Immunogenicity of Lyophilized MVA Vaccine for HIV-1 in Mice Model

Highly attenuated modified vaccinia Ankara（MVA） is sensitive to repeat freeze-thaw cycle and easy to lose activity. In order to make the activity of MVA vaccine remain stable during its manufacturing, storage, and administration, the lyophilization as a good option could be resorted to; through screening, the right stabilizer composition and its production procedure were obtained. The final moisture content of freezing-dried recombinant MVA-HIV vaccine was lower than 3%. It can be reconstituted quickly and shows regular physical appearance and stable potency. In vivo functional experiment, mice were divided randomly into the liquid vaccination group, the lyophilized vaccination group, and the control group. Having been DNA vaccine priming, the mice were boosted with a dose of 10^7 pfu MVA- HIV vaccine, which produced indistinguishable antibody titer and cytotoxic T-lymphocyte（CTL） level compared with those of liquid vaccination group （ P 〉 0.05 ）. These results demonstrate that lyophilized MVA vaccine can induce high immunogenicity in mice.

Montanide ISA-720 and Naloxone in HBsAg Vaccine Formulation:Cytokine Profiling and Monitoring of Long-Lasting Humoral Immune Responses

Objective This study aimed to investigate the effects of Montanide ISA-720 and Naloxone(NLX)in Hepatitis B surface antigen(HBsAg)vaccine formulation on cytokine and long-lasting antibody responses.Methods First,the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10mg/kg.The experimental mice were immunized three times at a 2-week interval,and then IL-4,IL-2,TNF-α,and IFN-γcytokines;long-lasting IgG antibody responses 220 days after the last shot;and IgG1/IgG2a isotypes were assessed by ELISA.Results The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups,whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses.In addition,NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio.Moreover,HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses,and NLX in Alum-and MON720-based vaccines induced long-lasting antibody responses.Conclusion NLX in Alum-based vaccine decreased IL-4 cytokine response,increased IL-2/IL-4 cytokine ratio,and improved long-lasting humoral immune responses in both vaccine formulations.Therefore,the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.

Promoting higher-valent pediatric combination vaccines in China: challenges and recommendations for action

Many countries have adopted higher-valent pediatric combination vaccines to simplify vaccination schedules and minimize health expenditures and social costs.However,China is conservative in the use of pediatric combina-tion vaccines.By reviewing and synthesizing quantitative and qualitative data,in this commentary we identify gaps and challenges to combination vaccine use and make recommendations for promoting use of higher-valent pediatric combination vaccines in China.Challenges are in four dimensions:(1)legislation and regulation,(2)immunization schedule design,(3)vaccine awareness and price,and(4)research and development capacity.To optimize the use of combination vaccines to reduce vaccine-preventable disease burden,we make recommendations that address key challenges:(1)develop policies and regulations to strengthen enforcement of the Vaccine Administration Law and remove regulatory hurdles that hinder combination vaccine research and development,(2)establish an evi-dence-informed policy-making mechanism for combination vaccines,(3)resolve immunization schedule conflicts between monovalent and combination vaccines,and(4)implement effective interventions to increase vaccine awareness and reduce price.

内源性IL-12决定人PBMC产生干扰素γ的水平(英)

