COVID-19 mortality paradox(United States vs Africa):Mass vaccination vs early treatment

The coronavirus disease 2019(COVID-19)mortality rate in 55 African countries is almost 4.5 times lower than in the coronavirus disease 2019(COVID-19)despite Africa having over 4.2 times more people.This mortality paradox is also evident when comparing Nigeria,a heavily populated,poorly vaccinated and weakly mandated country to Israel,a small,highly vaccinated and strictly mandated country.Nigeria has almost 4 times lower COVID mortality than Israel.In this Field of Vision perspective,I explain how this paradox has evolved drawing upon my academic,clinical and social experience.Since April 2020,I’ve developed and been using the Egyptian immune-modulatory Kelleni’s protocol to manage COVID-19 patients including pediatric,geriatric,pregnant,immune-compromised and other individuals suffering from multiple comorbidities.It’s unfortunate that severe acute respiratory syndrome coronavirus 2 is still evolving accompanied by more deaths.However in Africa,we’ve been able to live without anxiety or mandates throughout the pandemic because we trust science and adopted early treatment using safe,and effective repurposed drugs that have saved the majority of COVID-19 patients.This article represents an African and Egyptian tale of honor.

Accurate Diagnosis of SARS-CoV-2 JN.1 by Sanger Sequencing of Receptor-Binding Domain Is Needed for Clinical Evaluation of Its Immune Evasion

Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

A new DNA vaccine fused with the C3d-p28 induces a Th2 immune response against amyloid-beta

To enhance anti-amyloid-beta （Aβ） antibody generation and induce a Th2 immune response, we constructed a new DNA vaccine p（Aβ3-10 ）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant. In this study, we administered this adjuvant intramus-cularly to female C57BL/6J mice at 8-10 weeks of age. Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody, isotypes, and cytokines in splenic T cel s. A 3-（4,5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to detect the prolifera-tion rate of splenic T cel s. Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques. The p（Aβ3-10）10-C3d-p28.3 vaccine induced high titers of anti-amyloid-βantibodies, which bound to Aβplaques in APP/PS1 transgenic mouse brain tissue, demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease. Moreover, the vaccine elicited a pre-dominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes, indicating that the vaccine could shift the cel ular immune response towards a Th2 phenotype. This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile. Our results indicate that the p（Aβ3-10）10-C3d-p28.3 vaccine is a promising immunothera-peutic option for Aβvaccination in Alzheimer’s disease.

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)疫苗研究进展

2019冠状病毒病(COVID-19)是严重急性呼吸综合征冠状病毒2(SARS-CoV-2)感染引起的,SARS-CoV-2病毒毒株不断突变、进化。疫苗研究是控制COVID-19最直接、最有效的方式,根据制备机制不同,SARS-CoV-2疫苗分为灭活病毒疫苗、减毒活疫苗、mRNA疫苗、DNA疫苗、病毒载体疫苗、病毒样颗粒疫苗和蛋白亚单位疫苗等。其中,病毒蛋白亚单位疫苗因其安全性、有效性高具有广泛应用前景,病毒核衣壳蛋白免疫原性高,可变性较低,可作为疫苗制备新方向。我们通过梳理SARS-CoV-2疫苗研究进展、疫苗安全性、疫苗免疫效率等方面,总结疫苗研究的发展现状,并提出可能的研制策略,以期为今后防疫工作提供参考。

Evolution of Cervical Lesions Associated with Human Papillomavirus Infection after the Introduction of Vaccination

