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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

The congenital APOA1 K107del mutation disrupts the lipid-free conformation of monomeric APOA1 and impairs oligomerization

Apolipoprotein A-I (APOA1) oligomerization is thought to be essential for high-density lipoprotein (HDL) formation and metabolism. Naturally occurring mutations can disrupt normal APOA1 folding and self-association, leading to dysfunctional HDL formation and cardiovascular disease. The congenital APOA1 variant p.K131del (APOA1 K107del ) has been associated with cardiovascular pathologies such as low HDL-cholesterol levels and aortic amyloidosis, and multiple studies indicate structural changes in APOA1 conformation underlie associated dysfunction. In the current study, we confirmed that APOA1 K107del exhibits no notable defect in lipid-binding. However, using polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC), we found that loss of lysine 107 resulted in a remarkable shift in the distribution of APOA1 oligomers with a much higher proportion of monomers present in APOA1 K107del compared to wild-type APOA1. Further investigation using quantitative cross-linking revealed a major disruption of interactions in helical regions reported to participate in domain swaps necessary for proper self-association. This structural disruption appears to impair N- and C-termini interactions and dynamics that lead to non-specific aggregation. In conclusion, these findings support the hypothesis that lysine 107 is critical for proper folding and self-association of lipid-free APOA1 which could impact HDL biogenesis.

APOA1 (p.K131del)↗
DOE OSTI2022

Quantile-Specific Heritability of Inflammatory and Oxidative Stress Biomarkers Linked to Cardiovascular Disease

Purpose: Heritability (h 2 , the proportion of the phenotypic variance attributable to additive genetic effects) is traditionally assumed to be constant throughout the distribution of the phenotype. However, the heritabilities of circulating C-reactive protein, interleukin-6, plasminogen activator inhibitor type-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) concentrations depend upon whether the phenotype is high or low relative to their distributions (quantile-dependent expressivity), which may account for apparent gene–environment interactions. Whether the heritabilities of other inflammatory biomarkers linked to cardiovascular disease are quantile-dependent remain to be determined. Patients and Methods: Quantile-specific offspring-parent (β OP ) and full-sib regression slopes (β FS ) were estimated by applying quantile regression to the age- and sex-adjusted phenotypes of families surveyed as part of the Framingham Heart Study. Quantile-specific heritabilities were calculated as: h 2 =2β OP /(1+r spouse ) and h 2 ={(1+8r spouse β FS ) 0.5 – 1}/(2r spouse ). Results: Heritability (h 2 ± SE) of lipoprotein-associated phospholipase A2 (Lp-PLA 2 ) mass concentrations increased from 0.11 ± 0.03 at the 10 th percentile, 0.08 ± 0.03 at the 25 th , 0.12 ± 0.03 at the 50 th , 0.20 ± 0.04 at the 75 th , and 0.26 ± 0.06 at the 90 th percentile, or 0.0023 ± 0.0006 per each one-percent increase in the phenotype distribution (P linear trend = 0.0004). Similarly, h 2 increased 0.0029 ± 0.0011 (P linear trend = 0.01) for sP-selectin, 0.0032 ± 0.0009 (P linear trend = 0.0001) for soluble intercellular adhesion molecule 1 (sICAM-1), and 0.0026 ± 0.0006 for tumor necrosis factor receptor 2 (TNFR2) (P linear trend = 5.0 × 10 -6 ) per each one-percent increase in their distributions when estimated from β OP . Osteoprotegerin and soluble ST2 heritability also increased significantly with increasing percentiles of their distributions when estimated from β FS . Lp-PLA 2 activity, CD40 ligand, TNFα, interleukin-18, and myeloperoxidase heritability showed no significant quantile-dependence. Conclusion: The heritabilities of circulating Lp-PLA 2 -mass, sP-selectin, sICAM-1, TNFR2, osteoprotegerin and soluble ST2 concentrations are quantile-dependent, which may contribute to purported genetic modulations of: 1) sP-selectin’s relationships to venous thrombosis, pulmonary hypertension, type 2 diabetes and atorvastatin treatment; 2) sICAM-I’s relationships to brain abscess and atorvastatin treatment; and 3) Lp-PLA 2 ’s relationships to myocardial infarction and preeclampsia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Proteomic Determinants of Variation in Cholesterol Efflux: Observations from the Dallas Heart Study

