Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to the insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). Not only displays the pSpike/PP-sNp superior gene-transfection and favorable biocompatibility in the mouse airway, but pSpike/PP-sNp promotes a tripartite immunity consisting of systemic, cellular and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp might be a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Therapeutic Drugs Targeting 2019-nCoV Main Protease by High-Throughput Screening

2019 Novel Coronavirus (2019-nCoV) is a virus identified as the cause of the outbreak of pneumonia first detected in Wuhan, China. Investigations on the transmissibility, severity, and other features associated with this virus are ongoing. Currently, there is no vaccine or therapeutic antibody to prevent the infection, and more time is required to develop an effective immune strategy against the pathogen. In contrast, specific inhibitors targeting the key protease involved in replication and proliferation of the virus are the most effective means to alleviate the epidemic. The main protease of SARS-CoV is essential for the life cycle of the virus, which showed 96.1% of similarity with the main proteaseof 2019-nCoV, is considered to be an attractive target for drug development. In this study, we have identified 4 small molecular drugs with high binding capacity with SARS-CoV main protease by high-throughput screening based on the 8,000 clinical drug libraries, all these drugs have been widely used in clinical applications with guaranteed safety, which may serve as promising candidates to treat the infection of 2019-nCoV.

Progress and challenge of vaccine development against 2019 novel coronavirus (2019-nCoV) / 中华预防医学杂志

The outbreak of 2019 novel coronavirus (2019-nCoV) infection poses a serious threat to global public health. Vaccination is an effective way to prevent the epidemic of the virus. 2019-nCoV along with severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the same β-genus of coronavirus family. Base on the previous experience and the technical platform of developing SARS-CoV and MERS-CoV vaccines, scientists from all over the world are working hard and quickly on the related fields. There are substantial progress in these fields including the characterizing the 2019-nCoV virus, identification of candidate antigens and epitopes, establishment of animal models, characterizing the immune responses, and the design of vaccines. The development of 2019-nCoV vaccines cover all types: inactivated virus vaccine, recombinant protein vaccine, viral vector-based vaccine, mRNA vaccine, and DNA vaccine, et al. As of March 2020, two 2019-nCoV vaccines have entered phase I clinical trials. One is named as Ad5-nCoV developed by the Chinese Institute of Biotechnology of the Academy of Military Medical Sciences and Tianjin Cansino Biotechnology Inc. Ad5-nCoV is based on the replication-defective adenovirus type 5 as the vector to express 2019-nCoV spike protein. The another vaccine is mRNA-1273 developed by the National Institute of Allergy and Infectious Diseases and Moderna, Inc.. RNA-1273 is an mRNA vaccine expressing 2019-nCoV spike protein. Although the rapid development of 2019-nCoV vaccine, it still faces many challenges with unknown knowledge, including the antigenic characteristics of the 2019-nCoV, the influence of antigenic variation, the protective immune response of host, the protection of the elderly population, and the downstream manufacturing process of the new vaccine. The safety and efficacy of vaccines are the first priority for vaccine development and should be carefully evaluated.

Practice and evaluation of the elective course "Structural Biology" in biotechnological pharmaceutics teaching for biotechnology undergraduates / 中华医学教育探索杂志

Biotechnological Pharmaceutics is a compulsory course for biotechnology undergraduates in our school.We designed and implemented an elective course named Structural Biology to help students master technological principles through practice.This elective course included in-classroom lectures and experiments; during which we encouraged students to work together,and design,prepare,implement,and complete projects; examination score of Biotechnological Pharmaceutics was used to assess learning outcomes.The results showed that students who took this course gained higher score in the examination,indicating that the elective course is effective to improve the learning effect of Biotechnological Pharmaceutics for biotechnology undergraduates.

Practice and evaluation of the elective course "Structural Biology" in biotechnological pharmaceutics teaching for biotechnology undergraduates / 中华医学教育探索杂志

Biotechnological Pharmaceutics is a compulsory course for biotechnology undergraduates in our school. We designed and implemented an elective course named Structural Biology to help students master technological principles through practice. This elective course included in-classroom lectures and experiments; during which we encouraged students to work together, and design, prepare, implement, and complete projects; examination score of Biotechnological Pharmaceutics was used to assess learning outcomes. The results showed that students who took this course gained higher score in the examination, indicating that the elective course is effective to improve the learning effect of Biotechnological Pharmaceutics for biotechnology undergraduates.

