Abstract
Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.
INTRODUCTION
Influenza A viruses are enveloped RNA viruses belonging to the family Orthomyxoviridae and are divided into subtypes on the basis of serological and genetic differences in their major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Sixteen different HA (H1 to H16) and 9 NA (N1 to N9) subtypes have been identified among influenza A viruses, all of which have been found in avian species (1–3). Recently, a 17th subtype (H17) was identified in bats in Central America (4). An influenza A virus can cause a pandemic when a novel influenza virus spreads within a human population that has little or no preexisting immunity. Pandemics are often associated with higher morbidity and mortality rates than epidemics caused by seasonal influenza viruses (5, 6). Three influenza pandemics have occurred in the past century, in 1918 (caused by an H1N1 virus), in 1957 (H2N2), and in 1968 (H3N2), and one in this century, in 2009 (H1N1). These viruses were introduced either directly from an animal reservoir, as was the case of the 1918 and 2009 H1N1 pandemics (7, 8), or as a result of genetic reassortment of avian and human influenza viruses, as was the case in the 1957 H2N2 and 1968 H3N2 pandemics (9, 10).
H3 subtype influenza viruses have been isolated from humans, pigs, horses, dogs, cats, seals, and numerous avian species (11–18). Swine influenza viruses are prevalent in pigs worldwide. In 1998, a human influenza A H3N2 virus appeared in North American pigs, and since then, two genotypes of H3N2 viruses have been isolated from this population: a double reassortant virus which contains five gene segments derived from the classical swine lineage (NS, NP, M, PB2, and PA) and three genes from a human influenza virus (HA, NA, and PB1), and a triple reassortant virus containing three gene segments derived from the classical swine virus (NS, NP, and M), three from a human virus (HA, NA, and PB1), and two from an avian virus (PB2 and PA) (19). By the end of 1999, viruses antigenically and genetically related to the triple reassortant lineage were widespread in pigs in the United States (20), whereas the double reassortant virus did not spread efficiently among swine. Although swine influenza viruses are a common and important pathogen among pigs, human infections with swine-origin influenza viruses (SOIV) were rarely detected, and the cases that have occurred were associated with limited or no human-to-human transmission (20–25) until the 2009 pandemic H1N1 (pH1N1) virus emerged. Excluding the pandemic H1N1, from 1990 to 2010, 27 human infections were reported, 21 of which were caused by triple-reassortant influenza A viruses (13 subtype H1N1, 1 subtype H1N2, and 7 subtype H3N2) (26, 27).
Since 2010, an increasing number of human cases of swine-origin H3N2 influenza virus infections have been reported in the United States. These triple reassortant H3N2 viruses were similar to the H3N2 swine viruses circulating in the North American swine population since 1998 (27). These H3N2 viruses, which infect humans, are called variant (v) viruses. Due to the concern about the pandemic potential of these viruses, A/Minnesota/11/2010 and A/Indiana/10/2011 (H3N2v) were selected as vaccine candidates (28). From July 2011 to April 2012, 13 human influenza infections were caused by triple-reassortant H3N2 viruses containing the matrix gene derived from the 2009 pandemic H1N1 virus (29), which has been shown to promote aerosol transmission in ferrets (30). Remarkably, 305 human infections of A (H3N2v) in multiple states of the United States have been reported since July 2012 (31), resulting in a total of 319 H3N2v human cases from August 2011 to October 2012. So far, these viruses have shown limited ability for person-to-person transmission (32).
Equine H3N8 influenza viruses were first isolated in Miami in 1963 (A/eq/Miami/1/63) (17), and since then these viruses have circulated enzootically in horses, causing significant disease and economic burden worldwide (33). While normally confined to equidae, equine H3N8 influenza viruses crossed the species barrier and were isolated from racing greyhound dogs in the United States in 2004 (34). However, retrospective serological analysis suggests that H3N8 influenza viruses were circulating in racing greyhounds as early as 1999 (35). This virus has become enzootic in the United States (34, 36), and canine H3N8 infections have been reported in the United Kingdom and Australia (37–39). In addition, two H3N8 influenza viruses were isolated from pigs in central China during surveillance for swine influenza virus in 2004 to 2006 (40). Although naturally occurring transmission of equine influenza viruses 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 that received an intranasal dose of between 104.6 and 105.3 50% tissue culture infective doses (TCID50) of virus. However, illness occurred infrequently (12%), suggesting that this virus was more virulent for horses than for humans (41–43).
Low-pathogenicity H3N2 and H3N8 influenza viruses infect wild bird populations in Europe and the Americas (44, 45). An avian-origin H3N2 influenza virus was isolated from pet dogs in South Korea in 2007 (16), and an avian H3N8 influenza virus was isolated recently from sick seals in New England (11).
Prediction of future influenza pandemic strains is difficult; therefore, the best way to prepare for pandemics is to develop and evaluate the safety and immunogenicity of vaccines for different subtypes in preclinical and clinical studies. Very little information is available on the kinetics of replication and cross-reactivity of the immune response of swine, equine, and avian H3 influenza viruses in experimental animal models. The purpose of this study was to determine whether it was possible to select an animal H3 virus that would cross-protect against H3 viruses from other species. If this was not possible, we sought to select one representative virus from each species. We were also interested in developing mouse and ferret models to evaluate the immunogenicity and efficacy of the candidate animal H3 viruses. To this end, we selected 11 geographically and temporally distinct H3 influenza viruses that had been isolated from pigs, horses, and birds and evaluated the kinetics of their replication in mice and ferrets and compared their ability to induce a broadly cross-reactive antibody response. The selection of the viruses from the different species was based on a phylogenetic tree, including isolates available from the Influenza Research Database. We chose viruses from different HA clusters and within a cluster. We chose the isolate closest to the node, because we wanted to select viruses that were closer to an ancestral sequence. We have followed a similar approach with H2, H6, and H7 influenza viruses (46–48).