目的：ＩＦＮ－γ是由被有丝分裂原或抗原所激活的Ｔ细胞和ＮＫ细胞所产生，它具有广泛的免疫调节活性，现认为ＩＬ－１２（外源性）是诱导 ＩＦＮ－γ产生的强诱导剂，并可促进静息 ＣＤ４＋Ｔ细胞朝向 Ｔｈ１表型分化，即诱导细胞免疫。目的是为了解由ＰＢＭＣ产生的内源性ＩＬ－１２是否在体外可诱导ＩＦＮ－γ的产生及通过何机制诱导细胞免疫。方法：用抗ＣＤ３抗体、ＰＨＡ、抗ＣＤ３抗体加抗ＣＤ２８抗体和抗原（ＭＬＣ）来检测被刺激的ＰＢＭ细胞的ＩＦＮ－γ的产生，同时也用ＩＬ－１２和ＩＬ－１２Ｒβ１的中和抗体来抑制ＩＦＮ－γ的产生。结果：激活的人ＰＢＭ中ＩＦＮ－γ分泌依赖于内源性ＩＬ－１２的产生，而且激活的Ｔ细胞可诱导ＡＰＣ细胞产生ＩＬ－１２，此过程是通过Ｔ细胞表面的ＣＤ４０Ｌ和ＡＰＣ的ＣＤ４０相互作用而实现。结论：这些结果提示，内源性ＩＬ－１２在正常宿主抗细胞内抗原的感染反应中起重要作用，在某些形式的自身免疫性疾病和移植排斥反应的免疫病理发生中也起中心作用。

SARS康复者M抗原特异性记忆性T细胞免疫应答的探讨

目的探讨SARS患者康复后,外周血单个核细胞(PBMCs)中是否存在SARS-CoVM抗原特异性T细胞。方法从完全康复期SARS患者和健康人外周血中分离PBMCs,体外经M混合多肽刺激后,采用酶联免疫吸附试验(ELISA)、酶联免疫斑点试验(ELISpot)及流式细胞仪检测技术,分析抗原特异性T淋巴细胞的反应性。结果在未经任何抗原刺激的情况下,PBMCs几乎不分泌IFN-γ。当SARS-CoVM混合多肽刺激后,SARS康复期患者的PBMCs分泌大量的IFN-γ,并可检测出高频率的IFN-γ产生细胞。与健康刺激组及康复期SARS患者未刺激组相比,差异显著(P<0.05)。流式结果显示,SARS康复期患者PBMCs经M多肽刺激后,分别有0.11%CD4+和0.16%CD8+T淋巴细胞分泌IFN-γ。根据IFN-γ和IL-2的表达与否,可将CD4+T细胞分为三个亚群IFN-γ-IL-2+、IFN-γ+IL-2+和IFN-γ+IL-2-。结论SARS-CoV感染后,机体可以产生针对SARS-CoVM蛋白的抗原特异性细胞免疫反应,其免疫记忆可以在体内维持很长时间。

Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

Enterovirus （EV71） can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 （VP1）, plays a critical role in the pathogenicity of EVT1. High level expression and secretion ofVP 1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP 1 DNA vaccines, including wt-VP 1, tPA-VP 1, VP l-d, VP 1-hFc and VP 1 - mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different （P〈0.05）. In this study, we further investigated the protein lev- els of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc （hFc） to VP1. VP 1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VPI-hFc protein for additional studies.

Roles and relevance of mast cells in infection and vaccination

In addition to their well-established role in allergy mast cells have been described as contributing to functional regulation of both innate and adaptive immune responses in host defense. Mast cells are of hematopoietic origin but typically complete their differentiation in tissues where they express immune regulatory functions by releasing diverse mediators and cytokines. Mast cells are abundant at mucosal tissues which are portals of entry for common infectious agents in addition to allergens. Here, we review the current understanding of the participation of mast cells in defense against infection. We also discuss possibilities of exploiting mast cell activation to provide adequate adjuvant activity that is needed in high-quality vaccination against infectious diseases.

Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli.To determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source,and recombinant protein GST-Bad as a substrate.Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro.To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo,we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn,a dominant negative version of Cdk2,in Hela cells and determined the phosphorylation status of protein Bad.The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent.Moreover,etoposide also caused the phosphorylation of endogenetic Bad.In conclusion,here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

Decreased of BAFF-R expression and B cells maturation in patients with hepatitis B virus-related hepatocellular carcinoma

