Molecular Medicine Israel

Vaccination induces broadly neutralizing antibody precursors to HIV gp41


A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features


Broad vaccine protection against highly antigenically diverse viruses, such as human immunodeficiency virus (HIV), hepatitis C virus, influenza or the family of betacoronaviruses, has not been achieved in humans but will likely require induction of broadly neutralizing antibodies (bnAbs) that bind to conserved epitopes on otherwise variable membrane glycoproteins. Monoclonal bnAbs for each of the above pathogens have been discovered, and specific genetic and structural features of each bnAb allow binding to its cognate epitope1,2,3,4. To use known bnAbs as guides for the design of vaccines that elicit similar responses, strategies to induce bnAbs with predefined genetic properties and binding specificities are needed5,6,7. One such strategy, germline-targeting vaccine design, is predicated on molecular design of the ‘priming’ immunogen to first elicit responses from rare bnAb-precursor B cells with genetic properties needed for bnAb development. Following the prime, sequential boosting with immunogens of increasing similarity to the native glycoprotein aims to guide B cell maturation to produce bnAbs targeting the desired epitope8,9,10,11.

Germline-targeting priming in humans was demonstrated for the eOD-GT8 60mer immunogen targeting precursors for VRC01-class bnAbs specific for the HIV envelope CD4-binding site11, which was an advance toward the goal of developing precision vaccines that elicit prespecified classes of bnAbs. However, in contrast to the VH-dominant binding mode of VRC01-class bnAbs, most bnAbs to HIV and other viruses exhibit heavy chain complementarity determining region 3 (HCDR3)-dominant interactions with antigen, making it critical to demonstrate induction of HCDR3-dominant bnAb precursors by germline-targeting priming immunogens7. An effective HIV vaccine will need to induce several different classes of bnAbs for sufficient coverage against global isolates. Induction of HCDR3-dominant bnAbs to the membrane-proximal external region (MPER) of the HIV-1 envelope protein (Env) might be crucial due to the high breadth of neutralization provided by such bnAbs (for example, approximately 92–98% for bnAbs 10E8 (ref. 12), LN01 (ref. 13) and DH511 (ref. 14)), the relatively high epitope conservation that should reduce the potential of viral escape, and the strong protection by 10E8 in a passive nonhuman primate (NHP) immunization study despite relatively low potency against the challenge virus15. However, induction of MPER bnAbs faces challenges, including the recessed location of the MPER at the base of the Env trimer12,16, the need to induce antibodies with long HCDR3s bearing specific sequence motifs, and the lack of affinity of most MPER bnAb precursors for their peptide epitopes17,18,19. Furthermore, immune tolerance mechanisms block the induction of MPER bnAbs 2F5 and 4E10, potentially due to lipid reactivity20, raising concerns that other more potent MPER bnAbs, such as 10E8, might also face tolerance barriers21,22,23,24. Here, we developed and validated germline-targeting epitope scaffold nanoparticle priming immunogens to induce 10E8-class HCDR3-dominant bnAb-precursor responses. These immunogens represent candidates for human vaccination and demonstrate design and evaluation processes that could be applied to other bnAb targets.


10E8-class naive precursors are present in most humans

Structural12,16,25,26 and mutational12,18 data indicate that 10E8 binds to its MPER helical peptide epitope primarily through a germline DH3-3-encoded binding motif YxFW positioned near the tip of a long (22-amino acid (aa)) HCDR3, required to access the sterically occluded epitope at the base of full-length membrane-bound Env (Fig. 1a). The activity of 10E8 bnAb further requires a PP motif in the junction between D and J genes within the HCDR3, which could have arisen either during V(D)J recombination or somatic hypermutation (SHM), and germline-encoded HCDR1 and framework region 2 residues and somatically mutated HCDR2 residues within the gene encoding VH3-15. We therefore defined 10E8-class heavy chain precursors as heavy chains with a VH gene closely related to VH3-15 and an HCDR3 length of 21–24 aa with a YxFW motif at the equivalent position within the HCDR3 as 10E8 (Extended Data Fig. 1a). This definition allowed for diverse V–D and D–J junctions and did not require the PP motif that can arise during SHM. To determine if heavy chains with these properties were present in humans, we searched an ultradeep next-generation sequencing (NGS) dataset of primarily naive IgM heavy chains from 14 HIV-seronegative donors7,27. Heavy chains matching the 10E8-class properties were found in all donors, with a geomean frequency of 1:68,000 (Fig. 1b).

