Influenza pandemics are very difficult to anticipate, both with regard to timing and with regard to species origin, and can be devastating in their impact. The unpredictability of the emergence of new pandemic strains of influenza virus in human populations is a particularly significant concern at this time, because new influenza vaccines are difficult to produce with current technology and generally require at least 6 months to prepare and test before distribution (reviewed in references 23
, and 66
). Accordingly, there is great interest in understanding if and how the encounter with seasonal viruses and vaccines influences the ability of the immune system to respond to novel pandemic strains of influenza virus. In the study reported here, we have specifically evaluated the ability of a previously widely circulating seasonal strain of influenza A H1N1 virus to potentiate responses to the novel pandemic strain of H1N1 that arose in Mexico in the spring of 2009. In evaluating this issue, we examined two inbred mouse strains that express different MHC molecules and background genes for their CD4 T cell reactivities toward epitopes conserved in seasonal and pandemic influenza viruses, the impact of CD4 memory on B cell responses to the pandemic strain, and the ability of the encounter with a seasonal strain of influenza virus to protect against the pathological effects of the pandemic strain.
A number of important results were revealed by these studies. First, our studies revealed that different inbred mouse strains are differentially susceptible to the pathological consequences of infection with the H1N1 strain of the virus: A/J mice are more susceptible than BALB/c and C57BL/10 mice (not shown). Other studies have shown that the mouse genetic background can dramatically impact susceptibility to influenza A virus strains (1
). In one particularly systematic study of protective immunity to A/Puerto Rico/8/34 (A/PR8) (58
), a mouse-adapted laboratory strain of influenza virus, A/J and DBA/2 mice displayed the most susceptibility to infection, while C57BL/6, BALB/c, CBA, and FVB/NJ mice were among the more resistant strains and ultimately recovered from infection. This study suggested that the genetics of susceptibility are complex and involve multiple stages of influenza virus replication, the innate response, and the adaptive immune response. In the BALB/c mice that we have studied for this report, the activity of CD8 T cells does not appear to be responsible for the resistance to infection, since removal of these cells by antibody-mediated depletion did not render the mice more susceptible to the pathological effects of virus infection, such as weight loss and virus-induced mortality (data not shown). This finding is in agreement with recent studies showing that exacerbated inflammatory responses often characterize mouse strains highly susceptible to influenza infection (6
). Studies on human subjects have also suggested that susceptibility to influenza is shared by close relatives and is thus a heritable property (2
). In our studies, although we have not determined the mechanisms that underlie the rapid weight loss induced by A/California/04/09 infection, we have found that mice of the most susceptible strain, A/J, become highly resistant to virus-induced weight loss and mortality if they are preexposed to a nonpathogenic seasonal strain of H1N1 virus, suggesting that immunological memory overrides the natural susceptibility of this mouse strain to the pandemic virus. The results of CD8 depletion experiments indicate that this protection does not depend on CD8 T cells, although it is likely that in normal animals or humans, CD8 T cells contribute significantly to protection. It is likely that memory CD4 T cells can contribute to protection in the secondary challenge by multiple mechanisms. As discussed above, they may facilitate early production of neutralizing antibodies and are likely to provide a direct effector function in response to influenza (reviewed in references 37
). Recent studies (59
) suggest that memory CD4 T cells can induce rapid production of a number of innate inflammatory cytokines and chemokines within the lungs of influenza-infected animals, which may dramatically reduce early viral replication and facilitate the recruitment of other effector cells (64
). There is also evidence for direct cytotoxic effects of CD4 T cells in response to influenza (8
). Memory CD4 T cells of the appropriate specificity and phenotype are likely to contribute on multiple levels to increased resistance to future challenge with pathogenic strains of influenza virus and to contribute to more-effective responses to vaccination.