BACKGROUND Recent evidence has indicated the role of B cells and B cell-activating factor(BAFF)in the development of hepatocellular carcinoma(HCC).AIM To characterize circulating BAFF receptor expression and B cell subpopulations in patients with hepatitis B virus(HBV)-related HCC.METHODS Peripheral blood samples collected from 41 patients with chronic HBV infection(25 patients without HCC and 16 patients with HCC)and 9 healthy controls were assessed for BAFF receptors[BAFF-R(B cell-activating factor receptor),transmembrane activator and cyclophilin ligand interactor,B-cell maturation antigen]and B cell subpopulations by multicolor flow cytometry.RESULTS The frequency of BAFF-R expressing B cells to total B cells was significantly lower in patients with HCC(3.39%±2.12%)compared with the non-HCC group(5.37%±1.90%)and healthy controls(6.23%±2.32%),whereas there was no difference in transmembrane activator and cyclophilin ligand interactor and Bcell maturation antigen.The frequencies of CD27+Ig D+memory B cells,CD27+Ig Dclass-switched memory B cells and plasmablasts were significantly lower in the patients with HCC compared to patients without HCC(1.23±1.17 vs 3.09±1.55,P=0.001,0.60±0.44 vs 1.69±0.86,P<0.0001 and 0.16±0.12 vs 0.37±0.30,P=0.014,respectively).However,the ratio of na?ve and transitional B cell did not differ significantly between the three groups.In addition,decreased BAFF-R expression on B cells was significantly correlated with large tumor size and advanced tumor stage.CONCLUSION Our data demonstrated BAFF-R expression was reduced in B cells that involved with the frequencies of B cells maturation in patients with HCC.The depletion of BAFF-R might play an important role in the development of HCC in patients with chronic HBV infection.

Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

Two eukaryotic vectors expressing 9 tandem repeats of human MUCI（VNTR）, VR1012-VNTR, and pEGFP-VNTR, were constructed by cloning VNTR gene into VR1012 and pEGFP, respectively. VNTR stably expressing murine Lewis lung carcinoma（LLC） cell line（VNTR^＋ LLC） was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells. The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR^＋ LLC cells was confirmed by means of Western blotting. A syngenic graft tumor model was generated by subcutaneous injection of VNTR^＋ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell qumber dependent manner. VNTR mRNA expression in the tumor formed was confirmed by RT-PCR. After the third immunization mice were challenged subcutaneously with 5×10^5 VNTR^＋ LLC cells, a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone. Thus, the suppression of subcutaneous tumor was antigen-specific. This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

Construction of Recombinant Fowlpox Virus(rFpv) Expressing HIV-1 Gag-pol Protein

The induction of human immunodeficiency virus（HIV）-specific T-cell response is generally considered as critical to the development of effective immunity to HIV type 1 （ HIV-1 ）. Recombinant Avipoxvirus vectors are used widely for vaccination against HIV-1, where the induction of a cytotoxic CD8 ＋ T-cell（CTL） response seems to be an important component of protective immunity. A recombinant fowlpox virus（rFPV/Gag-pol） expressing the Gag-pol protein of HIV was constructed and characterized. The specific expression protein in CEF cells infected by recombinant fowlpox and the specific antibody in the sera of mice immunized with rFPV were analyzed via Western-blot.

Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein(HPV16 L1) in Pichia pastoris

In this study, a recombinant Pichia pastoris expression system was developed to express HPV16 L1 protein that was driven by a strong AOX1 promoter. HPV16L1 gene was cloned into vector pPICZ,αB. HPV16 L1 protein expression induced by methanol was screened by using sodium dedecyl sulfate-polyacrylamide gel electrophoresis （SDSPAGE） and Western blotting. The results indicate that the HPVl6 L1 protein is secreted by the recombinant P. pastoris, and the purified HPV16 L1 protein can self-assemble into vires-like particles（ VLPs）, which show a good immunogenicity and induces high-titer antibody in mice.