Background: The main objective of this study is to analyse the change in the type of lesions developed by HPV-infected patients after the introduction of the vaccine in three different periods;2002-2006 (years previous to the implementation of the vaccine in Spain), 2009-2011 (shortly after the vaccination) and 2020-2021 (years where the vaccine was well established) at a single hospital. Methods: This is an observational, descriptive, retrospective study based on the review of the results of the biopsies of patients with HPV lesions at a single large tertiary hospital, Hospital Clínico San Carlos, in Madrid, Spain. We have collected the data from three different time periods: 2002-2006, 2009-2011, 2020-2021 to try to understand the potential changes in these lesions after vaccine introduction. Results: In this time we have reviewed the data from 946 women. In these three periods, a decreasing trend in the rate of squamous cell carcinoma was noted, the rate of adenocarcinoma remains stable, and the rate of cervical intraepithelial neoplasia grades 2 - 3 (CIN 2-3) lesions shows an increasing trend. We have also found a change in the mean ages of the patients with these lesions, as this increased in the three lesions caused by HPV after the implementation of the vaccine. Our study indicates that the identification of other high risk serotypes, apart from 16 and 18, as well as those with indeterminate risk, has undergone a progressive increase, increasing from 24.24% and 14.11% respectively in 2002-2006 to 40.42% and 28.34% in 2020-2021. Conclusion: Our study confirms the effectiveness of the vaccines developed so far, against the HPV serotypes they contain. This is demonstrated by the evidence, in our population, of a decrease in the incidence of squamous cell carcinoma in uterine cervix. In parallel, an increase in the mean age of diagnosis has been verified, for both squamous cell carcinoma and its CIN 2-3 precursor lesions, as well as a change in the infective trend of HPV serotypes that are not included in the current vaccines.

布鲁氏菌分子标记、毒力基因缺失株YZ-2生物学特性及免疫原性研究

试验旨在考查布鲁氏菌分子标记、毒力基因缺失疫苗株YZ-2的生物学特性和免疫原性。对YZ-2菌落形态、生化特性、变异检查试验、毒力、遗传稳定性等生物学特性,以及免疫小鼠后体液免疫和细胞免疫水平进行试验,并与其亲本株S19进行比较。结果显示,YZ-2的形态及生化特性同亲本株S19相一致,但变异检查试验结果显示YZ-2由亲本株的光滑型变为粗糙型;毒力试验证实YZ-2的毒力与亲本株S19显著减弱,且YZ-2遗传稳定性良好。免疫原性试验证实,布鲁氏菌YZ-2在整体上具有与亲本株相似的免疫原性。结果表明,布鲁氏菌分子标记、毒力基因缺失疫苗株YZ-2的安全性较S19进一步提高,稳定性好,且具有与亲本株相近的免疫原性,有很好的应用前景。

布鲁菌半胱氨酸水解酶在甲硫氨酸循环中的催化活性研究

[目的]细菌的sah H基因编码S-腺苷同型半胱氨酸水解酶(Sah H),该酶参与细菌的甲硫氨酸循环(AMC),调控细菌的多种生理功能。[方法]通过构建重组表达质粒p ET28a-Bru-sah H和p ET28a-Pse-sah H,分别表达布鲁菌(Brucella abortus)S2308株和铜绿假单胞菌(Pseudomonas aeruginosa)PAO1株的Sah H重组蛋白Bru-Sah H和Pse-Sah H。将纯化后的Bru-Sah H、Pse-Sah H以及我们前期表达纯化的禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)的Pfs和Lux S蛋白,分别在体外催化S-腺苷同型半胱氨酸(SAH),通过对产物同型半胱氨酸(HCY)的浓度测定,评价不同重组蛋白的催化活性,并对催化底物时产生的自诱导分子2(AI-2)活性进行检测。[结果]Bru-Sah H和Pse-Sah H分别催化1 mmol·L-1SAH生成38和47μmol·L-1HCY,而APEC的Pfs和Lux S蛋白能催化相同浓度的SAH产生401μmol·L-1HCY。运用哈维弧菌BB170检测上述底物的AI-2活性,结果表明只有同时采用AEPC的Pfs和Lux S蛋白催化SAH,才能形成有活性的AI-2分子,而Bru-Sah H和Pce-Sah H均不能催化SAH形成活性AI-2分子。[结论]Bru-Sah H能催化SAH生成HCY,为进一步研究sah H在布鲁菌感染过程中的作用提供依据。

乳腺癌HER2／neu手性多肽疫苗的实验研究

目的研究具有局部手性的HER-2/neu多肽疫苗对细胞毒性T细胞(CTLs)的诱导作用。方法用固相法合成HER-2/neu手性肽疫苗并进行纯化,建立小鼠的乳腺癌模型,用上述手性肽做疫苗、重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)为佐剂,对小鼠进行免疫,分别应用MTT法、ELISPOT法、颗粒酶释放法测定T细胞的增殖,T细胞分泌IFN-γ,CTL对肿瘤细胞的杀伤活性。结果合成的手性HER-2/neu可明显的促进T细胞增殖,促进T细胞分泌IFN-γ及诱导CTL杀伤乳腺癌细胞。结论手性HER-2/neu多肽疫苗可在小鼠体内诱导特异细胞免疫,增加对乳腺癌细胞的杀伤作用。