High-density lipoproteins (HDLs) are promising targets for predicting and treating atherosclerotic cardiovascular disease (ASCVD), as they mediate removal of excess cholesterol from lipid-laden macrophages that accumulate in the vasculature. This functional property of HDLs, termed cholesterol efflux capacity (CEC), is inversely associated with ASCVD. HDLs are compositionally diverse, associating with >250 different proteins, but their relative contribution to CEC remains poorly understood. Our goal was to identify and define key HDL-associated proteins that modulate CEC in humans. The proteomic signature of plasma HDL was quantified in 36 individuals in the multi-ethnic population-based Dallas Heart Study (DHS) cohort that exhibited persistent extremely high (>=90th%) or extremely low CEC (<=10th%) over 15 years. Levels of apolipoprotein (Apo)A-I associated ApoC-II, ApoC-III, and ApoA-IV were differentially correlated with CEC in high (r = 0.49, 0.41, and —0.21 respectively) and low (r = —0.46, —0.41, and 0.66 respectively) CEC groups (p for heterogeneity (pHet) = 0.03, 0.04, and 0.003 respectively). Further, we observed that levels of ApoA-I with ApoC-III, complement C3 (CO3), ApoE, and plasminogen (PLMG) were inversely associated with CEC in individuals within the low CEC group (r = —0.11 to —0.25 for subspecies with these proteins vs. r = 0.58 to 0.65 for subspecies lacking these proteins; p < 0.05 for heterogeneity). These findings suggest that enrichment of specific proteins on HDLs and, thus, different subspecies of HDLs, differentially modulate the removal of cholesterol from the vasculature.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor

Abstract Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella‐like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well‐defined binding pockets and structural information on native interactions has hindered structure‐guided ligand discovery against Flpp3. Here, we used a combination of physics‐based and deep‐learning methods to design high‐affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24–110 nM. For the second site, an initial binder showed a dissociation constant ( K D ) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub‐nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X‐ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (Cα root‐mean‐square deviation (RMSD): 0.9 Å). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

Gokce‐Alpkilic, Gizem [Molecular Engineering and S↗
DOE OSTI2023

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids

Abstract Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP‐independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T‐component, or TatT, is predicted to be a periplasmic‐facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long‐chain fatty acids. Our bioinformatics, quantitative analyses of protein‐ligand interactions, and high‐resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

In-Depth Proteome Profiling of the Hippocampus of LDLR Knockout Mice Reveals Alternation in Synaptic Signaling Pathway

The low-density lipoprotein receptor (LDLR) is a major apolipoprotein receptor that regulates cholesterol homeostasis. LDLR deficiency is associated with cognitive impairment by the induction of synaptopathy in the hippocampus. Despite the close relationship between LDLR and neurodegenerative disorders, proteomics research for protein profiling in the LDLR knockout (KO) model remains insufficient. Therefore, understanding LDLR KO-mediated differential protein expression within the hippocampus is crucial for elucidating a role of LDLR in neurodegenerative disorders. In this study, we conducted first-time proteomic profiling of hippocampus tissue from LDLR KO mice using tandem mass tag (TMT)-based MS analysis. LDLR deficiency induces changes in proteins associated with the transport of diverse molecules, and activity of kinase and catalyst within the hippocampus. Additionally, significant alterations in the expression of components in the major synaptic pathways were found. Furthermore, these synaptic effects were verified using a data-independent acquisition (DIA)-based proteomic method. In conclusion, our data will serve as a valuable resource for further studies to discover the molecular function of LDLR in neurodegenerative disorders.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Lpp positions peptidoglycan at the AcrA-TolC interface in the AcrAB-TolC multidrug efflux pump

The multidrug efflux pumps of Gram-negative bacteria are a class of complexes that span the periplasm, coupling both the inner and outer membranes to expel toxic molecules. The best-characterized example of these tripartite pumps is the AcrAB-TolC complex of Escherichia coli. However, how the complex interacts with the peptidoglycan (PG) cell wall, which is anchored to the outer membrane (OM) by Braun’s lipoprotein (Lpp), is still largely unknown. In this work, we present molecular dynamics simulations of a complete, atomistic model of the AcrAB-TolC complex with the inner membrane, OM, and PG layers all present. We find that the PG localizes to the junction of AcrA and TolC, in agreement with recent cryo-tomography data. Free-energy calculations reveal that the positioning of PG is determined by the length and conformation of multiple Lpp copies anchoring it to the OM. The distance between the PG and OM measured in cryo-electron microscopy images of wild-type E. coli also agrees with the simulation-derived spacing. Finally, sequence analysis of AcrA suggests a conserved role for interactions with PG in the assembly and stabilization of efflux pumps, one that may extend to other trans-envelope complexes as well.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

exRNA-eCLIP intersection analysis reveals a map of extracellular RNA binding proteins and associated RNAs across major human biofluids and carriers

