Dennis R. Burton

Directed evolution of a stem-helix–targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats. Author Summary The persistent emergence of new SARS-CoV-2 variants of concern that evade neutralizing antibody responses and other zoonotic betacoronaviruses with pandemic potential have provided strong motivation to develop broadly neutralizing antibodies (bnAbs) that target more conserved regions of the spike protein in sarbecoviruses and other betacoronaviruses. Here, we employed a directed evolution strategy to engineer the sarbecovirus-neutralizing antibody CC65.1, which targets the conserved S2 stem helix, to enhance its binding affinity with the MERS-CoV stem helix region, thereby conferring MERS-CoV neutralization. High-resolution structural studies of engineered CC65.1 revealed that key mutations reshape the paratope to better accommodate and stabilize the MERS-CoV S2 stem helix, resulting in increased binding affinity and neutralization potency. This study emphasizes the critical role of affinity maturation in expanding neutralization breadth and provides valuable insights for design of bnAbs to prevent and treat pandemic threats by betacoronaviruses.

Vaccination elicits HIV broadly neutralizing antibodies in primates

The high antigenic diversity of HIV has been a major obstacle to development of a broadly protective vaccine. Nevertheless, protective HIV broadly neutralizing antibodies (bnAbs) exist and have been proposed as templates for vaccine development 1-6 . Germline-targeting is a conceptually radical vaccine design approach to elicit bnAbs, aiming to prime rare bnAb-precursor B cells possessing pre-determined human genetic and structural features shared with template bnAbs, and then guide B cell affinity maturation to potent bnAb evolution with heterologous boosters 7-11 . Although the approach has shown promise in clinical 12-17 and pre-clinical 18-34 studies, it faces many immunological challenges and, to date, has not succeeded in generating bnAbs in humans or nontransgenic animals. Here, we report an adjuvanted protein germline-targeting vaccine tested in outbred nonhuman primates that generated bnAb-class memory B cells and sera capable of neutralizing diverse HIV clinical isolates. bnAb lineages were generated in ≥50% of animals, achieving up to 67% neutralization breadth compared to the reference bnAb. Vaccine-induced bnAbs exhibited precise structural mimicry of human bnAb interactions with HIV envelope (Env), matching the germline-targeting predictions. Furthermore, serum bnAb activity developed in 44% of animals and in the most striking instance reached titers expected to confer protection against diverse HIV isolates. These results demonstrate proof of principle that germline-targeting vaccines can reproducibly elicit prespecified classes of bnAbs to prespecified epitopes under endogenous conditions, supporting further optimization of this approach for HIV vaccine development.

Structure and functional diversity of antibodies targeting the P. falciparum circumsporozoite protein C-terminal domain

Vaccine elicitation of HIV broadly neutralizing antibodies from genome-edited B cells in non-human primates and derived lymphoid organoids

Germline-targeting HIV immunogen induces cross-neutralizing antibodies in outbred macaques

SUMMARY Germline-targeting-(GT) is a promising strategy to activate rare broadly neutralizing antibody (bnAb)-producing B cells against HIV, but induction of such responses in outbred animals has not been achieved. Using antibody-guided structure-based design, we engineered a germline-targeting trimer immunogen Q23-APEX-GT2 that primes diverse V2-apex bnAb precursors. Q23-APEX-GT2 efficiently activated V2-apex-specific B cells in humanized knock-in mice and consistently elicited immunofocused antibody responses in rhesus macaques, priming multiple long CDRH3-loop bnAb-B cell lineages. Monoclonal antibodies from immunized macaques exhibited broad heterologous HIV trimer binding and cross-neutralization. Atomic-level structural studies confirmed precise epitope targeting and revealed CDRH3-paratope configurations that mirrored those of human V2-apex bnAbs. This study provides proof-of-principle for successful priming and maturation of authentic V2-apex bnAb precursors in outbred macaques, underscoring the potential of V2-apex-targeted vaccines. HIGHLIGHTS Engineered Q23-APEX-GT2 trimer to stimulate diverse V2-apex bnAb B cell precursors Q23-APEX-GT2 primed rare V2-apex bnAb B cells in mice and outbred rhesus macaques Q23-APEX-GT2 elicited immunofocused antibody responses and diverse V2-apex B cell lineages with desirable long-CDRH3 paratope properties Q23-APEX-GT2 alone induced V2-apex antibodies with broad HIV trimer binding and modest neutralization breadth Structural analysis confirmed bnAb site targeting, mirroring human and rhesus V2-apex bnAbs