S-O_2-1细菌核糖体制剂体外诱生白细胞介素的研究

用自制S-O_2-1细菌核糖体制剂,进行体外诱生白细胞介素1和2的研究。结果表明:S-O_2-1细菌核糖体制剂能诱导C_(57)BL/6小鼠的脾脏细胞分泌较低滴度的IL_2,腹腔粘附细胞分泌较高滴度的IL_1,其对IL_1的诱生效应高于同样实验条件下LPS(10μg/ml)的作用。

布鲁氏菌S-2与S-19株提取的脂多糖在人布鲁氏菌病诊断抗原中的应用

目的使用脂多糖(lipopolysaccharide,LPS)提取试剂盒提取布鲁氏菌S-19与S-2菌株中的脂多糖,检测这2种脂多糖的抗原性与诊断价值。方法用工业化生产的布鲁氏菌S-19与S-2菌株菌液用LPS纯化试剂盒通过裂解、纯化、洗涤的方法提取LPS,用提取的LPS进行斑点酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)与布鲁氏菌病阳性、正常阴性、疱疹病、血吸虫病患者血清反应,观察反应结果并判断诊断价值。结果斑点ELISA实验结果显示从S-19与S-2株中提取的LPS与布鲁氏菌病阳性血清能作出反应,反应颜色明显,诊断价值高。结论S-19与S-2菌株中提取的LPS能与布鲁氏菌病阳性血清反应,有较好的诊断价值。

抗原改造联合纳米肿瘤疫苗用以克服肿瘤新抗原短缺及在多种肿瘤类型上激起强效抗肿瘤免疫反应

Neoantigen cancer vaccines have been envisioned as one of the most promising means for cancer therapies.However,identifying neoantigens for tumor types with low tumor mutation burdens continues to limit the effectiveness of neoantigen vaccines.Herein,we proposed a "hit-and-run" vaccine strategy which primes T cells to attack tumor cells decorated with exogenous "neo-antigens".This vaccine strategy utilizes a peptide nanovaccine to elicit antigen-specific T cell responses after tumor-specific decoration with a nanocarrier containing the same peptide antigens.We demonstrated that a poly(2-oxazoline)s(POx) conjugated with OVA_(257-264) peptide through a matrix metalloprotease 2(MMP-2) sensitive linker could efficiently and selectively decorate tumor cells with OVA peptides in vivo.Then,a POx-based nanovaccine containing OVA_(257-264) peptides to elicit OVA-specific T cell responses was designed.In combination with this hit-and-run vaccine system,an effective vaccine therapy was demonstrated across tumor types even without OVA antigen expression.This approach provides a promising and uniform vaccine strategy against tumors with a low tumor mutation burden.

重组麻疹病毒载体SARS-CoV-2疫苗病毒滴度荧光定量PCR检测方法的建立、验证及应用

目的建立检测重组麻疹病毒载体严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)疫苗病毒滴度的荧光定量PCR(qPCR)法,并进行验证及初步应用。方法以重组质粒pUC57-S351为模板,扩增SARS-CoV-2 S基因,优化引物浓度,建立qPCR检测方法。对该方法的专属性、重复性进行验证,确定线性范围及检测限。应用建立的qPCR法对生物反应器连续培养感染后1~12 d的重组病毒S基因拷贝数进行检测。结果选择引物浓度为0.20μmol/L建立qPCR方法。该方法能特异性检测SARS-CoV-2 S基因拷贝数;模板浓度在2×10^(2)~2×10^(8)copies/μL之间,线性关系良好(R^(2)=0.995),检测下限为2×10^(2)copies/μL;6次重复检测重组病毒S基因拷贝数的变异系数(coefficient of variation,CV)为2.64%。应用建立的qPCR法检测生物反应器连续培养感染后1~12 d的重组病毒S基因拷贝数的结果与细胞病变法检测结果基本一致。结论建立的qPCR法专属性和重复性良好,可用于重组麻疹病毒载体SARS-CoV-2疫苗病毒滴度检测。

