The principle of vaccination is based on the ability to stimulate both T and B memory responses that provide immunity against the antigen target. Animal studies have clearly shown a role of antigen-specific T cells in protection from virus infections (28
). Evaluation of currently licensed influenza vaccines and those in the development pipeline would help to identify T memory/effector responses that could be harnessed or optimized to improve the vaccine immunogenicity and breadth of protection. In the current study we observed the following attributes associated with vaccination of young adults with live attenuated influenza A (H5N2) vaccine: (i) T memory/effector cellular immune responses; (ii) a correlation between postvaccine induction of IFN-γ+
CD4 and CD8 T cells measured by standard ICCS procedure and trogocytosis-positive CD4 and CD8 T cells identified by novel TRAP assay; and (iii) the comparability of HAI seroconversion rates, the gold standard method to evaluate immunogenicity elicited by influenza vaccines, with postvaccine n
-fold changes in T-cell levels.
Data in this study showed that two doses of live influenza A (H5N2) vaccine promoted both CD4 and CD8 memory T-cell responses in peripheral blood of healthy young subjects. It has been shown that about 90% of effector T cells are cleared from the host by apoptosis following elimination of the antigen; remaining T cells acquire resistance to apoptosis and develop into a memory phenotype (34
). LAIV strains are generally eliminated from the recipient within the first week after vaccine administration (2
). As in the current study, blood samples collected 21 days after vaccination were considered to be memory T cells.
He et al. (15
) observed cellular immunity in response to the live attenuated seasonal influenza vaccine FluMist. In their studies, they detected a significant increase in the GMPs of influenza A (H3N2)-specific IFN-γ+
CD4 and CD8 T cells only in children 5 to 9 years old and not in adults. Such data could be explained by the presence of existing immunity to the virus and thus greater baseline levels of virus-specific T cells in adults relative to children in their study. Our results showed that live attenuated influenza A (H5N2) vaccine was able to induce reliable increases in T-cell levels even in adults, due to the possibility that those subjects had not been vaccinated with A LAIV (H5N2) nor exposed to wild-type influenza H5 virus before their participation in the trial and had no preexisting immunity to influenza A (H5N2). We noted an inverse correlation between baseline levels and postvaccine n
-fold changes in the GMPs of influenza-specific CD4 and CD8 T cells that compare to the data of He et al. (14
) from exposure to seasonal FluMist LAIV. These authors have hypothesized that baseline levels of virus-specific T cells could be a predictive factor for the immunological outcome of vaccination.
We were able to show levels of influenza A (H5N2)-specific CD4 and CD8 T cells in peripheral blood of human clinical trial subjects prior to vaccination as well as in subjects who received only placebo vaccination. Recently, similar findings were observed by other investigators who had detected influenza A (H5N1)-specific T cells in subjects who had never been exposed to avian influenza viruses (18
). These authors showed that most of the identified influenza A (H5N1)-reactive T cells were specific to conserved viral proteins such as the influenza matrix or nucleoproteins, and a few identified conserved T-cell epitopes from the influenza hemagglutinin. Data supported the observation that seasonal influenza A (H3N2) and A (H1N1) virus infections were able to elicit significant levels of cross-reactive T cells to avian influenza H5 variant (18
). Based on these observations, we hypothesize that preexisting influenza A (H5N2)-specific T cells detected in subjects in our trial were most likely from exposure to seasonal influenza viruses or vaccines and likely recognized conservative, cross-reactive cellular epitopes from the virus.
