| Abstract: | A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-1.Vaccines have successfully stopped or limited infections by preventing pathogen transmission and have thereby contributed toward eradication of deadly diseases (1). Nonetheless, successful vaccination against highly variable pathogens, such as enveloped RNA viruses, remains a daunting task. For example, vaccination against influenza virus is only modestly effective because constant antigenic drift necessitates yearly updates of the vaccine (2). HIV (HIV-1) is also an RNA virus with a high mutation rate that creates extensive sequence diversity and facilitates immune evasion (3, 4). During HIV-1 infection, immune responses are often narrowly focused against a small subset of viruses (5). The development of broadly neutralizing antibodies (bnAbs) is relatively rare (5–15% of infections), and usually only arises after antigenic stimulation by constantly evolving viral variants over the course of 1–3 y or longer (6, 7). A vaccine that confers protection against the global diversity of HIV-1 is highly challenging and, as yet, an unachieved goal (8).Neutralizing antibodies (nAbs) are a correlate of protection for almost all successful vaccines (9, 10). The envelope glycoprotein (Env) is the only HIV-1–encoded surface protein and, as such, constitutes the sole bnAb target. A major problem in vaccine development is the intrinsic metastability of Env that is essential for its membrane-fusion function (11). To mediate cell entry, the Env trimer must undergo a series of complex, receptor-triggered conformational changes within and between its noncovalently associated subunits, gp120 and gp41, to progress from prefusion to fusion-active to postfusion forms (12). How to create stable recombinant Env proteins in a prefusion conformation relevant for bnAb recognition and elicitation has been an area of intensive research.We have previously described a cleaved, stabilized Env trimer from a clade A isolate, termed BG505 SOSIP.664 (13). This SOSIP trimer represents the paradigm for display of all known bNAb epitopes (except the membrane proximal external region, MPER, which was omitted due to its hydrophobicity), with minimal exposure of most non-nAb epitopes (14–16). This BG505 trimer has been extensively characterized and validated structurally in complexes with at least one member of each main bnAb class [trimer apex, PG9 (13); N332-epitope, PGT122 (17, 18); CD4 binding site, PGV04 (19); and cleaved gp41/gp120 interface, PGT151 (20)]. This Env trimer was recently shown to elicit strong, but narrow, nAb responses in animals against the autologous, neutralization-resistant (tier-2) virus (21).It is unlikely that the BG505 SOSIP.664 trimer by itself will achieve the goal of inducing bnAbs against heterologous tier-2 viruses (21). That task may be beyond the capabilities of any single Env protein. One possible strategy to drive a broader response is to use a diverse set of trimers derived from different HIV-1 isolates/clades, analogous to the trivalent/tetravalent seasonal influenza vaccines. The challenge an HIV vaccine faces is much more onerous, however, because of the far greater global sequence diversity of HIV-1 (3).We recently described the production and in vitro characterization of another native-like trimer, B41 SOSIP.664 (clade B) (16) to complement its clade A counterpart. As an immunogen, the B41 trimer also generated a strong, but narrow, tier-2 autologous nAb response (21). Here, we sought to identify additional trimers with comparable properties but based on clade C sequences. Clade C is responsible for over 50% of all new infections worldwide, predominantly in sub-Saharan Africa—the epicenter of the AIDS pandemic (22). Neutralization profiles of clade C viruses suggest their trimers may differ subtly from other clades. For example, despite possessing all putative N-linked glycans associated with the 2G12 epitope, many clade C viruses are resistant to this bnAb (23). Thus, clade C trimers may have atypical structural or glycosylation properties.Here, we screened a panel of 15 clade C env sequences to seek SOSIP.664 trimers with high yield, good thermal stability, native-like antigenicity (display of bnAb epitopes, occlusion of non-nAb epitopes), and native-like structure. We found two trimers, DU422 and ZM197M, with these desired properties. Cryo-electron microscopy (cryo-EM) of ZM197M SOSIP.664 in complex with VRC01 Fab confirmed that the trimers are native-like and provided valuable insights into the distinct neutralization profiles of clade C viruses. These high-quality clade C trimers can now contribute to vaccine strategies aimed at inducing a bnAb response. |
