In this study, we attempted to evaluate the immune responses to cross reactive T-cell and antibody epitopes, which together, can work towards the development of a potential universal vaccine. Using an immunoproteomics method, we identified and characterized five naturally presented epitopes that are conserved amongst various strains of influenza virus. Furthermore, we examined the efficacy of these epitopes in vivo in combination with a known universal antibody epitope, M2e. We demonstrate a strong T cell response, in addition to neutralization, across several strains. Together, these epitopes could potentially form the backbone of a universal influenza vaccine.
Although current influenza vaccines target humoral immunity, there is evidence that T-cell responses are extremely important for protection against influenza. Importantly, in addition to the role of CTLs in mediating viral clearance 
, CD8+ T cells in humans were shown to have cross-reactive acute 
and memory responses 
to different subtypes of influenza A virus. Although our work primarily focused on HLA-A2 supertype-specific T cell epitopes, we have also indicated that other major HLA supertype specific peptides can be identified using the same immunoproteomics approach, which could lead to the generation of a multi-epitope universal vaccine. Goodman et al
recently demonstrated a multi-epitope DNA prime/pox virus boost recombinant vaccine protected against influenza virus infection in a mouse influenza model 
indicating this is a valid and effective approach for the generation of a cross-reactive vaccine.
Although a vast number of predicted and immunologically characterized epitopes are reported in the literature (IEDB-AR, http://tools.immuneepitope.org
), successful vaccines based on these predicted epitopes have not yet been developed. Generally, motif-based predicted epitopes may activate CTL in the context of in vitro
assays, however, not all of the activated CTLs will recognize naturally processed antigenic peptides on infected cells due to differences between motif-predicted epitopes and those that are endogenously presented 
. Furthermore, comparison of the motif prediction method with direct mass spectrometry analysis of endogenously presented epitopes from virus infected cells revealed a high number of predicted epitopes were not processed and presented 
. Interestingly, we identified a previously reported epitope derived from NS1 protein (P5) 
, which was identified using motif-prediction and ex vivo
CTL analysis. While this study demonstrates that obtaining an epitope with this approach is possible, it is unlikely to work in a high throughput setting due to the numerous predicted peptide sequences, which vary in their binding affinities for the MHC molecule. Natural presentation of these peptides by infected cells would be difficult to examine without actually analyzing the MHC repertoire.
It is interesting to note that our peptide analysis of influenza infected cells did not yield a well studied motif predicted epitope derived from M1 protein (GILGFVFTL, M158–66
. This is concerning, although informative, and is most likely be due to the fact that this epitope is not well-presented on infected cells. Since epitopes presented by infected cells are critical for generating a clinically relevant protective T cell response, it is likely that this is the reason cellular immunity to seasonal influenza is limited and essentially non-protective 
. In addition, our epitope specific CTL analysis using PBMCs from healthy donors did not show an enhanced secondary response as one would expect from an influenza-exposed and vaccinated population. Specifically, if memory in the immunized population existed to the peptide epitopes we uncovered, a strong CTL response would have been illustrated. However, the response was moderate at best suggesting no preexisting immunity, i.e. CTL memory, specific for the identified epitopes was present in this population. Our results may also provide reason to believe that epitopes presented by infected cells may be subdominant epitopes 
and do not activate a strong CTL response as opposed to the dominant epitopes that generate a memory CTL response during infection or vaccination.
The data generated by in silico approaches may overestimate the number of conserved epitopes and may not necessarily identify all immunoreactive naturally presented epitopes. In addition, the motif prediction method may be limited in identifying subdominant epitopes, which are reported to activate T cells in secondary influenza virus specific responses 
. Furthermore, considering motif predicted epitopes are validated by screening circulating CTLs from virus infected individuals, it is important to note a study by Thomas et al 
where they demonstrated a CD8+ T cell immunodominance hierarchy that was suggested to be dependent on the concentration of the presented epitope, size of the available CD8+ T cell repertoire, activation after initial priming, and competition and cooperation between different epitope-specific responses. Additionally, Zhong et al. 
reported a large number of naturally processed and in vivo presented viral CTL epitopes in a mouse model system. In fact, comparison of the motif prediction method with direct mass spectrometry analysis of endogenously presented epitopes isolated from virus infected cells revealed a high number of predicted epitopes were not processed and presented by infected cells 
. These findings illustrate the complexity of CD8+ T cell based screening of functional epitopes and how this approach may miss hidden subdominant epitopes. Our results, in accordance with these previous studies, further emphasize the need to identify epitopes presented by infected cells and the potential benefit of immunizing individuals with those peptides in a vaccine formulation to gain clinically relevant broad protection.
M2e-specific antibodies have the potential to provide broad protective immunity across influenza A strains. Mice immunized intranasally with combined M2e and T cell determinants exhibited significant production of M2e-specific antibodies as well as decreased morbidity after influenza virus infection 
. In our study, we have demonstrated strong humoral and cell mediated immunity against influenza virus by immunization with both M2e and five conserved T cell epitopes. We did, however, observe a slight decrease in antibody response when the M2e peptide is immunized in combination with the T cell epitopes. This may be due to the competition of peptide uptake by the APCs. Ultimately, the most promising universal influenza vaccine candidate may come from such a combination of antigens.
In the viral pathogen field, due to the lack of knowledge of vital T cell epitopes, T cell responses to viral infection including influenza have not been routinely assessed in infected patients or in vaccinated individuals. Although several influenza specific T cell epitopes identified by motif prediction and overlapping peptide library screening methodologies exist, a comprehensive analysis of naturally presented T cell epitopes from infected cells, such as our study, has never been undertaken. We believe that these epitopes may serve as part of a universal vaccine candidate complementary to current vaccines and, if successful, could lead to a paradigm shift in how prophylactic and therapeutic antiviral vaccines are formulated.
This report is the first ever on naturally presented conserved influenza specific T cell epitopes identified by direct analysis of infected cells. Additionally, we have demonstrated that conserved epitope-specific CTLs could recognize multiple strain infected target cells and, when combined with a universal antibody epitope, could be useful in a vaccine formulation. Any universal vaccine that produces broad immunity against influenza would be a significant improvement over the current strain-specific vaccines that must be produced anew each year. Moreover, an epitope based vaccine would be produced synthetically, a major improvement over the current method of growing influenza vaccine in fertilized eggs. Therefore, it not only would generate a more effective immune response, but would also prove more economically sensible. The primary objective of this study is an attempt to create such a vaccine product that will complement the universal antibody based vaccines currently in development to combat influenza infection and pandemic.