We have previously shown that the formulation of a clade 1 A/Vietnam/1194/2004 H5N1 split-virion vaccine with an α-tocopherol containing oil-in-water emulsion-based Adjuvant System (AS03A
) induced broad clade 2 cross-reactive humoral immunity and reduced the amount of antigen required to produce a humoral response that meets the European Union Committee for Medicinal Products for Human Use and US Food and Drug Administration licensure criteria [11
]. Two doses of adjuvanted H5N1 split-virion vaccine containing 3.75 μg HA induced high levels of neutralizing and HI antibodies against the clade 1 vaccine strain, as well as a significant cross-reactive neutralizing antibody response against the clade 2 H5N1 isolates A/Indonesia/5/2005 (subclade 2.1), A/turkey/Turkey/1/2005 (subclade 2.2), and A/Anhui/1/2005 (subclade 2.3) [11
]. Here, we have investigated the CD4 T-cell and memory B-cell responses underlying these cross-reactive antibody responses. CD4 T-cell responses were detected by intracellular cytokine staining after stimulation with either split virus or overlapping peptides. The present study makes four points: First, we show that all responses, i.e., antibodies, B cell memory, and CD4 T cells, are increased when the vaccine is formulated with AS03A
adjuvant. Second, persistent neutralizing antibody responses are mirrored by long-lived memory B-cell responses. Third, vaccination induces polyfunctional and highly cross-reactive CD4 T-cell responses and results in long-lived central and effector CD4 T-cell memory. Fourth, H5N1-specific CD4 T and memory B cells are already detected before vaccination.
We observed stronger memory B-cell responses for the H5N1 antigen when the vaccine was formulated with adjuvant. Memory B-cell frequencies were increased after the first vaccination and rose further after the second vaccination. Since antibody-secreting plasma cells and memory B cells may represent independently regulated cell populations [29
], long-lived memory B cells may not play a direct role in the maintenance of antibody levels, but they could differentiate into plasma cells after booster vaccination or natural infection. Therefore, higher levels of memory B cells would predict a better ‘boostability’ of the response. We further observed increased H5N1-specific CD4 T-cell responses with the adjuvanted vaccine. The frequencies of antigen-specific, polyfunctional CD4 T cells increased strongly at day 21 post-vaccination with the adjuvanted vaccine. There appeared to be no increase in the response after the second vaccination, i.e., at day 42. A possible explanation for this perceived lack of further expansion is that the peak of the CD4 T-cell response occurred earlier after the second vaccination [30
] and that the day 42 time point measures the response in the contraction phase. Alternatively, it is possible that specific CD4 T cells may have migrated towards the injection site, thereby affecting CD4 T-cell frequencies in peripheral blood. The fact that the response levels at days 21 and 42 were nearly identical may suggest that the antigen-specific CD4 T cells underwent an expansion phase prior to the day 42 sampling time point. Further investigation of the kinetics of the response should shed further light on this question.
Phenotypically, the responding CD4 T-cell population consisted of both effector–memory-like cells (CD45RA−
) and central memory cells (CD45RA−
) with a tendency for CCR7−
effector–memory cells to dominate the response at day 180. Overall, the adjuvanted vaccine induced a significantly larger memory pool as compared to the non-adjuvanted vaccine. The functional profiles of the responding CD4 T cells were similar for both adjuvanted and non-adjuvanted vaccines, with responses being dominated by CD40L+
- and IL-2-producing cells. Fewer IFN-γ- and TNF-α-producing cells were evident. This bias towards IL-2-producing CD4 T cells was noted previously in healthy adults that received the hepatitis B virus, tetanus, or diphtheria vaccines [30
] and it has been suggested that this phenotype is typical for protein subunit vaccines in general [31
Overall, the H5N1-specific CD4 T-cell responses had a Th1 bias and very few IL-13-producing cells were detected at any time point. Confirming recently published results [32
], cross-reactive CD8 T-cell responses were measured at baseline, indicating the capacity of our assay to detect CD8 T-cell responses. However, no increased CD8 T-cell responses were observed after vaccination, which is in contrast to the responses usually induced by a viral infection [33
]. Indeed FluMist
® (MedImmune LLC, Gaithersburg, MD), which is a live-attenuated influenza vaccine, induces CD8 T-cell responses [20
]. Besides the antigen content, the most likely explanation for this is that protein antigens are efficiently presented through the MHC class II pathway but not via the MHC class I pathway. The induction of CD8 T-cell responses to protein antigen depends on the cross-presentation pathway which, in mice, critically depends on type-I IFN [34
] and specialized CD8α+
DCs, but which is not yet fully understood in humans.
