In April 2009 a novel H1N1 influenza virus spread throughout the USA and Mexico and then to the world. Its origins remain unknown but sequence analysis of the genome revealed that the new human virus was a reassortant between two swine viruses. There are no data to indicate whether this reassorting event occurred in pigs, in people or in laboratory culture [1
]. The new virus is variously called “pandemic”, “2009” or “swine-origin” H1N1 (swH1N1).
The influenza type A genome consists of eight segments of single stranded, negative-sense RNA, each coding for one or two proteins using alternative splicing or alternative reading frames. When two different influenza A viruses infect a cell there can be a reassorting of the genome segments, and this is considered to be the origin of new pandemic viruses. Genes coding for the major surface antigens of an animal or bird virus can become incorporated into an otherwise human virus, producing a reassortant virus that replicates in humans but has hemagglutinin (HA) and sometimes neuraminidase (NA) antigens that are new to the human immune system. The 2009 swH1N1 virus has a different origin. All of its genes are derived from swine influenza viruses; the NA and matrix genes are from the Eurasian swine virus lineage while the remainder are from a “US triple reassortant” virus [2
]. Although the HA of the new virus diverged from classical swine H1N1 HA around 1998, there is still considerable similarity with classical swine HA (such as the A/New Jersey/76 strain) [2
] and also with older human HAs as has been noted in comparing antigenic properties [5
]. Not surprisingly, older people were found to have more anti-swH1N1 antibodies than younger cohorts in samples taken before the pandemic, but the age cut-off defining “young” vs. “older” subjects varies widely in different reports, making it difficult to compare them [4
]. At least one study found anti-swH1N1 antibodies were higher in people who had been vaccinated in 1976 with the swine influenza vaccine [9
CDC estimated that approximately 60,000,000 people in the US were infected by swH1N1 in the first year of circulation. The disease caused by this virus is usually mild, but the virus was more commonly isolated from younger people than the elderly population that is usually at risk for complications from influenza infection, and pregnant women were found to be among the more severe cases. Estimates of infection rates using serology varied with age and location, and children seroconverted at higher rates than older people [8
Curiously, the seasonal vaccine was reported to provide little or no protection against swH1N1 infection [4
]. The lack of any cross-reactivity is an anomaly, given that the definition of the H1 subtype is that antibodies against one H1 HA will cross-react with other H1 HAs. There is also wide diversity in results from one study to another. It is well recognized that the hemagglutinin-inhibition (HAI) assay used to measure seroconversion is no longer a reliable indicator of vaccine responsiveness or recent infection, partly because high levels of pre-existing antibodies suppress further increase in the HAI titer, and partly because of inherent large errors in the method, which measures an end point from two-fold dilutions. In recent years HAI results have been confounded by varying specificity and affinity of receptor binding [11
]. Each virus of interest can be tested with several species of red blood cells, but even after optimizing the red cell species, the results are highly variable [13
]. The goal of our study was threefold: 1) to determine whether multiple and/or alternative B cell and T cell parameters are more reliable than HAI alone in assessing immune responses to vaccination and infection, 2) to look for evidence of cross-reactive antibodies to swH1N1 as a result of immunization with the seasonal vaccine, and 3) to compare the magnitude of immune responses to vaccination vs. infection. We applied our Native ELISA method [14
], HAI and a new microplate format NA inhibition (NAI) assay to investigate induction of antibodies, along with T cell proliferation and IFNγ production to measure T cell responses, in two sets of subjects: (i) those who were vaccinated first with seasonal trivalent subunit influenza vaccine (2009-2010 formulation) and secondly with monovalent swH1N1 vaccine and (ii) those who suffered an influenza-like illness during the pandemic in Oklahoma City in summer/fall of 2009. The results show that individuals vary in whether they respond with increased amount and/or affinity of antibodies and whether the increase is accompanied by robust T cell activity. Thus, multiple parameters are needed to assess the immune response to infection or vaccination. Half of the subjects showed an increase in antibodies that cross-reacted to swH1N1 after seasonal vaccination. Antibody affinity and NAI activity were higher after natural infection than after vaccination.