MATERIALS AND METHODS
Selection of viruses.
The H3 influenza viruses included in this study are listed in Table 1, along with their abbreviations. Viruses were provided by Richard Webby, St. Jude Children's Research Hospital, Memphis, TN (A/swine/Colorado/1/1977 [H3N2], A/swine/Ukkel/1/1984 [H3N2], A/equine/Romania/1/1980 [H3N8], and A/equine/Georgia/1/1981 [H3N8]); Christopher Olsen, University of Wisconsin–Madison, Madison, WI (A/swine/Iowa/H02AS8/2002 [H3N2]); Suzy Carman, University of Guelph (A/swine/Ontario/42729A/2001 [H3N3]); Debra Elton, Animal Health Trust, Newmarket, United Kingdom (A/equine/Newmarket/5/2003 [H3N8]); Nuria Busquets, UAB-IRTA, Universitat Autònoma de Barcelona, Barcelona, Spain (A/Anas plathyrhynchos/Spain/0454/2006 [H3N8]); John M. Pearce, U.S. Geological Survey, Anchorage, AK (A/northern pintail/Alaska/44228-129/2006 [H3N8]); Michael Osterholm, University of Minnesota, Minneapolis, MN (A/blue-winged teal/Texas/Sg-00079/2007 [H3N8] and A/mallard/Minnesota/Sg-00169/2007 [H3N8]); and Edward Dubovi, Cornell University, Ithaca, NY (A/canine/Florida/15592/2004 [H3N8] and A/canine/New York/1201262/11 [H3N8]). The HA amino acid sequence identity ranged from 85.7 to 92.4% for the swine viruses, 96.1 to 98.6% for the equine viruses, and 95.4 to 98.9% for the avian viruses (see Table S1 in the supplemental material). Among the equine viruses, A/equine/Romania/1/1980 belongs to the predivergent lineage, A/equine/Newmarket/5/2003 belongs to the American lineage sublineage Florida Clade 2, and A/equine/Georgia/1/1981 belongs to Florida Clade 1 (49).
Table 1.
Swine, equine, and avian H3 influenza viruses used in this study
| Name | Abbreviation | Subtype |
|---|---|---|
| A/swine/Colorado/1/1977 | sw/CO/77 | H3N2 |
| A/swine/Ukkel/1/1984 | sw/Ukk/84 | H3N2 |
| A/swine/Iowa/H02AS8/2002 | sw/IA/02 | H3N2 |
| A/swine/Ontario/42729A/2001 | sw/ONT/01 | H3N3 |
| A/equine/Romania/1/1980 | eq/ROM/80 | H3N8 |
| A/equine/Georgia/1/1981 | eq/GA/81 | H3N8 |
| A/equine/Newmarket/5/2003 | eq/Newm/03 | H3N8 |
| A/Anas plathyrhynchos/Spain/0454/2006 | an-plath/SP/06 | H3N8 |
| A/northern pintail/Alaska/44228–129/2006 | npin/AK/06 | H3N8 |
| A/blue-winged teal/Texas/Sg-00079/2007 | tl/TX/079/07 | H3N8 |
| A/mallard/Minnesota/Sg-00169/2007 | mal/MN/0169/07 | H3N8 |
Virus stocks were propagated in the allantoic cavities of 9- to 11-day-old specific-pathogen-free embryonated hen's eggs (Charles River Laboratories, Franklin, CT) at 35°C. Virus titers were determined in Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA) and calculated using the Reed and Muench method (50). Mouse and ferret experiments were conducted in biosafety level 2 laboratories at the National Institutes of Health (NIH) or at MedImmune. Experiments using A/Anas plathyrhynchos/Spain/0454/2006 (H3N8) were conducted in biosafety level 3 (BSL-3) containment, because this virus was originally isolated under BSL-3 conditions.
Animals.
Four- to 6-week-old female BALB/c mice (Taconic Farms, Inc., Germantown, NY) were used in all mouse experiments. Mouse studies were conducted at the NIH. Ten- to 12-week-old ferrets (Triple F Farms, Sayre, PA) were used in the ferret studies, and experiments were conducted at the NIH and at MedImmune. Animal experiments were approved by the NIH and MedImmune Animal Care and Use Committees, respectively. All ferrets used in this study were negative for hemagglutination inhibition (HAI) antibodies against currently circulating human H1N1 and H3N2 viruses.
Morbidity and mortality of H3 influenza viruses in mice.
To evaluate weight loss and mortality, groups of five female BALB/c mice were lightly anesthetized and inoculated intranasally (i.n.) with 50 μl containing 106 TCID50 of each virus. Mice were monitored daily for weight loss, and mortality was recorded over a period of 14 days. Mice that lost 25% of their body weight were sacrificed according to the Institutional Animal Care and Use Committee guidelines.
Kinetics of virus replication in mice.
