Four Gram-stain-negative, non-spore-forming, motile, short rod-shaped strains (LY29T, LY160, LY54T and LY1275) were isolated from soil of the Qinghai-Tibetan Plateau, China. Based on 16S rRNA gene sequences, strain pairs LY29T/LY160 and LY54T/LY1275 were identified as members of the genera Sphingomonas and Sphingosinicella, respectively, with highest similarities to Sphingomonas alba SE158T (97.8%) and Sphingomonas montis DRJ-4T (98.0%), respectively. In phylogenomic analyses based on 426 core gene sequences, LY29T and LY160 clustered with Sphingomonas rhizophila THG-T61T, while LY54T and LY1275 were closely related to Sphingosinicella humi QZX222T, Sphingosinicella flava UDD2T and Sphingosinicella vermicomposti KCTC 22446T. Digital DNA-DNA hybridization and average nucleotide identity values between the four strains and their closest relatives were determined to be below 24.2% and 80.49%, respectively, thus falling below the recommended species delineation thresholds (70% and 95-96%, respectively). The genomic DNA G+C contents of strains LY29T and LY54T were 64.8 and 66.8 mol%, respectively. Strain LY29T contained predominant fatty acids C16 (C16:1  ω7c and C16:1  ω6c) and C18 (C18:1  ω7c and C18:1  ω6c), while LY54T had iso-C16:0 as the major fatty acid. Both strains shared major polar lipids (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified sphingoglycolipid). Strain LY29T possessed ubiquinone Q-10 and homospermidine, whereas LY54T contained MK-9(H2) as the major quinone. Based on a polyphasic taxonomic approach, type strains LY29T (=GDMCC 1.4177T=JCM 36472T) and LY54T (=GDMCC 1.4236T=KCTC 8630T) represent novel species of the genera Sphingomonas and Sphingosinicella, for which the names Sphingomonas lindongxini sp. nov. and Sphingosinicella wutangchuni sp. nov. are proposed.

Wildlife serves as a natural reservoir for emerging viruses. Although virome studies of bats and rodents have advanced our understanding of viral diversity, the virome characteristics and zoonotic risks of wildlife inhabiting the Qinghai-Tibet Plateau-a global biodiversity hotspot-remain poorly understood. Here, we conducted a virome survey of 22 wild animal species from the Qinghai-Tibet Plateau using meta-transcriptomic sequencing. We characterized the diversity and distribution of wildlife-associated viruses and found that birds tended to harbor higher viral diversity and abundance than mammals. In addition, greylag goose (Anser anser) and Himalayan vulture (Gyps himalayensis) harbored a high proportion of cross-species viruses and occupied central positions in the transmission network. In total, we identified 36 viral genera with pathogenic potential to humans or animals. Among them, several viruses, including rotavirus A (RVA) and parainfluenza virus 5 (PIV-5), pose a high risk of zoonotic transmission. Notably, the plateau vole (Neodon fuscus) carried the highest number of high-risk viruses, and the RVA strains it harbors represent a distinct early evolutionary lineage. These findings highlight the public health relevance of wildlife on the Qinghai-Tibet Plateau and emphasize the need for continued surveillance of high-risk hosts and viruses.

Two novel Gram-stain-positive, non-spore-forming, strictly aerobic, regular rod-shaped bacteria (HY1910T and HY1908) were isolated from bat feces collected from Changshou District of Chongqing City (Altitude: 490 m; N: 30° 02′ 15″, E: 107° 07′ 4″, September 2011) and Chuxiong Yi Autonomous Prefecture, Yunnan province (Altitude: 1795 m; N: 25°09′10″, E: 102°04′39″, October 2013), respectively. Optimal growth is obtained at 28–30 °C (range, 10–37 °C) on BHI-5

BACKGROUND:Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) represents a novel bunyavirus that poses significant public health challenges. As a key prognostic indicator of clinical outcome, the viral load determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is relatively inaccurate and incomparable across different studies. Digital PCR (dPCR) has recently proved to be a more ideal tool for viral load assessment. OBJECTIVE:To develop a dPCR-based S-segment-specific method for SFTSV viral load monitoring and evaluate its performance in clinical samples. STUDY DESIGN:Specific dPCR was developed using primers/probes for the N region in the S segment of the SFTSV genome. The performance of dPCR was confirmed using serial dilutions of viral cultures, and dPCR viral load quantification was compared with the result of RT-qPCR in 166 suspected SFTS patients. RESULTS:DPCR demonstrated superior sensitivity with a detection limit of 190.5 copies/mL, high linearity, and good reproducibility. Six false negative samples were detected by dPCR among the 28 RT-qPCR negative samples. The correlation between RT-qPCR and dPCR was low at a low viral load level. Both dPCR and RT-qPCR were important risk factors for severity and mortality by the multivariate logistic regression analysis The accurate viral load based on dPCR has a strong predictive ability for patient outcomes and shows significant correlation with multiple host response markers. CONCLUSION:The results suggest that dPCR is a highly sensitive alternative to the measurement of SFTSV and should be considered for clinical utilization in patients with suspected SFTS.

