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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 145 records · Page 8
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Combining computational modeling and experimental library screening to affinity-mature VEEV-neutralizing antibody F5

Engineered monoclonal antibodies have proven to be highly effective therapeutics in recent viral outbreaks. However, despite technical advancements, an ability to rapidly adapt or increase antibody affinity and by extension, therapeutic efficacy, has yet to be fully realized. We endeavored to stand-up such a pipeline using molecular modeling combined with experimental library screening to increase the affinity of F5, a monoclonal antibody with potent neutralizing activity against Venezuelan Equine Encephalitis Virus (VEEV), to recombinant VEEV (IAB) E1E2 antigen. We modeled the F5/E1E2 binding interface and generated predictions for mutations to improve binding using a Rosetta-based approach and dTERMen, an informatics approach. The modeling was complicated by the fact that a high-resolution structure of F5 is not available and the H3 loop of F5 exceeds the length for which current modeling approaches can determine a unique structure. A subset of the predicted mutations from both methods were incorporated into a phage display library of scFvs. This library and a library generated by error-prone PCR were screened for binding affinity to the recombinant antigen. Results from the screens identified favorable mutations which were incorporated into 12 human-IgG1 variants. The best variant, containing eight mutations, improved KD from 0.63 nM (parental) to 0.01 nM. While this did not improve neutralization or therapeutic potency of F5 against IAB, it did increase cross-reactivity to other closely related VEEV epizootic and enzootic strains, demonstrating the potential of this method to rapidly adapt existing therapeutics to emerging viral strains.

affinity-maturation↗
DOE OSTI2020

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

We report analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Profiling B cell immunodominance after SARS-CoV-2infection reveals antibody evolution to non-neutralizing viral targets

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

antibody↗
DOE OSTI2021

Cross-reactive immunogenicity of group A streptococcal vaccines designed using a recurrent neural network to identify conserved M protein linear epitopes

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal sequence of the protein defines the more than 200 M types of Strep A and also contains epitopes that elicit opsonic antibodies, some of which cross-react with heterologous M types. Current efforts to develop broadly protective M protein-based vaccines are directed at identifying potential cross-protective epitopes located in the N-terminal regions of cluster-related M proteins for use as vaccine antigens. In this study, we have used a comprehensive approach using the recurrent neural network ABCpred and IEDB epitope conservancy analysis tools to predict 16 residue linear B-cell epitopes from 117 clinically relevant M types of Strep A (~88% of global Strep A infections). Furthermore, to examine the immunogenicity of these epitope-based vaccines, nine peptides that together shared ≥60% sequence identity with 37 heterologous M proteins were incorporated into two recombinant hybrid protein vaccines, in which the epitopes were repeated 2 or 3 times, respectively. The combined immune responses of immunized rabbits showed that the vaccines elicited significant levels of antibodies against all nine vaccine epitopes present in homologous N-terminal 1–50 amino acid synthetic M peptides, as well as cross-reactive antibodies against 16 of 37 heterologous M peptides predicted to contain similar epitopes. The epitope-specificity of the cross-reactive antibodies was confirmed by ELISA inhibition assays and functional opsonic activity was assayed in HL-60-based bactericidal assays. The results provide important information for the future design of broadly protective M protein-based Strep A vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A Size-Selectively Biomolecule-Immobilized Nanoprobe-Based Chemiluminescent Lateral Flow Immunoassay for Detection of Avian-Origin Viruses

Here, a signal-amplifiable nanoprobe-based chemiluminescent lateral flow immunoassay (CL-LFA) was developed to detect avian influenza viruses (AIV) and other contagious and fatal viral avian-origin diseases worldwide. Signal-amplifiable nanoprobes are capable of size-selective immobilization of antibodies (binding receptors) and enzymes (signal transducers) on sensitive paper-based sensor platforms. Particle structure designs and conjugation pathways conducive for antigen accessibility to maximum amounts of immobilized enzymes and antibodies have advanced. The detection limit of the CL-LFA using the signal-amplifiable nanoprobe for the nucleoprotein of the H3N2 virus was 5 pM. Sensitivity tests for low pathogenicity avian influenza H9N2, H1N1, and high pathogenicity avian influenza H5N9 viruses were conducted, and the detection limits of CL-LFA were found to be 10 3.5 50% egg infective dose (EID 50 )/mL, 10 2.5 EID 50 /mL, and 10 4 EID 50 /mL, respectively, which is 20 to 100 times lower than that of a commercial AIV rapid test kit. Moreover, CL-LFA demonstrated high sensitivity and specificity against 37 clinical samples. The signal-amplifiable probe designed in this study is a potential diagnostic probe with ultrahigh sensitivity for applications in the field of clinical diagnosis, which requires sensitive antigen detection as evidenced by enhanced signaling capacity and sensitivity of the LFAs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Evaluating 225 Ac and 177 Lu Radioimmunoconjugates against Antibody–Drug Conjugates for Small-Cell Lung Cancer

