HIV p24 as Scaffold for Presenting Conformational HIV Env Antigens

et al. (2012) HIV p24 as Scaffold for Presenting Conformational HIV Env Antigens. PLoS ONE 7(8): e43318. doi:10.1371/journal.pone.0043318 HIV p24 as Scaffold for Presenting Conformational HIV Env Antigens Maria Tagliamonte Daniela Marasco Alessia Ruggiero Angelo De Stradis Maria Lina Tornesello Maxim Totrov Franco Maria Buonaguro Luigi Buonaguro Roger Le Grand, Commissariat a l'Energie Atomique(cea), France Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs) represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4+ and CD8+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes. - Funding: The study was supported by the European Communitys Seventh Framework Programme Next Generation HIV-1 Immunogens inducing broadly reactive Neutralising antibodies NGIN (FP7/20072013) under grant agreement nu 201433. Dr. Tagliamonte is funded by the NGIN Programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following conflicts: Dr. Totrov is employee of Molsoft LLC. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Efforts to elicit protective immunity to HIV has resulted in unsatisfactory results [1]. In particular, elicitation of broadly reactive and cross-clade neutralizing antibodies (NAbs) is representing an unprecedented challenge for the intrinsic property of HIV to generate molecular and antigenic variants escaping the immune surveillance [2]. However, cross-reactive neutralizing antibodies targeting the envelope glycoprotein can indeed arise during the natural course of HIV-1 infection [3,4,5,6,7], as shown by the broadly neutralizing antibodies isolated from HIV-1infected individuals. In particular, b12 and 2G12 bind to conserved epitopes in the gp120 subunit [8,9]; 2F5 and 4E10 bind to conserved, contiguous epitopes in the gp41 subunit [10,11]. More recently, additional broadly neutralizing antibodies have been described, targeting either discontinuous epitopes in trimeric structures (PG9 and PG16) [12], the CD4 binding site (HJ16, VRC01/2 and VRC03) [13,14], or the V3 loop [15,16,17]. Strategies to elicit or expand such HIV broadly reactive and cross-clade NAbs are currently pursued by several groups, aiming at focusing the immune response on specific epitopes which can be either immunorecessive, cryptic or transiently exposed. To this goal, one of the optimal experimental strategies appears to be the selection of the minimal structural and antigenic epitopes, in order to isolate them from all other potential and confounding B-cell epitopes as well as from the shielding N-linked glycans within the whole HIV envelope glycoprotein [18,19,20,21]. Such minimal epitopes, indeed, can be grafted in a constrained status onto appropriate heterologous protein scaffolds to mimic their antibody-bound conformation and be possibly able to elicit the counterpart broadly neutralizing Nabs. Along such path, very recently the gp41 2F5-specific minimal epitope has been grafted on different protein scaffolds [22] inducing high titers of cross-reactive Ab response [23]. Similarly, the gp120 V3 loop has been grafted on a thioredoxin [24] or cholera toxin subunit (CTB) [25] scaffold, exhibiting high-affinity binding to a large panel of broad-neutralizing mAbs and inducing high titers of anti-V3 antibodies with broad-neutralization effect [25]. An additional relevant feature for a vaccine approach, aiming at an efficient induction of neutralizing antibodies, is to present B cell epitopes as dense, repetitive arrays mimicking the natural organization observed in viruses which induce highly protective neutralizing antibodies [26]. Densely repetitive B cell epitopes, indeed, induce also T cell-independent B cell activation in contrast to the same antigen presented in monomeric non-organized conformation, as shown in the Vescicular Stomatitis Virus (VSV) model [27]. In this perspective, Virus-Like Particles (VLPs) represent a highly attractive vaccine strategy, closely resembling authentic virions with a regular and rigid capsid structure presenting conformational viral epitopes as dense repetitive arrays [28,29,30,31,32]. However, antigen presentation on enveloped VLPs (i.e. HIV-VLPs) may be affected by a sparse and irregular distribution which reflects the structure of the authentic virions [33,34,35]. In order to overcome such limitation, non-enveloped particulate vaccines based on assembled chimeric HIV p24 Gag core protein can be prospected. Very recently, indeed, the hexameric structure of capsomers derived from in vitro assembling of recombinant HIV p24 capsid protein (p24 CA protein) has been described. HIV p24 CA proteins form homogenous populations of stable and soluble stand-alone capsomers, which assemble in vitro with the need of neither cellular membrane nor MA and NC gag viral proteins [36]. Based on such observations, the HIV p24 CA protein is a highly attractive molecule to be used as particulate protein scaffold for presenting dense repetitive arrays of minimal structural and antigenic HIV Env epitopes aiming at eliciting broadly NAbs [37]. Compared to other neutral structures, indeed, it would represent an invaluable advancement. In fact, besides the presentation of relevant Env neutralizing epitopes, it would provide also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4+ and CD8+ T cell responses. This would result in highly effective induction of both T cell-independent and T cell-dependent B cell activation, specific for both the grafted Env epitopes and the Gag-protein scaffold. In this regards, the present paper describes the design and characterization of HIV p24-based scaffold d (...truncated)