Predicting the binding of peptides to human leukocyte antigen (HLA) alleles is critical for identifying epitopes initiating immune responses in vaccine development and immunotherapies. Despite the progress in predicting binding peptides of 1% HLA class-I alleles with in silico prediction tools, it remains challenging to predict binding peptides for alleles lacking epitopes. To achieve the binding prediction on those zero-shot alleles, we developed a hierarchical progressive learning (HPL) framework that progressively learns sequence patterns of specific peptide-HLA complexes. HPL models improve the prediction performance for zero-shot HLA class-I alleles by 60.8% (1,414.0% for non-classical ones). We further developed an automated peptide mutation search program, which automatically identifies binding peptides for any target HLA class-I allele and mutates weak-binding or non-binding peptides to enhance binding affinity under the guidance of HPL models. Our program generated high-affinity binding candidates of any target allele with limited mutations in more than 38.1% of testing cases.

Most current HIV gut microbiota studies, based on 16S rRNA gene sequencing, are limited to the genus level. Since different genera encompass several to hundreds of species, in this study, we performed research at the species level based on the HGMAD database. This cross-sectional study investigated differences in gut microbiota between healthy control (HC) subjects and people with HIV (PWH) at the species level, characterized the specific enterotype and the correlation between gut microbiota patterns and immune status in PWH. We recruited 114 individuals in the PWH group and 58 individuals in the HC group, from whom fecal samples were collected for 16S rRNA gene sequencing of the V3–V4 region. Significant differences in α diversity and β diversity (p-values < 0.05) were observed between the two groups. Compared to the HC group, the PWH group exhibited increased opportunistic pathogens and decreased commensal bacteria. Additionally, 114 species or higher taxa were enriched, while 49 species or higher taxa were depleted in the PWH group relative to those in the HC group. The gut microbiota of the HC group was categorized into enterotype Bacteroides (ET-B) and enterotype Prevotella (ET-P), whereas that of the PWH group exhibited ET-B, ET-P and enterotype Enterobacter (ET-E). ET-E was a unique enterotype in the PWH group, in which the abundance of Enterobacter was significantly increased. The absolute number of CD4+T lymphocytes and CD4/CD8 ratio were lower in ET-E HIV-infected individuals compared to those in the other two enterotypes. Ten signatures in three enterotypes showed high accuracy for distinguishing high and low CD4+T-cell counts groups, as well as high and low CD4/CD8 ratio groups, and the areas under curves were 0.831 (95%CI: 0.734–0.928) and 0.815 (95%CI: 0.721–0.909). Spearman’s correlation analysis revealed that five signatures enriched in the ET-E group were positively correlated with the absolute number of CD8+T lymphocytes. To sum up, compared to the HC group, the gut microbiota of PWH group exhibit reduced microbial diversity, an overall distinct microbial structure, and increased opportunistic pathogens. Furthermore, a novel enterotype (ET-E) was identified in HIV patients. Signatures based on different enterotypes can accurately and effectively discern the immune status of PWH, suggesting that the microbial composition of HIV infection is associated with the immune status.

Developing an effective HIV vaccine is a momentous challenge. An exceptionally wide range of candidate HIV vaccines have been tested, yet many were poorly immunogenic, and of the select few that advanced into efficacy trials, only one demonstrated any efficacy. Here we report the results of the largest-scale cross-protocol immunogenicity comparison to date: 13 HIV vaccine trials (including 36 vaccine regimens) conducted across nine countries worldwide, strengthened by standardized trial designs, validated assays in centralized laboratories, and harmonized immunogenicity endpoints - providing an objective approach to identify the HIV vaccine candidate(s) with the best immunogenicity. A polyvalent DNA prime + protein boost regimen (HVTN 124) including Env immunogens of four subtypes, matched between prime and boost, achieved the best anti-V1V2 antibody responses by a large margin and also induced high CD4+ T-cell responses - two key immune responses implicated in HIV vaccine protection. Our results provide strong support to test this promising HIV vaccine design in more advanced phase clinical trials and will also guide the future design of additional HIV vaccines.Trial registration: ClinicalTrials.gov identifier: NCT01799954..Trial registration: ClinicalTrials.gov identifier: NCT02109354..Trial registration: ClinicalTrials.gov identifier: NCT02404311..Trial registration: ClinicalTrials.gov identifier: NCT02207920..Trial registration: ClinicalTrials.gov identifier: NCT02296541..Trial registration: ClinicalTrials.gov identifier: NCT03284710..Trial registration: ClinicalTrials.gov identifier: NCT02915016..Trial registration: ClinicalTrials.gov identifier: NCT02997969..Trial registration: ClinicalTrials.gov identifier: NCT03122223..Trial registration: ClinicalTrials.gov identifier: NCT03409276..Trial registration: ClinicalTrials.gov identifier: NCT02968849..Trial registration: ClinicalTrials.gov identifier: NCT03060629..Trial registration: ClinicalTrials.gov identifier: NCT00223080..

