Immunogenicity of a New HIV-1 DNA Construct in a BALB/c Mouse Model

Background: Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progres-sion. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. Objective: In the present study, a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. Methods: HIV-1 P24-Nef gene was cloned in pCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-γ evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Results: Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-γ (p<0.05). Cytotox-icity and lymphocyte proliferation responses of mice vaccinated with the candidate vac-cine were significantly increased compared to control groups (p<0.05). Conclusion: HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model.     

Degree and distribution of variability in the 5' untranslated, E1, E2/NS1 and NS5 regions of the hepatitis C virus (HCV)

Hepatitis C virus (HCV) shows a high degree of variability resulting in many different variants. In this work we described the variability of several subgenomic fragments from the 5' untranslated region (5'-UTR) and E1, E2/NS1 and NS5 regions comparing, for every position, all the sequences published in GenBank^® v. 88 (July 1995) as well as new sequences obtained in this work. Variability was determined in two ways. First, we analysed the degree and type of substitutions found in these regions. Second, we defined the most variable and conserved segments in each region and compared our prediction with previous studies. Our results confirm that HCV variability changes along the different regions. Although we found four variable domains in the 5'-UTR, this region was the only one to contain conserved domains. Envelope (E1, E2/NS1) and NS5 regions showed high variability throughout; however, we were able to define six and three hypervariable domains, respectively. The degree and distribution of variability established in this work is supported by the high number of sequences and the different types included in the study. Knowledge of how variability is distributed along the different regions of the HCV genome could be of use in the design of new diagnostic and therapeutic strategies against HCV infection.     

恶性疟原虫裂殖子表面蛋白-2DNA疫苗诱导小鼠T淋巴细胞的增殖

目的　为了探讨恶性疟原虫FCC - 1/HN株裂殖子表面蛋白 - 2 (MSP - 2 )DNA疫苗在小鼠体内诱导的免疫应答的特性及抗感染的保护性免疫机制。方法　将重组真核表达质粒pBK/MSP - 2经骨骼肌注射BALB/c小鼠 ,小鼠经DNA疫苗免疫 8w后 ,用流式细胞仪分析脾脏T淋巴细胞的分化 ,并体外培养脾脏细胞 ,用夹心ELISA法测定IFN -γ和IL - 2的产生。结果　与对照组相比 ,疫苗组CD+ 4CD8+ T淋巴细胞有显著性的增高 ,体外培养的脾脏细胞IFN -γ有一个高浓度的分泌。结论　恶性疟原虫FCC - 1/HNMSP - 2DNA疫苗诱导了一个TH1的免疫应答类型     

Point mutations in E2, NS3 and NS5A of hepatitis G virus

AIM To compare the point mutation deviations of HGV among E2, NS3 and NSSA.METHODS Seven patients with hepatic diseases from Japan and China were selected for this study. RNAwas extracted and amplified by semi-nested RT-PCR; and the PCR products were sequenced directly.RESULTS The point mutation deviations of HGV ia E2, NS3 and NS5A were 10% - 17%, 11% -23%,and 0% - 5%, in nuclcotide sequences and 4% - 12%, 0%, and 0% - 6% in amino acid sequencesrespectively.CONCLUSION The frequency of variation at the nucleotide level was in the order NS3>E2>NS5A, whileat the amino acid level the order was E2 >NS5A>NS3. The detected sequences from the N-terminus of E2may be the poorly conserved region of HGV.     

结核分枝杆菌感染对BALB/c小鼠B细胞发育分化的影响

目的 探讨结核分枝杆菌感染对小鼠B淋巴细胞分化发育的影响。方法 建立Mtb感染BALB/c小鼠模型,在感染早期(4周)及晚期(8周)分别取小鼠骨髓细胞及脾脏细胞,或经培养后,荧光抗体染色,用流式细胞术对感染早期及晚期小鼠的B细胞表型进行分析,设生理盐水处理的小鼠作为对照。结果 Mtb感染组与对照组比较,pro-B细胞(CD45R⁺CD43⁺)(4周:t=2.886,P<0.05)、未成熟B细胞(CD45R⁺IgM⁺IgD^-)(4周:t=4.760,P<0.05)减少,骨髓成熟B细胞(CD45R⁺IgM^+/-IgD⁺)(4周:t=-3.485,P<0.05;8周:t=-2.594,P<0.05)与脾脏成熟B细胞(CD45R⁺IgMlowIgDhi)(4周:t=-2.275,P<0.05;8周:t=-2.97,P<0.05)增多;小鼠脾脏B细胞活化分子CD69表达升高(4周:t=-2.271,P<0.05;8周:t=-2.052,P<0.05);小鼠脾脏记忆性B细胞 (CD45R⁺CD27⁺IgD^+/-)升高(4周:t=-4.203,P<0.05;8周:t=-5.280,P<0.05)。结论 Mtb感染能影响B细胞的分化发育,促使B细胞向成熟细胞分化,并能促使B细胞活化,使机体更有利于抗结核的感染。     

