Challenges in the Development of a Vaccine Against COVID-19

1.引言由严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的新型冠状病毒肺炎(COVID-19)目前已在全球大规模暴发,已成为重大的公共卫生危机事件[1]。严峻的疫情形势凸显了有效的治疗和预防方案的必要性。一些国家已经加快了开发安全有效疫苗的临床试验进程,从而遏制目前疫情的发展[2]。

Increase of CD4^+CD25^+ T cells in Smad3^(-/-) mice

AIM： To investigate the changes of lymphocyte subpopulations, especially CD4^＋CD25^ T regulatory cells in Smad3^-/- mice. METHODS： Hematological changes and changes of lymphocyte subpopulations were detected in Smad3＂/- mice using cell counter and flow cytometry, respectively, and compared to their littermate controls. RESULTS： The numbers of neutrophils and lymphocytes in peripheral blood were significantly increased in Smad3^-/- mice compared to littermate controls. CD19^＋ expressing cells in blood and spleen, and CD8^＋ T cells in thymus were all markedly decreased in Smad3^-/- mice. More important, Smad3^-/- mice had an increased population of CD4^＋CD25^＋ T cells in peripheral lymphoid tissues, including thymus, spleen, and lymph nodes. CONCLUSION： These observations suggest that the changes of lymphocyte subpopulations might play a role in susceptibility to inflammation of Smad3^-/- mice.

TRAF6 polymorphisms not associated with the susceptibility to and severity of sepsis ina Chinese population

BACKGROUND： The tumor necrosis factor recepter associated factor （TRAF） 6 is an important intracellular adapter protein that plays a pivotal role in activating multiple inflammatory and immune related processes induced by cytokines. TRAF6 represents a strong candidate susceptibility factor for sepsis. We investigated whether polymorphisms at the TRAF6 gene are associated with the susceptibility to and severity of sepsis.METHODS： A hospital-based case-control study was conducted with 255 patients with sepsis and 260 controls who were recruited from Zhengzhou, China. Haplotype tagging single nucleotide polymorphisms （htSNPs） were selected from the HapMap database and genotyped using the SNPstream genotyping platform. The associations with the susceptibility and disease severity of sepsis were estimated by logistic regression, and adjusted for age, sex, smoking, drinking, chronic diseases status, APACHEII score and critical illness status.RESULTS： A total of 13 TRAF6 SNPs were tagged by 7 htSNPs. Five htSNPs （rs5030490, rs5030411, rs5030416, rs5030445 and rs3740961） were genotyped in the case control study. Genotype frequencies of the htSNPs were conformed to the Hardy-Weinberg equilibrium in both patients and controls. No significant association was found between the 5 htSNPs and the susceptibility to and severity of sepsis. Compared with the main haplotype -11120A/-10688T/-9423A/805G/12967G, no certain haplotype was associated with the signi？ cantly susceptibility to or severity of sepsis.CONCLUSION： TRAF6 gene polymorphisms might not play a major role in mediating the susceptibility to and severity of sepsis in the Chinese population. A larger population-based case-control study is warranted.

Construction and evaluation of anti-gastrin immunogen based on P64K protein

AIM： To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria rneningitids and to compare their immunogenic effect. METHODS： G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21（DE3）. After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done, Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. RESULTS： G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84^th day after the first immunization, the titer of antibody against cross-linked protein reached 51 200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. CONCLUSION： The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.

Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer

BACKGROUND Due to the poor prognosis of gastric cancer(GC),early detection methods are urgently needed.Plasma exosomal circular RNAs(circRNAs)have been suggested as novel biomarkers for GC.AIM To identify a novel biomarker for early detection of GC.METHODS Healthy donors(HDs)and GC patients diagnosed by pathology were recruited.Nine GC patients and three HDs were selected for exosomal whole-transcriptome RNA sequencing.The expression profiles of circRNAs were analyzed by bioinformatics methods and validated by droplet digital polymerase chain reaction.The expression levels and area under receiver operating characteristic curve values of plasma exosomal circRNAs and standard serum biomarkers were used to compare their diagnostic efficiency.RESULTS There were 303 participants,including 240 GC patients and 63 HDs,involved in the study.The expression levels of exosomal hsa_circ_0079439 were significantly higher in GC patients than in HDs(P<0.0001).However,the levels of standard serum biomarkers were similar between the two groups.The area under the curve value of exosomal hsa_circ_0079439 was higher than those of standard biomarkers,including carcinoembryonic antigen,carbohydrate antigen(CA)19-9,CA72-4,alpha-fetoprotein,and CA125(0.8595 vs 0.5862,0.5660,0.5360,0.5082,and 0.5018,respectively).The expression levels of exosomal hsa_circ_0079439 were significantly decreased after treatment(P<0.05).Moreover,the expression levels of exosomal hsa_circ_0079439 were obviously higher in early GC(EGC)patients than in HDs(P<0.0001).CONCLUSION Our results suggest that plasma exosomal hsa_circ_0079439 is upregulated in GC patients.Moreover,the levels of exosomal hsa_circ_0079439 could distinguish EGC and advanced GC patients from HDs.Therefore,plasma exosomal hsa_circ_0079439 might be a potential biomarker for the diagnosis of GC during both the early and late stages.