BACKGROUND:Sequential multivalent immunizations are used to counter diversity in rapidly mutating viruses. Here, we evaluated the effect of HIV-1 immunogen formats on the binding profile of memory B-cells elicited in two independent Rhesus macaque trials. METHODS:In one trial, female Rhesus macaques were immunized with a multiclade HIV-1 gp120 envelope glycoprotein (Env) cocktail and bled two weeks post final immunization. In another trial, male and female Rhesus macaques were sequentially immunized with clonally-related Env glycoproteins: Four immunogens were administered as non-stabilized gp140 Envs and the fifth as a specially stabilized gp140 Env trimer (SOSIP); animals were bled before and after SOSIP immunization. Immunogen-binding peripheral memory B-cells were sorted and cultured at limiting dilution. Culture supernatants were assessed by ELISA for binding to individual immunogens. RESULTS:In the first trial, 81% (591/734) of B-cells cross-react with multiple Envs and most bind to all immunogens. In the second trial, 81% (331/410) of B-cells isolated before SOSIP administration react with all non-stabilized gp140 Env immunogens and 27% also cross-react with the yet-to-be-administered SOSIP-stabilized Env. However, after SOSIP administration, SOSIP-stabilized trimer-reactive B-cells increase to 86% (219/256) but most (82%) do not cross-react with the preceding immunogens. CONCLUSIONS:Multiclade and sequential regimens before SOSIP-stabilized Env immunization elicited B-cells that converge on shared epitopes. A change in immunogen format results in a divergent B-cell response that vastly fails to engage prior responses. Critically, B-cell priming with non-stabilized Env cannot modify the effect of the epitope immunodominance hierarchy in a SOSIP trimer. These results suggest that a change in immunogen format may cause off-target B-cell engagement, but also that B-cell repriming is possible despite pre-existing immunity.

ABSTRACT HIV-1 continues to pose a significant global health challenge, requiring ongoing research into effective prevention and treatment strategies. Understanding the B-cell repertoire that can be engaged upon vaccination in humans is crucial for the development of future preventive vaccines. In this study, peripheral blood mononuclear cells from HIV-negative participants in the multivalent HVTN124 human HIV-1 vaccine clinical trial were interrogated for HIV-reactive B cells using LIBRA-seq, a high-throughput B-cell mapping technology. We report the discovery of glycan-reactive antibodies, with one of these being capable of neutralizing diverse heterologous HIV-1 virus strains. Furthermore, isolated antibodies showed broad cross-reactivity against antigens from a variety of other pathogens. The emerging class of glycan-reactive virus-neutralizing antibodies with exceptional breadth of pathogen cross-reactivity may present an effective target for vaccination at the population level.IMPORTANCEUnderstanding how the human immune system recognizes and combats viruses is crucial for developing better vaccines and treatments. Here, through characterization of the B-cell receptor repertoires of participants in HVTN124, a multivalent HIV-1 vaccine human clinical trial, we discovered antibodies that recognize sugar molecules (glycans) on antigens from a range of unrelated viral families. In addition to their binding breadth, these antibodies can also neutralize multiple diverse strains of HIV-1. Our findings reveal an emerging and underappreciated mechanism for antibodies to counteract virus infection, potentially opening doors for developing vaccines that preferentially elicit glycan-reactive antibody species to broadly protect against different viruses.This study is registered with ClinicalTrials.gov as NCT03409276.

