Structure-Function Studies of the HIV-1 Envelope Glycoproteins

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The development of a protective and/or therapeutic HIV vaccine has been hampered by unprecedented challenges, primarily due to the unique properties of the HIV-1 envelope (Env) trimer, a cleverly engineered entry machinery that features an extraordinary assortment of immune-evasion tactics, including antigenic variation, heavy glycosylation of exposed surfaces and the so-called "conformational camouflage". Further insights into the complex structure-function relationships in the HIV-1 Env trimer and its protective shield may be critical to guide the rational design of a protective vaccine and other immunoprophylaxis measures. 1) Enhancement of broadly HIV-neutralizing antibodies. Three years ago, we reported the discovery of a second CD4-binding site in the HIV-1 Env trimer, that we defined as CD4-binding site 2 (CD4-BS2), opened new perspectives for vaccine and therapy. We also identified selected anti-CD4-BS antibodies, such as VRC03 and VRC06, which mimic the quaternary-binding mode of CD4 and establish contact with two adjacent gp120 protomers via an extended loop in their heavy chain framework region 3 (FR3). We proved the functional role of this quaternary contact by deleting the FR3 loops of VRC03 and VRC06, which resulted in a near-complete loss of binding and neutralization activity. Since the establishment of quaternary contact appears to bolster the trimer interaction both in CD4 and in selected antibodies, we hypothesized that it might likewise further improve the activity of some of the most potent bNAbs, all of which interact with a single gp120 protomer (e.g., VRC01, VRC07, N6). To validate this assumption, we rationally engrafted the extended FR3 loop of VRC03 onto different CD4-supersite bNAbs and tested the resulting chimeric antibodies against a wide panel of global HIV-1 strains (n = 208). FR3-loop chimerization enhanced the neutralizing activity of potent bNAbs against a majority of global HIV-1 strains. The interactive quaternary surface was delineated by solving the crystal structure of two FR3 loop-chimeric antibodies in complex with a soluble Env trimer. Furthermore, compared to unmodified antibodies, chimeric antibodies displayed a reduced autoreactivity and a prolonged in vivo half-life in both huFcRn transgenic mice and rhesus macaques. Due to their increased neutralizing potency and favorable biological and pharmacokinetic properties, FR3-loop-chimeric bNAbs are being considered for use in HIV prevention and treatment. We have recently extended these studies to other very potent bNAbs, including the N49P family from the IHV in Baltimore and antibody 1-18 from the Klein group in Germany. Interestingly, chimerization of the N49P antibodies was successful resulting in the most potent bNAbs against the CD4-BS ever reported so far. By contrast, modification of antibody 1-18 resulted in a loss of function, confirming that successful chimerization requires specific structural features that ar4 not common to all anti-CD4-BS antibodies. We are currently testing the efficacy of selected chimeric antibodies in humanized mouse models. 2) Structure-guided interdomain stabilization of the HIV-1 envelope trimer abrogates CD4 binding and improves immunogenicity. The inherent flexibility of the HIV-1 Env trimer and its ability to bind the CD4 receptor represent two of the major obstacles to the development of a vaccine capable of eliciting bNAbs. In particular, binding to CD4 not only induces conformational changes that rapidly compromise the native antigenic state of the trimer, but also occludes the CD4-binding site (CD4-BS), which is a critical antigenic target for antibody elicitation, and causes immunogen sequestration by CD4+ T cells, which lack antigen-presenting capabilities. To reduce the trimer flexibility and impair binding to CD4, we used a structure-guided approach to introduce neo-disulfide bonds bridging the inner and outer domains of gp120 (interdomain locks) in a region of molecular mimicry between gp120 and CD4 (SLWDQ). The neo-disulfide bridges effectively formed, as shown by mass-spectrometry analysis, and resulted in trimer stabilization in a native-like pre-fusion configuration. Of note, this design was successfully applied to both soluble trimers (SOSIP) and native full-length gp160 from different HIV-1 strains and clades. Interdomain-locked trimers showed increased thermal stability, reduced spontaneous misfolding, reduced or abrogated binding to non-neutralizing antibodies, enhanced binding to bNAbs and, most importantly, loss of CD4-binding activity. The crystal structure of an interdomain-stabilized trimer confirmed the formation of the expected disulfide bond and provided insights into the structural basis for CD4-binding impairment. 3) In vivo evaluation of an mRNA/VLP vaccine platform in macaques Using the knowledge accrued by structure-guided design of Env-based immunogens, we conducted an efficacy study in rhesus macaques (M. mulatta) using a messenger RNA (mRNA)-based vaccine with the aim of eliciting broadly neutralizing antibodies (bNAbs) against tier-2 HIV-1 strains of different clades of global epidemic relevance. Our vaccine was based on the combination of key technological advances, most notably the use of co-formulated mRNA encoding full-length (membrane-anchored) HIV-1 Env and SIV Gag in order to induce the in vivo production of virus-like particles (VLPs), which provide the best mimic of the native virus particles produced during natural infection. The vaccine was highly immunogenic and we documented for the first time the induction of bona fide tier-2 cross-neutralizing bNAbs, which were associated with protection from tier-2 heterologous challenge with the difficult-to-neutralize strain SHIV-AD8. These results provide a scientific foundation for a transition towards clinical experimentation.