Biology subjects

Gao, G. F.

Publications and source records attributed to Gao, G. F..

At least 19 recordsLinked to original sources

bioRxiv2025

Rational design of respiratory syncytial virus dimeric F-subunit vaccines in protein and mRNA forms

BackgroundRespiratory syncytial virus (RSV) poses a significant public health threat, particularly to children and the elderly. Two protein-based vaccines and one mRNA vaccine have been approved, all targeting the prefusion conformation of the fusion (F) glycoprotein. However, it has been reported that the F protein transitions to the post-fusion state during storage, resulting in a reduction of the vaccines immunogenicity. MethodsIn this study, we engineered novel pre-F-based antigens to preserve pre-F-specific immunodominant epitopes while eliminating sub-potent ones. Based on this, we constructed a series of single-chain dimers and selected the one with the highest expression yield and melting temperature (Tm). Next, we created a heterodimer, scDimer AB. Structural and protein characterization analyses were conducted to verify our design. All monomeric and scDimer antigens were used to immunize rodent models. Additionally, we prepared the antigens in mRNA form and immunized BALB/c mice. Finally, we combined both antigen forms, administering intramuscular mRNA priming followed by intranasal protein delivery in mice. In all immunization strategies, viral challenges were performed in animals to evaluate the immunologic protective effects. FindingsThrough rational design, we developed a monomeric and two single-chain dimeric (scDimer) proteins with the expected characteristics, including complete II, V, and O epitopes and a partial III epitope. The scDimers elicited stronger binding and neutralizing antibody responses in rodent models compared to the monomer, and they also boosted T cell responses when combined with appropriate adjuvants. After three doses of scDimer immunization, live RSV was barely detectable in the tissues of infected animals. The copies of RNA encoding N-gene were significantly reduced in the immunized groups compared to the PBS-injected control groups. We also engineered mRNA versions of the antigens and verified their protective efficacy in mice. Notably, there were no significant differences between intranasal boost and intramuscular two doses after RSV challenged, suggesting that intranasal boost provided equivalent protection to intramuscular vaccination and could reduce the risk of vaccine-enhanced disease (VED) potentially. InterpretationThe scDimer-based RSV vaccines effectively protected rodents from RSV infections, highlighting their clinical potential. Our antigen design removed certain suboptimal epitope regions, enhancing the efficiency of antigen presentation and increasing the proportion of the most potent pre-F-specific neutralizing antibodies. This approach provides a novel perspective for future vaccine design. FundingNational Key R&D Program of China, National Science Foundation of China, Young Scientists in Basic Research, Chinese Academy of Sciences, and Special Program of China National Tobacco Corporation. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe licensed respiratory syncytial virus (RSV) vaccines were based on RSV pre-F structure. We conducted a PubMed search for articles published in English from databases inception to March 31, 2025, using the search terms "RSV" (interchanged with "respiratory syncytial virus"), "pre-F" (interchanged with "prefusion F") and "vaccine". Our search revealed that most of the articles focus on combining pre-F with RSV G protein or other proteins to form viral-like particles (VLPs) or nanoparticles. The second-largest category of articles examined the antibodies level elicited by the pre-F antigens in animal model or some special cohorts. Other studies addressed topics such as optimization of injection dosage and immunization strategies, methodologies for detecting pre-F-specific antibodies, and the development of mRNA-LNP vaccines. Notably, only 4 articles described novel designs of RSV pre-F antigens, all of which were trimeric and based exclusively on the RSV A subtype F protein sequence. Added value of this studyTo our knowledge, our design is the first single-chain dimer version of RSV pre-F antigen. The engineered antigens demonstrated high yield, thermostability, and immunogenicity comparable to that of DS-Cav1. Additionally, we developed the first chimeric RSV-antigen covering both subtype A and B, thereby broadening the potential protective spectrum. We further validated the immunogenicity of our designs in mRNA form and as a protein-mRNA combination. This research emphasizes the potential of constructing and combining minimized, stable antigen modules, offering a novel approach for future vaccine development. Implications of all the available evidenceIn this study, we present a novel approach for constructing and combining minimized, stable antigen modules for future vaccine design. The strong immunogenicity of our antigens underscores their potential for clinical application. Furthermore, given the structural similarities between the RSV antigen and those of other Pneumoviruses, such as human metapneumovirus (hMPV), our design strategy could be extended to the development of multivalent vaccines.