In order to evaluate the kinetics of replication of the 11 H3 viruses in different tissues, groups of 20 female BALB/c mice were anesthetized and inoculated i.n. with 50 μl containing 106 TCID50 of each virus. Five mice from each group were euthanized 2, 3, 4, and 7 days postinoculation (dpi), and nasal turbinates (NTs), lungs, brain, and spleen were harvested and stored at −80°C. The frozen tissues were thawed, weighed, and homogenized in Leibovitz-15 (L-15) medium (Invitrogen-Gibco) containing a 2× concentration of antibiotic-antimycotic (penicillin, streptomycin, and amphotericin B) (Invitrogen-Gibco) to make 5% (wt/vol) (NT) or 10% (wt/vol) (lungs, brain, and spleen) tissue homogenates. Tissue homogenates were clarified by centrifugation at 1,500 rpm for 10 min and titrated in 24- and 96-well tissue culture plates containing MDCK cells. The virus titer for each organ was determined by the method described by Reed and Muench (50) and was expressed as log10 TCID50/gram of tissue.
Kinetics of virus replication in ferrets.
We evaluated the kinetics of replication of 10 of the H3 viruses and excluded the sw/IA/02 (H3N2) virus because of space constraints. Groups of ferrets were lightly anesthetized with isoflurane and inoculated i.n. with 500 μl containing 107 TCID50 of each virus. Three ferrets from each group were sacrificed 1, 3, or 5 dpi, and their NTs, lungs, and brain were harvested and stored at −80°C. Organs were thawed, weighed, and homogenized in L-15 medium as described above to make a 10% suspension of each tissue, and titers were determined by the Reed and Muench method (50) and expressed as log10 TCID50/gram of tissue. The body weight and temperature of these animals were measured prior to virus inoculation (day zero) and daily for 5 days. Fever was defined as a core body temperature of >103.8°F.
Generation of antisera. (i) Mice.
Postinfection antiserum was generated in mice against each of the 11 H3 influenza viruses. Groups of five 4- to 6-week-old BALB/c mice were lightly anesthetized and inoculated i.n. with 50 μl containing 106 TCID50 of each virus. All of the mice were bled before administration of virus and 14, 28, 35, and 42 dpi. Sera from each group of mice were pooled for neutralization assays.
(ii) Ferrets.
Postinfection antiserum was generated in one or two 10- to 12-week-old ferrets for each virus. Each animal was lightly anesthetized with isoflurane and subsequently inoculated i.n. with 500 μl containing 107 TCID50 of each virus. Animals were monitored for signs of morbidity and mortality. All ferrets were bled before administration of virus, and serum was collected 14 and 28 dpi.
Evaluation of the cross-reactivity of antibody against homologous and heterologous H3 viruses. (i) HAI.
Antibody titers in postinfection ferret sera were determined by HAI assays according to standard protocols (51). Nonspecific inhibitors were removed from serum by overnight treatment with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan). The HAI titer was recorded as the reciprocal of the highest dilution of serum that completely inhibited agglutination of turkey red blood cells. By convention, 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.
(ii) Neutralization.
Neutralizing antibody titers in pre- and postinfection mouse and ferret sera were determined in a microneutralization (MN) assay. Serial 2-fold dilutions of heat-inactivated serum were prepared starting from a 1:20 dilution. The neutralizing antibody 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 HAI titer and the titer generated against the heterologous virus.
RESULTS
Four swine, three equine, and four avian geographically and temporally distant H3 influenza viruses were selected for this study (Table 1). We focused on older swine H3 viruses because the human H3 HA from the mid-1990s became established in pigs in North America in 1998, and vaccines against these human H3 viruses are stored in repositories. HA amino acid sequence identity of the selected H3 influenza viruses is presented in Table S1 in the supplemental material. An alignment of the HA sequences of the 11 viruses with the HA sequence of the human influenza A/Aichi/2/1968 (H3N2) reference virus, showing the potential glycosylation sites, the antigenic sites A to E, and the receptor binding sites, is presented in Fig. S1.
Morbidity and mortality in mice and ferrets.
The virulence of a dose of 106 TCID50 of each H3 influenza virus was evaluated in mice, and the weight loss and mortality data for the swine, equine, and avian viruses are presented in Fig. 1A, B, and C, respectively. sw/CO/77, sw/Ukk/84, and sw/ONT/01 viruses induced significant weight loss, which peaked on day 3 for sw/ONT/01 (15.5%) and on day 6 for sw/CO/77 (16%) and sw/Ukk/84 (25%). Lethality was observed at 6 dpi (40% for sw/CO/77 and sw/ONT/01 and 60% for sw/Ukk/84) (Fig. 1A). Mice inoculated with sw/IA/02 did not develop significant weight loss or lethality, and neither did mice inoculated with the equine viruses (Fig. 1B). Interestingly, the avian npin/AK/06 induced significant weight loss (22%) by 3 dpi, with a return to baseline by 9 dpi without associated mortality, while the avian an-plath/SP/06 did not cause morbidity or mortality. Tl/TX/079/07 and mal/MN/0169/07 induced significant weight loss, which peaked on days 4 (20%) and 7 (25%), respectively; 40% of the mice inoculated with tl/TX/079/07 died between 3 and 4 dpi, and 60% of those inoculated with mal/MN/0169/07 died between 4 and 7 dpi (Fig. 1C).
Fig 1.
