Peter D. Kwong

Intranasal COVID-19 vaccines with the ability to induce broad and durable mucosal and systemic immunity would be useful as stand-alone vaccines or in combination with injectable vaccines. We evaluated in hamsters the breadth of immunity and durability of protection elicited by co-administration of two live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3) vectors expressing antigenically distinct prefusion-stabilized S proteins of the ancestral SARS-CoV-2 isolate (B/HPIV3/S-6P) or the BA.5/Omicron variant (B/HPIV3/S-BA.5-2P). These vectors are being developed as bivalent pediatric vaccines against HPIV3 and SARS-CoV-2 and are based on bovine PIV3 with the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins replaced by those of human PIV3. To broaden the S-specific antibody response, we evaluated co-administration of these B/HPIV3 S-expressing vectors. Each B/HPIV3 S-expressing vector induced robust serum anti-S IgG and IgA antibody levels to the antigen-matched S protein that were sustained for at least 5 months. Co-administration increased the breadth of the S-specific antibody response, spanning the breadth of the response elicited by each vector individually. Hamsters immunized intranasally with the mixture of vectors developed neutralizing antibodies to ancestral and recently circulating SARS-CoV-2 strains and were protected against BA.5/Omicron challenge 5 months after immunization, with no weight loss, SARS-CoV-2 challenge virus replication, or increase in host inflammatory cytokines in the airways after the challenge, indicating durable protection. Thus, intranasal co-administration of live-attenuated B/HPIV3 expressing antigenically distinct S proteins induced broad and durable antibody responses, and long-term protection against BA.5/Omicron challenge. This approach warrants further development and may better protect against emerging SARS-CoV-2 variants. IMPORTANCE: Intranasal SARS-CoV-2 vaccines that induce systemic and local mucosal immunity hold promise, especially as booster vaccines against SARS-CoV-2 variants. We developed live-attenuated bovine/human parainfluenza virus type 3 (B/HPIV3)-vectored vaccines as bivalent pediatric vaccines against PIV3 and SARS-CoV-2. Co-administration in a single dose of two B/HPIV3 vectors, one expressing the spike protein S from the ancestral Wuhan-Hu-1 strain and one expressing S of BA.5/Omicron, induced broad serum anti-S antibody responses that remained strong for at least 5 months. Hamsters challenged with the BA.5/Omicron strain 5 months after immunization were protected from weight loss, inflammatory responses, and challenge virus replication in the airways. Thus, in the hamster model, intranasal co-administration of live-attenuated B/HPIV3 expressing SARS-CoV-2 S elicits protection durable over several months, and combining two antigenically distinct B/HPIV3 S-expressing vectors substantially broadens the antibody response. This mucosal immunization approach may provide durable and broad protection against SARS-CoV-2.

HIV-1 envelope glycoprotein (Env), a gp120–gp41 trimer, undergoes coordinated conformational changes that drive membrane fusion and allow immune evasion by transiently concealing neutralization-sensitive epitopes. Most broadly neutralizing antibodies (bNAbs) target gp120, whereas a distinct subset recognizes conserved gp41 regions, such as the fusion peptide and the membrane-proximal external region; however, their impact on Env dynamics and associated neutralization mechanisms remains unclear. By using bioorthogonal tagging for single-molecule FRET, we monitored real-time bNAb-induced conformational sampling of Env on intact virions. Most gp41-directed bNAbs allosterically stabilized the prefusion-closed (PC) state, whereas the bivalent 10E8.4/iMab favored both PC and CD4-bound open (predominant) states. Antibodies redistributed the conformational populations of Env with modest kinetic effects, preserving a sequential transition pathway. These findings reveal two modes of neutralization for gp41-directed antibodies, fixing the PC conformation and opening it up – in both cases, with neutralization occurring via long-range allosteric control of Env dynamics.

Alphaviruses are enveloped, positive-sense, single-stranded RNA viruses of the Togaviridae family with substantial impact on human and animal health primarily through mosquito transmission. In huma...

Viral entry glycoproteins are often shielded from immune recognition by dense N ‐linked glycans that limit antibody access to protein epitopes. While glycan‐reactive antibodies with unusual architectures have been described, how canonical Y‐shaped antibodies engage these glycan‐rich surfaces remains poorly defined. Here, we characterize two human antibodies, VRC35 and VRC36, isolated from an HIV‐1‐infected donor, that recognize diverse glycosylated viral glycoproteins. Cryo‐electron microscopy structural analyses of these antibodies in complex with viral entry glycoproteins, including HIV‐1 envelope, influenza hemagglutinin, SARS‐CoV‐2 spike, and the Lassa virus glycoprotein complex, reveal adaptive Fab stoichiometries ranging from single‐Fab binding to dimeric and higher‐order assemblies are mediated by intra‐ and inter‐IgG interactions that depend on local glycan organization. Dense glycan clustering on HIV‐1 and influenza glycoproteins supports multivalent Fab assemblies and correlates with neutralization activity, whereas sparse glycan environments on SARS‐CoV‐2 and Lassa virus favor weak or heterogeneous engagement without neutralization. Structural and mutational analyses further demonstrate that homotypic Fab–Fab interactions stabilize multivalent engagement and contribute to neutralizing activity. Together, these findings define a structural framework in which viral glycan organization constrains antibody valency and engagement, while somatic hypermutation contributes to the acquisition of homotypic Fab‐Fab interactions that facilitate multivalent recognition of viral glycan shields.