布鲁杆菌S2株通过JNK-p53信号通路诱导小鼠BV-2小胶质细胞凋亡

目的研究布鲁杆菌S2株诱导BV-2小胶质细胞凋亡的机制,以发现神经型布鲁杆菌病药物治疗的新靶点。方法用布鲁杆菌S2株分别侵染BV-2小胶质细胞0、3、6、12、24 h,Western blot和RT-qPCR技术检测p-JNK、p53蛋白和mRNA表达水平,流式细胞术检测细胞凋亡,细胞免疫荧光技术检测p-JNK蛋白核转运。结果布鲁杆菌S2株能促进BV-2小胶质细胞p-JNK、p53蛋白及mRNA表达,并增加p-JNK蛋白核转运。布鲁杆菌S2株能诱导BV-2小胶质细胞凋亡。结论布鲁杆菌S2株通过激活BV-2小胶质细胞JNK,促进p53表达,诱导细胞凋亡。

A clinical trial of active immunotherapy with anti-idiotypic vaccine in nasopharyngeal carcinoma patients

OBJECTIVE: To investigate the effect of active immunotherapy with anti-idiotypic vaccine in patients with nasopharyngeal carcinoma (NPC). METHODS: Anti-idiotypic antibodies (2H4/5D3) bearing the internal image of the NPC antigen were used in active immunotherapy in NPC patients receiving radiotherapy. Antibodies and cytokine levels in patient sera were determined using ELISA before and after active immunotherapy. IL-2 mRNA expression in the peripheral blood mononuclear cells (PBMC) was measured by in situ hybridization. RESULTS: Nineteen patients with NPC at stage IV were treated with alum-precipitated 2H4 or 5D3. Neither hypersensitivity nor adverse side effects were observed. The levels of anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased. Human anti-mouse antibodies (HAMA) were seen in 19 patients of the experimental group; the levels of Ab1' did not increase in the control group. Serum IL-2, IFN-gamma and TNF-alpha levels were increased in most patients in the experimental group, while no differences were observed in Ab1' and cytokine levels between pre- and post-therapy in the control group. In addition, IL-2 mRNA expression in PBMCs from NPC patients was closely related to serum IL-2 (r = + 0.8829) levels by in situ hybridization. CONCLUSIONS: Anti-idiotype vaccine is safe for clinical active immunotherapy. Anti-idiotypic vaccine might be able to enhance humoral and/or cellular immunity in NPC patients receiving radiotherapy.

基于RBD抗原性的五种SARS-CoV-2血清型分类

新冠病毒(SARS-CoV-2)的不断进化带来了大量的变异株,特别是Omicron变异株及其众多的亚型.这些变异株表现出越来越强的免疫逃逸能力,使现有疫苗和治疗抗体的效力不断下降.目前,众多变异株已表现出血清交叉中和作用减弱的现象,表明新冠病毒可能已进化出多种血清型.因此,我们选取新冠病毒的主要抗原,即刺突(S)蛋白的受体结合域(RBD)对其进行血清分型.我们首先选择了23个具有代表性的新冠病毒毒株,涵盖了前Omicron变异株和Omicron变异株的多种亚型.通过对RBD抗原性的系统评估,我们将23种变异株分为5种血清型,每种血清型都包含了数种基因型不同的变异株.具体而言,Ⅰ型涵盖了所有前Omicron变异株(含两种亚型),而其余四种血清型均包含处于不同进化阶段的Omicron亚型.本文中的血清分型可以为新型变异株的快速评估奠定基础,并指导未来针对新冠病毒的广谱疫苗和中和抗体的开发.

