Biology subjects

Pancera, M.

Publications and source records attributed to Pancera, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-EM mapping of circumsporozoite protein epitope accessibility and visualization of antibody-induced shedding on Plasmodium falciparum sporozoites

The circumsporozoite protein (CSP) on the surface of the sporozoite stage of Plasmodium falciparum parasites is one of the major malaria antigens recognized by the adaptive immune system. Antibodies against CSP can provide protection from malaria infection following mosquito bite of the human host. Recent studies have shown how such antibodies recognize recombinant, truncated forms of Plasmodium falciparum CSP (PfCSP); however, much remains unknown about epitope accessibility and presentation of PfCSP on the parasite itself. Understanding the nature of the native antigen is of significant interest for optimization of immunogen design, since immunization with whole attenuated sporozoites has been shown to provide long-lasting protection against malaria disease. Here we use cryo-electron microscopy and tomography to investigate how antibodies against various PfCSP epitopes (N-terminal domain (NTD), Junction, central repeat, and C-terminal domain (CTD)) impact the PfCSP coat on whole Plasmodium falciparum sporozoites (PfSPZ). We observe that PfCSP by itself cannot be visualized on the surface of the cell by cryo-EM. However, by titrating in antibodies that recognize the Junction motif and central repeats, we delineate a boundary layer formed by PfCSP on the sporozoite outer membrane. The antibody concentration series also enabled us to determine conditions required to induce the dramatic CSP shedding reaction. By comparing matched monovalent FAb with IgG, we demonstrate that antibody bivalency is critical to driving this reaction. Additionally, we demonstrate that even at high concentrations, antibody against the NTD does not bind, indicating the absence or inaccessibility of this epitope on the PfCSP displayed by radiation-attenuated sporozoites, while the CTD can be bound by antibodies without producing a CSP shedding reaction. These studies shed light on the nature of the PfCSP coat in its native context on the parasite, thus revealing the sporozoites antigenic profile.

microbiology↗

bioRxiv2025

The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

The development of malaria blood-stage vaccines has been hampered by sequence variation in many Plasmodium falciparum proteins involved in erythrocyte invasion. In the past few years, asparagine-rich merozoite antigen (PfARMA) has emerged as a potential vaccine candidate due to its low amino acid sequence diversity and the association between anti-PfARMA antibody levels and protection to malaria. Here, we used samples from P. falciparum-exposed individuals to study naturally acquired B cell and antibody responses to PfARMA. B cell responses to PfARMA were dominated by IgM+ B cells that recognized the N-terminal intrinsically disordered region 1 (IDR1) of PfARMA. A human monoclonal antibody (hmAb) to IDR1 was non-neutralizing, while a second hmAb binding to the folded domain showed weak neutralizing activity. Both PfARMA-specific plasma IgM and IgG responses predominately targeted IDR1 and their levels increased with P. falciparum exposure. However, in contrast to previous reports, these antibody responses did not correlate with protection in age and exposure-matched children. Interestingly, approximately 30% of unexposed individuals had IgG that also targeted IDR1 and was polyreactive, binding to regions with high asparagine content. Finally, we determined that PfARMA is located in or near PfEBA-175+ micronemes. These data suggest that while IgG to the folded domain of PfARMA may inhibit parasite growth, antibody responses to PfARMA are primarily directed to IDR1 and may not directly contribute to protection against malaria.

immunology↗

bioRxiv2025

Anti-Malaria Antibody Engineering Broadens Recognition Motifs and Reveals New Homotypic Interactions that Enhance Protective Breadth