The 10E8 light chain contributes to binding of membrane-associated Env by contacting the virion lipid membrane and conformationally stabilizing HCDR3 (ref. 26). The range of germline light chains that have the potential to acquire mutations to mediate such contacts is unclear but could be large. In two paired heavy chain–light chain datasets28,29, human light chains within the VL3 family used by 10E8 were paired with VH3-15 heavy chains at a frequency of approximately 1:7.5, suggesting that the frequency of 10E8-class heavy chain–light chain precursors was approximately 1:510,000. Thus, 10E8-class precursors are present in healthy humans at a substantial frequency.

Germline-targeting immunogens bind 10E8-class precursors

Because the MPER region is sterically occluded at the base of full-length membrane-bound Env and absent from most soluble native-like trimers30, epitope scaffold immunogens were previously designed to conformationally stabilize and expose the C-terminal MPER helix26,31,32 (Fig. 1a). We prioritized one of these epitope scaffolds, T117v2 (ref. 26), for further optimization due to its favorable thermal stability, solubility and presentation of surfaces adjacent to the MPER graft that could be engineered to increase contacts with the YxFW motif in the 10E8 HCDR3. T117v2 bound strongly to mature 10E8 (Kd = 390 pM) but showed no binding (Kd ≥ 100 µM) to 52 10E8-class precursors identified in the NGS database search above paired with the inferred germline (iGL) 10E8 light chain (hereafter NGS precursors; Supplementary Table 1).

We then performed a multistate design and selection process aimed at developing T117v2-based immunogens with the following features: 10 µM affinity or better for the 10E8 unmutated common ancestor (UCA) and as many NGS precursors as possible to enable priming of diverse 10E8-class precursors7,11,33; an affinity gradient for 10E8-class antibodies with the highest affinity for mature 10E8 to favor affinity maturation toward mature 10E8 in vivo5,7,11,34; multivalent display of epitope scaffolds on single-component self-assembling nanoparticles to facilitate mRNA lipid nanoparticle (mRNA-LNP) delivery, improve trafficking to lymph nodes35 and increase B cell responses5; and N-linked glycosylation sites added to the scaffold and base nanoparticle to reduce off-target responses36. Using a combination of structure-based design, computational modeling and directed evolution via yeast surface display34, we iteratively optimized binding of T117v2 to 10E8 iGL, UCA and NGS precursors, resulting in a series of immunogens that we refer to as 10E8-GT (Extended Data Fig. 1b–h and Methods). After nine rounds of optimization, 10E8-GT9.2 bound with low affinity to a small subset (15%) of NGS precursors (geomean Kd = 22 µM; Fig. 2a, Extended Data Fig. 1h, and Supplementary Tables 1 and 2). Further optimization of a pocket designed to contact germline DH3-3-encoded residues at the tip of the 10E8 HCDR3 that are critical for 10E8 neutralization12 produced 10E8-GT10.1 and 10E8-GT10.2. These bound to more NGS precursors (22% and 60%, respectively), with geomean Kd values of 1.4 µM and 5.4 µM, respectively (Fig. 2a), but compared with T117v2 they bound weakly to mature 10E8 (Kd = 27 nM and Kd = 247 nM, respectively; Fig. 2a). Optimization of affinity for mature 10E8 generated 10E8-GT11 that had high affinity (Kd = 1.4 nM) for mature 10E8 but low affinity (geomean Kd = 12 µM) for a minority (6%) of NGS precursors (Fig. 2a,b and Extended Data Fig. 1). Finally, we simultaneously optimized the binding of mature 10E8 and NGS precursors to produce 10E8-GT12 (Extended Data Fig. 1). This final design engaged 46% of precursors with a geomean Kd of 4.3 µM and bound strongly (Kd = 1.0 nM) to mature 10E8 (Fig. 2a,b).

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