Serologic Cross-Reactivity with Pandemic (H1N1) 2009 Virus in Pigs, Europe

We tested serum samples from pigs infected or vaccinated with European swine influenza viruses (SIVs) in hemagglutination-inhibition assays against pandemic (H1N1) 2009 virus and related North American SIVs. We found more serologic cross-reaction than expected. Data suggest pigs in Europe may have partial immunity to pandemic (H1N1) 2009 virus.

from descendants of Hong Kong/68 pandemic virus, and a subtype H1N2 reassortant virus, which acquired H1 from human infl uenza (H1N1) viruses in the 1980s (5). We aimed to clarify whether pigs infected or vaccinated with these viruses have cross-reactive antibodies against pandemic (H1N1) 2009 virus or related North American SIVs, both of which have an antigenically distinct, classical H1 HA.

The Study
We tested 66 swine sera obtained from previous experimental studies (6). All pigs were negative for SIV antibodies at the start of the experiments. Serum samples were collected 3-4 weeks after the last infection or immunization. Immunization categories, experimental groups, and infl uenza virus strains used are shown in the Table. Twenty-seven pigs were intranasally (IN) inoculated with 7.0 log 10 50% egg infectious doses of swine/ Belgium/1/98 (avian-like swine infl uenza [H1N1]), swine/ Gent/7625/99 (reassortant infl uenza [H1N2] with humanlineage HA), swine/Flanders/1/98 (reassortant infl uenza [H3N2] with human-lineage HA), or some combination of the three. These viruses are representative of prevailing SIVs in western Europe. Two groups received a single inoculation with either SIV (H1N1) or SIV (H1N2). Three groups received dual, consecutive inoculations with 2 SIV subtypes at a 4-week interval.
Twenty-four pigs received 2 intramuscular (IM) doses of commercial, inactivated SIV vaccine, at a 4-week interval. One vaccine contained a classical swine-lineage subtype H1N1 virus, A/New Jersey/8/76; the remaining 3 contained various avian-like subtype H1N1 SIVs (Table). The vaccines contained various types of adjuvants.
Six pigs were fi rst inoculated IN with swine/ Belgium/1/98 (H1N1) followed by a single IM administration of New Jersey/8/76-based SIV vaccine 5 weeks later. Nine pigs were hyperimmunized against various European SIVs (Table)  Three European vaccines also induced cross-reactive antibodies to North American H1 SIVs, and 2 induced antibody titers >20 to pandemic (H1N1) 2009 virus in most pigs. HI titers against pandemic virus were lower with A/ New Jersey/8/76-based vaccine than with vaccine contain-ing an avian-like SIV (H1N1).
A single vaccination with A/New Jersey/8/76-based vaccine induced only minimal HI antibody titers in infl uenza-naive pigs, but existing antibody titers (data not shown) to European avian-like subtype H1N1 SIV in pigs previously infected with this virus dramatically increased, even though these viruses contain HA proteins of the avian and classical swine lineages respectively. All pigs had antibodies to the viruses with a classical swine H1 HA, including pandemic (H1N1) 2009 virus.
Finally, hyperimmunization with European SIVs of subtype H1N1, H3N2, or H1N2 resulted in high titers to the homologous viruses. Cross-reactions with viruses with a classical H1 were consistently observed with serum samples from pigs hyperimmune to avian-like European infl uenza (H1N1) viruses, but they were rare or absent after

Conclusions
A large antigenic and genetic distance exists between European H1 SIVs and viruses with a classical H1 HA (8,9). Nevertheless, pigs infected or vaccinated with European SIVs frequently have cross-reactive HI antibodies to pandemic (H1N1) 2009 virus and related North American SIVs. Two factors predispose for serologic cross-reactivity: 1) elevated antibody titers to European avian-like subtype H1N1 SIVs, as in hyperimmune swine serum and some postvaccination serum; and 2) infection with 2 European SIV subtypes.
Consecutive experimental infection of pigs with the antigenically distinct SIVs of subtypes H1N1 and H1N2 causes a strong boost of already existing HI antibody titers to the fi rst infecting virus, as shown by longitudinal investigations (6). These pigs may even develop low levels of cross-subtype-reactive HI antibodies to SIV (H3N2). In humans and in mice, sequential infections with infl uenza virus variants seemingly lead to a predominant antibody response against cross-reactive epitopes on the HA, which are shared with the fi rst infecting virus, whereas the response to strain-specifi c epitopes may be lower (10). Such cross-reactive antibodies may cause cross-recognition of viruses with a classical H1 HA in our dually infectionimmune pigs, but further studies with monoclonal antibody escape mutants are needed to understand cross-reactivity at the molecular level. Multiple SIV infections have become common in swine-dense regions of Europe since the introduction of the H1N2 subtype in the late 1990s (11).
The HI test will clearly fail to differentiate here between established SIVs and pandemic (H1N1) 2009 virus. This conclusion is further supported by preliminary HI tests on sera collected from fi nishing pigs in Belgium during 2007. Of 172 serum samples, 35% and 36%, respectively, were positive against swine/Iowa/2004 (H1N1) and swine/ Indiana/99 (H1N2); 95% of these positives had antibodies to >1 European SIV subtype.
Besides serum antibody to the HA, an infection with live infl uenza virus also stimulates mucosal immunity and cellular immune responses to highly conserved viral epitopes (12). These responses mean partial protection against a subsequent infection with an antigenically unrelated strain may occur in the absence of cross-reactive antibodies (13). In contrast, protection offered by inactivated infl uenza vaccines is almost entirely dependent on serum HI antibody titers. Two of the 4 vaccines used induced HI antibody titers against pandemic (H1N1) 2009 virus that are considered protective. Unexpectedly, avian infl uenza (H1N1) SIV-based vaccine gave higher antibody titers than the A/New Jersey/76-based vaccine. The amino acid homology between vaccine and test strains is thus a less reliable predictor of serologic cross-reactivity than one would assume. Our data suggest that preexisting immunity to established SIV strains may partially protect pigs in Europe against pandemic (H1N1) 2009 virus, but the extent of such protection needs to be assessed in well-controlled challenge experiments. Pandemic (H1N1) 2009 infection in pigs in Europe has so far been limited to countries where SIVs are absent or circulating at low levels. Whether the virus will become established in countries where SIVs are widespread remains to be seen.
The divergence between H1 of contemporary seasonal infl uenza (H1N1) viruses of humans and pandemic (H1N1) 2009 virus is approximately the same as that between SIVs in Europe and North America. However, cross-reactive HI antibody responses to pandemic (H1N1) 2009 virus have been almost exclusively detected in humans born before 1950 (14). These persons have been exposed to older variants of seasonal infl uenza (H1N1) viruses that are more closely related to classic swine infl uenza (H1N1) viruses and pandemic (H1N1) 2009 virus. The present epidemiologic situation in humans thus differs from that in pigs. Two antigenically distinct infl uenza (H1N1) viruses-seasonal and the 2009 virus-could cocirculate in humans in the future. This cocirculation will likely broaden serologic responses and protection against infl uenza but may also complicate interpretation of HI test results.