The monoclonal antibody L9 mediates high-level protection against malaria in children for up to 6 months in Africa. L9 preferentially binds with high affinity to the NVDP minor repeat on the P. falciparum circumsporozoite protein (PfCSP). Here, we sought to improve the affinity of L9 to enhance protection against rare strains with two spatially separated minor repeats or a single minor repeat. Site saturation mutagenesis and yeast display-screening identified a panel of affinity-improved variants. In vivo challenge showed one variant, L9_yd19, to be modestly more potent against a chimeric transgenic Plasmodium encoding PfCSP with two widely spaced minor repeats from a Kenyan parasite strain, with no loss in potency against the benchmark 3D7 strain with its standard complement of minor repeats. L9_yd19 also had high affinity against NANP major repeats and was protective against transgenic Plasmodium with PfCSP containing only NANP major repeats (NANP12). Cryo-EM studies revealed L9_yd19 to recognize PfCSP with two distinct homotypic interfaces, which combined to yield two trimeric layers of antibodies comprising asymmetric trimers that dimerized in a head-to-head fashion. These data reveal a new antibody mechanism that utilizes interfaces involving dual homotypic symmetry elements, a 2-fold and an asymmetric 3-fold, for potentially improved malaria prevention. HIGHLIGHTSO_LIL9 is a highly protective antimalarial antibody that preferentially binds the NVDP minor repeat on Plasmodium falciparum circumsporozoite protein (PfCSP) and also binds with low affinity to the NANP major repeat; due to these targeting preferences, it has shown reduced protection against designed transgenic malaria strains with only a single NVDP motif (Fig. 1). C_LIO_LIUsing yeast display, a panel of L9 variants were generated based on higher affinity against the minor NVDP and major NANP motifs to determine if they could improve protection against strains with fewer minor repeat regions or only containing major repeats (Figs 1-4). C_LIO_LIOne L9 variant, L9_yd19 showed enhanced protection against chimeric transgenic CSP variants with a single minor repeat or two minor repeats in which the spacing was separated; L9_yd19 also showed protection against chimeric transgenic CSP variants containing only the NANP major repeat (Figs. 4-5). C_LIO_LICryo-EM analyses revealed L9_yd19 recognition of CSP to comprise two distinct homotypic interfaces: a side-to-side interface within asymmetric antibody trimer and a head-to-head interface between antibody trimers related by 2-fold symmetry that combined to yield a higher-order complex comprising two trimeric layers of antibodies (Figs. 6-7) C_LIO_LIStructure-function studies reveal a new antibody-based structural mechanism with dual homotypic interfaces mediating protection against varying numbers and spacing of minor repeats and major repeats. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/679367v1_fig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1978620org.highwire.dtl.DTLVardef@e6b84forg.highwire.dtl.DTLVardef@14e750eorg.highwire.dtl.DTLVardef@1e6ff51_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Improving affinity to target the CSP minor repeat enhances L9 antibody protective breadth. (A) The CSP minor repeat (NVDP) minor repeat is the principal binding target of the potent anti-malarial antibody L9. Genetic polymorphisms are present among P. falciparum strains in the number and spacing of NVDP motifs contained in the CSP repeat regions, and the loss of NPNV epitopes reduces the in vivo protective potency of the L9 antibody. (B) To enhance L9 affinity against the CSP antigen, multi-mutation libraries were generated from the L9 template and selectively screened against CSP peptide repeat motifs using yeast display. Libraries were first selected against Peptide22 (Pep22), which contains two minor and two major repeats, and subsequently selected against NTDS_5/3, a truncated CSP molecule that presents peptides in a more native-like conformation. Identified L9 variants were characterized for binding affinity in vitro and for protective potency in vivo against a panel of malaria antigens. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/679367v1_fig4.gif" ALT="Figure 4"> View larger version (59K): org.highwire.dtl.DTLVardef@844f00org.highwire.dtl.DTLVardef@5ebe9borg.highwire.dtl.DTLVardef@33d634org.highwire.dtl.DTLVardef@1342c8c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO In vitro and in vivo characterization reveals functional improvement for affinity-enhanced L9 variants against multiple transgenic strains, including the Kenya2-3 KI strain and a major repeat-only transgenic. (A) Biolayer interferometry (BLI) analysis against Peptide 22 (containing 2 NVDPs) and full-length CSP (fl_CSP) of P. falciparum 3D7. L9_WT affinity is marked with a vertical line on the legend bar. L9 variants marked with an asterisk (*) were selected for detailed in vivo characterization. (B) In the transgenic sporozoite intravenous (IV) challenge model, mice were administered IgG from L9 variants or control antibodies (-2 h), followed by IV challenge with P. berghei sporozoites (PbSPZ) encoding different PfCSP knock-in sequences (0 h), and subsequent luciferin-based evaluation (42 h). Each group comprised n=10 mice, with animals receiving the indicated antibody dose. (C) Transgenic PbSPZ challenge. Liver infection burden was quantified, and data are shown as mean {+/-} standard deviation; mean liver burden total flux for L9_WT at 12.5 {micro}g/mouse is indicated by a dashed line. Statistical significance between L9_WT and each L9 variant group at 12.5 {micro}g were determined using the ordinary one-way ANOVA test with a two-tailed P value calculation and a Dunnetts multiple test correction. Statistical significance was also analyzed between L9_yd19 and MAM01 using an unpaired t test with a two-tailed P value calculation; the two groups were significantly different when tested against 1 NVDP KI PbSPZ. See also Table S1 and Fig. S5. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/679367v1_fig5.gif" ALT="Figure 5"> View larger version (33K): org.highwire.dtl.DTLVardef@58bed2org.highwire.dtl.DTLVardef@580075org.highwire.dtl.DTLVardef@1864220org.highwire.dtl.DTLVardef@15f6582_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5.C_FLOATNO Affinity correlations reveal that improved protection breadth correlates with affinity to multiple malaria antigens. Correlation between binding affinity to PfCSP Kenya2-3 strain, NTDS_5/3 or PfCSP 3D7 strain (x-axis, linear scale) and normalized liver burden against various transgenic PbSPZs (y-axis, linear scale). Pearsons correlation coefficient (R) was calculated using the median normalized liver burden values, with corresponding two-tailed P-values and 95% confidence intervals. A linear regression was fit based on a Pearsons correlation analysis. See also Fig. S6. C_FIG O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig6.gif" ALT="Figure 6"> View larger version (46K): org.highwire.dtl.DTLVardef@14bf2org.highwire.dtl.DTLVardef@12123d9org.highwire.dtl.DTLVardef@b8b17aorg.highwire.dtl.DTLVardef@18c0195_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Structural analyses of L9_yd19 and PfCSP (3D7 or Kenya2-3) reveals a new interaction mode. (A) CryoEM structure of L9_yd19 in complex with PfCSP (3D7) at 3.4 [A] resolution. Heavy chain mutations are shown in red and light chain mutations are shown in blue. 27 residue stretch of PfCSP is ordered and the rest of the PfCSP is disordered. (B) CryoEM structure of L9_yd19 in complex with PfCSP (Kenya2-3) at 3.3 [A] resolution. 2D classes show both three L9_yd19 Fabs (orange arrow) and six L9_yd19 Fabs (red arrow) bound to PfCSP (Kenya2-3). 3D reconstruction for six L9_yd19 Fabs bound to PfCSP (Kenya2-3) is shown. C_FIG O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/679367v1_fig7.gif" ALT="Figure 7"> View larger version (54K): org.highwire.dtl.DTLVardef@bbe52corg.highwire.dtl.DTLVardef@1c2c4b2org.highwire.dtl.DTLVardef@1ffc3a5org.highwire.dtl.DTLVardef@85ac9e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7.C_FLOATNO L9_yd19 engineered mutations stabilize a new head-to-head homotypic interface allowing up to six Fabs to bind to PfCSP (Kenya2-3). (A) Structural comparison of L9_yd19 bound to PfCSP-3D7 and PfCSP-Kenya2-3. PfCSP is shown in red sticks with the N- and C-termini labeled. (B) L9_yd19 in complex with PfCSP-Kenya2-3. Fabs (3,4), (2,5), and (1,6) are related by 2-fold (C2) symmetry. Critical residues generating the head-to-head homotypic interface are shown in insets. C_FIG