In-Silico Analysis and Protective Efficacy of the PcrV Recombinant Vaccine against Pseudomonas Aeruginosa in the Burned and PA-Infected BALB/c Mouse Model

Background: Pseudomonas aeruginosa is considered as the most severe cause of infections in burn patients and pneumonia infections. Objective: To study the protective effects of recombinant protein vaccine harboring the PcrV of P. aeruginosa in the mouse model of burn and respiratory infections. Methods: Recombinant protein vaccine harboring the PcrV was expressed in the E. coli BL-21 strain. Mice were immunized with the purified recombinant protein, and the antibody titer was measured in the sera obtained from the immunized mice. Immunized and control mice werechallenged by active and passive immunization. The microbial counts in the lung, skin, liver, spleen, and kidney were compared with the control mice. Results: Bioinformatics analysis indicated that the PcrV protein was conserved in 1552 clinical and environmental isolates. Also, the isoelectric point (pI), molecular weight, and Grand Average of Hydropathy (GRAVY) score were analyzed. Mice were injected with recombinant protein, and serum from immunized mice reacted strongly with recombinant antigen at a dilution of 1:64000. The survival rate of mice infected with 5xLD50 of the P. aeruginosa increased significantly up to 75% in the standard strains (PAO1 and PAK), and the number of bacteria, especially in the internal organs (kidney, spleen, and liver) significantly reduced compared to the mice immunized with placebo. Conclusions: Our results demonstrated that the PcrV protein could be an effective candidate vaccine for the generation of antibody response against P. aeruginosa infection.     

Ag85B核酸疫苗的免疫原性和保护性研究

扩增结核分枝杆菌的Ag85B基因并克隆于真核表达载体 pJW4 30 4 ,转染COS - 7细胞后 ,Westernblot检测表明该基因在细胞内得到了正确表达。用该质粒免疫C5 7BL/ 6小鼠 ,免疫三次后 ,用纯化的重组蛋白Ag85B检测小鼠血清中的抗体 ,抗体滴度达到 1:10 2 4 0 0 ,(γ -干扰素测定表明 ,免疫动物的干扰素含量达到 116 0 3± 10 4 pg /ml。实验结果说明Ag85B核酸疫苗在实验动物体内引起了较强的免疫应答。三次免疫后经静脉强毒攻击 ,免疫组小鼠肺脏和脾脏的细菌数比对照空载体组分别减少了 1 5和 15倍以上。本研究表明 ,该核酸疫苗能同时诱导机体产生细胞和体液免疫应答并获得较高的保护效率。     

HepG2细胞中HCV NS5A蛋白对HCV IRES启动蛋白翻译的影响

目的探讨HepG2细胞中HCV非结构蛋白5A（NS5A）对HCV IRES启动蛋白翻译的影响，以了解HCV的复制调控机制。方法将构建的表达双荧光素酶的双顺反子载体pCMV-Rluc-IRES-Fluc和含HCV NS5A基因的表达质粒pcDNA-NS5A共转染HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，细胞免疫荧光技术检测HCV-NS5A蛋白的表达，RT-PCR检测虫荧光素酶基因mRNA水平，并与相应对照做比较，以观察HCV NS5A对HCV IRES介导虫荧光素酶翻译水平的影响。结果转染pcDNA-NS5A的HepG2细胞中虫荧光素酶活性明显高于转染pCDNA3．I-3flag的对照组，并存在剂量依赖关系；而RT-PCR虫荧光素酶基因mRNA水平在两组间差异无统计学意义。转染pcDNA-NS5A的HepG2细胞质中可见HCV NS5A蛋白的表达。结论HCV NS5A蛋白对HCV IRES介导虫荧光素酶的翻译有正调节作用，并存在剂量依赖关系．     

