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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Investigation of microorganisms in cannabis after heating in a commercial vaporizer

There are concerns about microorganisms present on cannabis materials used in clinical settings by individuals whose health status is already compromised and are likely more susceptible to opportunistic infections from microbial populations present on the materials. Most concerning is administration by inhalation where cannabis plant material is heated in a vaporizer, aerosolized, and inhaled to receive the bioactive ingredients. Heating to high temperatures is known to kill microorganisms including bacteria and fungi; however, microbial death is dependent upon exposure time and temperature. It is unknown whether the heating of cannabis at temperatures and times designated by a commercial vaporizer utilized in clinical settings will significantly decrease the microbial loads in cannabis plant material. To assess this question, bulk cannabis plant material supplied by National Institute on Drug Abuse (NIDA) was used to assess the impact of heating by a commercial vaporizer. Initial method development studies using a cannabis placebo spiked with Escherichia coli were performed to optimize culture and recovery parameters. Subsequent studies were carried out using the cannabis placebo, low delta-9 tetrahydrocannabinol (THC) potency and high THC potency cannabis materials exposed to either no heat or heating for 30 or 70 seconds at 190°C. Phosphate-buffered saline was added to the samples and the samples agitated to suspend the microorganism. Microbial growth after no heat or heating was evaluated by plating on growth media and determining the total aerobic microbial counts and total yeast and mold counts. Overall, while there were trends of reductions in microbial counts with heating, these reductions were not statistically significant, indicating that heating using standard vaporization parameters of 70 seconds at 190°C may not eliminate the existing microbial bioburden, including any opportunistic pathogens. When cultured organisms were identified by DNA sequence analyses, several fungal and bacterial taxa were detected in the different products that have been associated with opportunistic infections or allergic reactions including Enterobacteriaceae, Staphylococcus, Pseudomonas, and Aspergillus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Ectomycorrhizal Plant-Fungal Co-invasions as Natural Experiments for Connecting Plant and Fungal Traits to Their Ecosystem Consequences

Introductions and invasions by fungi, especially pathogens and mycorrhizal fungi, are widespread and potentially highly consequential for native ecosystems, but may also offer opportunities for linking microbial traits to their ecosystem functions. In particular, treating ectomycorrhizal (EM) invasions, i.e., co-invasions by EM fungi and their EM host plants, as natural experiments may offer a powerful approach for testing how microbial traits influence ecosystem functions. Forests dominated by EM symbiosis have unique biogeochemistry whereby the secretions of EM plants and fungi affect carbon (C) and nutrient cycling; moreover, particular lineages of EM fungi have unique functional traits. EM invasions may therefore alter the biogeochemistry of the native ecosystems they invade, especially nitrogen (N) and C cycling. By identifying “response traits” that favor the success of fungi in introductions and invasions (e.g., spore dispersal and germination) and their correlations with “effect traits” (e.g., nutrient-cycling enzymes) that can alter N and C cycling (and affect other coupled elemental cycles), one may be able to predict the functional consequences for ecosystems of fungal invasions using biogeochemistry models that incorporate fungal traits. Here, we review what is already known about how EM fungal community composition, traits, and ecosystem functions differ between native and exotic populations, focusing on the example of EM fungi associated with species of Pinus introduced from the Northern into the Southern Hemisphere. We develop hypotheses on how effects of introduced and invasive EM fungi may depend on interactions between soil N availability in the exotic range and EM fungal traits. We discuss how such hypotheses could be tested by utilizing Pinus introductions and invasions as a model system, especially when combined with controlled laboratory experiments. Finally, we illustrate how ecosystem modeling can be used to link fungal traits to their consequences for ecosystem N and C cycling in the context of biological invasions, and we highlight exciting avenues for future directions in understanding EM invasion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dynamic subcanopy leaf traits drive resistance of net primary production across a disturbance severity gradient