immunology↗

bioRxiv2025

DEVELOPMENT OF A POTENT MONOCLONAL ANTIBODY FOR TREATMENT OF HUMAN METAPNEUMOVIRUS INFECTIONS

Human metapneumovirus (HMPV) is a major cause of respiratory infections, particularly among vulnerable populations, yet effective therapeutics remain unavailable. Monoclonal antibodies (mAbs) offer a promising approach for both treatment and prevention. Here, we describe the discovery and characterization of 4F11, a highly potent and broadly neutralizing mAb with demonstrated in vitro and in vivo efficacy against HMPV. Using cryo-electron microscopy, we defined a unique mechanism of binding HMPV employed by 4F11, which distinguishes it from previously characterized RSV and HMPV mAbs. 4F11 targets an epitope located at the apex of the prefusion F protein (site O) with a 1:1 stoichiometry, distinct from the 3:1 stoichiometry observed with other HMPV site O antibodies. Unlike other site O antibodies, which penetrate the glycan shield between Asn57 and Asn172, 4F11 binds vertically and directly interacts with the Asn172 glycan, representing a unique glycan-dependent mode of recognition. In vitro, 4F11 displayed high potency and broad neutralization across diverse HMPV strains. It also showed a low propensity for resistance development, with only a single escape mutation (K179E) identified, a mutation not found in any published HMPV sequence to date. Viruses rescued with the K179E escape mutation had significantly decreased fitness in vitro compared to wild-type virus. In a hamster challenge model, 4F11 significantly reduced viral loads in both the lungs and nasal turbinates. These findings highlight 4F11 as a promising candidate for therapeutic development, particularly for immunocompromised individuals and other high-risk groups.

immunology↗

bioRxiv2025

Structural basis for antibody cross-neutralization of dengue and Zika viruses

Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of Dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Mutagenesis studies of viral envelope proteins showed that the epitope specificity of F25.S02 is distinct from EDE1 bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ~4.2 [A] cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 [A] crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics.

immunology↗

bioRxiv2025

HPV16 neutralizing monoclonal antibodies show evidence for common developmental pathways and public epitopes

Antibodies to human papillomavirus (HPV) primarily recognize surface exposed residues on five loops of the major capsid protein (L1) that vary significantly among HPV types. We determined which loops were required for neutralization for 70 HPV16 specific human monoclonal antibodies (mAbs) cloned from participants who received an HPV vaccine, and we describe molecular features of those antibodies. Chimeric HPV16 pseudovirus (cpsV), each having one surface loop bearing multiple amino acid substitutions, were used to determine neutralization specificity. The HPV16-FG-loop was the loop most frequently required for neutralization (44 of 70, 62.9%), however, all other surface loops were used for neutralization by multiple mAbs: HI (13, 18.6%), DE (15, 21.4%), EF (six, 8.6%), BC (four, 5.7%). Antibodies that required multiple loops were common (17, 24.3%). Three mAbs (4.3%) required sequences on the c-terminus of L1 and for another three mAbs the neutralization specificity could not be determined. Two types of mAbs appeared to be overrepresented: ten mAbs used VH 2-70 IGHV paired with VL {lambda}1-40, having characteristic mutations in complementarity determining region two (CDRL2). Cryogenic electron microscopy (Cryo-EM) revealed that two of these antibodies bound five Fabs per pentamer interacting with all five L1-surface loops. The other type of mAbs that appeared to be overrepresented were ten mAbs using VH 4-34, seven of which also used DH3-16*02 with conserved CDRH3 sequences. Cryo-EM for one of these mAbs, that required the FG-loop for neutralization, was shown to bind one Fab per pentamer at the apex, interacting with the DE- and FG-loops, with sequences of the Fab CDRH3 inserted between the DE- and FG-loops from two protomers. These two types of mAbs were found repeatedly in the four participants suggesting that these antibodies shared developmental pathways and bound to similar immunodominant epitopes on the virus. HighlightsMost human mAbs recognized L1 surface loops but three of 70 recognized sequences on the C-terminal arm of L1 Some antibodies induced by HPV vaccination follow shared developmental pathways. Human monoclonal antibodies using VH 2-70/VL {lambda}1-40 were found in all participants and bound with at a stoichiometry of five Fabs per capsomer. Human monoclonal antibodies using the diversity gene segment D3-16*02 were found in all participants and one Fab was shown to bind with a stoichiometry of one Fab per capsomer. In briefA panel of 70 HPV16 specific human monoclonal antibodies (mAbs), cloned from memory B cells or plasmablasts following HPV vaccination, was characterized by determining the surface loops of the major capsid protein (L1) required for neutralization and examined for shared molecular features. All five L1 loops were found to be used for neutralization by one or more antibodies, but the most frequent target of these antibodies was the FG loop followed by the HI and DE loops. Ten antibodies paired the heavy chain variable gene VH 2-70 with the light chain variable gene VL {lambda}1-40 and these antibodies had conserved mutations in the CDRL2 region of VL {lambda}1-40. Mutating the CDRL2 back to the predicted germline sequence significantly reduced neutralization. Cryo-EM analysis of two VH2-70/VL{lambda}1-40 mAbs showed five Fabs binding per L1 pentamer and a conserved epitope with Fabs interacting with all five variable loops across two adjacent protomers. Seven other mAbs had a heavy chain composed of the variable region VH4-34 with the diversity gene D3-16*02 resulting in the sequence motif WSGYR in the CDRH3. Mutation of that sequence to alanine ablated HPV16 neutralization activity. A cryo-EM structure of one of these antibodies showed one Fab binding the pentamer apex with the WSGYR motif inserting between three loops from two protomers. Antibodies with paired VH 2-70/VL {lambda}1-40 and the antibodies with CDRH3 containing the WSGYR sequence, were found in all four study participants suggesting that such antibodies may be commonly elicited following HPV vaccination.