丙型肝炎病毒E1，E2/NS1，NS5蛋白基因在大肠杆菌内的表达及其产物的初步应用

目的在体外表达及纯化ＨＣＶ不同功能区的病毒蛋白，用于研究ＨＣＶ各功能区抗体的临床意义。方法将ＨＣＶＥ１，Ｅ２／ＮＳ１，ＮＳ５区的部分基因片段克隆到表达质粒ＰＭＡＬ－ＣＲＩ中ＭＢＰ编码基因的下游，在大肠杆菌中进行表达。结果表达的融合蛋白ＭＢＰ－Ｅ１，ＭＢＰ－Ｅ２／ＮＳ１，ＭＢＰ－ＮＳ５的分子量分别为５７０００、６５０００、６２０００，ＷｅｓｔｅｒｎＢｌｏｔ分析表明这些融合蛋白具有ＨＣＶ的抗原活性。将这三种融合蛋白纯化后作为抗原检测了丙型肝炎患者血清９５份，其中抗－ＨＣＶＥ１阳性率为１０．５％，抗－ＨＣＶＥ２／ＮＳ１为６３．２％，抗－ＨＣＶＮＳ５为５３．７％。结论本实验表达的ＨＣＶ融合蛋白在ＨＣＶ感染的血清学诊断中具有一定的价值。     

Preventive Effect of Hochu-ekki-to on Lipopolysaccharide-Induced Acute Lung Injury in BALB/c Mice

This study was designed to investigate the effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, on the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in female BALB/c mice by the intranasal administration of 0.1 mg/kg LPS. The mice were divided into a group receiving normal feed and another group receiving feed mixed with TJ-41 at a dose of 1 g/kg/day for 8 weeks before LPS challenge. In the bronchoalveolar lavage fluid, the preadministration of TJ-41 caused significant reduction in the absolute number of total cells, neutrophils, and macrophages. The preadministration of TJ-41 significantly inhibited increases in the serum level of keratinocyte chemoattractant (KC), which is a murine chemotaxin for neutrophils that corresponds to human interleukin-8, with respect to its concentration at 24 h after LPS challenge. Furthermore, the histopathologic findings indicated that alveolitis with leukocyte infiltration in the alveolar space was less severe in the TJ-41-treated mice than in the control mice. These findings indicated that the preadministration of TJ-41 could show an inhibitory effect on ALI in this experimental murine system associated with the suppression of chemokine production.     

Effect of interleukin-15 on the course of myocarditis in Coxsackievirus B3-infected BALB/c mice

OBJECTIVE:

Cytokines have an important role in both the initiation and perpetuation of viral myocarditis. Because a causative therapy of myocarditis is not yet well established and immunomodulation is a promising approach, the influence of interleukin (IL)-15, a proinflammatory cytokine, on the course of experimental myocarditis in Coxsackievirus B3 (CVB3)-infected mice was examined.

METHODS:

Hearts from CVB3-infected (n=14), sham-infected (n=14) and CVB3-infected BALB/c mice treated with IL-15 (n=6) or a competitive IL-15 fusion protein (n=6) were analyzed for hemodynamic function, cellular infiltrates and myocardial collagen content.

RESULTS:

Induction of myocarditis was associated with significant loss of body and heart weight, decreased left ventricular function, and increased collagen content and cellular infiltrates in the myocardium. Treatment of infected animals with IL-15 resulted in normalization of body and heart weight, and significantly improved systolic and diastolic left ventricular function, comparable with that of uninfected animals. This was paralleled by a significant reduction of myocardial collagen content to levels observed in animals without disease and by markedly reduced cellular infiltration of lymphocytes and macrophages in the myocardium. Inhibition of intrinsic IL-15 with IL-15 fusion protein tended to aggravate the disease.

CONCLUSIONS:

Treatment with IL-15 has a positive effect on CVB3-induced murine myocarditis and seems to be a promising approach to modifying clinical course, hemodynamics and histopathology of virus-induced myocarditis. Further studies are needed to identify the underlying mechanisms.     

