This study showed that pdm flu vaccination (A/California/7/2009) induced increased levels of A/H1N1/California/7/2009-specific Abs, but also strong immune cellular responses (measured by IFN-γ production in a whole-blood assay) directed against flu A antigens from H1N1, H3N2 and H5N1 strains, flu B strains, and the flu M1 matrix antigen.
We have not been able to include the pdm flu strain. However, increased IFN-γ production after vaccination suggested that A/California/7/2009-specific T-cell responses were induced by pdm flu vaccination. This would imply that pdm flu vaccine induced (cross)-reactive cellular responses did not only target the flu A H1N1 strains, but also H3N2, H5N1 and flu B strains. In agreement with this observation, we showed that participants who received the pdm flu vaccination exhibited also a significant increase in Ab titers directed against A/H1N1/Solomon Islands/3/2006 and A/H5N1/Vietnam/1203/2004, while participants who did not receive the pdm flu vaccine exhibited only a significant increase in A/H1N1/California/7/2009-specific Abs but not in Abs against the other flu A strains tested by the flu hemagglutination inhibition assay (data not shown).
The flu vaccine, studied in this report, represents a split virus vaccine, which is mainly composed of surface membrane glycoproteins, yet traces of M1 have also been detected  as part of the vaccine formulation. We tested M1 and M2 as targets for cellular immune responses in the current study. The M2 protein is a proton-selective ion channel protein, integral in the viral envelope of the influenza A virus. M2 brings protons into the virion core. Acidification of virus interior, leads to weakening of electrostatic interaction and leads to dissociation between M1 (matrix protein) and viral rib nucleoprotein (RNP) complexes . Traces of M1 in the split vaccine may be responsible for the strong anti-M1 directed responses defined by IFN-γ production, which has not been reported until now. Flu vaccines are produced and standardised based on their haemagglutinin and neuraminidase content, and traces of the M1 proteins, contained in the current standard vaccines, may in part be responsible for conferring protective immune responses between flu A strains, since the M1 protein is quite conserved . The M1-directed cellular immune responses, along with traces of M1 proteins in split vaccines may in part responsible for the cross-reactive immune responses against H5N1 associated with the pdm flu vaccine, since CD4+ and CD8+ T-cell responses directed against H5N1 have preferentially reported to focus on M1 or NP (nucleoproteins) .
In addition, we showed at study entry that previous seasonal flu vaccination (in 2006–2009) did not lead to an increased IFN-γ production in response to flu antigen components from 2006–2009 flu vaccines, but was instead associated with significantly lower IFN-γ production in response to flu antigen components from 2008–2009 flu vaccines (A/H1N1/Brisbane/59/2007, A/H1N1/Solomon Islands/3/2006 and B/Florida/4/2006). A similar trend was observed when analysing H1N1/Brisbane/59/2007- and A/H1N1/Solomon Islands/3/2006-specific antibodies. However, at the end of the study, we could not detect significant differences in IFN-γ production in response to flu antigens between previously vaccinated and unvaccinated participants. This suggests that after the flu season, previously vaccinated and unvaccinated participants were able to mount comparable immune responses (as measured by IFN-γ production in the whole-blood assay).
A recent report showed that individuals with a history of seasonal flu vaccination exhibited after natural pdm flu infection or pdm flu vaccination a skewed Ab response towards previously encountered flu antigens, further proving the impact of previous flu vaccination on subsequent infection or vaccination . Seasonal flu vaccination of children has been reported to interfere with the development of heterosubtypic immunity  and Ab responses to pandemic H1N1 appeared to be reduced in participants who received seasonal flu vaccination 3 months prior to vaccination with the pmd flu vaccine . The report by Skowronski and co-workers also suggested an association between the previous 2008–2009 flu vaccination and pandemic H1N1 illness in Canada ; the mechanisms underlying this finding are ill-defined and warrant further research to better understand the impact of seasonal flu vaccination, i.e. the potential ‘negative imprint’ of previous vaccinations on cellular immune memory responses.
Reservations about flu vaccination, including the notion that flu vaccination would not result in appropriate protection, have been discussed in the public domain [24–26]. One of the arguments is that flu vaccination may not protect against ILI symptoms during flu the season. This notion was corroborated in our study. A number of study participants, after pdm flu vaccination, experienced more ILI symptoms, perhaps due to the observed increase in prevalence of rhinovirus infection in pdm flu vaccinated participants as compared to non-pdm flu vaccinated participants. Other reasons may account for these observations, one of them being the low predictive value of the ILI case definition . Future prospective studies may address the question whether certain flu vaccines are able to increase cellular immune responses in the respiratory system, particularly after encounter with the wildtype flu. Increased ‘influenza-like’ symptoms may occur upon exposure to additional pathogens, such as rhino- or coronavirus, that stimulate ‘flu-primed’ innate or adaptive immune responses. This hypothesis is supported by the observation that infection with rhinovirus may have delayed the circulation of H1N1, most likely via activation of non-specific innate immune responses in the respiratory system .
Study participants who received the pdm flu vaccine showed similar IFN-γ production profiles in response to the flu antigens tested, irrespective of the experience of ILI symptoms. However, we observed that study participants who received the pdm flu vaccine and reported ILI symptoms (compared to non-pdm flu vaccinated participants with ILI symptoms), showed by the end of the study a comparable increase in A/California/7/2009-specific Abs and an increase in IFN-γ production in response to the flu A H1N1 and H3N2 strains. This immune status could not be observed in participants who reported no ILI symptoms, irrespective of whether or not they received the pmd flu vaccination. This suggests that some pmd flu vaccinated participants who reported ILI symptoms may have been exposed to the H3N2 seasonal flu supporting previous observations that pmd flu vaccination did not affect the rate of H3N2 infections . The pdmH1N1 strain dominated the flu season in Sweden in 2009–2010, but flu H3N2 and flu B were also present in 1.7% and 1.4% of the samples analysed by the Swedish National Influenza Centre .
The comparable increase in A/H1N1/California/7/2009-specific Abs in serum from unvaccinated and pdm flu vaccinated study participants who reported ILI symptoms also suggests that non-pdm flu vaccinated study participants may have been exposed to the pdm flu.
We speculate that the non-pdm flu vaccinated study participants who reported ILI symptoms had been exposed to the pdm flu. These individuals exhibited lower levels of IFN-γ production in response to the H1N1 and H3N2 flu antigens as compared to pmd flu vaccinated study participants who reported ILI symptoms. This would also fit with the hypothesis that IFN-γ production directed against related flu antigens upon natural by pdmH1N1 infection is short-lived as compared to IFN-γ induced by pdm flu vaccination. This notion was indeed corroborated: we showed that shortly after a positive pdmH1N1 PCR, IFN-γ production to most of the flu antigens was significantly increased, yet declined by the end of the study (May 2010). In contrast, we could detect significantly higher levels of IFN-γ production in response to M1 (which is not a designated vaccine component) at the end of the study in pdm flu vaccinated participants; this was not the case for study participants who tested positive for pdmH1N1 by PCR (Table 6).
Finally, our observation that study participants i) with a negative pdm flu PCR ii) absent pdm flu vaccination and iii) a negative ILI symptom report, showed increased IFN-γ production in response to most of the flu antigens, concomitant with the absence of A/H1N1/California/7/2009-specific Ab increase, suggests that these individuals may have been exposed to pdm flu, i.e. that they were ‘silently infected’ and developed a strong anti-pdm flu T-cell (but not a B-cell) response.