of previous exposure to seasonal H3N2 influenza viruses, and therefore, one dose of the H3N8 vaccine may be sufficient to confer protection.

In summary, we generated a candidate LAIV against an EIV and demonstrated that a single dose of the vaccine was highly immunogenic and efficacious in protecting mice and ferrets from challenge with the homologous and an antigenically distinct heterologous H3N8 virus from a different sublineage. Based on these promising preclinical data, careful clinical evaluation of the eq/GA/81 (H3N8) ca vaccine is warranted as part of pandemic preparedness efforts. We found evidence of cross-reactive antibodies in subjects >60 years of age that could be directed at the stalk domain of the HA protein. Although data for persons between 32 and 60 years of age are lacking, it appears that a proportion of the human population may be previously primed for a robust response to an equine influenza H3N8 vaccine.

ACKNOWLEDGMENTS

This research was supported by the Intramural Research Program of the NIAID, NIH, and was performed as part of a Cooperative Research and Development Agreement between the Laboratory of Infectious Diseases, NIAID, and MedImmune, LLC.

We thank Ian Moore, the staff of the Comparative Medicine Branch, NIAID, and the staff at MedImmune's Animal Care Facility for technical support for animal studies. We are grateful to Richard Webby and Debra Elton for providing the viruses used in this study and to JoAnn Suzich for reviewing the manuscript.

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