"Teaching reformation in biotechnological pharmaceutics based on ""Excellent Engineer Education and Training Plan""" / 中华医学教育探索杂志

Biotechnological pharmaceutics is an important course for pharmacy undergraduates , however there are many problems in current curriculum design and teaching methods. With the advantage of our platform National Engineering Research Center of Immunological Products, we tried to carry out the teaching reformation based on Excellent Engineer Education and Training Plan. We optimized the curriculum standards and teaching design, highlighted the combination ofBiotechnological pharmaceuticsand Engineering, strengthened the experiment teaching, tried the reformation of teaching method such as flipped classroom and PBL, strengthened the cultivation of innovative thinking and scientific research ability. Our teaching reformation is beneficial to cultivating the compound talents in the field of biotechnological pharmaceutics.

Anti-helicobacter pylori effect of total alkaloids of sophora alopecuroides in vivo / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Helicobacter pylori (H. pylori) infection could lead to most gastroduodenal diseases and is even identified as a carcinogen of gastric cancer. Total alkaloids of sophora alopecuroides (TASA) is widely used in herbal remedies to treat various infectious diseases, including stomach-associated diseases. This study is aimed at evaluating the antimicrobial activity of TASA on H. pylori-infected BALB/c mice mouse gastritis.</p><p><b>METHODS</b>Totally 120 BALB/c mice were orally inoculated with H. pylori Bacterial liquid to construct BALB/c mice H. pylori infection gastritis animal model, after the model was successfully created. We randomly assigned 100 infected mice into 10 treatment groups, the first group (normal saline); the second group (bismuth pectin); the third group (omeprazole); the fourth group (TASA 2 mg/d); the fifth group (TASA 4 mg/d); the sixth group (TASA 5 mg/d); the seventh group (TASA + bismuth pectin); the eighth group (TASA + omeprazole); the ninth group (bismuth pectin + clarithromycin + metronidazole); the tenth group (omeprazole + clarithromycin + metronidazole), 5 other non-infected mice as negative control. Mice were orally inoculated twice a day and 7 days continuously. Then the mice were killed 4 weeks after treatment, we used realtime PCR to detect 16sDNA of H. pylori to test both the colonization and the clearance mice of bacteria of each treatment. We applied hematoxylin and eosin (HE) staining and immunostaining of mice gastric mucosa to observe the general inflammation and related factors interleukin 8 (IL-8), cyclooxygenase 2 (COX-2), and nuclear factor-kappa B (NF-κB) expression change after treatments.</p><p><b>RESULTS</b>Firstly, we ensured that after 6-week intragastric administration, the bacteria colonization reached an exceed peak which is far higher than positive threshold (P < 0.001); secondly, after treatments, it is revealed that TASA combined with omeprazole or bismuth pectin showed promising antimicrobial activity against H. pylori as well as conventional triple therapy (P < 0.001); thirdly, HE staining showed that the inflammation on mice gastric mucosal membrane were also relieved obviously in TASA combined treatments and conventional triple therapy compared with normal saline treated mice, moreover, from immunohistochemistry results, H. pylori-induced IL-8, COX-2, and NF-κB were consistently suppressed in seventh, eighth, ninth, and tenth group to a certain extent.</p><p><b>CONCLUSION</b>These results open the possibility of taking TASA as an anti-inflammatory agent for H. pylori gastritis.</p>

Isolation and purification of recombinant VacA and Helicobacter pylori-secreted VacA and VacA-induced cell vacuolar change and apoptosis / 军事医学

Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.

