Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron.

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody.

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.

Protective human monoclonal antibodies target conserved sites of vulnerability on the underside of influenza virus neuraminidase.

Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.

Deep mutational scanning reveals functional constraints and antibody-escape potential of Lassa virus glycoprotein complex.

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals. It remains unclear if exposures to antigenically distant SARS-CoV-2 variants can overcome memory B cell biases established by initial SARS-CoV-2 encounters. We determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that targeted epitopes conserved between the BA.5 and ancestral spike. XBB exposures also elicited antibody responses that primarily targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low frequencies of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

An Antigenic Atlas of HIV-1 Escape from Broadly Neutralizing Antibodies Distinguishes Functional and Structural Epitopes.

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the virus's envelope (Env) protein and are themselves promising immunotherapies. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. Although structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here, we mapped how all possible single amino acid mutations in Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites near, but not in direct contact with, the antibody. The Env mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs's independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

MetaFluAD: meta-learning for predicting antigenic distances among influenza viruses.

Influenza viruses rapidly evolve to evade previously acquired human immunity. Maintaining vaccine efficacy necessitates continuous monitoring of antigenic differences among strains. Traditional serological methods for assessing these differences are labor-intensive and time-consuming, highlighting the need for efficient computational approaches. This paper proposes MetaFluAD, a meta-learning-based method designed to predict quantitative antigenic distances among strains. This method models antigenic relationships between strains, represented by their hemagglutinin (HA) sequences, as a weighted attributed network. Employing a graph neural network (GNN)-based encoder combined with a robust meta-learning framework, MetaFluAD learns comprehensive strain representations within a unified space encompassing both antigenic and genetic features. Furthermore, the meta-learning framework enables knowledge transfer across different influenza subtypes, allowing MetaFluAD to achieve remarkable performance with limited data. MetaFluAD demonstrates excellent performance and overall robustness across various influenza subtypes, including A/H3N2, A/H1N1, A/H5N1, B/Victoria, and B/Yamagata. MetaFluAD synthesizes the strengths of GNN-based encoding and meta-learning to offer a promising approach for accurate antigenic distance prediction. Additionally, MetaFluAD can effectively identify dominant antigenic clusters within seasonal influenza viruses, aiding in the development of effective vaccines and efficient monitoring of viral evolution.

PREDAC-CNN: predicting antigenic clusters of seasonal influenza A viruses with convolutional neural network.

Vaccination stands as the most effective and economical strategy for prevention and control of influenza. The primary target of neutralizing antibodies is the surface antigen hemagglutinin (HA). However, ongoing mutations in the HA sequence result in antigenic drift. The success of a vaccine is contingent on its antigenic congruence with circulating strains. Thus, predicting antigenic variants and deducing antigenic clusters of influenza viruses are pivotal for recommendation of vaccine strains. The antigenicity of influenza A viruses is determined by the interplay of amino acids in the HA1 sequence. In this study, we exploit the ability of convolutional neural networks (CNNs) to extract spatial feature representations in the convolutional layers, which can discern interactions between amino acid sites. We introduce PREDAC-CNN, a model designed to track antigenic evolution of seasonal influenza A viruses. Accessible at http://predac-cnn.cloudna.cn, PREDAC-CNN formulates a spatially oriented representation of the HA1 sequence, optimized for the convolutional framework. It effectively probes interactions among amino acid sites in the HA1 sequence. Also, PREDAC-CNN focuses exclusively on physicochemical attributes crucial for the antigenicity of influenza viruses, thereby eliminating unnecessary amino acid embeddings. Together, PREDAC-CNN is adept at capturing interactions of amino acid sites within the HA1 sequence and examining the collective impact of point mutations on antigenic variation. Through 5-fold cross-validation and retrospective testing, PREDAC-CNN has shown superior performance in predicting antigenic variants compared to its counterparts. Additionally, PREDAC-CNN has been instrumental in identifying predominant antigenic clusters for A/H3N2 (1968-2023) and A/H1N1 (1977-2023) viruses, significantly aiding in vaccine strain recommendation.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae.

Carbapenemase and extended ß-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Influenza virus antibodies inhibit antigen-specific de novo B cell responses in mice.