Across the globe, the forest carbon sink is increasingly vulnerable to an expanding array of low- to moderate-severity disturbances. However, some forest ecosystems exhibit functional resistance (i.e., the capacity of ecosystems to continue functioning as usual) following disturbances such as extreme weather events and insect or fungal pathogen outbreaks. Unlike severe disturbances (e.g., stand-replacing wildfires), moderate severity disturbances do not always result in near-term declines in forest production because of the potential for compensatory growth, including enhanced subcanopy production. Community-wide shifts in subcanopy plant functional traits, prompted by disturbance-driven environmental change, may play a key mechanistic role in resisting declines in net primary production (NPP) up to thresholds of canopy loss. However, the temporal dynamics of these shifts, as well as the upper limits of disturbance for which subcanopy production can compensate, remain poorly characterized. In this study, we leverage a 4-year dataset from an experimental forest disturbance in northern Michigan to assess subcanopy community trait shifts as well as their utility in predicting ecosystem NPP resistance across a wide range of implemented disturbance severities. Through mechanical girdling of stems, we achieved a gradient of severity from 0% (i.e., control) to 45, 65, and 85% targeted gross canopy defoliation, replicated across four landscape ecosystems broadly representative of the Upper Great Lakes ecoregion. We found that three of four examined subcanopy community weighted mean (CWM) traits including leaf photosynthetic rate (p = 0.04), stomatal conductance (p = 0.07), and the red edge normalized difference vegetation index (p < 0.0001) shifted rapidly following disturbance but before widespread changes in subcanopy light environment triggered by canopy tree mortality. Surprisingly, stimulated subcanopy production fully compensated for upper canopy losses across our gradient of experimental severities, achieving complete resistance (i.e., no significant interannual differences from control) of whole ecosystem NPP even in the 85% disturbance treatment. Additionally, we identified a probable mechanistic switch from nutrient-driven to light-driven trait shifts as disturbance progressed. Our findings suggest that remotely sensed traits such as the red edge normalized difference vegetation index (reNDVI) could be particularly sensitive and robust predictors of production response to disturbance, even across compositionally diverse forests. The potential of leaf spectral indices to predict post-disturbance functional resistance is promising given the capabilities of airborne to satellite remote sensing. We conclude that dynamic functional trait shifts following disturbance can be used to predict production response across a wide range of disturbance severities.

resistance↗
DOE OSTI2021

Deep Sequencing of MHC-Adapted Viral Lines Reveals Complex Recombinational Exchanges With Endogenous Retroviruses Leading to High-Frequency Variants

Experimental evolution (serial passage) of Friend virus complex (FVC) in mice demonstrates phenotypic adaptation to specific host major histocompatibility complex (MHC) genotypes. These evolved viral lines show increased fitness and virulence in their host-genotype-of-passage, but display fitness and virulence tradeoffs when infecting unfamiliar host MHC genotypes. Here, we deep sequence these viral lines in an attempt to discover the genetic basis of FVC adaptation. The principal prediction for genotype-specific adaptation is that unique mutations would rise to high frequency in viral lines adapted to each host MHC genotype. This prediction was not supported by our sequencing data as most observed high-frequency variants were present in each of our independently evolved viral lines. However, using a multi-variate approach to measure divergence between viral populations, we show that populations of replicate evolved viral lines from the same MHC congenic mouse strain were more similar to one another than to lines derived from different MHC congenic mouse strains, suggesting that MHC genotype does predictably act on viral evolution in our model. Sequence analysis also revealed rampant recombination with endogenous murine leukemia virus sequences (EnMuLVs) that are encoded within the BALB/c mouse genome. The highest frequency variants in all six lines contained a 12 bp insertion from a recombinant EnMuLV source, suggesting such recombinants were either being favored by selection or were contained in a recombinational hotspot. Interestingly, they did not reach fixation, as if they are low fitness. The amount of background mutations linked to FVC/EnMuLV variable sites indicated that FVC/EnMuLV recombinants had not reached mutation selection equilibrium and thus, that EnMuLV sequences are likely continuously introgressing into the replicating viral population. These discoveries raise the question: is the expression of EnMuLV sequences in mouse splenocytes that permit recombination with exogenous FVC a pathogen or host adaptation?

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Moving the needle: Employing deep reinforcement learning to push the boundaries of coarse-grained vaccine models

Highly mutable infectious disease pathogens (hm-IDPs) such as HIV and influenza evolve faster than the human immune system can contain them, allowing them to circumvent traditional vaccination approaches and causing over one million deaths annually. Agent-based models can be used to simulate the complex interactions that occur between immune cells and hm-IDP-like proteins (antigens) during affinity maturation—the process by which antibodies evolve. Compared to existing experimental approaches, agent-based models offer a safe, low-cost, and rapid route to study the immune response to vaccines spanning a wide range of design variables. However, the highly stochastic nature of affinity maturation and vast sequence space of hm-IDPs render brute force searches intractable for exploring all pertinent vaccine design variables and the subset of immunization protocols encompassed therein. To address this challenge, we employed deep reinforcement learning to drive a recently developed agent-based model of affinity maturation to focus sampling on immunization protocols with greater potential to improve the chosen metrics of protection, namely the broadly neutralizing antibody (bnAb) titers or fraction of bnAbs produced. Using this approach, we were able to coarse-grain a wide range of vaccine design variables and explore the relevant design space. Our work offers new testable insights into how vaccines should be formulated to maximize protective immune responses to hm-IDPs and how they can be minimally tailored to account for major sources of heterogeneity in human immune responses and various socioeconomic factors. Our results indicate that the first 3 to 5 immunizations, depending on the metric of protection, should be specially tailored to achieve a robust protective immune response, but that beyond this point further immunizations require only subtle changes in formulation to sustain a durable bnAb response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Antimicrobial Peptide Mimics for Clinical Use: Does Size Matter?