Study on correlation between helicobacter pylori infection-associated gastritis and the apoptotic genes in gastric mucosa / 重庆医学

Objective To investigate the correlation between Helicobacter pylori (HP) infection-associated gastritis and the ap-optotic genes in gastric mucosa .Methods Forty-five patients with chronic gastritis were registrated in our study from November 2013 to December 2014 .HP infection status in the patients was detected by using urease test and 13C-urea breath test .The degree of gastritis in the gastric mucosa with HP infection was confirmed via histopathology .qRT-PCR was used to measure the mRNA ex-pressions of Bax ,Bak and Bcl-2 in the gastric mucosa with HP infection and matched normal gastric mucosa .Person analysis was used to assess the correlation between the HP infection-associated gastritis and the mRNA expressions of Bax ,Bak and Bcl-2 in the gastric mucosa .Results Forty-five patients with HP infection in antrum and 45 patients (100% ) with chronic antrum gastritis were identified ,including 28 patients (62 .2% ) with light gastritis ,16 patients (35 .6% ) with moderate gastritis ,1 patient (2 .0% ) with severe gastritis .9 patients (20 .0% )with metaplasia ,5 patients(11 .1% ) with low grade intraepithelial neoplasms .The urease tests were negative in the gastric body of 45 patients ,6 patients (13 .3% )were mild chronic gastritis in the body ;Patient with meta-plasia and intrapithelial gastritis was not found .The Bax expression in the HP-infected gastric mucosa was markedly increased when compared with the normal gastric mucosa (P0 .05) .Conclusion HP infec-tion-associated gastritis positively correlated with the expressions of apoptotic genes in gastric mucosa ,suggesting that HP infection might result in increasing the Bax expression and further enhancing the cell apoptosis .

Optimization of innovation education system in medical undergraduate teaching practice / 中华医学教育探索杂志

This paper analyzed innovative education management system,innovative education resources,innovative education mode and innovative education evaluation standard through practicing‘integration of production,study and research’ teaching platform. Meanwhile,this paper explored the optimization system of innovative education in medical undergraduate teaching.

Practice and experience on carrying out the second class of structural biology for students major in biotechnology / 中华医学教育探索杂志

The research of structural biology is closely related to the field of biotechnology.Carrying out the second class of structural biology will be helpful in learning theoretical knowledge of biotechnology and in improving the teaching quality.Based on our experiences of carrying out the second classroom activities among students majoring in biotechnology,we believe that the selected teaching contents,reasonable classroom design,sufficient preparation before the class,flexible teaching methods and objective after-school summaries are essential for improving the teaching quality.

The characteristics of IL-22 in Helicobacter pylori infection / 中华微生物学和免疫学杂志

Objective To investigate the characteristics of IL-22 in Helicobacter pylori (H.pylori)infection.Methods Thirty H.pyloripositive and fifteen H.pylori negative gastric biopsy specimens were enrolled,IL-22 mRNA expression was detected by real-time PCR and the protein level of IL-22 was determined by ELISA in gastric tissue.The H.pyloriand cell coculture system was established and IL-22 expression was measured by real-time PCR to investigate the main source of IL-22 in gastric tissue.The IL-22-producing T cell was examined by FCM in the gastric mucosa tissue.Results Gastric mucosal IL-22 mRNA and protein levels were significantly higher in H.pylori-positive patients than uninfected patients (P＜0.05).A H.pyloriand cell coculture system was established successfully and gastric epithelial cell and T cell were the main source of IL-22 in gastric tissue.IL-22 was produced by CD4+ and CD8+ T cells and these T cells were increased in H.pylori-infected gastric mucosa (P＜0.05).Conclusion IL-22 took part in H.pyloriinfection induced immune response and increased IL-22-producing T cells was the important feature of H.pyloriinfection.

Experience of carrying out the second class of biotechnological pharmaceutics / 中华医学教育探索杂志

One of the aims of biotechnological pharmaceutics is to educate innovative highquality medical talents.The second classroom activities can be used as a complementation to ensure the teachiug effectiveness of the course.This study focuses on the experience of carrying out the second class of biotechnological pharmaceutics and tends to provide references for teachers who are going to carry out the second class.

The combination characteristics of EHEC O157 ∶H7 intimin and it's mutant intiminN916Y with translocated intimin receptor / 中华微生物学和免疫学杂志

Objective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.

Fusion expression and immunogenicity of Bordetella pertussis PTS1-FHA protein: implications for the vaccine development.