A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses

In 2020 and 2021,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),a novel coronavirus,caused a global pandemic.Vaccines are expected to reduce the pressure of prevention and control,and have become the most effective strategy to solve the pandemic crisis.SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2(ACE2)via the receptor-binding domain(RBD)of the surface spike(S)glycoprotein.In this study,a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells.The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody.Furthermore,the specific antibody titer and virus-neutralizing antibody(NAb)titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)_(3) and CpG.Our results showed that the virus-NAb lasted for more than six months in mice.To summarize,we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris,which stimulates neutralizing and protective antibody responses.A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

Biosynthesis and Immunological Evaluation of a Dual-Antigen Nanoconjugate Vaccine Against Brucella melitensis

Brucellosis,caused by Brucella,is one of the most common zoonosis.However,there is still no vaccine for human use.Although some live attenuated vaccines have been approved for animals,the protection effect is not ideal.In this study,we developed a dual-antigen nanoconjugate vaccine containing both polysaccharide and protein antigens against Brucella.First,the antigenic polysaccharide was covalently coupled to the outer membrane protein Omp19 using protein glycan coupling technology,and then it was successfully loaded on a nano-carrier through the SpyTag/SpyCatcher system.After confirming the efficient immune activation and safety performance of the dual-antigen nanoconjugate vaccine,the potent serum antibody response against the two antigens and remarkable protective effect in non-lethal and lethal Brucella infection models were further demonstrated through different routes of administration.These results indicated that the dual-antigen nanoconjugate vaccine enhanced both T helper 1 cell(Th1)and Th2 immune responses and protected mice from Brucella infection.Furthermore,we found that this protective effect was maintained for at least 18 weeks.To our knowledge,this is the first Brucella vaccine bearing diverse antigens,including a protein and polysaccharide,on a single nanoparticle.Thus,we also present an attractive technology for co-delivery of different types of antigens using a strategy applicable to other vaccines against infectious diseases.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder promote ferroptosis in oral squamous cell carcinoma

Objective: Ferroptosis is a novel cell death process which displays a promising role in cancer treatment.However, clinically available drugs targeting ferroptosis are rarely used, and yet there are no studies reporting on inducing ferroptosis via Chinese herbal extracts. Here we explored the tumor inhibition effects of Ganoderma lucidum(G. lucidum) on oral squamous cell carcinoma(OSCC). Specifically, we aimed to clarify the biological mechanism of components in the dietary, aqueous-soluble sporoderm-removed G. lucidum spore powder(A-GSP).Methods: Preliminary transcriptome analysis revealed the significant enrichment of the ferroptosis pathway.Cellular Fe2+, glutathione(GSH), malondialdehyde(MDA), reactive oxygen species(ROS) and lipid peroxide levels were measured to identify ferroptosis occurrence. Western blotting was used to measure ferroptosis-related proteins. Changes in mitochondria morphology and function were observed with transmission electron microscopy(TEM) and ATP detection assays. Ferroptosis inhibitor ferrostatin-1 was then used to verify the anti-tumor effects of A-GSP. Finally, nude mice xenograft models of oral cancer confirmed that A-GSP inhibited tumor growth.Results: A-GSP promoted ferroptosis in oral cancer cells by inducing Fe2+influx, GSH depletion, as well as lipid peroxide and ROS accumulation. Ferroptosis-related proteins exhibited corresponding changes, particularly Acylco A synthetase long chain family member 4(ACSL4) increase and glutathione peroxidase 4(GPX4) decrease. A-GSP considerably lowered mitochondrial volume and ridge number, while significantly decreasing ATP production. Ferrostatin-1 reversed all of these A-GSP-induced changes. In vivo, A-GSP exerted a ferroptosismediated tumor-suppressing effect without observable adverse reactions.Conclusions: Our findings demonstrate the therapeutic potential of A-GSP for treating patients with OSCC by targeting ferroptosis.