The search for efficient antimicrobial therapies that can alleviate suffering caused by infections from resistant bacteria is more urgent than ever before. Infections caused by multi-resistant pathogens represent a significant and increasing burden to healthcare and society and researcher are investigating new classes of bioactive compounds to slow down this development. Antimicrobial peptides from the innate immune system represent one promising class that offers a potential solution to the antibiotic resistance problem due to their mode of action on the microbial membranes. However, challenges associated with pharmacokinetics, bioavailability and off-target toxicity are slowing down the advancement and use of innate defensive peptides. Improving the therapeutic properties of these peptides is a strategy for reducing the clinical limitations and synthetic mimics of antimicrobial peptides are emerging as a promising class of molecules for a variety of antimicrobial applications. These compounds can be made significantly shorter while maintaining, or even improving antimicrobial properties, and several downsized synthetic mimics are now in clinical development for a range of infectious diseases. A variety of strategies can be employed to prepare these small compounds and this review describes the different compounds developed to date by adhering to a minimum pharmacophore based on an amphiphilic balance between cationic charge and hydrophobicity. These compounds can be made as small as dipeptides, circumventing the need for large compounds with elaborate three-dimensional structures to generate simplified and potent antimicrobial mimics for a range of medical applications. This review highlights key and recent development in the field of small antimicrobial peptide mimics as a promising class of antimicrobials, illustrating just how small you can go.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Multimerization of Ebola GPΔmucin on protein nanoparticle vaccines has minimal effect on elicitation of neutralizing antibodies

Ebola virus (EBOV), a member of the Filoviridae family of viruses and a causative agent of Ebola Virus Disease (EVD), is a highly pathogenic virus that has caused over twenty outbreaks in Central and West Africa since its formal discovery in 1976. The only FDA-licensed vaccine against Ebola virus, rVSV-ZEBOV-GP (Ervebo®), is efficacious against infection following just one dose. However, since this vaccine contains a replicating virus, it requires ultra-low temperature storage which imparts considerable logistical challenges for distribution and access. Additional vaccine candidates could provide expanded protection to mitigate current and future outbreaks. Here, we designed and characterized two multimeric protein nanoparticle subunit vaccines displaying 8 or 20 copies of GPΔmucin, a truncated form of the EBOV surface protein GP. Single-dose immunization of mice with GPΔmucin nanoparticles revealed that neutralizing antibody levels were roughly equivalent to those observed in mice immunized with non-multimerized GPΔmucin trimers. These results suggest that some protein subunit antigens do not elicit enhanced antibody responses when displayed on multivalent scaffolds and can inform next-generation design of stable Ebola virus vaccine candidates.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Single-cell and spatiotemporal transcriptomic profiling of brain immune infiltration following Venezuelan equine encephalitis virus infection

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naïve populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Single-nuclei transcriptome analysis of IgM+ cells isolated from channel catfish (Ictalurus punctatus) spleen

Catfish production is the primary aquaculture sector in the United States, and the key cultured species is channel catfish (Ictalurus punctatus). The major causes of production losses are pathogenic diseases, and the spleen, an important site of adaptive immunity, is implicated in these diseases. To examine the channel catfish immune system, single-nuclei transcriptomes of sorted and captured IgM + cells were produced from adult channel catfish. Three channel catfish (~1 kg) were euthanized, the spleen dissected, and the tissue dissociated. The lymphocytes were isolated using a Ficoll gradient and IgM + cells were then sorted with flow cytometry. The IgM + cells were lysed and single-nuclei libraries generated using a Chromium Next GEM Single Cell 3’ GEM Kit and the Chromium X Instrument (10x Genomics) and sequenced with the Illumina NovaSeq X Plus sequencer. The reads were aligned to theI. punctatusreference assembly (Coco_2.0) using Cell Ranger, and normalization, cluster analysis, and differential gene expression analysis were carried out with Seurat. Across the three samples, approximately 753.5 million reads were generated for 18,686 cells. After filtering, 10,637 cells remained for the cluster analysis. The cluster analysis identified 16 clusters which were classified as B cells (10,276), natural killer-like (NK-like) cells (178), T cells or natural killer cells (45), hematopoietic stem and progenitor cells (HSPC)/megakaryocytes (MK) (66), myeloid/epithelial cells (40), and plasma cells (32). The B cell clusters were further defined as different populations of mature B cells, cycling B cells, and plasma cells. The plasma cells highly expressedighmand we demonstrated that the secreted form of the transcript was largely being expressed by these cells. This atlas provides insight into the gene expression of IgM + immune cells in channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells.