Mutants of pertussis toxin (PT) S1 subunit and filamentous hemagglutinin (FHA) type I immunodominant domain from Bordetella pertussis (B. pertussis) are considered to be effective candidate antigens for acellular pertussis vaccines; however, the substantial progress is hampered in part for the lack of a suitable in vitro expression system. In this paper, the gene sequences of a S1 mutant C180-R9K/E129G (mS1) and a truncated peptide named Fs from FHA type I immunodominant domain were linked together and constructed to pET22b expression vector as a fusion gene; after inducing with IPTG, it was highly expressed in E. coli BL21 (DE3) as inclusion body. The fusion protein FsmS1 was purified from cell lysates and refolded successfully. The result of Western blotting indicate that it was able to react with both anti-S1 and anti-FHA McAbs; antiserum produced from New Zealand white rabbits immunized with this protein was able to recognize both native PT and FHA antigens as determined by western blotting. These data have provided a novel feasible method to produce PT S1 subunit and FHA type I immunodominant domain in large scale in vitro, which is implicated for the development of multivalent subunit vaccines candidate against B. pertussis infection.

Recombinant expression and biological activities of fusion protein EspA-Stx2B from enterohemorrhagic E. coil O157:H7 / 中华微生物学和免疫学杂志

Objective To clone the gene encoding protein of EspA and Stx2B from EHEC OI57:H7 by DNA recombinant technology, construct prokaryotic expression vector pET-28a ( + )-espAstx2B, express fusion protein of EspA-Stx2B and to analyze the biological and immunological characteristics of the fusion protein. Methods the sequence encoding the protein of EspA and Stx2B was amplified by PCR from the enterohemorrhagic Escherichia coli strain. The amplified products were connected with linker by recombinant technology and cloned into pET-28a( + ) vector. The vector was then transferred to the host cells E. Coli BL21 strain (DE3). Following, the protein expression was induced by IPTG. The expression quantities and style of fusion protein was then determined by SDS-PAGE. Its immunoreactivity was analyzed by Western blot. Finally, BALB/c mice were injected with the preliminarily purified recombination protein EspA-Stx2B, then oral challenged these mice with EHEC O157-SMR2 and counteracted toxic substances with O157 ultrasonic supernatant. Results The determination of the sequence encoding of the espA-stx2B fusion gene has 100% of consistency with the sequence from GenBank Sakai strain and contrivable linker. This fusion protein EspA-Stx2B was expressed as inclusion body formation and the percentage is approximately 40%. Western blot suggested the fusion protein has excellent immunoreactivity. Titer of antiserum of the mice to EspA-Stx2B increased evidently. EspA-Stx2B could not decrease bacterial number attached to the intestinal tract of mice based on fecal shedding of Oi57 in mice. In the test of death of BAI,B/c causing by conteracting toxic substances with O157 ultrasonic supernatant, immunoprotection of EspA-Stx2B rate was 66.7%. Conclusion A recombinant plasmid that has high performance on expression of EspA-Stx2B prorein was successfully constructed in present study, and the fusion protein has excellent immunoreactivity and immunogenicity. EspA-Stx2B could not decrease bacteria] number attached to the intestinal tract of mice based on fecal shedding of O157 in mice, but evidently decrease the mortality rate of the mice. The antiEspA and anti-Stx2B had immunoprotection effect by different means. These results may provide the foundation for the further development on EHEC O157:H7 double subunit vaccine.

Study on BALB/c mice by immunization with genetic engineering vaccines of enterohaemorrhagic Escherichia coil O157:117 / 中华微生物学和免疫学杂志

