Abstract.
T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4+ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4+ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4+ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence.

We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IAb and IAd. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4+ and CD8+ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4+ and CD8+ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

Introduction.
The development of an efficacious vaccine against human immunodeficiency virus 1 (HIV-1) still remains as the best long-term approach to control the acquired immunodeficiency syndrome (AIDS) pandemic since resource-poor endemic regions are not able to afford sustained antiretroviral therapy (ART). Clinically tested HIV-1 vaccines have shown no or modest efficacy so far [1], [2]. No vaccine strategy was able to induce broadly neutralizing antibodies and T-cell based vaccines have thus emerged as an alternative to counteract AIDS by limiting both viral transmission and disease progression [3]. Indeed, a recent study using non-human primates (NHP) has demonstrated that vaccine-induced virus-specific effector memory T-cell (TEM) responses can exert a profound early control on highly pathogenic simian immunodeficiency virus (SIV) infection after mucosal challenge, which has given more hope for the development of new T-cell based vaccines against HIV-1 [4].

The breadth of T-cell responses induced against HIV-1 has become a central goal in AIDS vaccine development after the STEP trial failure [1], [5]. In fact, different groups have shown that protection against SIV challenge is strongly associated with induction of either CD4+ or CD8+ T cells against multiple targets [6]–[9]. Thus, it is important to design novel vaccine platforms in order to broaden T-cell responses against HIV-1.

T-cell based vaccines against HIV-1 are frequently focused on the induction of CD8+ T-cell responses, which are known to be responsible for killing virus-infected targets [6], [10]–[12]. However, mounting evidence suggests that CD4+ T-cell responses may be important for controlling HIV-1 replication [13]. Although HIV-specific CD4+ T cells are preferentially targeted by the virus, the vast majority of these cells remains virus-free at any time in vivo [14], which may allow for their antiviral function. In fact, strong virus-specific CD4+ T-cell responses have been associated with natural control of HIV-1 infection [15], [16] and cytotoxic CD4+ T cells were shown to suppress viral replication in both SIV and HIV-1-infected cells [17], [18]. While the clinical associations of CD4+ T-cell responses with HIV-1 control must be carefully interpreted, due to a possible cause-effect issue, the finding that CD4+ T-cell depletion reduced vaccine-mediated protection [19] supports a direct role of such cells in HIV-1 immunity. Moreover, some groups have observed the association of vaccine-induced virus-specific CD4+ T-cell responses with protection against SIV challenge [7], [20], which further supports a protective role of CD4+ T cells. Therefore, it is important to explore the anti-viral immunity exerted by CD4+ T cells in order to develop novel vaccines against HIV-1/AIDS. It is possible that the induction of CD4+ T cells will be beneficent both due to the help provided to B cells and CD8+ T cells as well as due to direct effects on HIV-1-infected targets.

An important concern regarding AIDS vaccine development is how to elicit cellular immune responses to cover multiple HIV-1 circulating variants, which can differ by up to 20% within a subtype and show up to 35% of amino acid divergences between subtypes [21]. Artificially designed M-group consensus sequences display average distances to HIV-1 variants similar to those found intra-subtype and have been considered a potential alternative to circumvent the barrier posed by viral genetic diversity [22]. Indeed, studies have demonstrated that immunogens based on HIV-1 M-group consensus Env were able to provide broad cross-clade T-cell responses in both mice and macaques [23], [24], which suggests an important role for this strategy in HIV-1 vaccines.

The high polymorphism of human leukocyte antigens (HLA), which are responsible for determining the onset of T-cell responses, is also a challenge for vaccine development. It is expected that different HLA-bearing populations respond differently to the same immunogen and this may be decisive for the vaccine success. Vaccines encoding promiscuous peptides, each binding to multiple HLA molecules, may be a solution to this problem by allowing that multiple HLA molecules spread among the population contribute to the induction of broad T-cell responses in most of the immunized individuals. This would confer broader population coverage and enhance vaccine efficacy [25]–[27]. Thus, novel AIDS vaccines should be rationally designed to address both viral and host genetic diversity in order to confer immunity against multiple HIV-1 circulating variants in a population with diverse HLA alleles.

The inclusion of appropriate proteins in HIV-1 vaccines may be crucial for eliciting protective responses. While broad Gag- and Vif-specific responses have been correlated to vaccine-induced protection in SIV-challenged macaques [28], induction of Env-specific CD4+ T-cell responses contributed to enhanced SIV replication and accelerated progression to AIDS [29]. Env-specific CD8+ T-cell responses were also shown to be a strong predictor for disease progression in HIV-1-infected patients [30]. Furthermore, CD4+ T-cell responses targeting Gag and Env-specific epitopes were associated with spontaneous control of viral replication and progression to AIDS, respectively [31].

Recently, our group has designed a DNA vaccine encoding 18 conserved, multiple HLA-DR-binding epitopes from HIV-1 subtype B consensus sequence. This vaccine elicited broad, polyfunctional and long-lasting CD4+ T-cell responses in BALB/c and HLA class II transgenic mice [32], [33]. In this work we sought to develop a DNA vaccine that would be able to provide broad CD4+ T-cell immunity in a diverse HLA-bearing population, now targeting multiple HIV-1 M-group consensus peptides, potentially cross-reactive to a high proportion of circulating HIV-1 variants. In addition, we excluded Env peptides from our novel vaccine based on the evidence that Env-specific T-cell responses are frequently related to disease progression.

To accomplish our goals, we used the TEPITOPE algorithm [34], which has been successfully applied for in silico identification of promiscuous T-cell epitopes in the context of oncology, allergy, autoimmunity and infectious diseases [35]–[40], to scan the HIV-1 M-group consensus sequence. We identified 27 peptides from 7 different HIV-1 proteins (Gag, Pol, Nef, Vif, Vpr, Rev and Vpu), predicted to bind to multiple HLA-DR molecules and conserved among all M-group subtypes. The identified peptides bound in vitro to several HLA-DR, -DP and -DQ molecules and also to murine IAb and IAd molecules. The peptides were antigenic in natural infection, being recognized by peripheral blood mononuclear cells (PBMC) from HIV-1-infected patients. Finally, we designed a DNA vaccine (HIVBr27) encoding the 27 peptides in tandem and immunized BALB/c mice. HIVBr27 immunization elicited broad, cross-clade and polyfunctional CD4+ and CD8+ T-cell responses.