Immunology↗
DOE OSTI2025

Proteome-wide characterization of PTMs reveals host cell responses to viral infection and identifies putative antiviral drug targets

Post-translational modifications (PTMs) are biochemical modifications that can significantly alter protein structure, function, stability, localization, and interactions with other molecules, thereby activating or inactivating intracellular processes. A growing body of research has begun to highlight the role of PTMs, including phosphorylation, ubiquitination, acetylation, and redox modifications, during virus-host interactions. Collectively, these PTMs regulate key steps in mounting the host immune response and control critical host pathways required for productive viral replication. This has led to the conception of antiviral therapeutics that focus on controlling host protein PTMs, potentially offering pathogen-agnostic treatment options and revolutionizing our capacity to prevent virus transmission. On the other hand, viruses can hijack the host cellular PTM machinery to modify viral proteins in promoting viral replication and evading immune surveillance. PTM regulation during virus-host interactions is complex and poorly mapped, and the development of effective PTM-targeted antiviral drugs will require a more comprehensive understanding of the cellular pathways essential for virus replication. In this review, we discuss the roles of PTMs in virus infection and how technological advances in mass spectrometry-based proteomics can capture systems-level PTM changes during viral infection. Additionally, we explore how such knowledge is leveraged to identify PTM-targeted candidates for developing antiviral drugs. Looking ahead, studies focusing on the discovery and functional elucidation of PTMs, either on the host or viral proteins, will not only deepen our understanding of molecular pathology but also pave the way for developing better drugs to fight emerging viruses.

Immunology↗
DOE OSTI2025

Insights into regulatory T-cell and type-I interferon roles in determining abacavir-induced hypersensitivity or immune tolerance

Introduction Clinical use of several small molecule drugs may lead to severe T-cell-mediated idiosyncratic drug hypersensitivity reactions (iDHR) linked to HLA alleles, including abacavir (ABC) with HLA-B*57:01. Due to study limitations in humans, pathogenic networks in iDHR remain elusive. HLA transgenic murine models have been proposed to bridge knowledge gaps in tolerance and susceptibility to drugs. Methods Mice expressing HLA-B*57:01 and Foxp3-DTR/EGFP were generated to selectively deplete regulatory T-cells (Treg) with diphtheria toxin. ABC was administered for 8 days alone or together with cell- and cytokine-depleting antibodies. Cellular and transcriptomic responses were analyzed by RNA, flow cytometry and fluorescence methods. Results While CD8 + T-cell responses to ABC require HLA presentation, ABC also triggered mitochondrial stress in macrophagesin vitro, independently of HLA.In vivo, Treg were the primary mechanism of drug tolerance controlling HLA presentation and costimulation by antigen presenting cells. Treg ablation uncovered immune adverse events linked to activation and proliferation of both drug-specific and bystander CD8 + T-cells through CD28-mediated pathways with support from CD4 + non-Treg. Type-I interferon (IFN-I) and cellular-stress pathways influenced the fate of lymph node cells responding to ABC, implicating innate immune cells such as macrophages and plasmacytoid dendritic cells in the development of T-cell responses against the drug. IFN-I and IL-2 were necessary for CD8 + T-cell differentiation and ABC-induced adverse reactions. Conclusions This study unveils novel immune mechanisms driven by drug and host-related factors required forin vivoreactions and sheds light on potential biomarker and therapeutic targets for managing and preventing severe and life-threatening iDHR.

Immunology↗
DOE OSTI2025

Cross-species analysis of FcγRIIa/b (CD32a/b) polymorphisms at position 131: structural and functional insights into the mechanism of IgG- mediated phagocytosis in human and macaque