Construction of Recombinant Modified Vaccinia Ankara(MVA) Expressing Hepatitis B Virus Surface Antigen

The T lymphocyte response has been shown to be the determinant in the clearance of many viral infections. Hence, therapeutic vaccine candidates against HBV are designed to enhance this response of the immune system. Vaccinia virus vector-based vaccines have been proposed as excellent candidates to elicit long-term and strong T lymphocyte mediated immune responses. In this study, the recombinant MVA expressing HBV surface antigen has been constructed, which can elicit a potent T cell mediated response. The ELISA results for the surface protein in the medium of the recombinant MVA, strongly indicate that the recombinant virus has been successfully obtained.

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration(hALR), Expression and Purification of hALR

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration （hALR） serves as a hepatotruphic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chrumatography and this FAD-dependent sulthydryl oxidase activity was measured.

Adenovirus-mediated HSV-TK Gene Therapy Using hTERT Promoter in CNE Cells in vitro

Human telomerase reverse transcriptase（hTERT） activity was detected in human nasopharyngeal carcinoma celI（CNE） but not in human normal lung fibroblas t（CCD-11Lu）. Recombinant adenoviruses Ad-CMV-TK-enh and Ad-hTERT-TK-enh were constructed and infected into normal fibroblasts and nasopharyngeal carcinoma cells. Ad-CMV-TK-enh with 100 μmol/L of ganciclovir（GCV） caused 87% of CCD-11Lu cells death and 91% of CNE cells death, Ad-hTERT-TK-enh with 100 μmol/L of GCV caused 24% of CCD-11Lu cells death and 79% of CNE cells death. These results indicate that the Ad-hTERT-TK-enh with GCV may be a useful method in suppressing tumor growth in targeted nasopharyngeal carcinoma gene therapy.

Corrigendum to“3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation”[Virol Sin 38(2023)387–397]

Due to our negligence,the original version of this article,published online on Mar 14,2023,contained some mistakes in several Figs.In Fig.2D,the positions of the bands for protein 3A and 3B were incorrectly shifted.This has been modified in corrected Fig.2 as shown below.

3C^(pro)of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation

Foot-and-mouth disease virus(FMDV)has developed various strategies to antagonize the host innate immunity.FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms.The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γsignaling transduction.Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown.In this study,it was shown that FMDV replication was resistant to IFN-γtreatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes(ISGs).We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation.3C^(pro)expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences(GAS)promoter activity,without affecting the protein level,tyrosine phosphorylation,and homodimerization of STAT1.Finally,we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities.Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

SARS康复者特异性细胞免疫和免疫记忆T细胞功能的研究

目的 观察完全康复期严重急性呼吸综合征 (SARS)患者外周血针对SARS冠状病毒(SARS CoV)S抗原多肽的特异性细胞免疫反应。方法 分离外周血单个核细胞 (PBMC) ,经混合S抗原多肽刺激后 ,采用酶联免疫吸附试验 (ELISA)和酶联免疫斑点试验 (ELISPOT)检测 ,分析完全康复期SARS患者抗原特异性T淋巴细胞应答反应。结果 当SARS CoV的S抗原混合多肽刺激后 ,只有SARS康复期患者的PBMC分泌大量的IFN γ。同时 ,IFN γ产生细胞的阳性率也显著高于健康对照组 (P <0 .0 5)。此外 ,当用多克隆刺激剂 (PMA +ionomycin)刺激后 ,正常人和康复期SARS患者的PBMC产生等量的IFN γ及IFN γ产生细胞 ,两者经统计学处理 ,差异无统计学意义 (P >0 .0 5)。结论SRAS CoV不仅能诱导中和抗体的产生 ,而且还可诱导抗原特异性细胞免疫应答反应 ,并可在体内长期维持免疫记忆功能。

Fighting Ebola with ZMapp: spotlight on plant-made antibody

Currently,a large Ebola endemic is ongoing in West Africa.According to the World Health Organization,as of August19,the viral outbreak has led to 2240 suspected and confirmed cases and 1229 deaths in four countries:Guinea,Liberia,Nigeria,and Sierra Leone(http://www.cdc.gov/vhf/ebola/outbreaks/guinea/index.html).