immunology↗

bioRxiv2024

Reliable prediction of protein-protein binding affinity changes upon mutations with Pythia-PPI

Protein-protein interactions are essential for numerous biological functions, and predicting binding affinity changes caused by mutations is crucial for understanding the impact of genetic variations and advancing protein engineering. Although machine learning-based methods show promise in improving the prediction accuracy, the scarcity of experimental data remains a significant bottleneck. Here, we utilized multi-task learning and self-distillation to overcome the data limitation and improved the accuracy of deep learning-based protein binding affinity prediction. By incorporating a mutation stability prediction task, the model achieved state-of-the-art accuracy on the SKEMPI dataset and was subsequently used to predict binding affinity changes for millions of mutations, generating an expanded dataset for self-distillation. Compared with prevalent methods, Pythia-PPI increased the Pearson correlation between predictions and experimental data from 0.6447 to 0.7850 on the SKEMPI dataset, and from 0.3654 to 0.6051 on the virial-receptor dataset. These findings demonstrated Pythia-PPI to be a valuable tool for analyzing the fitness landscape of protein-protein interactions. We provided a web server at https://pythiappi.wulab.xyz for easy access to Pythia-PPI.

bioinformatics↗

bioRxiv2024

C-Reactive Protein Drives Potent Clearance of Blood Bacteria in the Liver by Activating the Complement System

Plasma C-reactive protein (CRP) is widely used as a biomarker for bacterial infections due to its massive induction during infections, however, the precise function of CRP in bacterial infections remains undefined. Here we show that CRP enables Kupffer cells (liver macrophages) to capture and eliminate a wide range of encapsulated bacteria from the bloodstream of mice and thereby provides rapid and effective immunity. Mechanistically, CRP binds to the structurally diverse capsular polysaccharides of major Gram-positive and -negative pathogens, and thereby activates complement C3 at the bacterial surface. The C3-opsonized microbes are in turn captured by C3 receptors on the surface of Kupffer cells, and eliminated in the liver sinusoids. Since CRP principally shares the functional features of antibodies in pathogen recognition/execution, CRP-based defense combines the broad spectrum of the innate immunity with the swiftness, potency and specificity of the adaptive immunity, which helps explain massive rise of CRP during systemic bacterial infections.

immunology↗

bioRxiv2023

Efficient inhibition of fusion inhibitor HY3000 peptide to SARS-CoV-2 emerging EG.5, EG.5.1 and BA.2.86 variants

SARS-CoV-2 continues to evolve and spread. Recently, the Omicron EG.5 lineage, bearing an additional F456L mutation in spike (S) protein compared to its ancestor XBB.1.9.2, and its sub-variant EG.5.1, which carries a further Q52H mutation, have raised concerns due to their increased prevalence and extended immune escape properties. Additionally, an alarming variant, BA.2.86, has also garnered global concern because it contains over 30 amino acid mutations in its S protein compared to BA.2, including more than 10 changes in receptor-binding domain (RBD), reminiscent of the appearance of the Omicron variant in late 2021. Therefore, there is an urgent need to assess the effectiveness of current vaccines and therapeutics against EG.5, EG.5.1 and BA.2.86. In our previous work, we reported the design and broad-spectrum antiviral activity of a peptide fusion inhibitor HY3000 against SARS-CoV-2 and its variants including XBB.1.5. Here, we continued to evaluate the inhibitory potency of the HY3000 peptide against the prevailing EG.5 and EG.5.1, as well as XBB.1.16, FL.1.5.1, FY.3 and BA.2.86. Our data indicated that the peptide retained its potent inhibitory activities against these variants, indicating its potential as a good virus fusion inhibitor with broad-spectrum therapeutic effect against current and future SARS-CoV-2 variants. Currently, the HY3000 has been finished in Phase II clinical trial in China and has also been approved to conduct clinical investigation by U.S. Food and Drug Administration (FDA), suggesting a good application prospect against the ongoing COVID-19.

