In the exploratory immunogenicity study, in agreement with the findings of other studies of children, a single dose of CAIV-T 107 generated the most consistent serum HAI antibody responses, although the level of response differed by vaccine strain and serostatus. In general, the HAI antibody levels for each of the three virus strains were higher among subjects who received CAIV-T 107 than among the other treatment groups. Specifically, among CAIV-T 107 recipients, serum HAI antibody responses to the A/H3N2 strain were greatest, followed by the B strain and then the A/H1N1 strain. TIV recipients exhibited the greatest antibody response to the A/H1N1 strain of all the treatment groups. Higher rates of seroconversion to the A/H3N2 virus strain and B strain for CAIV-T 107 recipients than for every other treatment group were statistically significant.
Levels of antibody to influenza virus in serum, as measured by the HAI assay, whether elicited by naturally acquired infection or through vaccination, have long been considered a correlate of protection against clinical influenza. More accurately, though, the studies to date investigating the role of serum antibody as a correlate have found that the protective levels of antibody have differed considerably (4
). A series of studies with healthy adults vaccinated with either TIV or LAIV followed by challenge with wild-type H1N1 or H3N2 viruses demonstrated that serum HAI antibody correlated with protection against viral replication after TIV but not LAIV vaccination (18
). This finding was supported by a more recent investigation that demonstrated that a LAIV containing A/Beijing/262/95(H1N1) elicited relatively low rates of seroconversion to A/New Caledonia/20/99(H1N1) virus in seronegative children, as measured by an HAI assay, that were markedly lower than the level of efficacy demonstrated in the community efficacy trial (37
). Another earlier trial found an association between serum HAI antibody and protection but found that some other factor was contributing to the protection of vaccinated subjects who were seronegative (7
). In that study, some vaccinated children had neither serum HAI antibody nor nasal wash immunoglobulin A, and it was postulated that an alternative immune mechanism, such as cellular immunity, may have contributed to protection by the live vaccine.
While there is no question from the previous investigations using CAIV-T or other LAIV in young children that serum HAI antibody is associated with protection against influenza, no linear correlation between protection from influenza and antibody titers has been established for any LAIV.
It is generally accepted that CMI has a significant role in recovery from clinical disease caused by influenza virus infection as well in the prevention of the development of influenza-associated complications, but it does not seem to contribute significantly to preventing infection (20
). However, the possible beneficial effects of the induction of T-cell-mediated immunity by vaccination in ameliorating the severity of influenza in humans remains largely unexplored (44
CMI can be differentiated into TH1 and TH2 types based on the production of specific cytokine profiles. For TH1 and TH2 responses, the major cytokines are IFN-γ and interleukin-4, respectively (60
). TH1 responses, through the production of IFN-γ, mediate the killing of organisms responsible for a variety of intracellular infections (60
), and as such IFN-γ is the cytokine that mediates protection. However, while NK cells of the innate immune system also are a significant source of IFN-γ (27
), it has been demonstrated that for influenza A viruses, this production is dependent on the generation of influenza A virus-specific T lymphocytes (30
IFN-γ has been measured using a variety of techniques, including flow cytometry and determination of IFN-γ concentrations in the supernatants of ex vivo-stimulated lymphocytes by enzyme-linked immunosorbent assay (29
), but it was the emergence of the ELISPOT assay that provided a simple measure of CMI, utilizing a low volume of blood, with the potential for large-scale application in the field. The volume of blood required has been identified as a critical factor in measuring CMI in very young children and infants (62
). However, there remain very few studies of CMI in very young children, and until this report, none have addressed the relationship between CMI and vaccine efficacy in a field setting.
Before proceeding with the application of the IFN-γ ELISPOT assay to the investigation of the role of CMI in the protection of very young children and infants against influenza in a community setting, we sought to confirm previous findings on the elicitation of IFN-γ in children following vaccination.
It has been observed previously that for children between the ages of 5 and 9 years, a single intranasal dose of live attenuated influenza virus vaccine (CAIV-T) elicited significant mean increases in the percentage of influenza A virus-reactive IFN-γ-positive cells in T-lymphocyte and NK cell subsets, as measured by flow cytometry following in vitro stimulation with influenza virus (31
), and that a single intramuscular dose of TIV in these older children elicited lower mean CMI responses than CAIV-T.