Weight loss in mice inoculated i.n. with 50 μl containing 106 TCID50 of swine (A), equine (B), and avian (C) viruses. Animals were monitored daily for weight loss, and mortality was recorded over a period of 14 days. Mice were euthanized when they lost 25% of their original body weight. A dagger indicates that a mouse died on the specified day.
Body weight and temperature were measured from days 0 to 5 after infection in the ferrets that were euthanized 5 days postinfection. No significant weight loss or temperature was observed in any group.
Replication kinetics in the respiratory tract of mice.
The kinetics of replication of each of the 11 viruses in the upper and lower respiratory tracts of mice are summarized in Fig. 2A and B, respectively. The swine and equine viruses replicated well in the upper respiratory tract, with peak titers ranging from 104.6 to 107.5 TCID50/g at 2 or 3 dpi. However, the avian influenza viruses replicated poorly in the NTs (Fig. 2A). With the exception of an-plath/SP/06, the swine, equine, and avian viruses replicated in mouse lungs, with peak titers from 104.1 to 108.0 TCID50/g between 2 and 4 dpi that declined by day 7. In general, the avian influenza viruses replicated to lower titers than the swine and equine viruses in the lungs of mice (Fig. 2B). None of the swine, equine, or avian H3 influenza viruses were isolated from the brain and/or spleen (data not shown).
Fig 2.
Replication kinetics of H3 influenza viruses in mice following i.n. inoculation of 106 TCID50/virus. Virus titers in the nasal turbinates (A) and lungs (B) of 5 mice per group sacrificed on 2, 3, 4, and 7 dpi are expressed as log10 TCID50/gram of tissue. Horizontal bars represent mean titers, and symbols represent titers from individual mice. The dashed horizontal line indicates the lower limit of detection, 101.8 and 101.5 TCID50 per gram for the NT and lungs, respectively.
Replication kinetics in the respiratory tract of ferrets.
The kinetics of replication of three H3 swine influenza viruses (sw/CO/77, sw/Ukk/84, and sw/ONT/01), the three H3 equine influenza viruses, and the four avian H3 influenza viruses in the respiratory tract of ferrets is displayed in Fig. 3. The swine influenza viruses replicated to high titers in the upper and lower respiratory tracts. Virus titers in the NTs of ferrets inoculated with sw/CO/77 and sw/ONT/01 peaked at 107.5 TCID50/g at 1 and 3 and at 1 dpi, respectively, and remained at similar levels at 5 dpi, while sw/Ukk/84 reached peak titers of 106.7 TCID50/g at 1 dpi and decreased to 103.9 TCID50/g by 5 dpi. Virus titers in the lungs of ferrets inoculated with sw/CO/77, sw/Ukk/84, and sw/ONT/01 peaked at 105.5, 106.4, and 108.0 TCID50/g at 5, 5, and 1 dpi, respectively, and peak titers in the lungs were similar to those of the NTs. Equine influenza virus replicated to moderate to high titers in the upper respiratory tract of ferrets, with peaks of 103.3 TCID50/g (eq/ROM/80), 105.4 TCID50/g (eq/GA/81), and 107.5 TCID50/g (eq/Newm/03) at 1, 5, and 3 dpi, respectively (Fig. 3A). Interestingly, the equine influenza viruses replicated poorly (102.5 TCID50/g) in the lungs of ferrets. All 4 avian H3 influenza viruses tested replicated well in the NTs and lungs, with peak titers ranging between 106.4 and 107.7 TCID50/g in the NTs and 104.4 and 107.6 TCID50/g in lungs. None of the swine, equine, or avian H3 influenza viruses were isolated from the brain and/or spleen (data not shown).
Fig 3.
Replication kinetics of H3 influenza viruses in ferrets following i.n. inoculation of 107 TCID50/virus. Virus titers in the nasal turbinates (A) and lungs (B) of 3 ferrets per group sacrificed on 1, 3, and 5 dpi are expressed as log10 TCID50/gram of tissue. Horizontal bars represent mean titers, and symbols represent titers from individual ferrets. The dashed horizontal line indicates the lower limit of detection, 101.5 TCID50 per gram for the NT and lungs.
Cross-reactive antibodies to homologous and heterologous H3 influenza viruses in mice and ferrets.
Postinfection sera were generated in mice and ferrets for the 11 selected H3 influenza viruses. To determine the cross-reactivity of the postinfection antisera raised against the selected H3 viruses, sera were evaluated by HAI and/or MN assays, and the cross-reactivity is represented in a checkerboard fashion. An antibody (Ab) titer that was 4-fold or lower than the homologous Ab titer was considered to be significantly different and indicative of a lack of cross-reactivity between viruses.
(i) Mice.
The cross-reactivity of mouse antisera against the selected viruses was assessed in MN assays (Table 2) but not in HAI assays, because a larger volume of serum is required for the latter. All of the viruses elicited neutralizing antibodies against the homologous viruses. No single virus elicited antibodies that cross-reacted with viruses from all 3 animal sources. The sw/Ukk/84, sw/IA/02, and sw/ONT/01 influenza viruses did not elicit cross-reactive antibodies against heterologous viruses isolated from the same species. Only mice inoculated with sw/CO/77 showed cross-reactive Ab against sw/Ukk/84 virus. Mice inoculated with sw/ONT/01 elicited cross-reactive antibodies against all avian H3 viruses included in this study, and sw/ONT/01 HA has a high amino acid sequence identity to avian HAs (see Table S1 in the supplemental material), suggesting that the HA of sw/ONT/01 was derived from an avian influenza virus. Mice inoculated with eq/ROM/80 and eq/GA/81 elicited antibodies that cross-reacted with heterologous viruses isolated from the same species. In addition, eq/ROM/80 and eq/GA/81 antisera cross-reacted with mal/MN/0169/07 and sw/Ukk/84, respectively. All 4 avian influenza viruses elicited cross-reactive antibodies against heterologous avian viruses and also against sw/ONT/01, and avian npin/AK/06 elicited cross-reactive antibodies against sw/Ukk/84 as well.