Immune responses to inactivated influenza virus adjuvanted with a different oil-in-water emulsion, MF59, have recently been described [35
]. Although a direct comparison between the adjuvants is complicated by the fact that different methodologies may have been used to measure immune responses, our data indicate that AS03A
-adjuvanted influenza vaccines induce strong HI responses and CD4 T-cell frequencies relative to those induced with the MF59-adjuvanted product.
It is interesting to note that H5-specific CD4 T cells and H5N1-specific memory B cells were detectable prior to vaccination. The identification of CD4 T-cell responses after peptide stimulation in day 0 samples clearly indicates that an H5-specific T-cell response exists before vaccination. However, since the split antigen used for the stimulation of B cells and CD4 T cells also contains the neuraminidase (NA) protein, it cannot be excluded that part of the pre-existing response was in fact specific for NA. Indeed there was a discrepancy between readily detected pre-existing H5N1-specific memory B cells and weaker pre-existing A/Vietnam-specific HI responses (detectable in only seven of 400 subjects (11)).
No correlations were observed between the frequencies of pre-existing H5N1-specific CD4 T cells and post-vaccination HI titers at days 42 or 180. Moreover, there was no correlation between the frequencies of pre-existing H5N1-specific CD4 T cells and post-vaccination CD4 T-cell responses, suggesting that at least part of the response is not dependent on pre-existing CD4 memory T cells.
Pre-existing memory CD4 T cells responded to peptide pools covering the HA sequences from the clade 1 A/Vietnam/1194/2004 vaccine virus as well as the clade 2 A/Indonesia virus. This suggests that the H5-cross-reactive CD4 T cells were induced by a previous infection with seasonal influenza. Thus, these results confirm recently published data showing that memory CD4 T cells, and also CD8 T cells, established by seasonal influenza A cross-react with H5N1 strains [32
]. The combined data raise the question on whether this memory T-cell pool expands after vaccination, instead of or in addition to the naïve T-cell repertoire. A further question is how this potential balance between the recruitment of naïve and memory responses is affected by the adjuvant. A related question is whether the adjuvant simply amplifies the response induced by the non-adjuvanted vaccine (i.e., more of the same) or whether it induces qualitative changes in the repertoire of the responding T cells (i.e., more but not the same). Recent data indicate that vaccine adjuvants change the clonal composition of antigen-specific CD4 T-cell populations responding to vaccines, favoring the selection of higher-affinity T cells [37
]. Thus, it seems likely that the adjuvant changes the T-cell response both quantitatively and qualitatively.
Adjuvantation of the split antigen vaccine with AS03A
resulted in a strong increase in the numbers of antigen-specific B cells and CD4 T cells. AS03A
does not contain a ‘classical’ TLR ligand and further studies into its mode of action are ongoing. Based on the results presented here and our recent data on the mode of action of the AS03A
Adjuvant System (Morel et al., manuscript submitted), we propose the following model: AS03A
induces a recruitment of neutrophils and monocytes at the injection site and induces the maturation of the recruited monocytes. Indeed AS03A
activates monocytes and macrophages but not dendritic cells (Morel et al.,
manuscript submitted). Our current data show that the presence of α-tocopherol in AS03 was required to achieve an optimal antibody response in mice immunized with HBsAg (Morel et al., manuscript submitted). Furthermore, the presence of α-tocopherol in the AS03A
Adjuvant System modulated the levels and kinetics of other cytokines and chemokines, including CCL2, CCL3, IL-6, CSF3, and CXCL1, increased the antigen loading in monocytes, and increased the recruitment of granulocytes in the draining lymph nodes (Morel et al., manuscript submitted). Thus, the ability of α-tocopherol to qualitatively and quantitatively modulate the innate immune response is correlated with a positive impact on the adaptive immune responses in pre-clinical models. Interestingly, IL-6 has been involved in the induction of follicular T helper cells [38
] which are essential to provide B-cell help and to promote germinal center reactions. This, in turn, could lead to increased antibody responses.