Antibody responses to influenza vaccines tend to be focused on epitopes encountered during prior influenza exposures, with little production of de novo responses to novel epitopes. To examine the contribution of circulating antibodies to this phenomenon, we passively transferred a hemagglutinin (HA)-specific monoclonal antibody (mAb) into mice before immunizing with whole inactivated virions. The HA mAb inhibited de novo HA-specific antibodies, plasmablasts, germinal center B cells, and memory B cells, while responses to a second antigen in the vaccine, neuraminidase (NA), were uninhibited. The HA mAb potently inhibited de novo antibody responses against epitopes near the HA mAb binding site. The HA mAb also promoted IgG1 class switching, an effect that, unlike the inhibition of HA responses, relied on signaling through Fc-gamma receptors. These studies suggest that circulating antibodies inhibit de novo B cell responses in an antigen-specific manner, which likely contributes to differences in antibody specificities elicited during primary and secondary influenza virus exposures.IMPORTANCEMost humans are exposed to influenza viruses in childhood and then subsequently exposed to antigenically drifted influenza variants later in life. It is unclear if antibodies elicited by earlier influenza virus exposures impact immunity against new influenza virus strains. Here, we used a mouse model to investigate how an anti-hemagglutinin (HA) monoclonal antibody (mAb) affects de novo B cell and antibody responses to the protein targeted by the monoclonal antibody (HA) and a second protein not targeted by the monoclonal antibody [neuraminidase (NA)]. Collectively, our studies suggest that circulating anti-influenza virus antibodies can potently modulate the magnitude and specificity of antibody responses elicited by secondary influenza virus exposures.

Armored bicistronic CAR T cells with dominant-negative TGF-ß receptor II to overcome resistance in glioblastoma.

Chimeric antigen receptor (CAR) T cells have shown significant efficacy in hematological diseases. However, CAR T therapy has demonstrated limited efficacy in solid tumors, including glioblastoma (GBM). One of the most important reasons is the immunosuppressive tumor microenvironment (TME), which promotes tumor growth and suppresses immune cells used to eliminate tumor cells. The human transforming growth factor ß (TGF-ß) plays a crucial role in forming the suppressive GBM TME and driving the suppression of the anti-GBM response. To mitigate TGF-ß-mediated suppressive activity, we combined a dominant-negative TGF-ß receptor II (dnTGFßRII) with our previous bicistronic CART-EGFR-IL13Rα2 construct, currently being evaluated in a clinical trial, to generate CART-EGFR-IL13Rα2-dnTGFßRII, a tri-modular construct we are developing for clinical application. We hypothesized that this approach would more effectively subvert resistance mechanisms observed with GBM. Our data suggest that CART-EGFR-IL13Rα2-dnTGFßRII significantly augments T cell proliferation, enhances functional responses, and improves the fitness of bystander cells, particularly by decreasing the TGF-ß concentration in a TGF-ß-rich TME. In addition, in vivo studies validate the safety and efficacy of the dnTGFßRII cooperating with CARs in targeting and eradicating GBM in an NSG mouse model.

Substitutions near the HA receptor binding site explain the origin and major antigenic change of the B/Victoria and B/Yamagata lineages.

Influenza B virus primarily infects humans, causing seasonal epidemics globally. Two antigenic variants-Victoria-like and Yamagata-like-were detected in the 1980s, of which the molecular basis of emergence is still incompletely understood. Here, the antigenic properties of a unique collection of historical virus isolates, sampled from 1962 to 2000 and passaged exclusively in mammalian cells to preserve antigenic properties, were determined with the hemagglutination inhibition assay and an antigenic map was built to quantify and visualize the divergence of the lineages. The antigenic map revealed only three distinct antigenic clusters-Early, Victoria, and Yamagata-with relatively little antigenic diversity in each cluster until 2000. Viruses with Victoria-like antigenic properties emerged around 1972 and diversified subsequently into two genetic lineages. Viruses with Yamagata-like antigenic properties evolved from one lineage and became clearly antigenically distinct from the Victoria-like viruses around 1988. Recombinant mutant viruses were tested to show that insertions and deletions (indels), as observed frequently in influenza B virus hemagglutinin, had little effect on antigenic properties. In contrast, amino-acid substitutions at positions 148, 149, 150, and 203, adjacent to the hemagglutinin receptor binding site, determined the main antigenic differences between the Early, Victoria-like, and Yamagata-like viruses. Surprisingly, substitutions at two of the four positions reverted in recent viruses of the Victoria lineage, resulting in antigenic properties similar to viruses circulating ∼50 y earlier. These data shed light on the antigenic diversification of influenza viruses and suggest there may be limits to the antigenic evolution of influenza B virus.