Table 2.
Cross-reactivity of postinfection mouse sera against H3 influenza viruses in a neutralization assaya
| Virus | Titer of mouse antisera raised against: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| sw/CO/77 | sw/Ukk/84 | sw/ONT/01 | sw/IA/02 | eq/ROM/80 | eq/GA/81 | eq/Newm/03 | an-plath/SP/06 | npin/AK/06 | tl/TX/079/07 | mal/MN/0169/07 | |
| sw/CO/77 | 254 | 32 | 10 | 10 | 10 | 10 | 10 | 10 | 113 | 57 | 10 |
| sw/Ukk/84 | 113 | 1,280 | 10 | 10 | 10 | 226 | 10 | 57 | 453 | 63 | 320 |
| sw/ONT/01 | 10 | 10 | 226 | 10 | 10 | 10 | 10 | 226 | 453 | 1,810 | 905 |
| sw/IA/02 | 10 | 16 | 10 | 160 | 113 | 28 | 113 | 10 | 226 | 320 | 453 |
| eq/ROM/80 | 10 | 10 | 10 | 10 | 905 | 453 | 28 | 10 | 10 | 57 | 57 |
| eq/GA/81 | 10 | 10 | 10 | 10 | 806 | 905 | 57 | 10 | 10 | 40 | 57 |
| eq/Newm/03 | 10 | 10 | 10 | 10 | 453 | 905 | 905 | 10 | 10 | 50 | 113 |
| an-plath/SP/06 | 40 | 10 | 806 | 10 | 160 | 10 | 10 | 403 | 1,613 | 5,120 | 3,225 |
| npin/AK/06 | 10 | 10 | 57 | 10 | 113 | 10 | 10 | 127 | 1,810 | 905 | 905 |
| tl/TX/079/07 | 10 | 10 | 320 | 10 | 57 | 10 | 10 | 453 | 905 | 3,620 | 1,810 |
| mal/MN/0169/07 | 10 | 10 | 127 | 10 | 226 | 113 | 10 | 640 | 1,810 | 3,620 | 3,620 |
Homologous neutralizing antibody titers are indicated in boldface. Underlined titers indicate cross-reactive antisera with titers within 4-fold of homologous titers. Mouse antisera were collected 42 days postinoculation. Antibody titers were determined in pooled sera from 5 mice per virus by microneutralization assay in MDCK cells.
(ii) Ferrets.
In an HAI assay, with the exception of sw/ONT/01, which elicited cross-reactive antibodies against an-plath/SP/06, none of the swine influenza viruses elicited cross-reactive antibodies against heterologous viruses isolated from the same or other species. Antisera from ferrets infected with eq/ROM/80 and eq/GA/81 virus showed cross-reactivity among themselves. tl/TX/079/07 elicited a cross-reactive antibody response against sw/ONT/01 but not to the other avian viruses. Finally, mal/MN/0169/07 elicited cross-reactive antibodies against sw/ONT/01, eq/ROM/8, eq/GA/81, and tl/TX/079/07 (Table 3).
Table 3.
Cross-reactivity of postinfection ferret sera against H3 influenza viruses in an HAI assaya
| Virus | Titer of ferret antisera raised against: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| sw/CO/77 | sw/Ukk/84 | sw/ONT/ 01 | sw/IA/02 | eq/ROM/80 | eq/GA/81 | eq/Newm/03 | an-plath/SP/06 | npin/AK/06 | tl/TX/079/07 | mal/MN/0169/07 | |
| sw/CO/77 | 5,120 | 640 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| sw/Ukk/84 | 320 | 5,120 | 10 | 10 | 10 | 40 | 10 | 10 | 10 | 10 | 20 |
| sw/ONT/01 | 10 | 10 | 320 | 10 | 10 | 10 | 10 | 10 | 10 | 320 | 320 |
| sw/IA/02 | 10 | 10 | 10 | 320 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| eq/ROM/80 | 10 | 10 | 10 | 10 | 320 | 320 | 20 | 10 | 10 | 10 | 80 |
| eq/GA/81 | 10 | 10 | 10 | 10 | 640 | 1,280 | 80 | 10 | 10 | 10 | 160 |
| eq/Newm/03 | 10 | 10 | 10 | 10 | 10 | 640 | 320 | 10 | 10 | 10 | 320 |
| an-plath/SP/06 | 10 | 10 | 160 | 10 | 10 | 10 | 10 | 160 | 10 | 10 | 10 |
| npin/AK/06 | 10 | 10 | 10 | 10 | 10 | 10 | 80 | 40 | 160 | 10 | 20 |
| tl/TX/079/07 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 80 | 640 | 160 |
| mal/MN/0169/07 | 10 | 10 | 10 | 10 | 10 | 20 | 10 | 10 | 10 | 20 | 320 |
Homologous neutralizing antibody titers are indicated in boldface. Underlined titers indicate cross-reactive antisera with titers within 4-fold of homologous titers. The lower limit of detection was assigned a value of 10. Sera from 1 or 2 animals were collected at 28 days postinoculation. Antibody titers were determined in sera from 1 or 2 ferrets per virus by HAI assay.
