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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Mechanism of glycoform specificity and in vivo protection by an anti-afucosylated IgG nanobody

Abstract Immunoglobulin G (IgG) antibodies contain a complex N -glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N -glycan and acts as a ‘glycan sensor’, forming hydrogen bonds with the afucosylated IgG N -glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Liquid Crystal‐Based Detection of Antigens with ELISA Sensitivity

Abstract A highly sensitive label‐free liquid crystal (LC)‐based technique is presented for detecting Immunoglobulin G (IgG) antigens used to uncover viral infections. The effectiveness, sensitivity, and selectivity of this detection method is demonstrated with goat IgG antigen at concentrations as low as 100 pg ml −1 , which is comparable to the sensitivity of the current enzyme‐linked immunosorbent assay (ELISA). The sensor is fabricated by decorating a transmission electron microscopy grid immobilized glass surfaces with antibodies; the target antigen is detected by a liquid crystal suspended onto the grid. This is different from previous methods where the antigen is detected either at the LC‐aqueous interface or in an LC sandwich cell with an antibody/antigen‐decorated substrate. This new approach has advantages such as easy sample preparation, higher sensitivity, and better storage capabilities. Binding the target antigen to the antibody results in a reorientation of the LC director that is detected optically. In addition to demonstrating the sensitivity, the physical principle of the detection is also discussed. This technique may apply to detect virtually any antigen of interest.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Characterization of the antispike IgG immune response to COVID-19 vaccines in people with a wide variety of immunodeficiencies

Research on coronavirus disease 2019 vaccination in immune-deficient/disordered people (IDP) has focused on cancer and organ transplantation populations. In a prospective cohort of 195 IDP and 35 healthy volunteers (HV), antispike immunoglobulin G (IgG) was detected in 88% of IDP after dose 2, increasing to 93% by 6 months after dose 3. Despite high seroconversion, median IgG levels for IDP never surpassed one-third that of HV. IgG binding to Omicron BA.1 was lowest among variants. Angiotensin-converting enzyme 2 pseudo-neutralization only modestly correlated with antispike IgG concentration. IgG levels were not significantly altered by receipt of different messenger RNA–based vaccines, immunomodulating treatments, and prior severe acute respiratory syndrome coronavirus 2 infections. While our data show that three doses of coronavirus disease 2019 vaccinations induce antispike IgG in most IDP, additional doses are needed to increase protection. Because of the notably reduced IgG response to Omicron BA.1, the efficacy of additional vaccinations, including bivalent vaccines, should be studied in this population.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Mapping brucellosis risk in Kenya and its implications for control strategies in sub-Saharan Africa

In Sub-Saharan Africa (SSA), effective brucellosis control is limited, in part, by the lack of long-term commitments by governments to control the disease and the absence of reliable national human and livestock population-based data to inform policies. Therefore, we conducted a study to establish the national prevalence and develop a risk map for Brucella spp. in cattle to contribute to plans to eliminate the disease in Kenya by the year 2040. We randomly generated 268 geolocations and distributed them across Kenya, proportionate to the area of each of the five agroecological zones and the associated cattle population. Cattle herds closest to each selected geolocation were identified for sampling. Up to 25 cattle were sampled per geolocation and a semi-structured questionnaire was administered to their owners. We tested 6,593 cattle samples for Brucella immunoglobulin G (IgG) antibodies using an Enzyme-linked immunosorbent assay (ELISA). We assessed potential risk factors and performed spatial analyses and prevalence mapping using approximate Bayesian inference implemented via the integrated nested Laplace approximation (INLA) method. The national Brucella spp. prevalence was 6.8% (95% CI: 6.2–7.4%). Exposure levels varied significantly between agro-ecological zones, with a high of 8.5% in the very arid zone with the lowest agricultural potential relative to a low of 0.0% in the agro-alpine zone with the highest agricultural potential. Additionally, seroprevalence increased with herd size, and the odds of seropositivity were significantly higher for females and adult animals than for males or calves. Similarly, animals with a history of abortion, or with multiple reproductive syndromes had higher seropositivity than those without. At the herd level, the risk of Brucella spp. transmission was higher in larger herds, and herds with a history of reproductive problems such as abortion, giving birth to weak calves, or having swollen testes. Geographic localities with high Brucella seroprevalence occurred in northern, eastern, and southern regions of Kenya all primarily characterized by semi-arid or arid agro-ecological zones dominated by livestock pastoralism interspersed with vast areas with mixed livestock-wildlife systems. The large spatial extent of our survey provides compelling evidence for the widespread geographical distribution of brucellosis risk across Kenya in a manner easily understandable for policymakers. Our findings can provide a basis for risk-stratified pilot studies aiming to investigate the cost-effectiveness and efficacy of singular and combined preventive intervention strategies that seek to inform Kenya’s Brucellosis Control Policy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Single-dose mucosal replicon-particle vaccine protects against lethal Nipah virus infection up to 3 days after vaccination