In examining the peptide specificity of the elicited CD4 T cells, we found that a surprisingly large fraction of the CD4 T cell response was dedicated to genetically conserved peptides that either were identical in the seasonal and pandemic strains or differed by only a single amino acid. Significantly, reactivity to conserved peptides, which are likely to be recalled upon challenge with the pandemic strain, was not limited to the highly conserved NP, NS1, and M1 proteins but was also readily apparent for conserved segments of HA and NA. Recent studies on responses to vaccinia virus suggest that CD4 T cell help to antigen-specific B cells may be best conveyed by T cells of the same protein specificity as the antigen-specific B cells (53
). This “linked” CD4 T cell help for B cells is probably due to the fact that for some viruses, the antigen internalized through the immunoglobulin receptor in peripheral lymphoid tissue may be an isolated viral protein rather than an infectious virus particle, thus leading to the display of peptides derived from that single protein on the cell surface for recruitment of cognate CD4 T cell help. If this is true for influenza virus, the memory HA- and NA-specific CD4 T cells we have detected here that are reactive to conserved segments of HA and NA may be particularly important in facilitating a protective antibody response to the HA and NA proteins. Our studies revealed that mice previously exposed to the seasonal strain displayed an accelerated CD4 T cell response that was readily detectable at 5 days after infection with the pandemic strain, much earlier than that observed in the primary response. Although the pools of peptides tested in the secondary challenge were unselected and were composed of all viral peptides, it is likely that most of the rapidly responding CD4 T cells were specific for conserved epitopes and thus represented the recall of memory cells. Our preliminary experiments (not shown) using individual peptides representing either new or conserved epitopes suggest that memory CD4 T cells are preferentially expanded in the recall response. Although our experiments described here have shown preferential reactivity to cross-reactive CD4 T cell epitopes during secondary responses to related influenza viruses, they have not explicitly evaluated the phenomenon of “original antigenic sin”. This phenomenon was described many decades ago to explain preferences in antibody responses to influenza (13
) and has been studied intermittently by others since, to examine biases in antibody and T cell responses in challenge experiments toward the strain of influenza virus that was originally encountered (28
). Although this is a potentially important complication of “preemptive” vaccination for protection from novel pandemic influenza virus strains, in order to address this issue appropriately, one would need to compare carefully both the affinity and the fine specificity of CD4 T cells elicited in the primary response and the secondary challenge, which has not been done here.
Additionally, and importantly, mice preexposed to a seasonal influenza virus displayed accelerated production of neutralizing-antibody responses to the new challenge pandemic strain, despite the apparent lack of serological cross-reactivity between the HA molecules expressed by the two viruses. The combined CD4 T cell data and serological analysis suggest that an expanded population of primed CD4 T cells can facilitate new B cell responses that have not been encountered before. According to this model, there may be limiting numbers of CD4 T cells capable of providing help during T-B cell interactions, as has recently been suggested by adoptive transfer experiments (50
). In addition to providing help during challenge with live infection by the novel virus, helper cells in the memory population of CD4 T cells specific for HA and NA may be critical for “dose-sparing” efforts that are needed for rapid deployment of limited doses of vaccines to novel influenza virus strains (reviewed in references 3
, and 54
). The relative contribution of this early antibody production to protection during the secondary challenge is not known at this time, but the local production of neutralizing antibody may help limit viral loads during replication in the lung.
The studies reported here also revealed that the CD4 T cell responses elicited by each virus include specificities unique to the respective strain. This result indicates that responses to the pandemic virus can be distinguished from responses derived from long-term memory. This will allow studies of subclinical encounters with this pandemic strain and analyses of unique phenotypic characteristics of CD4 T cells elicited by this virus. Recent studies on CD8 T cell differentiation suggest that the gene expression patterns and functionality of T cells continue to evolve with each restimulation (69
), suggesting that CD4 T cells specific for epitopes that are boosted repeatedly through life, such as the conserved epitopes, may be quite distinct from those specific for novel epitopes that have expanded only once, and it will be important to determine whether this is the case for responses to influenza. At this time, it is not clear whether the most “experienced” memory CD4 T cells are the most useful for facilitating antibody responses or for contributing to other, less well characterized effector functions of CD4 T cells during the immune response to influenza. Dissection of this issue will be facilitated by the availability of well-characterized peptide reagents that allow distinction between novel and long-term memory CD4 T cells. Additionally, if the CD4 T cells elicited by this particularly novel virus display unique functional characteristics, the use of these peptide reagents would allow phenotypic characterization of the pandemic-virus-specific CD4 T cells.
In conclusion, we have found that CD4 T cells displaying broad peptide and antigen specificity are elicited by seasonal viruses and that these CD4 T cells are specific both for epitopes that are shared with the pandemic strain and for epitopes unique to one of the strains. The genetically conserved epitopes are found within all of the viral proteins tested, and CD4 T cells specific for these epitopes show rapid recall responses during a secondary challenge. This recall response is associated with accelerated neutralizing-antibody production and protection from lethal infection with the pandemic H1N1 strain of influenza virus. In humans in whom multiple class II molecules are expressed, we would expect an even broader repertoire of CD4 T cells and a correspondingly broader repertoire of influenza virus-specific cells expanded by each encounter with seasonal vaccines or viruses. A substantial fraction of the immune responses to seasonal and pandemic influenza viruses will be cross-reactive, and thus, a seasonal influenza virus can elicit cross-reactive memory cells that can be mobilized for different roles in protection against infection with pandemic strains, or that can be used to facilitate rapid antibody responses to novel vaccine candidates.