Cross-reactivity was also determined in MN assays using all 11 H3 influenza viruses (Table 4). No single virus elicited antibodies that cross-reacted with viruses from the 3 animal sources. In concordance with HAI assay, sw/CO/77, sw/Ukk/84, and sw/IA/02 influenza viruses did not elicit cross-reactive antibodies against heterologous viruses isolated from the same species. However, sw/ONT/01 showed cross-reactivity against an-plath/SP/06 and tl/TX/079/07. Antisera from ferrets inoculated with both eq/ROM/80 and eq/GA/81 showed cross-reactivity against themselves as well as against eq/Newm/03 and sw/IA/02. Interestingly, eq/Newm/03 serum cross-reacted with eq/GA/81 and all 4 avian viruses, but not with the eq/ROM/80 virus. An-plath/SP/06 and npin/AK/06 elicited cross-reactive antibodies against heterologous viruses isolated from avian species and against sw/ONT/01 and sw/IA/02, respectively. Finally, tl/TX/079/07 elicited cross-reactive antibodies against heterologous viruses isolated from the same species and also against sw/ONT/01 and eq/ROM/80, and the avian mal/MN/0169/07 antiserum showed cross-reactivity against tl/TX/079/07, an-plath/SP/06, sw/IA/02, and sw/ONT/01 but not against the avian npin/AK/06. The concordance between the cross-neutralization titers using postinfection sera from mice and ferrets is displayed in Table 5; overall, 85% concordance was noted between the ferret and mouse MN assay results. We observed a high concordance (89 to 94%) in cross-neutralization titers against heterologous viruses isolated from the same species and a lower degree of concordance (78%) between HAI and cross-neutralization assays with ferret antisera (Table 6).
Table 4.
Cross-reactivity of postinfection ferret sera against H3 influenza viruses in a neutralization assay
| Virus | Titer of ferret antisera raised against: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| sw/CO/77 | sw/Ukk/84 | sw/ONT/01 | sw/IA/02 | eq/ROM/80 | eq/GA/81 | eq/Newm/03 | an-plath/SP/06 | npin/AK/06 | tl/TX/079/07 | mal/MN/0169/07 | |
| sw/CO/77 | 2,560/5,120a | 640/160 | 10b | 10/10 | 10/10 | 40/40 | 10 | 10 | 10 | 25/20 | 57/80 |
| sw/Ukk/84 | 320/508 | 10,240/12,902 | 10 | 10/10 | 101/127 | 63/113 | 10 | 10 | 10 | 16/28 | 50/160 |
| sw/ONT/01 | 10/10 | 10/10 | 320 | 10/10 | 10/10 | 10/10 | 57 | 202 | 28 | 806/508 | 254/1,613 |
| sw/IA/02 | 113/50 | 113/50 | 10 | 226/226 | 226/202c | 226/508 | 57 | 10 | 226 | 101/127 | 453/508 |
| eq/ROM/80 | 10/10 | 113/160 | 10 | 10/10 | 640/806 | 1,280/453 | 80 | 10 | 10 | 101/113 | 50/54 |
| eq/GA/81 | 10/10 | 202/57 | 10 | 10/10 | 905/1,810 | 1,613/1,280 | 226 | 10 | 10 | 10 | 28/226 |
| eq/Newm/03 | 10/10 | 160/57 | 10 | 28/20 | 806/806 | 806/640 | 453 | 10 | 10 | 40/57 | 113/905 |
| an-plath/SP/06 | 10 | 10 | 640 | 10 | 10 | 10 | 113 | 640 | 101 | 1,280/905 | 226/1,810 |
| npin/AK/06 | 10/10 | 10/10 | 57 | 10/10 | 10/10 | 57/10 | 226 | 160 | 226 | 403/403 | 113/1016 |
| tl/TX/079/07 | 10/10 | 10/10 | 320 | 10/10 | 20/57 | 57/10 | 113 | 254 | 113 | 1016/1280 | 320/3620 |
| mal/MN/0169/07 | 10/10 | 10/10 | 57 | 28/10 | 57/113 | 127/50 | 113 | 226 | 226 | 1280/1280 | 905/7241 |
Sera from 1 or 2 animals were collected 28 days postinoculation. Homologous neutralizing antibody titers are indicated in boldface.
The lower limit of detection was assigned a value of 10.
Underlined titers indicate cross-reactive antisera with titers within 4-fold of homologous titers.
Table 5.
Concordance of neutralization titers between postinfection mouse and ferret antiseraa
| Virus | Concordance with ferret antiserum against: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| sw/CO/77 | sw/Ukk/84 | sw/ONT/01 | sw/IA/02 | eq/ROM/80 | eq/GA/81 | eq/Newm/03 | an-plath/SP/06 | npin/AK/06 | tl/TX/079/07 | mal/MN/0169/07 | |
| sw/CO/77 | + | + | + | + | + | + | + | + | + | + | + |
| sw/Ukk/84 | − | + | + | + | + | − | + | + | − | + | + |
| sw/ONT/01 | + | + | + | + | + | + | + | + | + | + | + |
| sw/IA/02 | + | + | + | + | − | − | + | + | − | + | − |
| eq/ROM/80 | + | + | + | + | + | + | + | + | + | − | + |
| eq/GA/81 | + | + | + | + | + | + | − | + | + | + | + |
| eq/Newm/03 | + | + | + | + | + | + | + | + | + | + | + |
| an-plath/SP/06 | + | + | + | + | + | + | − | + | + | + | + |
| npin/AK/06 | + | + | − | + | + | + | − | + | + | + | − |
| tl/TX/079/07 | + | + | + | + | + | + | − | + | + | + | + |
| mal/MN/0169/07 | + | + | − | + | − | + | − | + | + | + | + |
A plus sign indicates concordant results; a minus sign indicates discordant results.