microbiology↗

bioRxiv2023

SARS-CoV-2 Omicron BA.2.86: less neutralization evasion compared to XBB sub-variants

The continual emergence and circulation of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have caused a great challenge for the coronavirus disease 2019 (COVID-19) pandemic control. Recently, Omicron BA.2.86 was identified with more than 30 amino acid changes on the spike (S) protein, compared to Omicron BA.2 or XBB.1.5. The immune evasion potential of BA.2.86 is of great concern. In this study, we evaluated the neutralizing activities of sera collected from participants and mice. Participants were divided into five groups according to their vaccination (inactivated vaccine, protein subunit vaccine ZF2001 or ZF2202-A) and infection (Omicron BF.7/BA.5.2) status. ZF2202-A is ZF2001 vaccines next-generation COVID-19 vaccine with updated bivalent Delta-BA.5 RBD-heterodimer immunogen. BALB/c mice were immunized with XBB.1.5 RBD-homodimer, BA.5-BA.2, Delta-XBB.1.5 or BQ.1.1-XBB.1.5 RBD-heterodimers protein vaccine candidates for evaluating the neutralizing responses. We found that Omicron BA.2.86 shows stronger immune evasion than BA.2 due to >30 additional mutations on S protein. Compared to XBB sub-variants, BA.2.86 does not display more resistance to the neutralizing responses induced by ZF2001-vaccination, BF.7/BA.5.2 breakthrough infection or a booster dose of ZF2202-A-vaccination. In addition, the mouse experiment results showed that BQ.1.1-XBB.1.5 RBD-heterodimer and XBB.1.5 RBD-homodimer induced high neutralizing responses against XBB sub-variants and BA.2.86, indicating that next-generation COVID-19 vaccine should be developed to enhance the protection efficacy against the circulating strains in the future.

immunology↗

bioRxiv2023

Neutralization of SARS-CoV-2 EG.5/EG.5.1 by sera from ZF2001 RBD-dimer and its next-generation vaccines

SARS-CoV-2 Omicron EG.5 and EG.5.1 are surging in several areas of the world, including China. Compared with XBB.1, EG.5 contains additional mutations of F456L and S486P in the spike protein receptor binding domain (RBD) and its subvariant EG.5.1 carries a further spike mutation Q52H. The immune escape potential of EG.5/EG.5.1 is of great concern. In this study, we evaluated the neutralization activities of sera from participants who received COVID-19 inactivated vaccines, protein subunit vaccine ZF2001 or a booster vaccination of Delta-BA.5 RBD-heterodimer protein vaccine, and participants who had a breakthrough infection during a wave of BF.7/BA.5.2 circulation in December 2022. Neutralization profiles elicited by bivalent RBD-heterodimer vaccine candidates containing XBB.1.5 antigen were evaluated in a murine model. We found that EG.5 and EG.5.1 displayed similar immune evasion potential to XBB.1 and XBB.1.5. The Delta-BA.5 RBD-heterodimer booster induced higher neutralizing titers against the tested XBB subvariants, including EG.5 and EG.5.1, than breakthrough infection by BF.7 or BA.5.2. In addition, Delta-XBB.1.5 and BQ.1.1-XBB.1.5 RBD-heterodimer vaccines induced high neutralizing activities against XBB sub-variants in a murine model, suggesting that next-generation COVID-19 vaccines with updated components must be developed to enhance the protection efficacy against the circulating SARS-CoV-2 strains.

immunology↗

bioRxiv2022

De novo design of site-specific protein interactions with learned surface fingerprints

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1, and CTLA-4. Several designs were experimentally optimized while others were purely generated in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

bioengineering↗

bioRxiv2022

Minor differences in peptide presentation between chicken MHC class I molecules can explain differences in disease susceptibility

