mtx-COBRA: Subcellular localization prediction for bacterial proteins.

BACKGROUND: Bacteria can have beneficial effects on our health and environment; however, many are responsible for serious infectious diseases, warranting the need for vaccines against such pathogens. Bioinformatic and experimental technologies are crucial for the development of vaccines. The vaccine design pipeline requires identification of bacteria-specific antigens that can be recognized and can induce a response by the immune system upon infection. Immune system recognition is influenced by the location of a protein. Methods have been developed to determine the subcellular localization (SCL) of proteins in prokaryotes and eukaryotes. Bioinformatic tools such as PSORTb can be employed to determine SCL of proteins, which would be tedious to perform experimentally. Unfortunately, PSORTb often predicts many proteins as having an "Unknown" SCL, reducing the number of antigens to evaluate as potential vaccine targets. METHOD: We present a new pipeline called subCellular lOcalization prediction for BacteRiAl Proteins (mtx-COBRA). mtx-COBRA uses Meta's protein language model, Evolutionary Scale Modeling, combined with an Extreme Gradient Boosting machine learning model to identify SCL of bacterial proteins based on amino acid sequence. This pipeline is trained on a curated dataset that combines data from UniProt and the publicly available ePSORTdb dataset. RESULTS: Using benchmarking analyses, nested 5-fold cross-validation, and leave-one-pathogen-out methods, followed by testing on the held-out dataset, we show that our pipeline predicts the SCL of bacterial proteins more accurately than PSORTb. CONCLUSIONS: mtx-COBRA provides an accessible pipeline that can more efficiently classify bacterial proteins with currently "Unknown" SCLs than existing bioinformatic and experimental methods.

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

An mpox virus mRNA-lipid nanoparticle vaccine confers protection against lethal orthopoxviral challenge.

Mpox virus (MPXV) caused a global outbreak in 2022. Although smallpox vaccines were rapidly deployed to curb spread and disease among those at highest risk, breakthrough disease was noted after complete immunization. Given the threat of additional zoonotic events and the virus's evolving ability to drive human-to-human transmission, there is an urgent need for an MPXV-specific vaccine that confers protection against evolving MPXV strains and related orthopoxviruses. Here, we demonstrate that an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved MPXV surface proteins involved in virus attachment, entry, and transmission can induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. Compared with modified vaccinia virus Ankara (MVA), which forms the basis for the current MPXV vaccine, immunization with an mRNA-based MPXV vaccine generated superior neutralizing activity against MPXV and VACV and more efficiently inhibited spread between cells. We also observed greater Fc effector TH1-biased humoral immunity to the four MPXV antigens encoded by the vaccine, as well as to the four VACV homologs. Single MPXV antigen-encoding mRNA vaccines provided partial protection against VACV challenge, whereas multivalent vaccines combining mRNAs encoding two, three, or four MPXV antigens protected against disease-related weight loss and death equal or superior to MVA vaccination. These data demonstrate that an mRNA-based MPXV vaccine confers robust protection against VACV.

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization.

While neutralizing antibodies that target the HIV-1 fusion peptide have been elicited in mice by vaccination, antibodies reported thus far have been from only a single antibody class that could neutralize ~30% of HIV-1 strains. To explore the ability of the murine immune system to generate cross-clade neutralizing antibodies and to investigate how higher breadth and potency might be achieved, we tested 17 prime-boost regimens that utilized diverse fusion peptide-carrier conjugates and HIV-1 envelope trimers with different fusion peptides. We observed priming in mice with fusion peptide-carrier conjugates of variable peptide length to elicit higher neutralizing responses, a result we confirmed in guinea pigs. From vaccinated mice, we isolated 21 antibodies, belonging to 4 distinct classes of fusion peptide-directed antibodies capable of cross-clade neutralization. Top antibodies from each class collectively neutralized over 50% of a 208-strain panel. Structural analyses - both X-ray and cryo-EM - revealed each antibody class to recognize a distinct conformation of fusion peptide and to have a binding pocket capable of accommodating diverse fusion peptides. Murine vaccinations can thus elicit diverse neutralizing antibodies, and altering peptide length during prime can improve the elicitation of cross-clade responses targeting the fusion peptide site of HIV-1 vulnerability. IMPORTANCE The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence. We found that variation in peptide length during prime elicits enhanced neutralizing responses in mice and guinea pigs. We identified vaccine-elicited murine monoclonal antibodies from distinct classes capable of cross-clade neutralization and of diverse fusion peptide recognition. Our findings lend insight into improved immunogens and regimens for HIV-1 vaccine development.

Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant in mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the Omicron lineage has resulted in diminished Coronavirus Disease 2019 (COVID-19) vaccine efficacy and persistent transmission. In this study, we evaluated the immunogenicity and protective efficacy of two, recently authorized, bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary two-dose immunization series in mice, both bivalent vaccines induced greater neutralizing antibody responses against Omicron variants than the parental, monovalent mRNA-1273 vaccine. When administered to mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced broadly neutralizing antibody responses. Whereas most anti-Omicron receptor binding domain antibodies in serum induced by mRNA-1273, mRNA-1273.214 and mRNA-1273.222 boosters cross-reacted with the antecedent Wuhan-1 spike antigen, the mRNA-1273.214 and mRNA-1273.222 bivalent vaccine boosters also induced unique BA.1-specific and BA.4/5-specific responses, respectively. Although boosting with parental or bivalent mRNA vaccines substantially improved protection against BA.5 compared to mice receiving two vaccine doses, the levels of infection, inflammation and pathology in the lung were lowest in animals administered the bivalent mRNA vaccines. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and confers protection in mice against a currently circulating SARS-CoV-2 strain.

Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant.

The emergence of SARS-CoV-2 variants in the Omicron lineage with large numbers of substitutions in the spike protein that can evade antibody neutralization has resulted in diminished vaccine efficacy and persistent transmission. One strategy to broaden vaccine-induced immunity is to administer bivalent vaccines that encode for spike proteins from both historical and newly-emerged variant strains. Here, we evaluated the immunogenicity and protective efficacy of two bivalent vaccines that recently were authorized for use in Europe and the United States and contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary immunization series in BALB/c mice, both bivalent vaccines induced broader neutralizing antibody responses than the constituent monovalent vaccines (mRNA-1273 [Wuhan-1], mRNA-1273.529 [BA.1], and mRNA-1273-045 [BA.4/5]). When administered to K18-hACE2 transgenic mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced greater breadth and magnitude of neutralizing antibodies compared to an mRNA-1273 booster. Moreover, the response in bivalent vaccine-boosted mice was associated with increased protection against BA.5 infection and inflammation in the lung. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and protection against currently circulating SARS-CoV-2 strains.

C3-Symmetric Aromatic Core of Griffithsin Is Essential for Potent Anti-HIV Activity.

Lectins, carbohydrate-binding proteins of nonimmune origin, bind to carbohydrates and glycan shields present on the surfaces of cells and viral spike proteins. Lectins thus hold great promise as therapeutic and diagnostic proteins, exemplified by their potent antiviral activities and the desire to engineer synthetic carbohydrate receptors based on lectin recognition principles. Here, we describe a new carbohydrate-binding architectural motif─namely, a C3-symmetric tyrosine-based aromatic core, present in the therapeutic lectin griffithsin (GRFT). By using structure-based amino acid substitutions, X-ray crystallography, molecular dynamics (MD) simulations, and HIV-1 neutralization assays, we show that this core is critical for potent (pM) antiviral activity and nanomolar binding to the glycan shield largely consisting of high mannose glycans. Crystal structures and MD simulations show that CH-π interactions stabilize the aromatic cluster to maintain the three pseudo-symmetric carbohydrate-binding sites, nonaromatic amino acid substitutions (Tyr to Ala) abrogate antiviral activity, and increasing the aromatic CH-π edge-to-centroid interface via a Tyr to Trp substitution yields a GRFT variant with improved potency and increased residence time of Man-9 observed in MD simulations. NMR titrations of a Tyr-to-Ala variant indicate that disruption of the aromatic prevents the intermolecular crosslinking between two equivalents of Man-9 and one carbohydrate-binding face observed in wild-type GRFT and known to be critical for picomolar potency of this lectin. This C3-symmetric aromatic core defines a new recognition motif for the design of carbohydrate receptors and suggests principles for engineering known lectins to have increased affinity and stability.