Interest in the use of 225 Ac for targeted alpha therapies has increased dramatically over the past few years, resulting in a multitude of new isotope production and translational research efforts. However, 225 Ac radioimmunoconjugate (RIC) research is still in its infancy, with most prior experience in hematologic malignancies and only one reported preclinical solid tumor study using 225 Ac RICs. In an effort to compare 225 Ac RICs to other current antibody conjugates, a variety of RICs are tested against intractable small-cell lung cancer (SCLC). Here we directly compare, in vitro and in vivo , two promising candidates of each α or β - category, 225 Ac and 177 Lu, versus pyrrolobenzodiazepine (PBD) nonradioactive benchmarks. The monoclonal antibody constructs are targeted to either delta like 3 protein (DLL3), a recently discovered SCLC target, or CD46 as a positive control. An immunocompromised maximum tolerated dose assay is performed on NOD SCID mice, along with tumor efficacy proof-of-concept studies in vivo . We overview the conjugation techniques required to create serum-stable RICs and characterize and compare in vitro cell killing with RICs conjugated to nonspecific antibodies (huIgG1) with either native or site-specific thiol loci against tumor antigen DLL3-expressing and nonexpressing cell lines. Using patient-derived xenografts of SCLC onto NOD SCID mice, solid tumor growth was controlled throughout 3 weeks before growth appeared, in comparison to PBD conjugate controls. NOD SCID mice showed lengthened survival using 225 Ac compared to 177 Lu RICs, and PBD dimers showed full tumor suppression with nine out of ten mice. The exploration of RICs on a variety of antibody-antigen systems is necessary to direct efforts in cancer research toward promising candidates. However, the anti-DLL3-RIC system with 225 Ac and 177 Lu appears to be not as effective as the anti-DLL3-PBD counterpart in SCLC therapy with matched antibodies and portrays the challenges in both SCLC therapy as well as the specialized utility of RICs in cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication. Though lipopolysaccharide (LPS) has been shown to act as a receptor for tailocins, the breadth of factors involved in tailocin sensitivity, and the mechanisms behind resistance to self-intoxication, remain unclear. Here, we employed genome-wide screens in four non-model pseudomonads to identify mutants with altered fitness in the presence of tailocins produced by closely related pseudomonads. Our mutant screens identified O-antigen composition and display as most important in defining sensitivity to our tailocins. In addition, the screens suggest LPS thinning as a mechanism by which resistant strains can become more sensitive to tailocins. Furthermore, we validate many of these novel findings, and extend these observations of tailocin sensitivity to 130 genome-sequenced pseudomonads. This work offers insights into tailocin–bacteria interactions, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Broadly cross-reactive human antibodies that inhibit genogroup I and II noroviruses

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins

The human metapneumovirus (hMPV) fusion (F) protein is essential for viral entry and is a key target of neutralizing antibodies and vaccine development. The prefusion conformation is thought to be the optimal vaccine antigen, but previously described prefusion F proteins expressed poorly and were not well stabilized. Here, we use structures of hMPV F to guide the design of 42 variants containing stabilizing substitutions. Through combinatorial addition of disulfide bonds, cavity-filling substitutions, and improved electrostatic interactions, we describe a prefusion-stabilized F protein (DS-CavEs2) that expresses at 15mg/L and has a melting temperature of 71.9°C. Crystal structures of two prefusion-stabilized hMPV F variants reveal that antigenic surfaces are largely unperturbed. Importantly, immunization of mice with DS-CavEs2 elicits significantly higher neutralizing antibody titers against hMPV A1 and B1 viruses than postfusion F. The improved properties of DS-CavEs2 will advance the development of hMPV vaccines and the isolation of therapeutic antibodies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular basis of differential HLA class I-restricted T cell recognition of a highly networked HIV peptide

Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Affinity-matured homotypic interactions induce spectrum of PfCSP structures that influence protection from malaria infection

The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Targeting of intracellular oncoproteins with peptide-centric CARs

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

In vivo human T cell engineering with enveloped delivery vehicles

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.

42 ENGINEERING↗
DOE OSTI2022

Crystal structure of ultra-humanized anti-pTau Fab reveals how germline substitutions humanize CDRs without loss of binding’

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology “Augmented Binary Substitution” (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Structures of respiratory syncytial virus G bound to broadly reactive antibodies provide insights into vaccine design

Abstract Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease in infants and older adults. The attachment glycoprotein (RSV G) binds to the chemokine receptor CX3CR1 to promote viral entry and modulate host immunity. Antibodies against RSV G are a known correlate of protection. Previously, several broadly reactive, high-affinity anti-RSV G human monoclonal antibodies were isolated from RSV-exposed individuals and were shown to be protective in vitro and in vivo. Here, we determined the structures of three of these antibodies in complex with RSV G and defined distinct conformational epitopes comprised of highly conserved RSV G residues. Binding competition and structural studies demonstrated that this highly conserved region displays two non-overlapping antigenic sites. Analyses of anti-RSV G antibody sequences reveal that antigenic site flexibility may promote the elicitation of diverse antibody germlines. Together, these findings provide a foundation for next-generation RSV prophylactics, and they expand concepts in vaccine design for the elicitation of germline lineage-diverse, broadly reactive, high-affinity antibodies.

Science & Technology - Other Topics↗