immunology↗

bioRxiv2024

Broadly inhibitory antibodies against severe malaria virulence proteins

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasites polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDR1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDR1 domains, representing five of the six subclasses of CIDR1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDR1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDR1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

immunology↗

bioRxiv2022

Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the CSP minor (NPNV) repeats, which is stabilized by a novel set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts CSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum. One sentence summaryThe L9 light chain is crucial for potency by conferring multivalent, high affinity binding to the NPNV minor repeats of PfCSP.

immunology↗

bioRxiv2022

Cross-Protective Antibodies Against Common Endemic Respiratory Viruses

Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) are a major cause of death, morbidity, and health care costs worldwide, and they can exact a significant toll on immunocompromised patients, the elderly, and those with underlying lung disease. There is an unmet medical need for safe and effective medications for many of the viruses responsible for common respiratory viral infections in vulnerable patient populations. While a protective monoclonal antibody exists for RSV, clinical use is limited to high-risk infant populations. Here, we present the discovery, in vitro characterization, and in vivo efficacy testing of two cross-neutralizing monoclonal antibodies, one targeting both HPIV3 and HPIV1 and the other targeting both RSV and HMPV. The 3x1 antibody is capable of targeting multiple parainfluenza viruses; the MxR antibody shares features with other previously reported monoclonal antibodies that are capable of neutralizing both RSV and HMPV. We obtained structures using cryo-electron microscopy of these antibodies in complex with their antigens to 3.62 [A] resolution for 3x1:HPIV3 and to 2.24 [A] for MxR:RSV, providing a structural basis to corroborate our in vitro characterization of binding and neutralization. Together, a cocktail of 3x1 and MxR could have clinical utility in providing broad protection against four of the respiratory viruses that cause significant morbidity and mortality in at-risk individuals.

microbiology↗

bioRxiv2022

Immunization with a self-assembling nanoparticle vaccine displaying EBV gH/gL protects humanized mice against lethal viral challenge

Summary/AbstractEpstein-Barr virus (EBV) is a cancer-associated pathogen responsible for 140,000 deaths per year. EBV is also the etiological agent of infectious mononucleosis and is associated with multiple sclerosis and rheumatoid arthritis. Thus, an EBV vaccine could alleviate significant morbidity and mortality. EBV is orally transmitted and has tropism for both epithelial cells and B cells which are present in the oral cavity. Therefore, a prophylactic vaccine would need to prevent infection of both cell types. Passive transfer neutralizing monoclonal antibodies targeting the viral gH/gL glycoprotein complex prevent experimental EBV infection in humanized mice and rhesus macaques, suggesting that gH/gL is an attractive vaccine candidate. Here, we produced and evaluated the immunogenicity of several nanoparticle immunogens displaying gH/gL with distinct valencies and geometries. After one or two immunizations, all nanoparticles elicited superior binding and neutralizing titers relative to monomeric gH/gL. Antibodies elicited by a computationally designed self-assembling nanoparticle that displays 60 copies of the gH/gL protein conferred protection against a lethal dose of EBV in a humanized mouse challenge model, whereas antibodies elicited by monomeric gH/gL did not. Taken together, these data motivate further development of gH/gL nanoparticle vaccine candidates for EBV.

immunology↗

bioRxiv2021

Characterization of a vaccine-elicited human antibody with sequence homology to VRC01-class antibodies that binds the C1C2 gp120 domain