The highly polymorphic classical major histocompatibility complexes (MHCs) can confer resistance or susceptibility to diseases. The chicken MHC is known to confer decisive resistance or susceptibility to various economically-important pathogens, including the iconic oncogenic herpesvirus that causes Mareks disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The BF2*1901 is more highly expressed and more thermostable than the closely-related BF2*1501, but the data for peptide repertoire available did not obviously correlate as expected. Here, we confirm for newly-developed chicken lines that the chicken haplotype B15 confers resistance to MD compared to B19. Using gas phase sequencing of peptide pools, and using immunopeptidomics involving mass spectroscopy, we find that the BF2*1901 binds a greater variety of amino acids in some anchor positions than BF2*1501. However, by X-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Though the self-peptides bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows it to tightly accept many more peptides overall, correlating with the expected hierarchies for expression level, thermostability and MD resistance. Moreover, the 2 helix of BF2*1901 is higher than BF2*1501, potentially reducing the number of T cell clones that can recognize this fastidious class I molecule. IMPORTANCEDisease susceptibility mechanism is complicated, but chicken infection of Mareks disease (MD) is one of ideal models, considering the only one highly expressed classical MHC class I, BF2. The different susceptibility of the two close alleles BF2*1901 and BF2*1501 with minor difference of expression and thermostability is still unfathomed. Here, we confirm B15 chicken confers resistance to MD compared to B19. But the BF2*1901 binds a broader variety of anchoring peptides than BF2*1501. This mystery was solved by the structural determination of the two molecules with one similar peptide. The wider and deeper peptide-binding groove of BF2*1501 allows it to tightly accept many more peptides overall, which is concordant to the expected hierarchies for expression level, thermostability and MD resistance.

immunology↗

bioRxiv2021

Reduced sera neutralization to Omicron SARS-CoV-2 by bothinactivated and protein subunit vaccines and the convalescents

Omicron variant continues to spread all over the world. There are lots of scientific questions remaining to be answered for such a devastating variant. There are a dozen of vaccines already in clinical use. The very urgent scientific question would be whether or not these vaccines can protect Omicron variant. Here, we tested the sera from both convalescents and vaccine recipients receiving either inactivated or protein subunits vaccines (CoronaVac from Sinovac, or BBIBP-CoV from Sinopharm, or ZF2001 from Zhifei longcom) for the binding antibody titers (ELISA) and neutralization antibodies titers (pseudovirus neutralization assay). We showed that Omicron do have severe immune escape in convalescents, with 15 of 16 were negative in neutralization. By contrast, in vaccinees who received three jabs of inactivated or protein subunit vaccine, the neutralizing activity was much better preserved. Especially in the ZF2001 group with an extended period of the second and third jab (4-6 months) remains 100% positive in Omicron neutralization, with only 3.1-folds reduction in neutralizing antibody (NAb) titer. In this case, we proposed that, the multi-boost strategy with an extended interval between the second and third jab for immune maturation would be beneficial for NAb against devastating variants such as Omicron.

immunology↗

bioRxiv2021

Structure selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to concentrate the antibody response to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and two variants of concern (VOC), B.1.351 and B.1.617.2. These findings demonstrate that immunogens built on structure selection can focus the response to conserved sites of vulnerability shared between wildtype virus and VOCs and induce neutralizing antibodies across variants.

immunology↗

bioRxiv2021

KIRCLE: An Analysis of Variations in KIR Genes in The Cancer Genome Atlas and UK Biobank