In the exploratory study described in this paper, despite the fact that it was conducted with younger children, aged just 6 months to <36 months, and despite differences in methodology (use of an IFN-γ ELISPOT assay for CMI detection; reporting median values in defining responses), we observed similar findings. In our studies, a single intranasal dose of 107
FFU of CAIV-T consistently elicited significant CMI responses, while TIV elicited negligible responses. Although the latter observation differs from the report by He et al. in that they observed strong CMI responses to TIV in children aged 6 months to <5 years, they did not explore CMI responses elicited by CAIV-T in this younger age group (31
). In both studies, the LAIV strains were provided by MedImmune, Inc., an identical single intranasal dose of CAIV-T was used, and the vaccine strains were based on the 6:2 cold-adapted temperature-sensitive reassortant influenza viruses originally described previously (8
From the exploratory study, despite the small numbers of subjects, it was clear that CAIV-T elicited more-frequent and higher-magnitude CMI responses than TIV and as such represented the optimal vaccine for further evaluation of the relevance of these observations to vaccine efficacy.
It has been observed previously that responses elicited by LAIV, but not TIV, were inversely proportional to prevaccination levels of influenza virus-specific antibody for all age groups, suggesting a strong role of previous exposure to the antigen in eliciting IFN-γ responses upon reexposure (23
). One notable exception was that in a previous report, children aged 6 months to <5 years, with little evidence of previous exposure, were most responsive to TIV (31
). There was no LAIV comparison; thus, those authors encouraged future studies to compare responses to TIV and a LAIV (e.g., CAIV-T) in this age group, as reported here.
In our study, the ability of CAIV-T to elicit CMI responses in children was similarly strongly affected by previous exposure to related or antigenically similar influenza viruses, either through vaccination or through natural exposure, as measured by prevaccination serum HAI levels.
Responses to TIV were elicited only in those children with detectable levels of preexisting antibody against influenza virus. This is consistent with the adult experience in that TIV seems to boost CMI and antibody responses only in those previously exposed to natural infection (28
). In this study, the advantage of the CAIV-T 107
dose level was that it elicited strong CMI responses among children with no detectable previous exposure, who potentially had the highest susceptibility to influenza virus infection.
This difference may be explained through the known persistence of live vaccine virus in immunologically naïve children following administration of CAIV-T, which may contribute to the maintenance of memory cells (3
). Persistence of live influenza vaccine viruses has been observed previously: 67.4% of all children aged 6 to <36 months shed vaccine virus over a 7-day period following a single dose of CAIV-T 107
Since the frequency of T lymphocytes specific for a given infectious organism is low in children who have not previously encountered that pathogen, vaccination can cause the numbers of antigen-specific T lymphocytes to increase manyfold, leading to the formation of a population of long-lived memory T cells capable of responding rapidly to future infection in this population (10
). It was evident that both parenteral vaccination (TIV) and live attenuated virus vaccination could restimulate systemic CMI memory responses toward influenza virus that had initially been generated through natural infection, confirming that identifying the role of CMI in protection would prove difficult in these age groups (20
). Therefore, for evaluation of the role of CMI in protection against clinically manifested, culture-confirmed influenza virus infection, very young children, who had the least prior exposure to influenza virus through vaccination or natural exposure and the maximal documented CMI responses after vaccination with CAIV-T, appeared the most appropriate candidate population. Further, the IFN-γ ELISPOT assay, as used in our exploratory study, appeared to provide comparable results across age groups, as reported previously, despite methodological differences.
For young children, we were able to demonstrate that CMI plays a significant role in protection against community-acquired clinical influenza virus infection, as determined by measuring differences in culture-confirmed clinical influenza attack rates across a range of CAIV-T doses. These ranged from a dose demonstrated to elicit suboptimal CMI (105
FFU) to the established protective dose (107
FFU). It has been reported that a certain threshold of effector TH1 CD8+
T (killer) lymphocytes may be required for protection against specific infections. For example, in a rodent malaria model, a threshold level of about 400 IFN-γ-secreting peptide-specific SFC/106
splenocytes is required to protect against sporozoite challenge (59
The estimated protection curve from this investigation of CAIV-T indicated that the majority of infants and young children with ≥100 SFC/106 PBMC were protected against clinical influenza, establishing this as a possible target level for protection in future influenza vaccine development.