Bioinformatics Identification of ZNFs/LINC00520/miR-181d/BCL2 Axis as a Novel Network in Cisplatin-Resistant Lung Adenocarcinoma Cells

Background: Resistance to cisplatin (DDP) leads to poor prognosis in patients with Lung Adenocarcinoma (LUAD) and limits its clinical application. It has been confirmed that autophagy promotes chemoresistance and, therefore, novel strategies to reverse chemoresistance by regulating autophagy are desperately needed. Methods: The differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs), and mRNAs (DEmRNAs) between A549 and A549/DDP cell lines were identified using the limma package in R, after gene expression profiles were obtained from Gene Expression Omnibus (GEO) database. By combining Autophagy-Related Genes (ARGs) from Human Autophagy Database (HADb), the interactions lncRNA-miRNAs and the interactions miRNAs-mRNAs respectively predicted by miRcode and miRDB/Targetscan database, the autophagy-related ceRNA network was constructed. Then, extraction of ceRNA subnetwork and Cox regression analyses were performed. A prognosis-related ceRNA subnetwork was constructed, and the upstream Transcription Factors (TFs) regulating lncRNAs were predicted by the JASPAR database. Finally, the expression patterns of candidate genes were further verified by quantitative real-time polymerase chain reaction (qRT-PCR) experiments. Results: A total of 3179 DEmRNAs, 180 DEmiRNAs, and 160 DElncRNAs were identified, and 35 DEmRNAs were contained in the HADb. Based on the ceRNA hypothesis, we established a ceRNA network, including 10 autophagy-related DEmRNAs, 9 DEmiRNAs, and 14 DElncRNAs. Then, LINC00520, miR-181d, and BCL2 were identified to construct a risk score model, which was confirmed to be a well-predicting prognostic factor. Furthermore, 5 TF ZNF family members were predicted to regulate LINC00520, whereas the RT-PCR results showed that the 5 ZNFs were consistent with the bioinformatics analysis. Finally, a ZNF regulatory LINC00520/miR-181d/BCL2 ceRNA subnetwork was constructed. Conclusions: An ZNFs/LINC00520/miR-181d/BCL2 axis as a novel network in DDP-resistant LUAD has been constructed successfully, which may provide potential therapeutic targets for LUAD.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma(HCC) tissues and associated with hepatocarcinogenesis.However,the exact mechanism of EZH2 up-regulation in HCC has not been determined.In this study,we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein(HBx) in regulating the expression of mEZH2.Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner.To further investigate the mechanism of mEZH2 overexpression,the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid.The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid,and deleting the-486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%.The-486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found.Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells.These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor,thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Drosophila Eyes Absent Homologue 2 is up-regulated in lung adenocarcinoma

Objective： Lung cancer has emerged as a leading cause of cancer death in the world. Eyes Absent （EYA） is an important and conserved transcriptional regulator of development. The aim of the present study was to identify the expression of Drosophila Eyes Absent Hemologue 2 （EYA2） in non-small cell lung cancer （NSCLC） and to investigate their correlation with clinical parameters. Methods： Fresh, paired lung samples （n = 59） of NSCLC were obtained by surgical resection at the Department of Thoracic Surgery of the People＇s Liberation Army General Hospital. Expression of EYA2 were examined by Western blot and immunohistochemical analysis in specimens of NSCLC and paired normal lung tissue. Clinical data, pathologic result and Ki67 expression were collected and subsequent correlation with EYA2 expression was analyzed. Results： EYA2 expression was found located in cytoplasm and nucleus, but mostly in cytoplasm. The expression of EYA2 increased in NSCLC by Western blot and immunohistochemistry, which was correlated with histology type, but not correlated with gender, age, pTNM stage, histological differentiation and lymph node metastasis. Compared with normal lung tissue, the expression of EYA2 significantly was up-regulated in lung adenocarcinoma, while no significant difference in lung squamous cell carcinoma. Expression of EYA2 was uncorrelated with expression of Ki67 in NSCLC. Conclusion： Expression of EYA2 was augmented in lung adenocarcinoma. EYA2 is likely participating in tumorigenesis and development of lung adenocarcinoma as transcriptional activator.