SARS-CoV-2 δ变异株S抗原含量检测方法的建立及验证

目的 建立严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)δ变异株S抗原含量的检测方法,并进行验证。方法 采用δ变异株重组表达的受体结合域(receptor binding domain,RBD)蛋白分别免疫山羊和家兔,制备抗RBD多克隆抗体。以制备的两种抗RBD多克隆抗体为包被抗体和一抗,HRP标记的羊抗兔IgG抗体为酶标抗体,建立用于检测S抗原含量的双抗体夹心ELISA法。棋盘滴定法确定包被抗体和一抗,同时优化3种抗体稀释度。验证方法的准确性、精密性、线性范围及专属性。采用建立的方法检测SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及其中间品的S抗原含量。结果 兔抗RBD多克隆抗体及羊抗RBD多克隆抗体的效价分别为1∶32 000和1∶64 000,蛋白浓度分别为2.26和5.41 mg/mL。最佳包被抗体为羊抗RBD多克隆抗体,一抗为兔抗RBD多克隆抗体,包被抗体、一抗及酶标抗体最佳稀释度分别为1∶4 000、1∶8 000和1∶16 000。40、20、10 U/mL3种浓度S抗原内部参考品重复6次检测结果的回收率为83.5%~103.0%;重复6次测定3批SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液抗原含量的CV均<7.5%,2名实验员对3批上述疫苗原液重复检测3次结果的CV均<7.5%;S抗原含量理论值在10~40 U/mL范围内,与测定值呈良好的线性关系,线性回归方程为:y=0.942 3 x+0.049 2,R~2=0.995 4,定量限为10 U/mL;建立的方法与甲型肝炎灭活疫苗(人二倍体细胞)、四价流感病毒裂解疫苗、Vero细胞宿主蛋白、Vero细胞培养上清液等无交叉反应。SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及中间品3次检测结果的CV均<15.0%。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性、专属性,可用于SARS-CoV-2 δ株灭活疫苗(Vero细胞)原液及中间品中S抗原的定量检测。

Protective effect of DNA-mediated immunization with a combination of SAG1 and IL-2 gene adjuvant against infection of Toxoplasma gondii in mice

OBJECTIVE: To characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis. METHODS: Mice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally. RESULTS: Significant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival. CONCLUSION: Humoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

表达新冠病毒S基因重组狂犬病病毒的构建及其免疫原性

【背景】新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)在全球流行已近3年,除对人类造成了巨大伤害,也影响了人类的伴侣动物。人的COVID-19疫苗已在全球应用,但动物用的新冠病毒疫苗却鲜有报道。【目的】研制兽用新冠病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)和狂犬病病毒(rabies virus,RABV)的二联苗。【方法】将合成的SARS-CoV-2 S基因和S1基因分别克隆至RABV弱毒疫苗株rHEP-Flury基因组G与L基因间,并将2个重组质粒分别与辅助质粒共转染至BHK-21细胞中,拯救重组病毒rHEP-nCOV-S和rHEP-nCOV-S1。通过RT-PCR、Western blotting和荧光抗体染色,验证重组病毒、确证S和S1蛋白在RABV中成功表达。再将重组病毒接种NA细胞及成年小白鼠,测定病毒的体外生长特性、重组病毒的致病性及免疫原性。【结果】免疫荧光结果显示,转染7 d后细胞上清均出现了绿色免疫荧光,表明已成功拯救嵌合SARS-CoV-2 S和S1基因的重组病毒RABV rHEP-nCOV-S和rHEP-nCOV-S1,并且rHEP-nCOV-S1的增殖和扩散能力强于亲本株rHEP-Flury,但rHEP-nCOV-S与亲本株无显著差异。Western blotting结果显示,在目的位置处均出现72 kDa和144 kDa特异性条带,表明S和S1蛋白在重组RABV中高效表达。重组病毒免疫6周KM小鼠后,小鼠的体重变化与亲本RABV基本一致,重组病毒诱导小鼠产生狂犬中和抗体。【结论】本研究拯救出了嵌合SARS-CoV-2 S/S1基因的重组RABV,为动物COVID-19载体疫苗的研发奠定了基础。

严重急性呼吸综合征冠状病毒2 S蛋白DNA疫苗超螺旋构型纯化工艺的建立

目的 建立严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-CoV-2)S蛋白DNA疫苗(pDRVI3.0-S)超螺旋构型的纯化工艺。方法 以pDRVI3.0-S超螺旋构型含量及质粒回收率为指标,通过正交试验对质粒DNA亲和层析的硫酸铵浓度(2.0、2.3、2.5 mol/L)、氯化钠浓度(0、0.3、0.5 mol/L)及上样载量(1.0、1.5、2.0 g/L)进行优化。结果 硫酸铵浓度为2.3 mol/L,氯化钠浓度为0.5 mol/L,上样载量为1 g/L时,pDRVI3.0-S的超螺旋含量最高,可达92%以上。结论 成功建立了含SARS-CoV-2 S蛋白DNA疫苗(pDRVI3.0-S)超螺旋构型的纯化工艺,为该疫苗的规模化生产提供了试验依据。