Although the role of RNA binding proteins (RBPs) in extracellular RNA (exRNA) biology is well established, their exRNA cargo and distribution across biofluids are largely unknown. To address this gap, we extend the exRNA Atlas resource by mapping exRNAs carried by extracellular RBPs (exRBPs). This map was developed through an integrative analysis of ENCODE enhanced crosslinking and immunoprecipitation (eCLIP) data (150 RBPs) and human exRNA profiles (6,930 samples). Computational analysis and experimental validation identified exRBPs in plasma, serum, saliva, urine, cerebrospinal fluid, and cell-culture-conditioned medium. exRBPs carry exRNA transcripts from small non-coding RNA biotypes, including microRNA (miRNA), piRNA, tRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), Y RNA, and lncRNA, as well as protein-coding mRNA fragments. Computational deconvolution of exRBP RNA cargo reveals associations of exRBPs with extracellular vesicles, lipoproteins, and ribonucleoproteins across human biofluids. Overall, we mapped the distribution of exRBPs across human biofluids, presenting a resource for the community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineered Membrane Vesicle Production via oprF or oprI Deletion Has Distinct Phenotypic Effects in Pseudomonas putida

Membrane vesicle (MV) production is a natural phenomenon in Gram-negative bacteria and represents an emerging synthetic biology tool for the secretion of biomolecules or bioproducts. Manipulation of membrane components has proven successful in enhancing MV production. However, the impact of membrane disruptions on strain fitness and protein composition warrants further investigation for the use of MVs in industrial bioprocesses. Here, we identify and characterize two genetic engineering strategies for inducing hypervesiculation-deletion of genes for the outer membrane porin OprF or the lipoprotein OprI-in the commonly used platform Pseudomonas putida KT2440. Deletion of oprI generated up to a 1.5-fold increase in MVs, larger MVs with a greater proportion of outer membrane proteins, and no significant impact on strain fitness compared to wild type. In contrast, deletion of oprF, relative to wild type, generated up to a 4-fold increase in MVs but diminished growth, permeabilized membranes, and increased cytosolic protein packaging. Both hypervesiculation phenotypes increased nontargeted and MV-targeted mNeonGreen extracellular signal by up to 6-fold, demonstrating vesiculation as a mechanism for protein secretion. Despite increased blebbing of MVs from gene deletions, proteins involved in membrane biosynthesis were not elevated relative to wild type. Overexpression of gpsA, which initiates glycerophospholipid biosynthesis, in the ΔoprF background improved the membrane integrity by 37% and maintained MV formation, highlighting the importance of membrane biosynthesis in restoring the membrane in hypervesiculating strains. Together, this study provides genetic engineering strategies with corresponding phenotypic outcomes toward providing a synthetic biology toolset for MV deployment in P. putida.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Electricity from lignocellulosic substrates by thermophilic Geobacillus species

Given our vast lignocellulosic biomass reserves and the difficulty in bioprocessing them without expensive pretreatment and fuel separation steps, the conversion of lignocellulosic biomass directly into electricity would be beneficial. Here we report the previously unexplored capabilities of thermophilic Geobacillus sp. strain WSUCF1 to generate electricity directly from such complex substrates in microbial fuel cells. This process obviates the need for exogenous enzymes and redox mediator supplements. Cyclic voltammetry and chromatography studies revealed the electrochemical signatures of riboflavin molecules that reflect mediated electron transfer capabilities of strain WSUCF1. Proteomics and genomics analysis corroborated that WSUCF1 biofilms uses type-II NADH dehydrogenase and demethylmenaquinone methyltransferase to transfer the electrons to conducting anode via the redox active pheromone lipoproteins localized at the cell membrane.

09 BIOMASS FUELS↗
DOE OSTI2023

Evidence of excited-state lifetime enhancement in dimyristoyl-phosphocholine nanodiscs by ultraviolet absorption spectroscopy

Ultraviolet absorption spectroscopy is an analytical tool that is commonly utilized to determine protein concentrations, primarily due to characteristic absorption of tryptophan and tyrosine at 280 nm. Accurate concentration determination is essential to the accuracy of many biophysical techniques, and ultraviolet absorption provides a convenient and rapid method to assess protein concentration. However, the widespread usage of this method assumes that ultraviolet absorption is the same for individual amino acids in aqueous solution (where the molar extinction coefficient was measured) as for the amino acid in the conformational environment of the protein of interest. We demonstrate that additional considerations may be necessary for the membrane scaffold protein in dimyristoyl-phosphocholine nanodiscs through ultraviolet absorption spectroscopy of nanodiscs, liposomes, the membrane scaffold protein, and a mixture of liposomes and the non-nanodisc-associated protein. The aromatic amino acids of the membrane scaffold protein absorb significantly less light at 280 nm when associated with lipids in a lipoprotein assembly, which we plausibly attribute to an enhancement of chromophore excited-state lifetimes due to reduced intramolecular motion in the nanodisc. We caution that using the molar absorptivity of the membrane scaffold protein alone to determine nanodisc concentrations may not yield accurate results. Instead, the molar absorptivity of each nanodisc formulation should be explored independently to account for the unique conformational environment of each nanodisc.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