Heterologous expression of the maize transcription factor ZmbHLH36 enhances abiotic stress tolerance in Arabidopsis

Basic helix-loop-helix(bHLH)transcription factors are widely distributed in eukaryotes,and in plants,they regulate many biological processes,such as cell differentiation,development,metabolism,and stress responses.Few studies have focused on the roles of bHLH transcription factors in regulating growth,development,and stress responses in maize(Zea mays),even though such information would greatly benefit maize breeding programs.In this study,we cloned the maize transcription factor gene ZmbHLH36(Gene ID:100193615,GRMZM2G008691).ZmbHLH36 possesses conserved domains characteristic of the bHLH family.RT-qPCR analysis revealed that ZmbHLH36 was expressed at the highest level in maize roots and exhibited different expression patterns under various abiotic stress conditions.Transgenic Arabidopsis(Arabidopsis thaliana)plants heterologously expressing ZmbHLH36 had significantly longer roots than the corresponding non-transgenic plants under 0.1 and 0.15 mol L^(−1) NaCl treatment as well as 0.2 mol L^(−1) mannitol treatment.Phenotypic analysis of soil-grown plants under stress showed that transgenic Arabidopsis plants harboring ZmbHLH36 exhibited significantly enhanced drought tolerance and salt tolerance compared to the corresponding non-transgenic plants.Malondialdehyde contents were lower and peroxidase activity was higher in ZmbHLH36-expressing Arabidopsis plants than in the corresponding non-transgenic plants.ZmbHLH36 localized to the nucleus when expressed in maize protoplasts.This study provides a systematic analysis of the effects of ZmbHLH36 on root growth,development,and stress responses in transgenic Arabidopsis,laying a foundation for further analysis of its roles and molecular mechanisms in maize.

Construction of a bivalent vaccine candidate against HAdV4/HAdV7 based on capsid-display strategy via Red-homologous recombination and counter-selection methodology

Human adenoviruses(HAdvs)are major respiratory pathogens.Specifically,human adenovirus type 4(HAdV4)and human adenovirus type 7(HAdV7)are known for causing fever and pneumonia,with docu-mented cases of fatalities among the population.In recent years,HAdV4/HAdv7 has been implicated in caus-ing substantial outbreaks,leading to increased morbidity in multiple countries.Most HAdV4 and HAdV7 infections have been reported in North America,Asia,Europe,Africa,South America,Oceania,and the Middle East.Most fatalities occurred in North America(the United States)and Asia(China and Singapore).Engineered recombinant adenoviruses have played a crucial role as vaccine vectors.In this study,we con-structed a recombinant adenovirus,Ad4ITRmut-Ad7E3,and evaluated it in vitro and in vivo.We observed that the replication rate of Ad4ITRmut-Ad7E3 was lower than that of the RI-67 strain,indicating that the mutation of inverted terminal repeats(ITRs)weakened the replication ability of HAdV4.Immunization of BALB/c mice with the bivalent Ad4ITRmut-Ad7E3 vaccine strain,administered by intraperitoneal injection and oral gavage,resulted in the elicitation of neutralizing antibodies targeting HAdV4 and HAdv7.This finding not only pro-vides a novel method and technique for the efficient construction of a polyvalent recombinant adenovirus vac-cine candidate against HAdV4 and HAdv7 but also against other prevalent adenovirus serotypes such as HAdV3,HAdV11,HAdV14,and HAdv55,from various regions.

