Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

Vaccine-mediated protection against Merbecovirus and Sarbecovirus challenge in mice.

The emergence of three highly pathogenic human coronaviruses-severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, Middle Eastern respiratory syndrome (MERS)-CoV in 2012, and SARS-CoV-2 in 2019-underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines protect against severe COVID-19, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor-binding domains (RBDs), which elicited live-virus neutralizing antibody responses. The trivalent RBD scNP elicited serum neutralizing antibodies against bat zoonotic Wuhan Institute of Virology-1 (WIV-1)-CoV, SARS-CoV, SARS-CoV-2 BA.1, SARS-CoV-2 XBB.1.5, and MERS-CoV live viruses. The monovalent SARS-CoV-2 RBD scNP vaccine only protected against Sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both Merbecovirus and Sarbecovirus challenge in highly pathogenic and lethal mouse models. This study demonstrates proof of concept for a single pan-sarbecovirus/pan-merbecovirus vaccine that protects against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

Vaccine-mediated protection against merbecovirus and sarbecovirus challenge in mice.

The emergence of three distinct highly pathogenic human coronaviruses - SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019 - underlines the need to develop broadly active vaccines against the Merbecovirus and Sarbecovirus betacoronavirus subgenera. While SARS-CoV-2 vaccines are highly protective against severe COVID-19 disease, they do not protect against other sarbecoviruses or merbecoviruses. Here, we vaccinate mice with a trivalent sortase-conjugate nanoparticle (scNP) vaccine containing the SARS-CoV-2, RsSHC014, and MERS-CoV receptor binding domains (RBDs), which elicited live-virus neutralizing antibody responses and broad protection. Specifically, a monovalent SARS-CoV-2 RBD scNP vaccine only protected against sarbecovirus challenge, whereas the trivalent RBD scNP vaccine protected against both merbecovirus and sarbecovirus challenge in highly pathogenic and lethal mouse models. Moreover, the trivalent RBD scNP elicited serum neutralizing antibodies against SARS-CoV, MERS-CoV and SARS-CoV-2 BA.1 live viruses. Our findings show that a trivalent RBD nanoparticle vaccine displaying merbecovirus and sarbecovirus immunogens elicits immunity that broadly protects mice against disease. This study demonstrates proof-of-concept for a single pan-betacoronavirus vaccine to protect against three highly pathogenic human coronaviruses spanning two betacoronavirus subgenera.

RAB11FIP5-Deficient Mice Exhibit Cytokine-Related Transcriptomic Signatures.

Rab11 recycling endosomes are involved in immunological synaptic functions, but the roles of Rab11 family-interacting protein 5 (Rab11Fip5), one of the Rab11 effectors, in the immune system remain obscure. Our previous study demonstrated that RAB11FIP5 transcripts are significantly elevated in PBMCs from HIV-1-infected individuals, making broadly HIV-1-neutralizing Abs compared with those without broadly neutralizing Abs; however, the role of Rab11FiP5 in immune functions remains unclear. In this study, a RAB11FIP5 gene knockout (RAB11FIP5 -/-) mouse model was employed to study the role of Rab11Fip5 in immune responses. RAB11FIP5 -/- mice exhibited no perturbation in lymphoid tissue cell subsets, and Rab11Fip5 was not required for serum Ab induction following HIV-1 envelope immunization, Ab transcytosis to mucosal sites, or survival after influenza challenge. However, differences were observed in multiple transcripts, including cytokine genes, in lymphocyte subsets from envelope-immunized RAB11FIP5 -/- versus control mice. These included alterations in several genes in NK cells that mirrored observations in NKs from HIV-infected humans expressing less RAB11FIP5, although Rab11Fip5 was dispensable for NK cell cytolytic activity. Notably, immunized RAB11FIP5 -/- mice had lower IL4 expression in CD4+ T follicular helper cells and showed lower TNF expression in CD8+ T cells. Likewise, TNF-α production by human CD8+ T cells correlated with PBMC RAB11FIP5 expression. These observations in RAB11FIP5 -/- mice suggest a role for Rab11Fip5 in regulating cytokine responses.

Immune checkpoint modulation enhances HIV-1 antibody induction.

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.

Initiation of HIV neutralizing B cell lineages with sequential envelope immunizations.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env- upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.

Development of a recombinant yellow fever vector expressing a HIV clade C founder envelope gp120.

Development of a HIV-1 vaccine is a major global priority. The yellow fever virus (YFV) attenuated vaccine 17D is among the most effective of currently used vaccines. However, the stability of the YFV17D vector when carrying non-flavivirus genes has been problematic. We have constructed and expressed HIV-1 Env in YFV17D with either single transmembrane (STM) or double transmembrane (DTM) YFV E protein domains for the development of anti-HIV antibodies. Here we describe modifications of the YFV17D vector such that HIV-1 Env gp120 is expressed in up to 5 passages in Vero cells. Immunization with recombinant YFV17D vector prime followed by HIV-1 CH505 TF gp120 protein boosts were able to induce neutralizing antibodies for a HIV-1 tier 1 isolate in mice. This modified YFV vector may be a starting point for constructing HIV-1 vaccine candidate priming vectors.

HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques.

Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response.IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41 immunodominance in memory B cells of both adult and neonatal RMs indicated that early vaccination could not overcome gp41 dominant responses.

Immunodominance of Antibody Recognition of the HIV Envelope V2 Region in Ig-Humanized Mice.