Table 6.
Concordance of results from neutralization and HAI assays using postinfection ferret antiseraa
| Virus | Concordance with ferret antiserum against: |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| sw/CO/77 | sw/Ukk/84 | sw/ONT/01 | sw/IA/02 | eq/ROM/80 | eq/GA/81 | eq/Newm/03 | an-plath/SP/06 | npin/AK/06 | tl/TX/079/07 | mal/MN/0169/07 | |
| sw/CO/77 | + | + | + | + | + | + | + | + | + | + | + |
| sw/Ukk/84 | + | + | + | + | + | + | + | + | + | + | + |
| sw/ONT/01 | + | + | + | + | + | + | + | − | + | + | + |
| sw/IA/02 | + | + | + | + | − | − | + | + | − | + | − |
| eq/ROM/80 | + | + | + | + | + | + | + | + | + | − | − |
| eq/GA/81 | + | + | + | + | + | + | + | + | + | + | − |
| eq/Newm/03 | + | + | + | + | − | + | + | − | + | + | − |
| an-plath/SP/06 | + | + | − | + | + | + | − | + | − | − | − |
| npin/AK/06 | + | + | + | + | + | + | + | + | + | − | + |
| tl/TX/079/07 | + | + | − | + | + | + | − | − | + | + | + |
| mal/MN/0169/07 | + | + | + | + | + | + | − | − | − | − | + |
A plus sign indicates concordant results; a minus sign indicates discordant results.
In summary, all of the selected H3 viruses replicated in the respiratory tract of mice and ferrets without adaptation. In general, avian viruses replicated to lower titers in the NTs and lungs of mice, and equine viruses showed poor replication in the lower respiratory tract of ferrets. Although none of the viruses elicited antibodies that cross-reacted with viruses from the 3 animal sources and swine viruses did not show cross-reactivity among themselves, the equine and avian influenza viruses elicited cross-reactive antibodies against heterologous viruses of the same or other species. Based on the replicative capacity in the upper and/or lower respiratory tract of mice and ferrets and the homologous and cross-reactive antibody response, we selected one equine (eq/GA/81) and one avian (tl/TX/079/07) virus as vaccine candidates for further development.
DISCUSSION
In the last century, influenza virus pandemics were caused by an animal influenza virus that adapted to infect humans (the 1918 pandemic), by reassortment between an avian influenza virus and a circulating human influenza virus (1957 and 1968 pandemics), or by a reassortant virus bearing a mix of swine, avian, and human influenza virus genes (the 2009 swine influenza pandemic) (7–10). Since 1968, human H3N2 influenza viruses have been circulating worldwide, and the population has been infected by and/or vaccinated against these viruses. However, H3 subtype influenza viruses have been isolated from humans, pigs, horses, dogs, cats, seals, and numerous avian species, and these viruses are antigenically and genetically distinct from human H3 viruses (11–18). Based on the emergence of a swine-origin H1N1 virus as a pandemic strain in 2009 despite the ongoing circulation of human H1 viruses, we believe it is necessary to consider the development of H3 animal influenza vaccines. So far, swine, equine, and avian H3 influenza viruses all have crossed the species barrier to mammals, including humans, dogs, and seals. Clearly the infections by H3N2v viruses are of concern, but we did not include these viruses in our analysis, because vaccines against these viruses are already under development. In the present study, we selected and studied 11 geographically and temporally distinct H3 influenza viruses, which were isolated from pigs, horses, and birds; we evaluated the kinetics of replication of these viruses in mice and ferrets and analyzed their ability to induce a cross-reactive antibody response. We were unable to identify a single virus that elicited an antibody response that cross-reacted across all sources.
The majority of the swine influenza viruses (sw/CO/77, sw/Ukk/84, and sw/ONT/01) caused significant weight loss and were lethal in mice. Although these viruses elicited moderate to high homologous titers, the Abs did not cross-react well with the heterologous swine viruses. The pattern of Ab reactivity in ferrets was similar to that in mice. Since it is unlikely that any vaccine virus prepared ahead of a pandemic will exactly match the pandemic virus, the ideal candidate for a pandemic vaccine should elicit cross-reactive antibodies against as many antigenically distinct viruses within a subtype as possible. The diversity of the swine H3 viruses is such that we were unable to select a swine influenza virus as a vaccine candidate. The selected swine viruses also show many amino acid substitutions in antigenic sites (see Fig. S1 in the supplemental material) that may explain the lack of cross-reactivity of antisera raised against viruses within this group. An expanded analysis of additional swine viruses is needed.