Broadly HIV-1 neutralizing VRC01-class antibodies bind the CD4-binding site of the HIV-1 envelope (Env) and contain VH1-2*02-derived heavy chains paired with light chains expressing five amino acid long CDRL3s. Their unmutated forms do not recognize Env or neutralize HIV-1. The lack of elicitation of VRC01-class antibodies in human clinical trials could potentially be due to the absence of activation of the corresponding naive B cells by the vaccine Env immunogens. To address this point directly, we examined Env-specific BCR sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed only one displayed sequence homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in both the CDRH3 and CDRL3 were necessary. Our findings support the use of specifically designed immunogens to activate VRC01-class B cells in future human vaccine trials.

immunology↗

bioRxiv2021

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAbs ontogeny and mechanisms of binding PfCSP to neutralize sporozoites will facilitate vaccine development. Here, we isolated mAbs clonally related to L9 and showed that this B-cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9{kappa}) with clonally-related heavy chains resulted in chimeric mAbs that cross-linked two NVDP, cross-reacted with NANP, and more potently neutralized sporozoites compared to their original light chain. Structural analyses revealed that chimeric mAbs bound the minor repeat motif in a type-1 {beta}-turn seen in other repeat-specific antibodies. These data highlight the importance of L9{kappa} in binding NVDP on PfCSP to neutralize SPZ and suggest that PfCSP-based immunogens might be improved by presenting [≥]2 NVDP.

immunology↗

bioRxiv2021

Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit.

Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74[A] crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development.

immunology↗

bioRxiv2021

Structurally related but genetically unrelated antibody lineages converge on an immunodominantHIV-1 Env neutralizing determinant following trimer immunization

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 ug/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a {beta}-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide an explanation for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Author summaryNHPs immunizations with an HIV-1 immunogen (native-like tier 2 clade C 16055 strain) elicit potent HIV-1 tier 2 autologous polyclonal neutralizing antibodies. To understand the basis of the autologous neutralization, we determined structures of antibodies isolated from the vaccinated NHPs in complex with their epitopes. Our structural analysis reveals that the V2 hypervariable region, unique to 16055, is immunodominant and targeted by antibodies from diverse lineages. Additionally, vaccine-elicited V2 NAbs use different binding angles to avoid Env N-glycan shield and the more negatively charged paratope displays potent autologous neutralizing function. In summary, detailed analysis of how vaccine-elicited monoclonal antibodies interact with the target antigen provide valuable information for the design of immunogens aimed to elicit more broadly HIV-neutralizing antibodies. The use of cocktail/prime-boost sequential regimens that include a range of sequence variation combined with the removal/shielding of unwanted immunodominant epitopes will likely be needed to reach this goal.

biochemistry↗

bioRxiv2021

Isolation and Characterization of Cross-Neutralizing Coronavirus Antibodies from COVID-19+ Subjects

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterized 198 antibodies isolated from four COVID19+ subjects and identified 14 SARS-CoV-2 neutralizing antibodies. One targeted the NTD, one recognized an epitope in S2 and twelve bound the RBD. Three anti-RBD neutralizing antibodies cross-neutralized SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency rather than the antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. The anti-S2 antibody also neutralized SARS-CoV-1 and all four cross-neutralizing antibodies neutralized the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

immunology↗

bioRxiv2020

Antibody binding to SARS-CoV-2 S glycoprotein correlates with, but does not predict neutralization

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals as well as S-specific monoclonal antibodies were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to binding of S glycoprotein in the context of viral particles remains to be established. Here we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA and neutralization assays we observed a strong correlation between these two parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of viral particles is required but not sufficient to mediate neutralization. Altogether, our results highlights the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.

microbiology↗

bioRxiv2020

PROTECTIVE ANTIBODIES AGAINST HPIV3 INFECTION

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. Unfortunately, an effective vaccine or therapeutic is not currently available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, or spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3 neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Importantly, prophylactic administration of one of these antibodies, named PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

immunology↗

bioRxiv2020

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain (RBD). The structure reveals CV30s epitope overlaps with the human ACE2 receptor binding site thus providing the structural basis for its neutralization by preventing ACE2 binding.

immunology↗