Four strains (LJ668 T /LJ756 and CY350 T /CY353) belonging to the genus Chryseobacterium were isolated from the Qinghai–Tibet Plateau soils. Cells were aerobic, Gram-stain-negative, rod-shaped, yellow-pigmented, non-motile and non-spore-forming. According to the 16S rRNA gene sequence alignment, the strain pair LJ668 T /LJ756 shared the highest similarities to Chryseobacterium aahli T68 T (98.09%) and Chryseobacterium limigenitum SUR2 T (98.09%), and the other one (CY350 T /CY353) to Chryseobacterium aquaticum KCTC 12483 T (97.98/98.13%). The phylogenetic analyses based on 16S rRNA gene and genomic sequences indicated that these four strains generated two separate clades in genus Chryseobacterium . The digital DNA–DNA hybridization and average nucleotide identity values of four novel isolates with other members of the genus Chryseobacterium were far below 70.0% and 95.0–96.0 % thresholds. The genomic DNA G+C content of LJ668 T and CY350 T is both 35.0 mol%. The major cellular fatty acids of the two pair strains are iso -C 15 : 0 and summed feature 3 (C 16 : 1 ω7c and/or C 16 : 1 ω6c). MK-6 is the only respiratory quinone for strains LJ668 T and CY350 T . The polar lipids of strain LJ668 T are phosphatidylethanolamine (PE), L1–5 and AL1–5, and the strain CY350 T is PE, L1–4, AL1–2, diphosphatidylglycerol, phosphoglycolipid and GL1–3. According to the results of genotypic, phenotypic and biochemical analyses, these four strains represent two novel species of the genus Chryseobacterium , for which the names Chryseobacterium chendengshani sp. nov. and Chryseobacterium wangxinyae sp. nov. are proposed. The type strains are LJ668 T (=CGMCC 1.17165 T =JCM 33877 T ) and CY350 T (=GDMCC 1.3790 T =KCTC 25690 T ), respectively.

HIV-1 continues to pose a significant global health challenge, requiring ongoing research into effective prevention and treatment strategies. Understanding the B cell repertoire that can be engaged upon vaccination in humans is crucial for the development of future preventive vaccines. In this study, PBMCs from HIV-negative participants in the multivalent HVTN124 human HIV-1 vaccine clinical trial were interrogated for HIV-reactive B cells using LIBRA-seq, a high-throughput B cell mapping technology. We report the discovery of glycan-reactive antibodies capable of neutralizing diverse heterologous HIV-1 virus strains. Further, isolated antibodies showed broad cross-reactivity against antigens from a variety of other pathogens, while remaining mostly negative on autoreactivity assays. The emerging class of glycan- reactive virus-neutralizing antibodies with exceptional breadth of pathogen cross- reactivity may present an effective target for vaccination at the population level.

Ubiquitin-like proteins (UBLs) constitute a family of evolutionarily conserved proteins that share similarities with ubiquitin in 3D structures and modification mechanisms. For most UBLs including small-ubiquitin-like modifiers (SUMO), their modification sites on substrate proteins cannot be identified using the mass spectrometry-based method that has been successful for identifying ubiquitination sites, unless a UBL protein is mutated accordingly. To identify UBL modification sites without having to mutate UBL, we have developed a dedicated search engine pLink-UBL on the basis of pLink, a software tool for the identification of cross-linked peptide pairs. pLink-UBL exhibited superior precision, sensitivity, and speed than "make-do" search engines such as MaxQuant, pFind, and pLink. For example, compared to MaxQuant, pLink-UBL increased the number of identified SUMOylation sites by 50 ∼ 300% from the same datasets. Additionally, we present a method for identifying small-molecule modifications of UBLs. This method involves antibody enrichment of a UBL C-terminal peptide following enrichment of a UBL protein, followed by LC-MS/MS analysis and a pFind 3 blind search to identify unexpected modifications. Using this method, we have discovered nonprotein substrates of SUMO, of which spermidine is the major one for fission yeast SUMO Pmt3. Spermidine can be conjugated to the C-terminal carboxylate group of Pmt3 through its N1 or also likely, N8 amino group in the presence of SUMO E1, E2, and ATP. Pmt3-spermidine conjugation does not require E3 and can be reversed by SUMO isopeptidase Ulp1. SUMO-spermidine conjugation is present in mice and humans. Also, spermidine can be conjugated to ubiquitin in vitro by E1 and E2 in the presence of ATP. The above observations suggest that spermidine may be a common small molecule substrate of SUMO and possibly ubiquitin across eukaryotic species.

Two Gram-stain-positive, oxidase-negative, non-motile, facultative anaerobic, α-hemolytic, coccus-shaped bacteria (zg-86T and zg-70) were isolated from the respiratory tracts of marmots (Marmota Himalayana) on the Qinghai-Tibet Plateau of China. Phylogenetic analysis of the 16S rRNA gene and 545 core genes revealed that these two strains belong to the Streptococcus genus. These strains were most closely related to Streptococcus respiraculi HTS25T, Streptococcus cuniculi CCUG 65085T, and Streptococcus marmotae HTS5T. The average nucleotide identity (ANI) and digital DNA‒DNA hybridization (dDDH) were below the threshold for species delineation. The predominant cellular fatty acids (CFAs) in this novel species were C16:0, C18:0, and C18:1ω9c, whereas the primary polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and an unknown phosphoglycolipid (PGL). The optimal growth conditions for the strains were 37 °C, pH 7.0, and 0.5

BACKGROUND:An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS:HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS:Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION:Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING:US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.