Introduction Antibodies play a critical role in immunity in part by mediating clearance of pathogens and infected cells by antibody-dependent cellular phagocytosis (ADCP) through engagement of Fc gamma receptors (FcγRs) on innate immune cells. Among these, FcγRIIa (CD32a) is a key activating receptor expressed on macrophages, dendritic cells, and other antigen-presenting cells. Its affinity for IgG and ability to mediate ADCP is influenced by allelic polymorphisms. In humans, a single amino acid polymorphism at position 131, where histidine (H) is substituted with arginine (R), leads to decreased IgG1 and IgG2 subclass binding affinity and, consequently, lower efficiency of phagocytic responses. Rhesus macaques ( Macaca mulatta ), which are widely used as nonhuman primate models, exhibit a similar polymorphism at position 131 of FcγRIIa, but with arginine replaced by proline (P). Here, we investigated structure-function relationships associated with the FcγRIIa polymorphism at position 131 in both species, specifically with respect to IgG1 and IgG2. Methods We determined the structures of complexes formed by each variant with IgG1 Fc and those formed by the higher affinity variant with IgG2 Fc for both species by x-ray crystallography and linked these structures to affinity and activity using SPR and an ADCP assay. We also determined the structure of human inhibitory FcγRIIb (CD32b) in complex with IgG1 Fc by x-ray crystallography. Results Through analysis of these structures, our studies reveal that FcγRIIa engagement is minimally influenced by Fc glycan composition, distinguishing it from FcγRIIIa whose affinity is strongly influenced by glycan-composition. Comparative structures of human and macaque FcγRIIa variants demonstrate species- and allele-specific differences in Fc binding, but our functional assays showed only minimal allele-specific effects in humans. In contrast, allele-specific effects in macaques were highly significant; the macaque P 131 variant showing uniformly reduced IgG affinity. Conclusion These insights highlight fundamental interspecies and allelic distinctions that are critical for interpreting FcγRIIa-mediated effector functions in macaque models and for optimizing translational antibody and vaccine design.

Tolbert, William D.↗
DOE OSTI2026

Discovering novel therapeutic V H Hs for emerging viruses: perspectives from VEEV selection strategies

Introduction: Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V H Hs). Methods: In vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of V H Hs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus. Results: Here we report four novel “human” V H Hs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V H Hs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V H Hs. Discussion: Though non-neutralizing, these V H Hs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V H Hs to perform viral “sponging” which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Editorial: Towards the rapid and systematic assessment of vaccine technologies

The COVID-19 pandemic highlighted both the extraordinary potential of modern vaccinology and persistent challenges in how vaccine technologies are assessed. While vaccines can be developed and deployed at unprecedented speed, our ability to predict efficacy in a population is constrained by methodological difficulties, underreporting of negative results, and limited comparability across studies. This editorial introduces a Research Topic that brings together an interdisciplinary collection of experimental, computational, and theoretical contributions spanning multiple pathogens and vaccine platforms. Across these contributions, emerging themes emphasize the need for standardized immunogenicity metrics, transparent reporting including negative findings, and harmonized experimental protocols to support meaningful comparisons. This editorial highlights community practices and shared commitments – supported by researchers, funders, and journals – that could strengthen reproducibility, transparency, and cumulative learning in vaccine research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Soil Microbes Trade-Off Biogeochemical Cycling for Stress Tolerance Traits in Response to Year-Round Climate Change

Winter air temperatures are rising faster than summer air temperatures in high-latitude forests, increasing the frequency of soil freeze/thaw events in winter. To determine how climate warming and soil freeze/thaw cycles affect soil microbial communities and the ecosystem processes they drive, we leveraged the Climate Change across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the northeastern United States, where replicate field plots receive one of three climate treatments: warming (+5°C above ambient in the growing season), warming in the growing season + winter freeze/thaw cycles (+5°C above ambient +4 freeze/thaw cycles during winter), and no treatment. Soil samples were taken from plots at six time points throughout the growing season and subjected to amplicon (rDNA) and metagenome sequencing. We found that soil fungal and bacterial community composition were affected by changes in soil temperature, where the taxonomic composition of microbial communities shifted more with the combination of growing-season warming and increased frequency of soil freeze/thaw cycles in winter than with warming alone. Warming increased the relative abundance of brown rot fungi and plant pathogens but decreased that of arbuscular mycorrhizal fungi, all of which recovered under combined growing-season warming and soil freeze/thaw cycles in winter. The abundance of animal parasites increased significantly under combined warming and freeze/thaw cycles. We also found that warming and soil freeze/thaw cycles suppressed bacterial taxa with the genetic potential for carbon (i.e., cellulose) decomposition and soil nitrogen cycling, such as N fixation and the final steps of denitrification. These new soil communities had higher genetic capacity for stress tolerance and lower genetic capacity to grow or reproduce, relative to the communities exposed to warming in the growing season alone. Our observations suggest that initial suppression of biogeochemical cycling with year-round climate change may be linked to the emergence of taxa that trade-off growth for stress tolerance traits.

54 ENVIRONMENTAL SCIENCES↗