How lipidomics can transform our understanding of virus infections

Traditionally, lipids have been thought of as molecules that provide structure and energy, but their functional relevance in disease mechanisms is increasingly clear, and with advancements in mass spectrometry technologies, lipidomics analyses is a tool gaining recognition. Beyond the utility of lipoprotein and fatty acid profiles in clinical diagnosis and chemotaxonomic identifications, respectively, lipidomics analysis that combines knowledge across disciplines and can resolve exact lipid structures has the potential to advance clinical diagnoses and associated treatment, differentiate pathogen types (e.g., virus vs. bacterial vs. fungal), detect natural vs lab-grown viruses, and perhaps distinguish a pathogen from a benign biological agent (i.e., threat potential). As new viruses emerge and localized outbreaks expand, lipidomics will accelerate our understanding of viral infections and aid in maximizing the discovery of novel biomarkers of pathogenicity and host response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of coagulation factor VIII bound to a patient-derived anti-C1 domain antibody inhibitor

The development of pathogenic antibody inhibitors against coagulation factor VIII (FVIII) occurs in ~30% of congenital hemophilia A patients receiving FVIII replacement therapy as well as in all cases of acquired hemophilia A. KM33 is an anti-C1 domain antibody inhibitor previously isolated from a severe hemophilia A patient. In addition to potently blocking FVIII binding to von Willebrand factor and phospholipid surfaces, KM33 disrupts FVIII binding to lipoprotein receptor-related protein 1 (LRP1), which drives FVIII hepatic clearance and antigen presentation in dendritic cells. Here, we report on the structure of FVIII bound to NB33, a recombinant derivative of KM33, by single-particle cryo-electron microscopy. Furthermore, structural analysis reveals the NB33 epitope localizes to FVIII residues R2090-S2094 and I2158-R2159 which constitute membrane-binding loops in the C1 domain. Further analysis reveals multiple FVIII lysine and arginine residues, previously shown to mediate binding to LRP1, dock onto an acidic cleft at the NB33 variable domain interface, thus blocking a putative LRP1 binding site. Together, these results demonstrate a novel mechanism of FVIII inhibition by a patient-derived antibody inhibitor and provide structural evidence toward engineering FVIII with reduced LRP1-mediated clearance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Omega-3 fatty acids attenuate cardiovascular effects of short-term exposure to ambient air pollution

Background: Exposure to air pollution is associated with elevated cardiovascular risk. Evidence shows that omega-3 polyunsaturated fatty acids (omega-3 PUFA) may attenuate the adverse cardiovascular effects of exposure to fine particulate matter (PM 2.5 ). However, it is unclear whether habitual dietary intake of omega-3 PUFA protects against the cardiovascular effects of short-term exposure to low-level ambient air pollution in healthy participants. In the present study, sixty-two adults with low or high dietary omega-3 PUFA intake were enrolled. Blood lipids, markers of vascular inflammation, coagulation and fibrinolysis, and heart rate variability (HRV) and repolarization were repeatedly assessed in 5 sessions separated by at least 7 days. This study was carried out in the Research Triangle area of North Carolina, USA between October 2016 and September 2019. Daily PM 2.5 and maximum 8-h ozone (O 3 ) concentrations were obtained from nearby air quality monitoring stations. Linear mixed-effects models were used to assess the associations between air pollutant concentrations and cardiovascular responses stratified by the omega-3 intake levels. Results: The average concentrations of ambient PM 2.5 and O 3 were well below the U.S. National Ambient Air Quality Standards during the study period. Significant associations between exposure to PM 2.5 and changes in total cholesterol, von Willebrand factor (vWF), tissue plasminogen activator, D-dimer, and very-low frequency HRV were observed in the low omega-3 group, but not in the high group. Similarly, O 3 -associated adverse changes in cardiovascular biomarkers (total cholesterol, high-density lipoprotein, serum amyloid A, soluable intracellular adhesion molecule 1, and vWF) were mainly observed in the low omega-3 group. Lag-time-dependent biphasic changes were observed for some biomarkers. Conclusions: This study demonstrates associations between short-term exposure to PM 2.5 and O 3 , at concentrations below regulatory standard, and subclinical cardiovascular responses, and that dietary omega-3 PUFA consumption may provide protection against such cardiovascular effects in healthy adults.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Relationship Between Radiation Dose and Markers of Insulin Resistance and Inflammation in Atomic Bomb Survivors