The first approval of malaria vaccines RTS,S/AS01 and R21/Matrix M marked a major milestone. Both vaccines present a portion of the Plasmodium falciparum circumsporozoite protein (PfCSP) containing an immunodominant major repeat region, which may limit breadth to other protective epitopes, limiting efficacy and durability. Using B cell receptor (BCR) knock-in mice, we found that the R21-included PfCSP epitope elicited robust B cell responses to the immunodominant major repeats but not to other highly protective epitopes, the minor repeat and junction. We then defined a minimal peptide capable of eliciting minor repeat–specific B cell responses and generating highly protective antibodies (Abs). We characterized these Abs bioinformatically and structurally to identify protective traits, which informed the design of variant Abs with improved affinity. Finally, we demonstrated that vaccination combining the R21-included PfCSP epitope, the minimal minor repeat peptide, and a junctional region immunogen elicited balanced B cell and Ab responses, enhancing protection in vivo. Broadening responses through immunofocusing may overcome immunogenic gaps in current malaria vaccines.

Efficient priming of B cell precursors is a rate-limiting step in the induction of V2 apex broadly neutralizing antibodies (bNAbs)^1,2. Here, we describe a novel germline-targeted HIV-1 Env (CAP256.OPT4) that increases the efficiency of V2 apex bNAb precursor priming by 30-400 fold compared with wild-type HIV-1 Envs and induces - in >90% of macaques - neutralization breadth that includes N130-containing viruses. Using three different delivery platforms - persistently replicating simian human immunodeficiency viruses (SHIVs), protein nanoparticles, and mRNA - we show bNAb priming as early as 4 weeks post-infection or immunization, and neutralization breadth in plasma by 12 weeks. In 14 SHIV-infected macaques, neutralization breadth reached as high as 90% on a 21-virus panel with potency as great as 1:20,000 (50% inhibitory dilution, ID₅₀). Monoclonal bNAbs isolated from these animals were similarly broad and potent, with cryo-EM structures representing three distinct lineages revealing canonical needle-like HCDR3 binding. Env-Ab coevolution and structural analyses identified five key residues and loop features under positive selection and temporally associated with neutralization breadth. Importantly, prime-boost immunogens designed to capture these features induced broad and potent neutralization of globally diverse viruses including those containing N130 glycan. Further, rhesus bNAbs were not restricted to IGHD3-15*01 heavy chain alleles. These results expand the utility of the rhesus model for HIV-1 vaccine design and provide a molecular blueprint for inducing V2 apex bNAbs in rhesus and humans.

Enterovirus D68 (EV-D68) is a picornavirus that causes biennial outbreaks of respiratory disease in young children that can progress to rare but severe complications, including acute flaccid myelitis (AFM). EV-D68 virus-like particles (VLPs) elicit potent neutralizing antibodies that are protective in animal models. Here, we report the isolation and characterization of monoclonal antibodies elicited by EV-D68 VLPs in nonhuman primates (NHPs). We identified five potently neutralizing mAbs targeting overlapping epitopes near the capsid fivefold axis of symmetry formed by pentamers of viral protein 1. Cryo–electron microscopy structures of mAbs 1E11 and 5H03 in complex with VLP revealed epitopes on the capsid that bridge the sialic acid binding site and the proteinaceous entry receptor major facilitator superfamily domain-containing protein 6 binding site. We further characterized the mechanisms by which these mAbs neutralize EV-D68 and found that mAbs can disrupt multiple steps in the EV-D68 life cycle, including promoting premature uncoating. Antibodies elicited by VLP immunization of NHP protected as well as a best-in-class human mAb in a mouse challenge model, although single–amino acid mutations in the capsid enabled viral escape. Our results demonstrate that EV-D68 VLP–elicited mAbs target major viral sites of vulnerability, provide insight into their mechanisms of neutralization, and reinforce the potential for VLP-based vaccines as a countermeasure for EV-D68.

Accurate calculation of energy changes upon mutation is a key requirement for the effective use of computational methods in protein design. In this study, we applied free energy perturbation (FEP) calculations to predict the effects of mutations on the binding free energy between the immunoglobulin subtype G (IgG) antibody fragment-crystallizable (Fc) region and the neonatal Fc receptor (FcRn), an interaction that is primarily responsible for antibody half-life. We assembled an extensive experimental dataset of Fc-FcRn binding affinities for wild-type ( wt ) and mutant complexes, including values from literature and from newly measured results. Starting from a crystal structure of the M252Y/S254T/T256E (“YTE”) Fc variant bound to FcRn, we prepared all-atom models of human IgG1-subtype wt and YTE variant Fc-FcRn complexes, adding explicit hydrogens and assigning protonation states for key ionizable residues. Initial results using standard FEP protocols to compute relative binding free energies were promising but exhibited multiple outliers. By accounting for coupling effects for FEP mutations near key histidine residues, we improved the results for several outliers, suggesting such coupling as an important approach for pH-sensitive systems. Further, upon determining new crystal structures of four Fc variants at multiple pH values, we observed subtle conformational changes in unbound Fc; by accounting for these conformational changes in FEP calculations, we additionally improved agreement with experiment. The detailed structural and energetic analyses of the Fc-FcRn system we present here thus provide an accurate energy-calculation framework to enable rational in silico design of novel Fc variants. Significance: The ability to determine changes in binding affinity upon mutation is critical to structure-based protein design. In this study, we demonstrate a successful computational approach using free energy perturbation (FEP) calculations on the antibody Fc-FcRn complex, a medically relevant system with implications for both therapeutic and prophylactic antibody use. Our successful calculation of accurate binding energies across a wide range of cases speaks to the power of the FEP methodology in navigating the free energy landscapes of dynamic molecular complexes. Furthermore, we show that accurate Fc-FcRn affinity calculations required careful consideration of conformational flexibility between bound and unbound states, contributing to our functional understanding of a system that will be important for future rational antibody-design efforts.