BackgroundNatural killer (NK) cells represent a critical component of the innate immune systems response against cancer and viral infections, among other diseases. To distinguish healthy host cells from infected or tumor cells, killer immunoglobulin receptors (KIR) on NK cells bind and recognize Human Leukocyte Antigen (HLA) complexes on their target cells. Just like the HLAs they bind, these KIRs exhibit high allelic diversity in the human population. ResultsIn order to better understand these immunoreceptors, we have developed KIRCLE, a novel method for genotyping individual KIR genes from whole exome sequencing data, and used it to analyze approximately 60,000 patient samples in The Cancer Genome Atlas and UK Biobank. We were able to assess population frequencies for different KIR alleles and demonstrate that, similar to HLA alleles, individuals KIR alleles correlate strongly with their ethnicities. In addition, we observed associations between different KIR alleles and HLA alleles, including HLA-B*53 with KIR3DL2*013 (Fishers Exact FDR = 7.64e-51). Finally, we showcased statistically significant associations between KIR alleles and various clinical correlates, including peptic ulcer disease (Fishers Exact FDR = 0.0429) and age of onset of atopy and various KIR alleles (Mann-Whitney-U FDR = 0.0751). ConclusionsKIR polymorphism and NK cells play a critical role in many diseases, often through their interactions with HLA complexes. Peptic ulcer disease and atopy are just two diseases in which NK cells may play a role beyond their "classical" realm of anti-tumor and anti-viral responses.

bioinformatics↗

bioRxiv2021

Neutralization of recombinant RBD-subunit vaccine ZF2001-elicited antisera to SARS-CoV-2 variants including Delta

SARS-CoV-2 variants brought new waves of infection worldwide. In particular, Delta variant (B.1.617.2 lineage) has become predominant in many countries. These variants raised the concern for their potential immune escape to the currently approved vaccines. ZF2001 is a subunit vaccine received emergency use authorization (EUA) in both China and Uzbekistan, with more than 100-million doses administrated with a three-dose regimen. The tandem-repeat dimer of SARS-CoV-2 spike protein receptor binding domain (RBD) was used as the antigen. In this work, we evaluated the neutralization of ZF2001-elicited antisera to SARS-CoV-2 variants including all four variants of concern (Alpha, Beta, Gamma and Delta) and other three variants of interest (Epsilon, Eta and Kappa) by pseudovirus-based assay. We found antisera preserved majority of the neutralizing activity against these variants. E484K/Q substitution is the key mutation to reduce the RBD-elicited sera neutralization. Moreover, ZF2001-elicited sera with a prolonged intervals between the second and third dose enhanced the neutralizing titers and resilience to SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A tandem-repeat dimeric RBD protein-based COVID-19 vaccine ZF2001 protects mice and nonhuman primates

A safe, efficacious and deployable vaccine is urgently needed to control COVID-19 pandemic. We report here the preclinical development of a COVID-19 vaccine candidate, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and NHPs, and also elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 g or 50 g ZF2001 prevented infection with SARS-CoV-2 in lung, trachea and bronchi, with milder lung lesions. No evidence of disease enhancement is observed in both models. ZF2001 is being evaluated in the ongoing international multi-center Phase 3 trials (NCT04646590) and has been approved for emergency use in Uzbekistan.

immunology↗

bioRxiv2021

Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat RBD of SARS-CoV-2 spike protein protects mice against COVID-19

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus vectored vaccines expressing spike (S) protein have advanced into phase 3 trials, with three approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD) or dimeric tandem-repeat RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular (Th1-based) immune responses. AdC7-RBD-tr2 showed higher antibody responses compared with both AdC7-S and AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.

immunology↗

bioRxiv2021

Neutralization of SARS-CoV-2 VOC 501Y.V2 by human antisera elicited by both inactivated BBIBP-CorV and recombinant dimeric RBD ZF2001 vaccines

Recently, the emerged and rapidly spreading SARS-CoV-2 variant of concern (VOC) 501Y.V2 with 10 amino acids in spike protein were found to escape host immunity induced by infection or vaccination. Global concerns have been raised for its potential to affect vaccine efficacy. Here, we evaluated the neutralization activities of two vaccines developed in China against 501Y.V2. One is licensed inactivated vaccine BBIBP-CorV and the other one is recombinant dimeric receptor-binding domain (RBD) vaccine ZF2001. Encouragingly, both vaccines largely preserved neutralizing titres, with slightly reduction, against 501Y.V2 authentic virus compare to their titres against both original SARS-CoV-2 and the currently circulating D614G virus. These data indicated that 501Y.V2 variant will not escape the immunity induced by vaccines targeting whole virus or RBD.