Objective To investigate immunoprophylactic potential of genetic engineering vaccines of enterohaemorrhagie Escherichia coli O157:H7 in BALB/c mice after immunization with these vaccines. Methods Sixty BALB/c mice (3 weeks old) were randomized averagely into 5 groups. Group 1-3 were im-munized respectively with IntiminC300, Stx2B and HIyAN436, group 4 with a combination of these three vaccines, and group 5 with PBS. Each mouse was immunized with vaccine(100 μg)and Al(OH)3 adjuvant (100 μg) for 3 times. After 7 d of the second and third immunization, serum of each mouse was collected and the different antibodies were detected. After 10 d of the last immunization, all mice were given drinking water containing streptomycin for 3 d before and following oral challenge with O157:H7 (109 CFU), and treated with clinical, microbiological and pathological examination. Results The three vaccines elicited high titer antiserum, and some mice were died after infection with O157. The livability of group 1-4 was re-spectively 73%, 64%, 36% and 91%. And these vaccines depressed fecal and colon shedding with O157. Condusion IntiminC300, Stx2B and HIyAN436 have certain protective efficacy for infection of O157, and combined immunization was more effective than single vaccine.

PCR-RFLP and bioinformatic assay of urease B gene from different helicobacter pylori strains / 医学研究生学报

Objective:To analyze the polymorphism of urease B gene(ureB) from different Hp strains. Methods:The sequences of ureB gene from 14 different Hp strains were amplified by PCR to analyze PCR-RFLP with HaeⅢ digestion.At the same time,the sequence of ureB gene was analyzed with biochemical software to compare the homology of nucleotide and amino acid sequence and to profile the phylogenetic tree. Results: The results of 1.7 kb ureB gene digested with HaeⅢ showed there were 2-5 band types in 14 different Hp strains and formed five distinct RFLP types.The two reference strains had the same RFLP type as 3 clinical isolates while the other clinical isolates and 3 isolates from animal model belong to 4 different RFLP types respectively.The nucleotide sequences and putative amino acide sequences of Hp ureB were compared.The various ureB sequences had high homologies(more than 96.6% and 98% in nucleotide sequences and amino acide sequences respectively) among 14 Hp strains.Particularly,there was the highest homologies(100%) between CCS9801、CCS9806、M3 and M10.Phylogenetic tree analysis showed that two reference strains and other isolates from clinical patients and Hp-infected mice model were located in two different lineage respectively in phylogenetic tree of nucleotide sequences while there were some variance in phylogenetic tree of amino acide sequences. Conclusion: Hp ureB was high conserved and homologous in the gene level as well as in the protein level.

microRNAs and the regulation in immune response / 医学研究生学报

MicroRNAs (miRNA) are a newly discovered class of endogenous,evolutionarily conserved small noncoding RNAs involved in posttranscriptional gene regulation. Functionally speaking,miRNAs act as key regulators in a wide variety of biological processes,including cell proliferation,cell differentiation,apoptosis,metabolism,developmental timing,signal transduction and tumor. Recent publications have provided compelling evidence that a range of miRNAs are involved in the regulation of immunity,including the innate immunity,proliferation of monocytes and neutrophils,the development and differentiation of B and T cells,infection and immunity,and the release of inflammatory mediators. In this review,we examine what is presently known of the function and mechanism of these miRNAs in the regulation of the innate and acquired immune response.

Purification and characterization of non-fusion recombinant human interleukin-18 expressed in E. coli / 第三军医大学学报

Objective To develop the purification method of rhIL 18 expressed in E coli Methods The harvested cells were disrupted using lysozyme and sonication The rhIL 18 inclusion bodies were extracted by centrifugation Washed inclusion bodies were solubilized in 8 mol/L urea The solubilized inclusion bodies were first purified by ion exchange chromatography under denaturing conditions After renaturation, gel filtration chromatography was used for further purification Samples were analyzed by SDS PAGE, HPLC, Western blotting and N terminal amino acid sequencing The biological activity of purified rhIL 18 was also measured Results It was found that rhIL 18 was expressed intracellularly in E coli as insoluble inclusion bodies The purity of rhIL 18 in extracted inclusion bodies was above 80% The final purified rhIL 18 was of high purity and exhibited the mass of molecule 19?10 3 by SDS PAGE The sequence of 15 amino acid residues form NH2 terminus of the purified rhIL 18 was consistent with the predicted sequence The purified rhIL 18 also induced IFN ? production of Con A stimulated human PBMC Conclusion The established method for purifying rhIL 18 is simple and effective and might be useful in the development of the large scaled purification process of rhIL 18