Sudhanshu Vrati and Dr

Sudhanshu Vrati and Dr. its Supporting Information files. Abstract An ideal HIV-1 Env immunogen is usually expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is usually efficiently cleaved when expressed around the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design. Introduction The envelope glycoprotein (Env) of human immunodeficiency virus type-1 (HIV-1) is usually a surface molecule that mediates virion entry into target cells. It undergoes conformational changes following engagement of the primary receptor, CD4 and subsequently binds to the co-receptor, which ultimately leads to virus-cell membrane fusion and entry of the virus into target cells [1C12]. Due to its essential role in viral infectivity, Env is the primary BRAF inhibitor target of neutralizing antibodies (nAb) and therefore is the focus of global HIV-1 vaccine immunogen design efforts. In natural HIV-1 infections, approximately 10C20% of chronically infected individuals develop neutralization breadth. Serum antibodies from these individuals can potently neutralize an array of HIV-1 isolates of different clades. This broad neutralizing activity, generated BRAF inhibitor during years of natural infection, is usually mediated by monoclonal antibodies whose target epitopes have been mapped to various sub-regions of the Env such as the CD4 binding site (CD4-bs), various glycans and the membrane proximal external region (MPER) [13C21]. Many of these broadly neutralizing antibodies (bnAb) are dependent on Env conformation and the recently isolated bnAb, PGT151 is also cleavage specific [22, 23]. In the absence of anti-retroviral therapy, passive immunization BRAF inhibitor with a combination of bnAbs can suppress viremia and is protective sequence as an alternate approach to increase expression of 4-2.J41 Env. As expected, codon-optimized 4-2.J41 showed an increase in expression around the cell surface. Interestingly, such modification in JRFL Env EIF4G1 has been reported to result in a reduction in the level of cleavage [39]. However, in the case of 4-2.J41, codon-optimization did not compromise the cleavage efficiency of this Env. In addition to retention of cleavage efficiency, codon-optimization is advantageous over Env cytoplasmic tail-truncation as a method for increasing its expression as this system allows for use of a single plasmid for delivery of the immunogen. In summary, in this study we have identified and characterized a clade C Env, 4-2.J41, which is naturally and efficiently cleaved around the cell surface. This Env selectively exposes epitopes for only neutralizing antibodies and BRAF inhibitor presents native trimeric conformation in the membrane-anchored form. It is now apparent that a trimeric Env protein that is fully cleaved and assumes a native conformation would be a better immunogen for eliciting neutralizing antibodies. However, JRFL was the only naturally cleaved Env available on which such immunogens could be designed. Recent characterizations of a modified, soluble form of BG505 Env trimer have further bolstered the importance of a cleavage-competent Env in immunogen design. One obvious yet important implication of this study is usually that future structural and immunological analysis of 4-2.J41 Env would provide opportunities to validate the information available as well as gain new insights on naturally cleaved Envs that is so far restricted to one Env, JRFL. From a vaccine perspective, since clade C of HIV-1 virus is responsible for almost half of BRAF inhibitor global infections, antibodies induced by this clade C Env could be effective against a larger pool.