Prior to this study, little was known about human arenavirus-specific CD4+ T cells. To our knowledge, only 6 human CD4+ T cell epitopes derived from a single arenavirus species, LASV, had been previously described [41, 42]. These earlier studies relied on identifying epitopes from T cell clones generated from LASV antibody (Ab) positive individuals. Because PBMC from arenavirus-immune donors were not available, we utilized HLA-DRB1*0101 transgenic mice to carry out an extensive screen of 299 arenavirus-derived peptides that bound with high affinity to HLA-DRB1*0101. Furthermore, we bypassed the requirement for BSL-4 containment for most arenavirus species considered by developing a panel of 24 rVACV vectors that expressed the different arenavirus antigens of interest. Utilizing this strategy, we identified 37 different CD4+ T cell epitopes from 7 pathogenic arenavirus species. Two of these epitopes (GTOV NP191-205 and MACV NP191-205) overlapped with the orthologous LASV NP190-202 epitope defined with T cell clones from LASV-immune individuals , suggesting that our approach identified CD4+ T cell epitopes relevant for humans. In the future, we plan to examine CD4+ T cell responses in arenavirus-immune human donors in order to assess the degree of overlap between arenavirus-specific responses recognized in HLA transgenic mice and humans. However, it has been demonstrated with other infectious pathogens, such as Mycobacterium tuberculosis, that HLA class II transgenic mice recognize the same pathogen-derived T cell epitopes as humans [43, 44].
Given that CD4+ T cells are essential for maintaining effective CTL responses during an arenavirus infection , knowledge of human CD4+ T cell epitopes is a crucial step in the development of an arenavirus vaccine that induces effective T cell-mediated immunity. As it is not likely that separate vaccines for each arenavirus species will be generated, one strategy might be the development of a multivalent vaccine that targets multiple species within the Arenaviridae family. This approach has proven itself in the case of the currently licensed vaccines against Streptococcus pneumoniae (i.e. Pneumovax 23), which contains capsular polysaccharide antigens from 23 of the most prevalent pneumococcal serotypes , and human papillomavirus (i.e. Gardasil), which consists of capsid proteins derived from 4 different serotypes prominently associated with disease .
Herein, we defined a set of CD4+ T cell epitopes from 7 different arenaviruses, with promiscuous binding characteristics. Together, these epitopes afforded ~83% coverage of the general population, on average, and thus, could be incorporated into a multivalent vaccine targeting multiple arenavirus species and ethnicities. For our analyses, we have excluded coverage at the HLA-DRB3, DRB4, and DRB5 loci, and also used a conservative threshold of 200 nM to define biologically relevant binding. Previously, we have shown that over 90% of known HLA-DR epitopes have an affinity < 1000 nM . Thus, our coverage may represent a low estimate.
In similar studies, we described the identification of a collection of human class I-restricted CD8+ T cell epitopes derived from the same 7 arenaviruses that afforded 60% coverage of the general population [11, 14, 47]. When immunized as a peptide cocktail, the CD8+ T cell epitopes protected HLA transgenic mice against challenge with rVACVs expressing either Old or New World arenavirus GPC . It is tempting to speculate that combining arenavirus-specific CD4+ and CD8+ T cell epitopes into a single multivalent vaccine might enhance the protective capacity of the CD8+ T cell response. Furthermore, we showed previously that cross-reactive T cell recognition of orthologous peptides derived from different arenavirus species further increased the coverage afforded by the CD8+ T cell epitopes. Likewise, we defined several CD4+ T cell epitopes that shared orthologous sequences between two or more arenavirus species (i.e. JUNV/WWAV GPC46-60, GTOV/JUNV/MACV L391-405, JUNV/MACV L1491-1505, JUNV/MACV L1711-1725, and GTOV/MACV NP191-205), suggesting the potential for CD4+ T cell cross-reactivity in vivo, and thus even greater virus and population coverage.
Three of the CD4+ T cell responses identified in this study, (MACV Z21-35, JUNV GPC46-60, and WWAV GPC46-60), were directed against regions that contained nested human CD8+ T cell epitopes. Our previous study defined a HLA-A*1101-restricted epitope, MACV Z27-36, and a HLA-A*0201-restricted epitope, WWAV GPC42-50 . Overlapping murine CD4+ and CD8+ T cell epitopes have been described in both H-2b and H-2d mice infected with LCMV [48, 49, 50], and influenza virus infection of H-2b mice . Thus, it seems that epitope sharing between both murine and human CD4+ and CD8+ T cells might be a general phenomenon amongst viruses with small proteomes. These overlapping epitopic regions could be of particular importance when designing a multivalent vaccine strategy that targets both arenavirus-specific CD4+ and CD8+ T cell responses.
The ability of T cell-based vaccine candidates to induce protective immunity against infection has largely been associated with the capacity of antigen-specific CD4+ and CD8+ T cells to produce multiple effector functions simultaneously (reviewed in ). A recent study evaluating the protective efficacy of vaccine formulations against Leishmania major found that vaccine-elicited protection was best correlated with the concurrent release of IFN-γ, TNF-α, and IL-2 by antigen-specific CD4+ T cells . Similarly, several studies have demonstrated a strong association between the maintenance of highly polyfunctional T cell responses and non-progressive HIV infection [53, 54, 55]. Here, we showed that a substantial percentage of the LCMV-specific CD4+ T cells produced a polyfunctional response, characterized by simultaneous release of IFN-γ, TNF-α, and IL-2, following peptide stimulation. Finally, we demonstrated that the LCMV-specific CD4+ T cells expressed CD40L following peptide stimulation, indicating their propensity to provide CD8+ T cell help [35, 36, 37]. Taken together, these data suggest that the identified CD4+ T cell epitopes might help induce a protective cell-mediated immune response in a vaccination setting.