Structure-guided design of native-like HIV Env Single Chain Trimers for enhanced stability, immunogenicity, and versatile vaccine delivery

Engineering HIV antibodies with enhanced breadth and potency of neutralization through multistate affinity maturation

Broadly neutralizing antibodies (bnAbs) against HIV hold promise as therapeutic and prophylactic agents, but realizing this potential requires antibodies that function across the antigenic heterogeneity of the HIV envelope glycoprotein (Env). Although numerous bnAbs have been isolated from infected individuals, their breadth and potency may not be sufficient to tackle global viral diversity, motivating efforts to further improve their neutralization capacity. Here, we address this challenge using a multistate antibody engineering approach integrating deep mutational scanning with combinatorial library screening across diverse Env variants. This strategy enables identification of mutation patterns that confer improved binding across antigenically distinct targets. Starting from one of the best-in-class CD4-binding site bnAbs, we performed iterative optimization to increase binding affinity across diverse Env variants. The resulting lead candidate exhibited improved breadth and up to 100-fold higher potency against pseudoviruses from large cross-clade historical and contemporary panels while maintaining biophysical and pharmacokinetic profiles conducive to clinical development. Structural and molecular dynamics analyses revealed a unique tri-tyrosine aromatic triad and reinforced electrostatic contacts that stabilized the bnAb/Env interface. These findings demonstrate that systematic in vitro engineering can generate bnAbs with enhanced breadth and potency, providing a generalizable strategy for developing therapeutic antibodies against highly diverse pathogens.

Common cold embecovirus imprinting primes broadly neutralizing antibody responses to SARS-CoV-2 S2

HIV broadly neutralizing antibody precursors to the Apex epitope induced in nonhuman primates

Group 1 and 2 broadly neutralizing antibodies retain neutralizing activity against highly mutated SARS-CoV-2 variants like BA.2.86 and JN.1 by targeting conserved spike receptor-binding domain epitopes.

Rapid acquisition of HIV-1 neutralization breadth in a rhesus V2 apex germline antibody mouse model after a single bolus immunization

Current vaccine strategies to elicit broadly neutralizing antibodies (bnAbs) against HIV-1 generally propose complex, multi-boost immunization regimens. In rhesus macaques, SHIV infection has been observed to rapidly drive the development of some classes of bnAbs that share structural similarities with those in humans. Here, we generated a knockin mouse model with B cells bearing the unmutated common ancestor (UCA) of the V2 apex-targeted bnAb lineage, V033-a. A single immunization of mice with a germline-targeting native-like trimer was sufficient to recapitulate the ontogeny of the mature rhesus bnAb in knockin mice-including rare, disfavored somatic mutations-leading to the induction of antibodies that exhibited potent neutralization against both autologous and heterologous tier 2 viruses. A boost with Env escape mutant trimers further improved breadth and potency, and cryo-EM structure revealed the structural basis for heterologous neutralization breadth. Non-human primate and mouse models can thus combine with structure to serve as a platform for identifying and confirming immunogens that streamline HIV-vaccination regimens.

mEM detects more epitopes than conventional methods, mapping polyclonal antibodies in 90 minutes.

Combining a rhesus cytomegalovirus/SIV vaccine with a neutralizing antibody to protect against SIV challenges in rhesus macaques

A vaccine is considered essential for controlling the HIV pandemic and ultimately eradicating AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have shown the ability to protect against the simian counterpart of HIV, SIV, in rhesus macaques. In this study, we provide preliminary evidence that combining these orthogonal antiviral mechanisms can offer increased protection against SIV. Specifically, the replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced by the presence of a passively administered neutralizing antibody at incompletely protective levels. This report encourages studies involving larger cohorts of macaques and alternative methods for administering neutralizing antibodies.