Structure of an influenza group 2-neutralizing antibody targeting the hemagglutinin stem supersite.

Several influenza antibodies with broad group 2 neutralization have recently been isolated. Here, we analyze the structure, class, and binding of one of these antibodies from an H7N9 vaccine trial, 315-19-1D12. The cryo-EM structure of 315-19-1D12 Fab in complex with the hemagglutinin (HA) trimer revealed the antibody to recognize the helix A region of the HA stem, at the supersite of vulnerability recognized by group 1-specific and by cross-group-neutralizing antibodies. 315-19-1D12 was derived from HV1-2 and KV2-28 genes and appeared to form a new antibody class. Bioinformatic analysis indicated its group 2 neutralization specificity to be a consequence of four key residue positions. We specifically tested the impact of the group 1-specific N33 glycan, which decreased but did not abolish group 2 binding of 315-19-1D12. Overall, this study highlights the recognition of a broad group 2-neutralizing antibody, revealing unexpected diversity in neutralization specificity for antibodies that recognize the HA stem supersite.

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2.

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host-receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface-expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.

GLYCO: a tool to quantify glycan shielding of glycosylated proteins.

MOTIVATION: Glycans play important roles in protein folding and cell-cell interactions-and, furthermore, glycosylation of protein antigens can dramatically impact immune responses. While there have been attempts to quantify the glycan shielding or coverage of a protein surface, none of the publicly available tools analyzes glycan shielding computationally at an atomistic level. RESULTS: Here, we developed an in silico approach, GLYCO (GLYcan COverage), to quantify the glycan shielding of a protein surface. The software provides insights into glycan-dense/sparse regions of the entire protein surface or a subset of the protein surface. GLYCO calculates glycan shielding from a single coordinate file or from multiple coordinate files, for instance, as obtained from molecular dynamics simulations or by nuclear magnetic resonance spectroscopy structure determination, enabling analysis of glycan dynamics. Overall, GLYCO provides fundamental insights into the glycan shielding of glycosylated proteins. AVAILABILITY AND IMPLEMENTATION: GLYCO is freely available at GitHub (https://github.com/myungjinlee/GLYCO). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Extended antibody-framework-to-antigen distance observed exclusively with broad HIV-1-neutralizing antibodies recognizing glycan-dense surfaces.

Antibody-Framework-to-Antigen Distance (AFAD) - the distance between the body of an antibody and a protein antigen - is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition.

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies.

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4ß7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs.

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

Sequence-Signature Optimization Enables Improved Identification of Human HV6-1-Derived Class Antibodies That Neutralize Diverse Influenza A Viruses.

Sequence signatures of multidonor broadly neutralizing influenza antibodies can be used to quantify the prevalence of B cells with virus-neutralizing potential to accelerate development of broadly protective vaccine strategies. Antibodies of the same class share similar recognition modes and developmental pathways, and several antibody classes have been identified that neutralize diverse group 1- and group 2-influenza A viruses and have been observed in multiple human donors. One such multidonor antibody class, the HV6-1-derived class, targets the stem region of hemagglutinin with extraordinary neutralization breadth. Here, we use an iterative process to combine informatics, biochemical, and structural analyses to delineate an improved sequence signature for HV6-1-class antibodies. Based on sequence and structure analyses of known HV6-1 class antibodies, we derived a more inclusive signature (version 1), which we used to search for matching B-cell transcripts from published next-generation sequencing datasets of influenza vaccination studies. We expressed selected antibodies, evaluated their function, and identified amino acid-level requirements from which to refine the sequence signature (version 2). The cryo-electron microscopy structure for one of the signature-identified antibodies in complex with hemagglutinin confirmed motif recognition to be similar to known HV6-1-class members, MEDI8852 and 56.a.09, despite differences in recognition-loop length. Threading indicated the refined signature to have increased accuracy, and signature-identified heavy chains, when paired with the light chain of MEDI8852, showed neutralization comparable to the most potent members of the class. Incorporating sequences of additional class members thus enables an improved sequence signature for HV6-1-class antibodies, which can identify class members with increased accuracy.

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1-3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria.

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope-CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.