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Salivary Antibodies against Multiple Environmental Pathogens Found in Individuals Recreating at an Iowa Beach

Detecting environmental exposures and mitigating their impacts are growing global public health challenges. Antibody tests show great promise and have emerged as fundamental tools for large-scale exposure studies. Here, we apply, demonstrate and validate the utility of a salivary antibody multiplex immunoassay in measuring antibody prevalence and immunoconversions to six pathogens commonly found in the environment. The study aimed to assess waterborne infections in consenting beachgoers recreating at an Iowa riverine beach by measuring immunoglobulin G (IgG) antibodies against select pathogens in serially collected saliva samples. Results showed that nearly 80% of beachgoers had prior exposures to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to norovirus GI.1 (59.41%), norovirus GII.4 (58.79%) and Toxoplasma gondii (22.80%) and over half (56.28%) of beachgoers had evidence of previous exposure to multiple pathogens. Of individuals who returned samples for each collection period, 6.11% immunoconverted to one or more pathogens, largely to noroviruses (GI.1: 3.82% and GII.4: 2.29%) and T. gondii (1.53%). Outcomes of this effort illustrate that the multiplex immunoassay presented here serves as an effective tool for evaluating health risks by providing valuable information on the occurrence of known and emerging pathogens in population surveillance studies.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

Non-NASA Center↗
NASA NTRS1989

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Non-NASA Center↗
NASA NTRS1992

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Non-NASA Center↗
NASA NTRS2010

Fluorogenic Cell-Based Biosensors for Monitoring Microbes

Fluorogenic cell-based sensor systems for detecting microbes (especially pathogenic ones) and some toxins and allergens are undergoing development. These systems harness the natural signaltransduction and amplification cascades that occur in mast cells upon activation with antigens. These systems include (1) fluidic biochips for automated containment of samples, reagents, and wastes and (2) sensitive, compact fluorometers for monitoring the fluorescent responses of mast cells engineered to contain fluorescent dyes. It should be possible to observe responses within minutes of adding immune complexes. The systems have been shown to work when utilizing either immunoglobulin E (IgE) antibodies or traditionally generated rat antibodies - a promising result in that it indicates that the systems could be developed to detect many target microbes. Chimeric IgE antibodies and rat immunoglobulin G (IgG) antibodies could be genetically engineered for recognizing biological and chemical warfare agents and airborne and food-borne allergens. Genetic engineering efforts thus far have yielded (1) CD14 chimeric antibodies that recognize both Grampositive and Gram-negative bacteria and bind to the surfaces of mast cells, eliciting a degranulation response and (2) rat IgG2a antibodies that act similarly in response to low levels of canine parvovirus.

Curtis, Theresa↗
DOE OSTI2025

Depletion of the Protein Hydration Shell with Increasing Temperature Observed by Small-Angle X-ray Scattering and Molecular Simulations

The hydration shell is an integral part of proteins since it plays key roles in conformational transitions, molecular recognition, and enzymatic activity. While the dynamics of the hydration shell have been described by spectroscopic techniques, the structure of the hydration shell remains less understood due to the lack of hydration shell-sensitive structural probes with high spatial resolution. We combined temperature-ramp small-angle X-ray scattering (T-ramp SAXS) from 255 to 335 K with molecular simulations to demonstrate that the hydration shells of the IgG-binding domain of Protein G (GB3) and the villin headpiece are remarkably temperature-sensitive. For proteins in the folded state, T-ramp SAXS data and explicit-solvent SAXS predictions consistently demonstrate decays of protein contrasts and radii of gyration with increasing temperature, which are shown to reflect predominantly temperature-sensitive, depleting hydration shells. The depletion is caused not merely by enhanced disorder within the hydration shells but also by partial displacements of surface-coordinated water molecules. Together, T-ramp SAXS and explicit-solvent SAXS calculations provide a novel structural view of the protein hydration shell, which underlies temperature-dependent processes such as cold denaturation, thermophoresis, or biomolecular phase separation.

electron density↗
DOE OSTI2022

Structure-Based Design and Antigenic Validation of Respiratory Syncytial Virus G Immunogens