Env-antibody coevolution identifies B cell priming as the principal bottleneck to HIV-1 V2 apex broadly neutralizing antibody development

Broadly neutralizing antibodies (bNAbs) are rarely elicited during HIV-1 infection. To identify obstacles to bNAb development, we longitudinally studied 122 rhesus macaques infected by one of 16 different simian-human immunodeficiency viruses (SHIVs). We identified V2 apex as the most common bNAb target and a subset of Envs that preferentially elicited these antibodies. In 10 macaques, we delineated Env-antibody coevolution from B cell priming to bNAb development. Antibody phylogenies revealed permissive developmental pathways guided by evolving Envs that contained few mutations in or near the V2 apex C-strand, which were a sensitive indicator of apex-targeted responses. The absence of such mutations reflected a failure in bNAb priming. These results indicate that efficiency of B cell priming, and not complexities in Env-guided affinity maturation, is the primary obstacle to V2 apex bNAb elicitation in SHIV-infected macaques and identify specific HIV-1 Envs to advance as novel vaccine platforms. One sentence summary: B cell priming is the primary bottleneck to HIV-1 V2 apex bNAb elicitation.

Molecular parameters governing antibody FcγR signaling and effector functions in the context of HIV envelope

Antibody effector functions contribute to the immune response to pathogens and can influence the efficacy of antibodies as therapeutics. To date, however, there is limited information on the molecular parameters that govern fragment crystallizable (Fc) effector functions. In this study, using AI-assisted protein design, the influences of binding kinetics, epitope location, and stoichiometry of binding on cellular Fc effector functions were investigated using engineered HIV-1 envelope as a model antigen. For this antigen, stoichiometry of binding was found to be the primary molecular determinant of FcγRIIIa signaling, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis, while epitope location and antibodybinding kinetics, at least in the ranges investigated, were of no substantial impact. These findings are of importance for informing the development of vaccination strategies against HIV-1 and, possibly, other viral pathogens.

Longitudinal analysis of drift in the circulating human antibody repertoire over four years

The human immune system is a sophisticated network of cells and molecules that plays a vital role in safeguarding the body against a broad range of foreign agents. One of the key components of the immune system is the circulating antibody repertoire, a vast collection of different antibodies that recognize and eliminate foreign agents specifically. The diversity and specificity of the circulating antibody repertoire are essential for effective immunity. In a study of ten healthy donors, we previously demonstrated the accessible human antibody repertoire to be on the order of 10 15 -10 18 distinct members, although an individual will only sample a fraction of that repertoire at a given time point. The mode of generation of the naïve antibody repertoire through random recombination before antigen contact suggests that the composition of the circulating antibody repertoire may drift over time, but the magnitude of this drift is poorly understood. Here, we used high-throughput sequencing to analyze two donors from our original study, approximately four years later. Our results reveal conservation of the size, overall diversity, and gross features of the circulating antibody repertoire, such as V- and J-gene usage and CDRH3 length distribution, over time but suggest substantial changes to fine features of the repertoire, such as combinations of V, J, D gene segments and P and N sequences, which may influence primary responses to newly encountered pathogens as well as to immunization procedures.

Combining a rhesus cytomegalovirus/SIV vaccine with neutralizing antibody to protect against SIV challenge in rhesus macaques

A vaccine is widely regarded as necessary for the control of the HIV pandemic and eventual eradication of AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have both been shown capable of vaccine protection against the simian counterpart of HIV, SIV, in rhesus macaques. Here we provide preliminary evidence that combining these orthogonal antiviral mechanisms can provide increased protection against SIV challenge such that replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced in the presence of passively administered incompletely protective levels of neutralizing antibody. The report invites studies involving larger cohorts of macaques and alternate routes of providing neutralizing antibody.

