Polyfunctional CD4⁺ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Several arenaviruses within the Arenaviridae family are rodent-borne human pathogens. Infection outcomes can range from subclinical disease to central nervous system damage [1], aseptic meningitis [2], congenital deformities [3, 4], and severe hemorrhagic fever (reviewed in [5]). Mortality among patients with arenaviral hemorrhagic fever ranges from 15 to 30% [6, 7]. Accordingly, arenavirus infections are considered a serious human public health problem. Despite the pathogenicity of arenaviruses, there are no licensed vaccines available and the live attenuated Junin virus (JUNV) vaccine, Candid #1, only has investigational new drug status in the U.S. [8]. Moreover, antiviral therapies are limited to the use of hyperimmune plasma [1] or the guanosine analogue ribavirin, which can lead to adverse side effects such as thrombocytosis, severe anemia, and birth defects [9, 10]. Because of their pathogenicity and the lack of vaccines and antivirals to prevent and treat infection, arenaviruses are also regarded as a potential bioterrorism threat, and as such are classified as Class A pathogens. Thus, there is a need to develop novel prophylactic vaccination strategies to combat arenavirus infection.

Several studies have reported a beneficial role for both T cell and antibody-mediated immunity in countering arenavirus infections. Vaccine strategies aimed at generating a CD8⁺ T cell-mediated response confer protection against virus challenge in murine [11–14], guinea pig [15, 16], and non-human primate [17] models of infection of two Old World arenaviruses, Lassa virus (LASV) and lymphocytic choriomeningitis virus (LCMV). In humans, cell-mediated immunity also seems to be critical for protection against LASV infection, as neutralizing antibodies appear several weeks or months after viral clearance [18, 19], and treatment of infected patients with hyperimmune plasma does not protect against disease [20]. In contrast, antibody-mediated immunity seems to play an important role protecting against New World arenavirus infection, as administration of immune plasma at an early infection stage significantly reduces morbidity and mortality [21]. T cell responses might also be involved in countering New World arenavirus infection as, similar to LASV infection, neutralizing antibodies often appear several weeks after resolution of infection of the New World arenavirus, JUNV [22]. JUNV-specific T cell responses have also been detected in patients vaccinated with Candid #1 [8].

An important component in developing protective CD8⁺ T cell and antibody-mediated immunity is the generation of effective CD4⁺ T cell help. Several previous studies, conducted in murine models of LCMV infection, have demonstrated that virus-specific CD4⁺ T cells play an essential role in priming optimal CD8⁺ T cell responses in vivo. Infection of mice lacking CD4⁺ T cells (either by transient depletion or knock-out) with LCMV Armstrong led to the failure of LCMV-specific CD8⁺ T cells to expand upon antigen re-encounter, thus demonstrating that CD4⁺ T cell help is required for secondary memory CTL expansion during acute virus infection [23]. In mouse models of chronic LCMV infection, it has long been established that CD4⁺ T cell help is required to control viremia [24, 25]. However, it was recently demonstrated that IL-21, produced by antigen-specific CD4⁺ T cells, is the critical helper factor needed to sustain effector CD8⁺ T cell activity and contain viremia during chronic LCMV infection [26–28]. In addition to providing help, in the case of LCMV infection, CD4⁺ T cells have been shown to have direct effector function mediated by cytokine secretion and cytolytic activity [29].

Given the importance of CD4⁺ T cells, a critical step in the development of a vaccine strategy against arenaviruses is the definition of arenavirus-specific CD4⁺ T cell epitopes with the capacity to induce help. However, one of the major obstacles in designing an arenavirus vaccine is the genetic diversity found amongst the different members of the Arenaviridae family, as well as the variability within a single arenavirus species. To overcome this challenge, it might be possible to combine antigens or epitopes derived from several arenavirus species in the same vaccine, and thus provide effective multivalent protection.

Thus, the goal of the present study was to identify HLA class II-restricted CD4⁺ T cell epitopes derived from arenaviruses associated with disease in humans that have the competency to provide help in a vaccination setting. We specifically targeted the 4 viral proteins (glycoprotein precursor (GPC), RNA-dependent RNA polymerase (L), nucleocapsid protein (NP), and zinc-finger binding protein (Z)) that are encoded by the 7 different species, including Guanarito virus (GTOV), JUNV, LASV, LCMV, Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). Because of the high degree of HLA class II polymorphism expressed in the human population, we focused on defining HLA-DRB1*0101-restricted CD4⁺ T cell epitopes, as these epitopes have demonstrated broad reactivity with other HLA-DR molecules [30], and thus have the potential to provide extensive population coverage across different ethnicities ([31] and J. Sidney and A. Sette, unpublished observations). Here, we tested whether it was possible to define HLA-DRB1*0101-restricted CD4⁺ T cell responses from the 7 arenaviruses of interest that provide broad population coverage, and demonstrate a polyfunctional phenotype with the potential to impart help.

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 NP_191-205 and MACV NP_191-205) overlapped with the orthologous LASV NP_190-202 epitope defined with T cell clones from LASV-immune individuals [41], 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 [23], 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 [45], and human papillomavirus (i.e. Gardasil), which consists of capsid proteins derived from 4 different serotypes prominently associated with disease [46].

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 [30]. 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 [14]. 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 GPC_46-60, GTOV/JUNV/MACV L_391-405, JUNV/MACV L_1491-1505, JUNV/MACV L_1711-1725, and GTOV/MACV NP_191-205), 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 Z_21-35, JUNV GPC_46-60, and WWAV GPC_46-60), were directed against regions that contained nested human CD8⁺ T cell epitopes. Our previous study defined a HLA-A*1101-restricted epitope, MACV Z_27-36, and a HLA-A*0201-restricted epitope, WWAV GPC_42-50 [14]. Overlapping murine CD4⁺ and CD8⁺ T cell epitopes have been described in both H-2^b and H-2^d mice infected with LCMV [48–50], and influenza virus infection of H-2^b mice [51]. 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 [52]). 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 [33]. Similarly, several studies have demonstrated a strong association between the maintenance of highly polyfunctional T cell responses and non-progressive HIV infection [53–55]. 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 [35–37]. 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.

In conclusion, the identification of CD4⁺ and CD8⁺ T cell epitopes from 7 different arenavirus species might lead to the development of a T cell-based vaccine strategy protecting against Old and New World arenavirus infection responsible for hemorrhagic fever and aseptic meningitis in humans. Promiscuous epitopes with the capacity to bind to multiple alleles within a HLA supertype are of particular relevance in generating a vaccine that targets a broad range of ethnicities within the general population. The validation of both CD4⁺ and CD8⁺ T cell epitopes in arenavirus-immune individuals, and their formulation in a multivalent construct would also be the logical next steps in the further exploration of this concept.