Reinherz: bNAbs Against Clade C
A vaccine capable of eliciting antibodies neutralizing a broad range of HIV-1 strains must target a region of the HIV-1 virus that is highly conserved across strains. The Reinherz consortium is aimed at eliciting antibodies directed against a region of the viral gp41 envelope protein known as the membrane proximal external region (MPER). Structural information on this lipid-embedded region will be exploited for vaccine immunogen design and delivery to the immune system in the form of lipid-coated nanoparticles. This vehicle system has the advantage of concomitantly facilitating delivery of adjuvants including Toll-like receptor (TLR) ligands. In addition, nanoparticles can be used to further deliver both T helper and cytotoxic T lymphocyte (CTL) epitopes in the nanoparticle core in future iterations.
1. Structural analysis of HIV-1 MPER from clade C viruses with and without the appended transmembrane (TM) gp41 segment in lipid environments.
2. Creation of MPER nanoparticles consisting of a polymer core particle encapsulated by an outer lipid vesicle or "skin" for MPER presentation to the immune system.
3. Elicitation of humoral immune responses to MPER immunogens, assessment of antibody titer, specificity and HIV-1 neutralizing activity.
Although infrequently elicited during the course of natural infection and rarely, if at all, upon conventional vaccination, the membrane-proximal external region (MPER) of the HIV-1 glycoprotein of Mr 41,000 (gp41) envelope protein subunit is the target of several human broadly neutralizing antibodies (BNAbs): 4E10, 2F5, Z13e1 and most recently, 10E8. How these BNAbs bind to their lipid-embedded epitopes and mediate antiviral activity is unclear, but this information may offer important insight into a worldwide health imperative. The Reinherz research team, including Gerhard Wagner, Jim Sun, Likai Song and Mikyung Kim, has utilized EPR and NMR techniques to define the manner in which these BNAbs differentially recognize viral membrane-encrypted residues configured within the L-shaped helix–hinge–helix MPER segment. Two distinct modes of antibody-mediated interference of viral infection were identified. 2F5, like 4E10, induces large conformational changes in the MPER relative to the membrane. However, although 4E10 straddles the hinge and extracts residues W672 and F673, 2F5 lifts up residues N-terminal to the hinge region, exposing L669 and W670. Detailed analysis of 2F5 extraction using the above techniques in conjunction with hydrogen exchange mass spectrometry (HX-MS) with John Engen demonstrates that 2F5 recognition is stepwise, involving a paratope more extensive than the core binding site contacts alone, and dynamically rearranging via an apparent CDRH3 scoop-like movement essential for MPER extraction from the viral membrane. Core epitope recognition on the virus requires induction of conformational changes in both the MPER and paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on antibody combining-site plasticity. In contrast, Z13e1 effects little change in membrane orientation or conformation, but rather immobilizes the MPER hinge through extensive rigidifying surface contacts. Thus, BNAbs disrupt HIV-1 MPER fusogenic functions critical for virus entry into human CD4 T cells and macrophages either by preventing hinge motion or by perturbing MPER orientation. HIV-1 MPER features, important for targeted vaccine design, have been revealed, with implications extending to BNAb targets on other viral fusion proteins. 10E8 also binds to W672 and F673 but with a different extraction profile to that of 4E10.
Recent progress was made in NMR spectroscopic studies to characterize three clade C MPER segments in a lipid environment. These clade C peptides all share the typical helix-hinge-helix motif first observed in a HxB2 peptide from clade B. We showed that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via inter-converting structures while at the same time affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix-capping, thereby disrupting hinge function.
In addition, the Irvine group's work on phospholipid-enveloped biodegradable microparticles and nanoparticles includes multilamellar vesicles, polymer nanoparticles and stealth liposomes as vaccines displaying MPER segments on lipid. In collaborative efforts within the group, specific anti-MPER antibodies have been generated with broad specificity for clade B and C sequences. Improvement in affinity through more optimal T follicular helper cell elicitation, and other particle and immunogen tuning efforts are required to generate useful neutralizing antibodies, however. The impact of surface MPER density and adjuvant choice are key elements of this investigation that are now well studied.
Inclusion of the TM segment of gp41 with the MPER in microparticle display and its impact on immunogenicity is a focus of intense interest for the Consortium. While structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity, by contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed MPER as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while preserving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent exposed MPER tryptophan residue (W680) was immunodominant, focusing immune responses despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for future immunogen design aimed at targeting B cell antibody responses, including induction of BNAbs.