microbiology↗

bioRxiv2020

Body site-specific and disease-specific virulome in the human microbiome

Human body habitats are home to a diverse array of microbes, and within these microbial ecosystems, there are exchanges of genetic material, including virulence factors (VFs). Little is known about the diversity and abundance of VFs in different body sites and different types of diseases. We developed a virulome analysis pipeline using the species-specific sequence identity inferred from intraspecies ANI values to precisely assign reads to virulence factors. We characterized the human virulome from four body habitats, including the gut, oral cavity, skin, and vagina. Specifically, the diversity and abundance of VFs in the oral cavity were significantly higher than those in other body sites, including stool. We highlight the importance of sex-specific analysis when studying the human virulome. We analyzed data from more than 4,000 samples across healthy and diseased subjects and 13 types of diseases from different metagenomic sequencing studies to characterize the disease-specific virulome. Atherosclerotic cardiovascular disease (ACVD) has a more diverse virulome than other diseases tested. Notably, many VFs, including genes for secretion systems and toxins, are more abundant in diseased subjects than in healthy controls. We present, to our knowledge, the most comprehensive healthy and diseased virulome dataset yet created.

genomics↗

bioRxiv2020

A Mycobacterium tuberculosis effector protein attacks host innate immunity by acting as an unusual ubiquitinating enzyme

Protein kinase G (PknG), a eukaryotic type serine-threonine protein kinase (STPK) in Mycobacterium tuberculosis (Mtb), is secreted into the cytosol of infected macrophages to promote intracellular survival of mycobacteria and has been considered as a promising therapeutic target for tuberculosis (TB) treatment. However, the molecular details of Mtb PknG-host intracellular interactions remain obscure. Here, we demonstrate that PknG serves as both the ubiquitin-activating enzyme (E1) and the ubiquitin ligase (E3) to promote ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-{beta}-activated kinase 1 (TAK1), and thus inhibits the NF-{kappa}B-mediated host innate immune responses. Surprisingly, PknG promotes the attachment of ubiquitin (Ub) to ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), instead of a usual C86-Ub thiol-ester bond, and then promotes the discharge of Ub from UbcH7 by acting as an isopeptidase before attaching Ub to its substrates TRAF2 and TAK1. These results demonstrate that Mtb PknG promotes ubiquitination of the key components of the host innate immunity by acting as an unusual ubiquitinating enzyme to suppress innate immunity. Our findings provide a potential TB treatment via targeting unconventional ubiquitinating activities of PknG. SignificanceMycobacterium tuberculosis (Mtb) protein kinase G (PknG), which is critical for Mtb intracellular survival, is a promising target for tuberculosis (TB) treatment. However, the molecular mechanisms underlying PknG-host interactions remain largely unclear. Here we demonstrate that PknG serves as both the ubiquitin-activating enzyme and the ubiquitin ligase to promote the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-{beta}-activated kinase 1 (TAK1), thus inhibiting NF-{kappa}B signaling activation. PknG promotes the attachment of ubiquitin to ubiquitin-conjugating enzyme UbcH7 via an isopeptide bond, instead of a usual thiol-ester bond, and releases the ubiquitin from UbcH7 by acting as an isopeptidase. These findings provide important information for rational development of TB treatment via targeting unconventional ubiquitinating activity of PknG.

microbiology↗

bioRxiv2020

Recombinant SARS-CoV-2 RBD molecule with a T helper epitope as a built in adjuvant induces strong neutralization antibody response

Without approved vaccines and specific treatment, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading around the world with above 20 million COVID-19 cases and approximately 700 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of Spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5), and prokaryotic expression, chromatography purification and the rational renaturation of the protein were performed. The antigenicity and immunogenicity of S1-4 protein was evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency of it was measured by virus neutralization test in Vero E6 cells with SARS-CoV-2. S1-4 protein was prepared to high homogeneity and purity by prokaryotic expression and chromatography purification. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity compared with S1-5 protein. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralization antibody GMT 256. The candidate subunit vaccine molecule could stimulate strong humoral and Th1 and Th2-type cellular immune response in mice, giving us solid evidence that S1-4 protein could be a promising subunit vaccine candidate.

bioengineering↗