The major challenge confronting those performing complex immunological studies with human infants and very young children is obtaining a sufficient volume of blood or drawing blood frequently enough to perform an expanded range of assays in order to further characterize the immune responses, including serum HAI antibody responses (13
In this trial, ethical considerations at the trial sites prevented additional blood draws or increased blood volume to permit characterization of the serum antibody responses. However, it had been observed previously following influenza and hepatitis B surface antigen vaccination that there is no correlation between serum antibody responses and CMI as measured by IFN-γ levels (31
). Further, the presence of serum HAI antibody was associated with a reduced influenza B vaccine virus take in young children, although not with a linear correlation (8
). The existence of another factor contributing to protection of vaccinated subjects who were seronegative by serum HAI antibody was also postulated by the authors of that study. Given the previously documented specific antibody responses elicited by CAIV-T vaccine in young children, it is reasonable to attribute a considerable part of the unaccounted-for protection identified in this study to specific antibodies.
CAIV-T has been demonstrated to elicit cross-reactive antibody and to confer a substantial degree of protection against heterologous, drifted influenza viruses (6
). At least four field efficacy trials have demonstrated that immunization with a LAIV can protect against antigenically drifted influenza virus strains, in addition to providing protection against homologous influenza virus strains (6
). In a pivotal efficacy field trial of CAIV-T in children, the drifted variant A/H3N2/Sydney/5/97 caused the majority of disease in year 2 of the study (6
). The formulation of CAIV-T used that season contained A/Wuhan as its H3N2 antigen; Wuhan and Sydney were significantly different antigenically, as determined by ferret antisera. CAIV-T was 86% efficacious at preventing culture-confirmed influenza due to A/H3N2/Sydney/5/97. In the same year, an effectiveness study of CAIV-T in adults demonstrated significant reductions in days of work lost among vaccinated adults versus placebo recipients (48
However, in this study, although CMI responses to the homologous (B/Yamagata-lineage) virus were elicited by the CAIV-T 107
dose level, these do not appear to be effective in contributing to protection against clinical illness with a significantly divergent influenza B virus (49
). A previous efficacy trial performed with children aged 6 to <36 months attending day care demonstrated that CAIV-T showed efficacy against influenza B viruses antigenically similar to the vaccine but also showed reduced efficacy by the B/Yamagata-lineage vaccine virus against circulating wild-type B/Victoria/87-lineage viruses (B/Hong Kong/1351/02-like and B/Hong Kong/330/01-like viruses) (67
Modern influenza B viruses are derived from one of two lineages: either B/Yamagata or B/Victoria/87. The CAIV-T formulation used in this trial contained a B/Yamagata-lineage vaccine virus. The influenza B epidemic that occurred during the trial comprised influenza B/Victoria/87-lineage viruses (B/Hong Kong/1351/02-like and B/Hong Kong/330/01-like viruses). These are not considered drift strains and as such may share fewer common antigens (17
This investigation represents the most thorough exploration of the role of CMI in protecting humans from influenza following vaccination. The efficacy evaluation is the single largest demonstration for any infectious disease of a quantitative role of CMI in protection against culture-confirmed infection, irrespective of pathogen, especially in children. It can be concluded from these studies that the ELISPOT assay for IFN-γ is a sensitive and reproducible measure of CMI and memory immune responses in all age groups, from young children to elderly adults, with application in a field setting. Further, the assay enabled the detection of previous exposure as well as the identification of immune responses following vaccination that might not otherwise be observable.
These findings contribute significantly to our understanding of the immune correlates of protection in influenza, as well as furthering our understanding of the requirements for generating and sustaining cellular immune responses. Further, because these findings can be used to guide predictions of the likelihood of protection induced following influenza vaccination, they define the requirements for future development of vaccines against influenza, especially those for use in children.