Pseudomonas aeruginosa infections and improper storage conditions influence the performance of 1,3‐β‐D‐glucan in diagnosis of invasive fungal infections

Background:The association between 1,3-β-D-glucan(BDG)levels and in-fections caused by Pseudomonas aeruginosa or Streptococcus pneumoniae,and the stability of BDG under different storage conditions are unclear.Methods:Strains of Pseudomonas aeruginosa and S.pneumoniae were grown in medium and human serum.The BDG concentrations in culture superna-tants were measured.The specificity and stability of BDG were also evaluated.Results:P.aeruginosa produced high levels of BDG in Luria-Bertani medium(>4×10^(4)pg/mL)and human serum(527.0 pg/mL),whereas S.pneumoniae produced low levels of BDG in THY medium(175.6 pg/mL)and human serum(78.3 pg/mL).The BDG produced by these two bacteria was specifically degraded by 1,3-β-D-glucanase.BDG was degraded when stored at different temperatures,decreasing by 22.5%and 9.3%at−20℃and−70℃,respectively,for 63 days;by 30.7%at 4℃for 12 days;and by 12.6%and 22.0%at 37℃for 6 and 12 h.Conclusion:BDG false-positivity must be considered in patients with bacteremia caused by P.aeruginosa when diagnosing invasive fungal infection.Human serum samples for the BDG test in medical facilities should be tested as soon as possible or stored at low temperatures before testing.

Effects of traditional Chinese medicine on treatment outcomes in severe COVID-19 patients:a single-centre study

As the search for effective treatments for COVID-19 continues,the high mortality rate among critically ill patients in Intensive Care Units(ICU)presents a profound challenge.This study explores the potential benefits of traditional Chinese medicine(TCM)as a supplementary treatment for severe COVID-19.A total of 110 critically ill COVID-19 patients at the Intensive Care Unit(ICU)of Vulcan Hill Hospital between Feb.,2020,and April,2020(Wuhan,China)participated in this observational study.All patients received standard supportive care protocols,with a subset of 81 also receiving TCM as an adjunct treatment.Clinical characteristics during the treatment period and the clinical outcome of each patient were closely monitored and analysed.Our findings indicated that the TCM group exhibited a significantly lower mortality rate compared with the non-TCM group(16 of 81 vs 24 of 29;0.3 vs 2.3 person/month).In the adjusted Cox proportional hazards models,TCM treatment was associated with improved survival odds(P<0.001).Furthermore,the analysis also revealed that TCM treatment could partially mitigate inflammatory responses,as evidenced by the reduced levels of proinflammatory cytokines,and contribute to the recovery of multiple organic functions,thereby potentially increasing the survival rate of critically ill COVID-19 patients.

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins:haemagglutinin,neuraminidase,and matrix protein(M1 and M2).Among them,M2 protein functions as an ion channel,important for virus uncoating in endosomes of virus-infected cells and essential for virus replication.In an effort to explore potential new functions of M2 in the virus life cycle,we used yeast two-hybrid system to search for M2-associated cellular proteins.One of the positive clones was identified as human Hsp40/Hdj1,a DnaJ/Hsp40 family protein.Here,we report that both BM2(M2 of influenza B virus)and A/M2(M2 of influenza A virus)interacted with Hsp40 in vitro and in vivo.The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40.Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^(IPK) that is a cellular inhibitor of PKR.PKR is a crucial component of the host defense response against virus infection.We therefore attempted to understand the relationship among M2,Hsp40 and p58^(IPK) by further experimentation.The results demonstrated that both A/M2 and BM2 are able to bind to p58^(IPK)in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^(IPK),and consequently induce cell death.These results suggest that influenza virus M2 protein is involved in p58^(IPK)mediated PKR regulation during influenza virus infection,therefore affecting infected-cell life cycle and virus replication.

Orthogonal modular biosynthesis of nanoscale conjugate vaccines for vaccination against infection

Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.

Hepatitis C Virus Infection Downregulates the Ligands of the Activating Receptor NKG2D

Natural killer （NK） cells are a major component of the host innate immune defense against various pathogens. Several viruses, including hepatitis C virus （HCV）, have developed strategies to evade the NK-cell response. In our study, we found HCV infection could trigger DNA damage response by both ataxia telangiectasia mutated （ATM） and ATM- and Rad3-related （ATR） pathways. Recent reports had revealed that NKG2D ligands （NK cell- activating ligands） were upregulated when a major DNA damage checkpoint pathway was activated. However, here we found that DNA damage response was activated but NKG2D ligands were downregulated upon HCV infection. Further studies showed that the protease NS3/4A of HCV which had been shown relation with immune invasion contributed to the reduced expression of NKG2D ligands. These findings provide a novel insight into the mechanisms evolved by HCV to escape from the NK cell response. Cellular ＆ Molecular Immunology. 2008;5（6）： 475-478.

Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd 'host/ Asd+ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the challenges of the above three Shigella strains.