immunization categories , experimental groups , and influenza virus strains used are shown in the table .
twenty - seven pigs were intranasally ( in ) inoculated with 7.0 log10 50% egg infectious doses of swine / belgium/1/98 ( avian - like swine influenza [ h1n1 ] ) , swine / gent/7625/99 ( reassortant influenza [ h1n2 ] with human - lineage ha ) , swine / flanders/1/98 ( reassortant influenza [ h3n2 ] with human - lineage ha ) , or some combination of the three
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 ( i m ) 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 ) .
phylogenetic lineage of the hemagglutinin gene : c , classical swine ; hu , human ; av , avian . 
infection with swine / belgium/1/98 ( h1n1 ) , swine / gent/7625/99 ( h1n2 ) , or swine / flanders/1/98 ( h3n2 ) . 
the first 3 vaccines are bivalent ( subtypes h1n1 , h3n2 ) ; the fourth is trivalent ( subtypes h1n1 , h3n2 , h1n2 ) . 
# infection with swine / belgium/1/98 ( h1n1 ) .  * * each individual pig was hyperimmunized with a different european siv isolate : subtype h1n1 viruses were swine / finistre/2899/82 , swine / belgium/1/98 , and swine / gent/132/2005 ; subtype h1n2 viruses were swine / scotland/410440/94 , swine / gent/7625/99 , and swine / gent/177/2002 ; subtype h3n2 viruses were swine / gent/1/84 , swine / flanders/1/98 , and swine / gent/131/2005 .
six pigs were first inoculated in with swine / belgium/1/98 ( h1n1 ) followed by a single i m administration of new jersey/8/76-based siv vaccine 5 weeks later .
nine pigs were hyperimmunized against various european sivs ( table ) by in inoculation , followed by an i m innoculation with the same virus in combination with freund s complete adjuvant 4 weeks later .
all serum samples were examined in hemagglutination - inhibition ( hi ) assays against the european subtype h1n1 , h1n2 , and h3n2 viruses listed above ; 3 north american sivs with a classical h1 ; and a / california/04/2009 , a prototype pandemic ( h1n1 ) 2009 virus ( 7 ) . the north american sivs included swine / iowa / h04ys2/2004 ( triple reassortant influenza [ h1n1 ] ) , swine / ontario/11112/04 ( reassortant influenza [ h1n1 ] ) , and swine / indiana/9k035/99 ( triple reassortant influenza [ h1n2 ] ) .
low amino acid homology was present in the ha1 region of the ha gene between the european h1 sivs used for infection of pigs and the north american h1 sivs ( range 70%75% ) or the pandemic ( h1n1 ) 2009 virus ( 69%72% ) .
when compared with avian - like influenza ( h1n1 ) strains in european vaccines , the new jersey/8/76 vaccine strain was more closely related to north american sivs ( 77%81% vs. 92%94% ) and pandemic ( h1n1 ) 2009 virus ( 72%75% vs. 90% ) .
hi antibodies induced by a single infection with european subtype h1n1 or h1n2 sivs did not cross - react with north american h1 sivs or with pandemic ( h1n1 ) 2009 virus .
in contrast , consecutive infections with 2 european subtypes frequently induced cross - reactive antibodies , even though european viruses do not contain a classical swine h1 ha .
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 containing an avian - like siv ( h1n1 ) . a single vaccination with a / new jersey/8/76-based vaccine induced only minimal hi antibody titers in influenza - 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 influenza ( h1n1 ) viruses , but they were rare or absent after hyperimmunization with subtype h1n2 or h3n2 strains .
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 first 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 influenza virus variants seemingly
lead to a predominant antibody response against cross - reactive epitopes on the ha , which are shared with the first infecting virus , whereas the response to strain - specific epitopes may be lower ( 10 ) .
such cross - reactive antibodies may cause cross - recognition of viruses with a classical h1 ha in our dually infection 
immune 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 finishing 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 influenza 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 influenza 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 influenza ( 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 influenza ( 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 influenza ( h1n1 ) viruses that are more closely related to classic swine influenza ( h1n1 ) viruses and pandemic ( h1n1 ) 2009 virus .
two antigenically distinct influenza ( h1n1 ) viruses  seasonal and the 2009 virus  could cocirculate in humans in the future .
this cocirculation will likely broaden serologic responses and protection against influenza but may also complicate interpretation of hi test results .
 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 . 
The Study
Conclusions