The equine viruses selected for this study replicated well in the upper and lower respiratory tracts of mice without causing significant weight loss or mortality. eq/ROM/80 and eq/GA/81 viruses elicited cross-reactive antibodies against the heterologous equine viruses, and these antibodies also cross-reacted with an avian and swine virus, respectively. Interestingly, eq/Newm/03 did not elicit cross-reactive antibodies in mice against any other H3 influenza virus included in this study. All avian viruses elicited cross-reactive Ab against heterologous viruses isolated from avian species, and all cross-reacted with sw/ONT/01. In agreement with Kawaoka et al. (52), the equine viruses replicated to high titers in the upper respiratory tract and to surprisingly low titers in the lower respiratory tract of ferrets. Several studies on the distribution of the sialic acid receptors in the respiratory tract of horses have shown that sialic acid-α2,3-galactose (SAα2,3gal) receptors are found on the surface of ciliated epithelial cells in the nasal mucosa, trachea, and bronchi (38, 53–55). The distribution of SAα2,3gal and SAα2,6gal receptors in the ferret respiratory tract is similar to that of humans, with SAα2,6gal receptors in the upper respiratory tract extending into the lower respiratory tract as far as the bronchioles and SAα2,3 and SAα2,6gal receptors in the lower respiratory tract distal to the respiratory bronchioles (56). Equine influenza viruses contain an amino acid combination of 226Q/228G (see Fig. S1 in the supplemental material), which is known to preferentially bind SAα2,3gal receptors; therefore, we expected that the equine viruses would replicate in the lower respiratory tract of ferrets. Further studies are needed to understand the restricted replication of the equine viruses in the lower respiratory tract of ferrets. In the ferret, eq/ROM/80 and eq/GA/81 viruses elicited cross-reactive antibodies against the heterologous equine viruses, but in contrast to the response in mice, the eq/Newm/03 virus elicited cross-reactive antibodies against eq/GA/81 and the four avian H3 influenza viruses in ferrets. eq/ROM/80 and eq/Newm/03 share 96.1% identity in HA amino acid sequence (see Table S1); the lack of cross-reactivity may be due to differences in antigenic site A (135G/R), B (189N/Q), or C (276T/I) (see Fig. S1). Although naturally occurring transmission of equine influenza viruses to humans has not been reported, experimental studies conducted in the 1960s showed that equine influenza viruses could infect humans; although they did not cause disease, the infections elicited a significant neutralizing Ab response (42, 43). Although the equine virus that has become established in dogs (34, 37, 38) does not yet appear to pose a threat to humans, the fact that equine influenza viruses can cross the species barrier argues for the possible need for control strategies for humans. Of the three equine viruses evaluated in this study, eq/GA/81 (H3N8) is the best choice for a vaccine candidate, because it elicited cross-reactive antibodies against heterologous equine influenza viruses and replicated to titers similar to or higher than those of eq/ROM/80 virus in the upper respiratory tract of mice and ferrets, respectively. The ability of postinfection ferret sera collected following primary infection with eq/ROM/80, eq/GA/81, and eq/Newm/03 viruses to neutralize infectivity of two canine influenza viruses, A/Canine/Florida/15592/2004 and A/Canine/New York/1201262/11, was assessed. We found that sera from ferrets infected with eq/ROM/80 did not cross-react with these canine viruses, presumably because the canine strains were derived from a more recent American equine lineage, although sera from ferrets infected with eq/GA/81 and eq/Newm/03 showed low-titer neutralizing activity (titers of 101 and 57 for eq/GA/81 against A/Canine/Florida/15592/2004 and A/Canine/New York/1201262/11, respectively, and 57 for eq/Newm/03 against A/Canine/Florida/15592/2004 and A/Canine/New York/1201262/11). Although the purpose of this study was not to select a canine vaccine candidate, this finding suggests that vaccination with the eq/GA/81 vaccine candidate elicits some cross-reactive antibodies against the canine viruses.
Birds are the reservoir from which viruses can infect humans and pigs. In this study, all of the avian viruses elicited cross-reactive antibodies against the heterologous avian viruses and against some swine and equine H3 influenza viruses. Although H3N8 influenza viruses have not been isolated from humans, coinfection of pigs with these and circulating human influenza viruses could result in emergence of a reassortant virus. The recent emergence of an H3N8 avian influenza virus in seals is of concern, because these viruses have acquired mutations that reflect adaptation to mammalian hosts and that increase virulence and transmissibility in avian H5N1 viruses infecting mammals (57–59). We have selected tl/TX/079/07 (H3N8) as a vaccine candidate.
In summary, while H3N2v viruses present the most immediate threat among swine H3 viruses, equine and avian H3 viruses should not be ignored, because both have crossed the species barrier. Based on the replicative capacity in the upper and/or lower respiratory tract and the cross-reactive antibody response against the greatest number of heterologous H3 influenza viruses tested in both mice and ferrets, we have selected the eq/GA/81 and tl/TX/079/07 influenza viruses as H3N8 candidates for vaccine development. We have also established basic information on animal viruses and virus replication in mice and ferrets that will be of value in evaluating the efficacy of these vaccines.
Supplementary Material
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 the staff of the Comparative Medicine Branch, NIAID, and the staff at MedImmune's Animal Care Facility for excellent technical support for animal studies. We thank Gabriel I. Parra for assistance with phylogenetic analyses. We are grateful to Richard Webby, Christopher Olsen, Debra Elton, Nuria Busquets, John M. Pearce, Michael Osterholm, Suzy Carman, and Edward J. Dubovi for providing the viruses used in this study.
Footnotes
Published ahead of print 10 April 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.03520-12.
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