In our study, a novel TRAP assay modified for human T-cell assessment was utilized to determine influenza A (H5N2)-specific trogocytosis-positive CD4 and CD8 T cells. It was suggested that the TRAP assay was comparable to the ICCS assay with added benefits of technical ease and reduced time (results can be obtained within a day) (8
). In addition, the TRAP assay permits the detection of reactive CD4 and CD8 T and B cells in a single assay and provides ability to detect early intercellular interactions such as trogocytosis between APCs and activated lymphocytes within the first minute of interaction. Studies from in vitro
and in vivo
animal models have shown that TRAP assays are comparable to previously published methods such as ICCS and cellular staining with MHC multimers (3
). We have shown dynamic changes in influenza-specific T cells measured by TRAP in vaccinated subjects compared with the placebo group. However, our data obtained by the TRAP assay did not correlate with ICCS results; the times of peak cell levels were different (days 42 and 63, respectively), and there was no correlation between individual levels of trogocytosis+
T cells. Discrepancies between ICCS and TRAP assays seem to depend on different techniques for the measurement of activated T cells. The ICCS method evaluates the terminal step of T-cell activation, namely, the production of cytokines, while the TRAP assay determines early events, i.e., trogocytosis, a plasma membrane exchange between effector and target cells, which begins during the very first minutes of cell-cell interaction (16
). Moreover, within the CD3+
T-cell gate, we were able to detect by ICCS CD4 cells producing IFN-γ which are predominantly related to the Th1 subset, whereas by TRAP assay we obviously estimated both Th1 and Th2 cell subsets, as all the CD4 T-cell subsets were considered to be trogocytosis positive (16
). This suggestion corresponds to levels of CD4 T cells measured by TRAP that were higher than those estimated by ICCS. A different timeline of CD4 effector/memory Th1/Th2 cell development and maintenance may provide the observed decrease of trogocytosis-positive cells at day 63 (versus day 42), which has not been found by the ICCS assay (22
We analyzed the relationship between humoral immune responses and levels of trogocytosis-positive CD4 T cells, including Th2 cells, which are known to be responsible for humoral immune responses (22
). We noted an inverse correlation between baseline trogocytosis-positive CD4 cells and antibody levels measured by HAI. The influence of CD4 T-cell levels on antibody production has also been shown in the mouse model by adoptive transfer of influenza-specific CD4 T cells followed by measurement of humoral immunity in transfer-recipient animals (6
). Since He et al. (14
) proposed that baseline levels of T cells could be a predictive marker of postvaccine cellular immune response, we hypothesized that baseline levels of trogocytosis-positive CD4 T cells could be a predictive indicator of both cellular and humoral immunogenicity in response to a vaccine. We consider these findings to be a significant selection factor to be considered for subjects participating in clinical trials.
The immunogenicity raised in response to influenza vaccines has traditionally been evaluated by serologic parameters measured in the hemagglutination inhibition (HAI) assay, including geometric mean titer in pre- and postvaccination specimens, seroconversion rate (typically a ≥4-fold increase in HAI titer), and a postvaccination protection threshold (e.g., an HAI titer of >1:40) (5
). However, anti-hemagglutination antibody production may not reflect vaccine efficacy as it does not measure the full scope of the immune response, in particular cellular immunity or memory responses. For prevention from and control of influenza infection, the WHO recommends the development and optimization of assays to determine cellular immunity and the development of appropriate criteria to measure vaccine immunogenicity (7
). In our study using a novel influenza A (H5N2) vaccine, we observed a significant discrepancy between the HAI-based immunogenicity results and data obtained from ICCS and TRAP assays used to measure postvaccine memory T-cell immunity. All subjects with negative postvaccine antibody response measured by HAI had significant changes in GMPs of IFN-γ-producing and/or trogocytosis-positive cells. Based on these findings, we highlight the need for measurement of cellular immunity to be able to more clearly evaluate effectiveness of influenza vaccines and to guide us to methods to improve the breadth of immunogenicity that can be achieved in future generations of influenza vaccines.
In summary, this study shows that immunization of healthy young adults with live attenuated A (H5N2) influenza vaccine induced an increase in influenza-specific CD4 and CD8 memory/effector T-cell levels as measured by a standard ICCS method, intracellular IFN-γ staining, and by a novel TRAP assay that detects activated trogocytosis-positive cells. Results obtained by both ICCS and TRAP assays are additive to HAI data in providing additional information about the immunogenicity of influenza vaccines. Baseline levels of influenza-specific CD4 and CD8 T cells target the postvaccine cellular immune response and can be considered as a predictive factor for vaccine effectiveness.