The saponin monophosphoryl lipid A nanoparticle adjuvant induces dose-dependent HIV vaccine responses in nonhuman primates

Induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play a role as drivers of such responses. Despite advances in vaccine strategies, development of a safe and effective HIV vaccine remains a significant challenge. Use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and effect of multiple variables - including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition - on the establishment of Env-specific memory T and B cell (TMem and BMem) responses and long-lived plasma cells in nonhuman primates (NHPs). Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest- versus the lowest-SMNP-dose group. Similarly, stronger vaccine responses were induced by the highest SMNP dose in CD40L+OX40+ CD4+ TMem (11-fold), IFN-γ+ CD4+ TMem (15-fold), IL21+ CD4+ TMem (9-fold), circulating T follicular helper cells (TFH; 3.6-fold), BM plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier 2 neutralizing antibodies were only observed in the higher-SMNP-dose groups. These investigations highlight the dose-dependent potency of SMNP and its relevance for human use and next-generation vaccines.

Passive infusion of an S2-Stem broadly neutralizing antibody protects against SARS-CoV-2 infection and lower airway inflammation in rhesus macaques

The continued evolution of SARS-CoV-2 variants capable of subverting vaccine and infection-induced immunity suggests the advantage of a broadly protective vaccine against betacoronaviruses (β-CoVs). Recent studies have isolated monoclonal antibodies (mAbs) from SARS-CoV-2 recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other β-CoVs. Many of these mAbs target the conserved S2 stem region of the SARS-CoV-2 spike protein, rather than the receptor binding domain contained within S1 primarily targeted by current SARS-CoV-2 vaccines. One of these S2-directed mAbs, CC40.8, has demonstrated protective efficacy in small animal models against SARS-CoV-2 challenge. As the next step in the pre-clinical testing of S2-directed antibodies as a strategy to protect from SARS-CoV-2 infection, we evaluated the in vivo efficacy of CC40.8 in a clinically relevant non-human primate model by conducting passive antibody transfer to rhesus macaques (RM) followed by SARS-CoV-2 challenge. CC40.8 mAb was intravenously infused at 10mg/kg, 1mg/kg, or 0.1 mg/kg into groups (n = 6) of RM, alongside one group that received a control antibody (PGT121). Viral loads in the lower airway were significantly reduced in animals receiving higher doses of CC40.8. We observed a significant reduction in inflammatory cytokines and macrophages within the lower airway of animals infused with 10mg/kg and 1mg/kg doses of CC40.8. Viral genome sequencing demonstrated a lack of escape mutations in the CC40.8 epitope. Collectively, these data demonstrate the protective efficiency of broadly neutralizing S2-targeting antibodies against SARS-CoV-2 infection within the lower airway while providing critical preclinical work necessary for the development of pan-β-CoV vaccines.

Modulation of antigen delivery and lymph node activation in nonhuman primates by saponin adjuvant saponin/monophosphoryl lipid A nanoparticle

Saponin-based vaccine adjuvants are potent in preclinical animal models and humans, but their mechanisms of action remain poorly understood. Here, using a stabilized HIV envelope trimer immunogen, we carried out studies in nonhuman primates (NHPs) comparing the most common clinical adjuvant aluminum hydroxide (alum) with saponin/monophosphoryl lipid A nanoparticles (SMNP), an immune-stimulating complex-like adjuvant. SMNP elicited substantially stronger humoral immune responses than alum, including 7-fold higher peak antigen-specific germinal center B-cell responses, 18-fold higher autologous neutralizing antibody titers, and higher levels of antigen-specific plasma and memory B cells. Positron emission tomography and computed tomography imaging in live NHPs showed that, unlike alum, SMNP promoted rapid antigen accumulation in both proximal and distal lymph nodes (LNs). SMNP also induced strong type I interferon transcriptional signatures, expansion of innate immune cells, and increased antigen-presenting cell activation in LNs. These findings indicate that SMNP promotes multiple facets of the early immune response relevant for enhanced immunity to vaccination.