Abstract Context In recent studies of childhood cancer survivors, diabetes has been considered a late effect associated with high therapeutic doses of radiation therapy. Our recent study of atomic bomb (A-bomb) survivors also suggested an association between radiation dose and diabetes incidence, with exposure city and age at exposure as radiation dose effect modifiers. Insulin resistance mediated by systemic inflammation and abnormal body composition has been suggested as a possible primary mechanism for the incidence of diabetes after total body irradiation; however, no studies have examined low to moderate radiation exposure (<4 Gy) and insulin resistance in A-bomb survivors. Objective To examine the association between radiation dose and markers of inflammation and insulin resistance. Methods This study investigated 3152 survivors who underwent a health examination between 2008 and 2012 and who were younger than 15 years at exposure. Multivariate linear regression analyses were used to evaluate the radiation effects on levels of markers of inflammation and insulin resistance. Results Radiation dose was significantly and positively associated with levels of C-reactive protein, triglycerides, homeostasis model assessment of β-cell function (HOMA-β), and HOMA of insulin resistance (HOMA-IR) after adjustment for relevant covariates including sex, city, and age at exposure. Adiponectin and high-density lipoprotein cholesterol levels were also associated significantly and negatively with radiation dose. However, city was not a dose modifier of the radiation response on these markers of inflammation and insulin resistance. Conclusion Insulin resistance might be a possible factor in radiation-related diabetes incidence in A-bomb survivors.

Endocrinology & Metabolism↗
DOE OSTI2023

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism’s energy needs likely exceed the modest output from glycolysis alone. Recently, building on our structure-function studies of T . pallidum lipoproteins, we proposed a “flavin-centric” metabolic lifestyle for the organism that partially resolves this conundrum. As a part of the hypothesis, we have proposed that T . pallidum contains an acetogenic energy-conservation pathway that catabolizes D-lactate, yielding acetate, reducing equivalents for the generation and maintenance of chemiosmotic potential, and ATP. We already have confirmed the D-lactate dehydrogenase activity in T . pallidum necessary for this pathway to operate. In the current study, we focused on another enzyme ostensibly involved in treponemal acetogenesis, phosphotransacetylase (Pta). This enzyme is putatively identified as TP0094 and, in this study, we determined a high-resolution (1.95 Å) X-ray crystal structure of the protein, finding that its fold comports with other known Pta enzymes. Further studies on its solution behavior and enzyme activity confirmed that it has the properties of a Pta. These results are consistent with the proposed acetogenesis pathway in T . pallidum , and we propose that the protein be referred to henceforth as TpPta.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nanolipoprotein particle (NLP) vaccine confers protection against Yersinia pestis aerosol challenge in a BALB/c mouse model

Introduction: Yersinia pestis is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Y. pestis results in a rapidly progressing illness that can only be successfully treated with antibiotics given shortly after symptom onset. Live attenuated or whole cell inactivated vaccines confer protection against bubonic plague, but pneumonic plague has been more difficult to prevent. Novel effective subunit vaccine formulations may circumvent some of these shortfalls. Here, we compare the immunogenicity generated by an advanced subunit vaccine (F1V fusion protein) and a nanolipoprotein particle (NLP)-based vaccine. Methods: The NLP, a high-density lipoprotein mimetic, provides a nanoscale delivery platform for recombinant Y. pestis antigens LcrV (V) and F1. BALB/c mice were immunized via subcutaneous injection twice, three or four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling, and mice were challenged with aerosolized Y. pestis CO92. Results: Both formulations induced a strong IgG response against the F1 and V proteins, along with a robust memory B cell response and a balanced cell-mediated immune response as evidenced by both Th1- and Th2-related cytokines. The NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the F1V vaccine. As with F1V, the inclusion of Alhydrogel (Alu) in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Mice that received two doses of F1:V:NLP + Alu and CpG were completely protected from a challenge with approximately eight median lethal doses of aerosolized Y. pestis CO92 and this protection confirmed the well-documented synergy between the F1 and V antigens in context of pneumonic plague. The NLPs have defined regions of polarity that facilitates the incorporation of a wide range of adjuvants and antigens with distinct physicochemical properties and are an excellent candidate platform for the development of multi-antigen vaccines.

F1↗