In the RV144 gp120 HIV vaccine trial, decreased transmission risk was correlated with Abs that reacted with a linear epitope at a lysine residue at position 169 (K169) in the HIV-1 envelope (Env) V2 region. The K169 V2 response was restricted to Abs bearing Vλ rearrangements that expressed aspartic acid/glutamic acid in CDR L2. The AE.A244 gp120 in AIDSVAX B/E also bound to the unmutated ancestor of a V2-glycan broadly neutralizing Ab, but this Ab type was not induced in the RV144 trial. In this study, we sought to determine whether immunodominance of the V2 linear epitope could be overcome in the absence of human Vλ rearrangements. We immunized IgH- and Igκ-humanized mice with the AE.A244 gp120 Env. In these mice, the V2 Ab response was focused on a linear epitope that did not include K169. V2 Abs were isolated that used the same human VH gene segment as an RV144 V2 Ab but paired with a mouse λ L chain. Structural characterization of one of these V2 Abs revealed how the linear V2 epitope could be engaged, despite the lack of aspartic acid/glutamic acid encoded in the mouse repertoire. Thus, despite the absence of the human Vλ locus in these humanized mice, the dominance of Vλ pairing with human VH for HIV-1 Env V2 recognition resulted in human VH pairing with mouse λ L chains instead of allowing otherwise subdominant V2-glycan broadly neutralizing Abs to develop.

Tissue memory B cell repertoire analysis after ALVAC/AIDSVAX B/E gp120 immunization of rhesus macaques.

The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV­1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env­specific B cell clonal lineages were absent in the terminal ileum. Env­specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.

HIV-1 Envelope Mimicry of Host Enzyme Kynureninase Does Not Disrupt Tryptophan Metabolism.

The HIV-1 envelope protein (Env) has evolved to subvert the host immune system, hindering viral control by the host. The tryptophan metabolic enzyme kynureninase (KYNU) is mimicked by a portion of the HIV Env gp41 membrane proximal region (MPER) and is cross-reactive with the HIV broadly neutralizing Ab (bnAb) 2F5. Molecular mimicry of host proteins by pathogens can lead to autoimmune disease. In this article, we demonstrate that neither the 2F5 bnAb nor HIV MPER-KYNU cross-reactive Abs elicited by immunization with an MPER peptide-liposome vaccine in 2F5 bnAb VHDJH and VLJL knock-in mice and rhesus macaques modified KYNU activity or disrupted tissue tryptophan metabolism. Thus, molecular mimicry by HIV-1 Env that promotes the evasion of host anti-HIV-1 Ab responses can be directed toward nonfunctional host protein epitopes that do not impair host protein function. Therefore, the 2F5 HIV Env gp41 region is a key and safe target for HIV-1 vaccine development.

Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity.

Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

Antigenicity and immunogenicity of transmitted/founder, consensus, and chronic envelope glycoproteins of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) vaccine development requires selection of appropriate envelope (Env) immunogens. Twenty HIV-1 Env glycoproteins were examined for their ability to bind human anti-HIV-1 monoclonal antibodies (MAbs) and then used as immunogens in guinea pigs to identify promising immunogens. These included five Envs derived from chronically infected individuals, each representing one of five common clades and eight consensus Envs based on these five clades, as well as the consensus of the entire HIV-1 M group, and seven transmitted/founder (T/F) Envs from clades B and C. Sera from immunized guinea pigs were tested for neutralizing activity using 36 HIV-1 Env-pseudotyped viruses. All Envs bound to CD4 binding site, membrane-proximal, and V1/V2 MAbs with similar apparent affinities, although the T/F Envs bound with higher affinity to the MAb 17b, a CCR5 coreceptor binding site antibody. However, the various Envs differed in their ability to induce neutralizing antibodies. Consensus Envs elicited the most potent responses, but neutralized only a subset of viruses, including mostly easy-to-neutralize tier 1 and some more-difficult-to-neutralize tier 2 viruses. T/F Envs elicited fewer potent neutralizing antibodies but exhibited greater breadth than chronic or consensus Envs. Finally, chronic Envs elicited the lowest level and most limited breadth of neutralizing antibodies overall. Thus, each group of Env immunogens elicited a different antibody response profile. The complementary benefits of consensus and T/F Env immunogens raise the possibility that vaccines utilizing a combination of consensus and T/F Envs may be able to induce neutralizing responses with greater breadth and potency than single Env immunogens.

Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion.

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Induction of antibodies in rhesus macaques that recognize a fusion-intermediate conformation of HIV-1 gp41.

A component to the problem of inducing broad neutralizing HIV-1 gp41 membrane proximal external region (MPER) antibodies is the need to focus the antibody response to the transiently exposed MPER pre-hairpin intermediate neutralization epitope. Here we describe a HIV-1 envelope (Env) gp140 oligomer prime followed by MPER peptide-liposomes boost strategy for eliciting serum antibody responses in rhesus macaques that bind to a gp41 fusion intermediate protein. This Env-liposome immunization strategy induced antibodies to the 2F5 neutralizing epitope 664DKW residues, and these antibodies preferentially bound to a gp41 fusion intermediate construct as well as to MPER scaffolds stabilized in the 2F5-bound conformation. However, no serum lipid binding activity was observed nor was serum neutralizing activity for HIV-1 pseudoviruses present. Nonetheless, the Env-liposome prime-boost immunization strategy induced antibodies that recognized a gp41 fusion intermediate protein and was successful in focusing the antibody response to the desired epitope.

Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies.

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.