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of children, the elderly, and immunocompromised individuals. Currently, there are no FDA-approved RSV vaccines. The RSV G glycoprotein is used for viral attachment to host cells and impairment of host immunity by interacting with the human chemokine receptor CX3CR1. Antibodies that disrupt this interaction are protective against infection and disease. Nevertheless, development of an RSV G vaccine antigen has been hindered by its low immunogenicity and safety concerns. A previous study described three engineered RSV G proteins containing single-point mutations that induce higher levels of IgG antibodies and have improved safety profiles compared to wild-type RSV G. However, it is unclear if the mutations affect RSV G protein folding and display of its conformational epitopes. In this study, we show that the RSV G S177Q protein retains high-affinity binding to protective human and mouse monoclonal antibodies and has equal reactivity as wild-type RSV G protein to human reference immunoglobulin to RSV. Additionally, we determined the high-resolution crystal structure of RSV G S177Q protein in complex with the anti-RSV G antibody 3G12, further validating its antigenic structure. These studies show for the first time that an engineered RSV G protein with increased immunogenicity and safety retains conformational epitopes to high-affinity protective antibodies, supporting its further development as an RSV vaccine immunogen. Respiratory syncytial virus (RSV) causes severe lower respiratory diseases of children, the elderly, and immunocompromised populations. There currently are no FDA-approved RSV vaccines. Most vaccine development efforts have focused on the RSV F protein, and the field has generally overlooked the receptor-binding antigen RSV G due to its poor immunogenicity and safety concerns. However, single-point mutant RSV G proteins have been previously identified that have increased immunogenicity and safety. In this study, we investigate the antibody reactivities of three known RSV G mutant proteins. We show that one mutant RSV G protein retains high-affinity binding to protective monoclonal antibodies, is equally recognized by anti-RSV antibodies in human sera, and forms the same three-dimensional structure as the wild-type RSV G protein. Our study validates the structure-guided design of the RSV G protein as an RSV vaccine antigen.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1987

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Hamilton, Robert G.↗
DOE OSTI2023

Structural and immunological differences in Plasmodium falciparum sexual stage transmission-blocking vaccines comprised of Pfs25-EPA nanoparticles

Development of a malaria vaccine that blocks transmission of different parasite stages to humans and mosquitoes is considered critical for elimination efforts. A vaccine using Pfs25, a protein on the surface of zygotes and ookinetes, is under investigation as a transmission-blocking vaccine (TBV) that would interrupt parasite passage from mosquitoes to humans. The most extensively studied Pfs25 TBVs use Pichia pastoris-produced recombinant forms of Pfs25, chemically conjugated to a recombinant carrier protein, ExoProtein A (EPA). The recombinant form of Pfs25 first used in humans was identified as Pfs25H, which contained a total of 14 heterologous amino acid residues located at the amino- and carboxyl-termini including a His6 affinity tag. A second recombinant Pfs25, identified as Pfs25M, was produced to remove the heterologous amino acid residues and conjugated to EPA (Pfs25M-EPA). Here, monomeric Pfs25M was characterized biochemically and biophysically for identity, purity, and integrity including protein structure to assess its comparability with Pfs25H. Although the biological activities of Pfs25H and Pfs25M, whether generated by monomeric forms or conjugated nanoparticles, appeared similar, fine-mapping studies with two transmission-blocking monoclonal antibodies detected structural and immunological differences. In addition, evaluation of antisera generated against conjugated Pfs25H or Pfs25M nanoparticles in nonhuman primates identified polyclonal IgG that recognized these structural differences.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/ neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1 A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryoelectron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1994

Effect of radiation and age on immunoglobulin levels in rhesus monkeys (Macaca mulatta)

We report the results of a study on the immunoglobulin levels of rhesus monkeys (Macaca mulatta) in a colony consisting of the survivors of monkeys that received a single whole-body exposure to protons, electrons or X rays between 1964 and 1969. This colony has been maintained to assess the long-term effects of ionizing radiation on astronauts and high-flying pilots. Of the original 358 monkeys that were retained for lifetime studies, 129 (97 irradiated and 32 controls) were available for our study. We found no significant difference between the irradiated and control monkeys in mean levels of IgA, IgG and IgM, irrespective of the radiation treatment. The availability of these aged monkeys provided a unique opportunity to compare their immunoglobulin levels to those of other monkeys of various ages, and thus assess the effect of age on immunoglobulin levels. We found that only the IgA levels increase with age.

Non-NASA Center↗
NASA NTRS1988

Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO3- uptake by more than 90% but had no effect on NO2- uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO3- uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3- uptake. The results present the possibility that NO3- uptake and NO3- reduction in the PM of barley roots may be related.

NASA Discipline Number 61-10↗
NASA NTRS1987

Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

Hamilton, Robert G.↗