Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

et al. (2012) Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein. PLoS ONE 7(1): e30233. doi:10.1371/journal.pone.0030233 Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross- Linked to a HIV-1 Envelope Glycoprotein Antu K. Dey 0 Brian Burke 0 Yide Sun 0 Klara Sirokman 0 Avishek Nandi 0 Karin Hartog 0 Ying Lian 0 Anthony R. Geonnotti 0 David Montefiori 0 Michael Franti 0 Gre goire Martin 0 Andrea Carfi 0 Pascal 0 Kessler 0 Loc Martin 0 Indresh K. Srivastava 0 Susan W. Barnett 0 Zhiwei Chen, The University of Hong Kong, Hong Kong 0 1 Novartis Vaccines and Diagnostics, Cambridge, Massachusetts, United States of America, 2 Department of Surgery, Duke University Medical Center , Durham , North Carolina, United States of America , 3 CEA, iBiTecS , Service d'Inge nierie Mole culaire des Prote ines , Gif sur Yvette , France The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved ''CD4 induced'' (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-27312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application. - Funding: This work was supported by the NIH HIVRAD (HIV Research and Design) grant, # 5P01 AI066287. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AKD, BB, YS, KS, AN, KH, AC and SWB are employees of Novartis Vaccines & Diagnostics (NVD); AC and SWB are shareholders of NVD. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is the lone viral gene product exposed on surface of the virus and therefore is the major target of HIV-1-specific neutralizing antibodies (NAbs). This trimeric glycoprotein mediates receptor binding and viral entry through its interactions with both CD4 and CCR5/CXCR4. These characteristics make Env a logical candidate as a component of an HIV-1 vaccine. Such a vaccine will likely need to elicit broadly cross-neutralizing antibodies to be effective. However, due to extensive intra- and inter-subtype sequence variability of Env, anti-Env antibody response are generally isolate-specific. To be effective against diverse isolates, the NAbs induced by a prospective vaccine will need to recognize multiple Env variants, as patients are unlikely to encounter viruses that match the vaccine strain during natural infection. Therefore, the ideal method of generating a broad, cross-reactive NAb response would be to target highly conserved regions in Env [1]. Unfortunately, many conserved epitopes of Env are occluded or transiently exposed, and therefore are poorly immunogenic. Several monoclonal antibodies (MAbs) have been isolated from patients with broad, potent-neutralizing activity [2,3,4,5,6,7], however attempts to elicit similarly potent and broadly NAbs by vaccination using recombinant forms of Env as antigens have, at best, met limited success [8,9]. The antibodies generated in this manner are primarily strain-specific and have limited breadth in their neutralizing activity. Designing immunogens that are able to elicit antibodies that are broadly cross-neutralizing is a challenge as accessible immunodominant variable regions redirect antibody responses away from the desired, but often masked conserved regions [1,10,11,12,13]. Thus, many studies involving Env have focused on attempting to dampen the immunogenicity of the highly variable regions and/or to increase the immunogenicity of the desired conserved epitopes [8,14,15,16]. One such conserved region that has been under evaluation is the conformational epitope that Env forms upon binding with the receptor CD4. This CD4-induced (CD4i) epitope is highly conserved [10]. In fact, many CD4i antibodies are quite potent, even against the highly divergent HIV-2, in the presence of sCD4, indicating the conserved nature of this epitope [10]. This make the CD4i epitope(s) an attractive target for vaccine development. A potential concern that CD4i antibodies, as whole IgG, face steric hindrance in accessing the epitope [17] and hence not effective in neutralizing primary isolates remain. However, broadly crossreactive neutralizing antibodies were elicited in rhesus macaques by using covalently cross-linked HIV-1 Env-CD4 receptor complexes [18]. DeVico et al. also showed in a SHIV-challenged model that antibodies to CD4i sites in HIV-1 gp120 correlated with the control of SHIV challenge in macaques vaccinated with cross-linked and single-chain gp120CD4 complexes [19]. Importantly, it was shown recently that humans infected with HIV-1 generate CD4i antibodies during the course of infection [20,21,22,23]. Additionally, it was observed that there is a correlation between the presence of CD4i NAbs and protection from disease [19,22]. Also in human clinical trials focusing on induction of Env-specific antibodies, either using protein alone, viral vector prime-protein boost or DNA prime-protein boost, analysis of serum antibody response show elicitation of neutralizing antibodies to CD4i sites [24,25]. Therefore, in an effort to create improved Env antigens that would effectively display cryptic CD4i epitope(s) without using the native CD4 molecule, which could present safety concerns in a vaccine preparation due to potential autoimmune reactions, studies using peptide mimetics of CD4 were performed [26,27,28,29]. These were based on the scyllatoxin-scaffold and several generations were evaluated to (...truncated)