泡球蚴感染对BALB/c小鼠肝细胞ERK1/2和p38表达变化的初步研究

目的通过检测ERK1/2和p38在泡球蚴感染小鼠肝组织中的表达,探讨其在肝泡球蚴病发生中的作用。方法采用半定量逆转录聚合酶链反应（RT-PCR）及免疫组织化学的方法检测16例感染组小鼠和16例对照组小鼠肝组织中的ERK1/2和p38的表达水平。结果感染组ERK1/2表达水平较对照组有统计学差异（P〈0.05）,p38的表达在感染组与对照组无统计学差异。结论在泡球蚴感染早期,肝细胞增殖占主导。     

白细胞介素2和15基因对柯萨奇病毒VP1 DNA疫苗诱导免疫应答的影响

目的:观察白细胞介素2(IL 2)、白细胞介素15(IL 15)基因对柯萨奇病毒(CVB3)VP1DNA疫苗诱导 免疫应答的影响,以及该疫苗在小鼠受到致死量CVB3攻击时的保护作用。方法:采用雄性4～6周龄Balb/c小 鼠100只,随机分为5组,并分别肌内注射0.9%氯化钠溶液、pcDNA3、pcDNA3 VP1、pcDNA3 VP1加pcDNA3 IL 2和pcDNA3 VP1加pcDNA3 IL 15,每周注射1次,共3次,每次免疫后第6天取血清,用微量中和试验检测 CVB3中和抗体,3次免疫后用800TCID50CVB3感染小鼠,观察小鼠的生存时间和生存率。结果:pcDNA3 VP1、pcDNA3 VP1加pcDNA3 IL 2、pcDNA3 VP1加pcDNA3 IL 15组均诱导小鼠产生了中和抗体,抗体滴度 随免疫次数增加而提高。病毒攻击后,各组的生存率差异无统计学意义,pcDNA3 VP1加pcDNA3 IL 2、pcD NA3 VP1加pcDNA3 IL 15组较其他3组生存时间明显延长。结论:IL 2、IL 15基因对CVB3VP1DNA疫苗诱 导小鼠产生免疫应答和免疫保护有一定的增强作用。     

Distribution of Trypanosoma cruzi stage-specific epitopes in cardiac muscle of Calomys callosus, BALB/c mice, and cultured cells infected with different infective forms

To examine whether distinct parasite infective forms or the mammalian host could affect the distribution of Trypanosoma cruzi stage-specific epitopes defined by monoclonal antibodies (Mabs) raised against mammalian-stage parasite forms, immunofluorescence studies followed the intracellular life cycle of the parasite in the cardiac muscle of Calomys callosus and BALB/c mice in the acute phase of the disease and in LLC-MK(2) cultured cells. Animals and cells were infected either with tissue-culture derived trypomastigotes (TCT) or bloodstream trypomastigotes (BT) from the Y strain of T. cruzi. Samples were examined under confocal fluorescence microscopy after labeling with Mabs 2C2, 1D9, 2B7, 3G8, 3B9, and 4B9 that react with carbohydrate epitopes on Ssp-4, a major amastigote surface glycoprotein; Mab 4B5 that identifies a noncarbohydrate epitope on all intracellular parasites stages, and Mab 3B2 that also recognizes a noncarbohydrate epitope expressed only in flagellated forms. Samples were double labeled with DAPI to visualize parasites' kinetoplasts and nuclei. Most of the Mabs used in this work displayed a surface labeling pattern on amastigotes present in Calomys and mice hearts, and in LLC-MK(2) cultured cells infected with BT or TCT. Mab 2B7, however, displayed a marked polymorphic distribution in antigen expression between both mammalian hosts, independent on the infective form. Beyond the polymorphic distribution of amastigote surface epitopes, Calomys, and mice heart sections presented several inflammatory cells around amastigotes and trypomastigotes nests.     

Immune response in susceptible BALB/c mice immunized with DNA encoding Lipophosphoglycan 3 of Leishmania infantum

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN‐γ and IL‐10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co‐administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime‐boost approach with a powerful adjuvant to elicit a robust parasite‐specific protective Th1 response.     

Phylogenetic and 2D/3D Analysis of HCV 1a NS4A Gene/Protein in Pakistani Isolates

Background:

The nonstructural protein NS4A of hepatitis C virus is composed of 54 amino acids. This small size protein has vital role in many cellular functions. The most important reported function is being a cofactor of viral enzymes serine protease and helicase.

Objectives:

The objective of this study was to analyze the phylogenetic variation, its impact in terms of translation and any functional change in protein structure at primary 2D/3D structure using computational tools from Pakistani patients isolates.

Materials and Methods:

Patient sera infected with Hepatitis C virus, genotype 1A, were obtained from Molecular Diagnostics lab, CEMB, University of the Punjab Lahore by using BD Vacutainer collection tubes (Becton Dickenson).

Results:

Phylogenetic analysis of the gene revealed that Pakistani 1a HCV strains are in the start of third cluster and there is a difference between inter Pakistani isolates at primary, secondary and tertiary levels.

Conclusions:

Mutations were present in the central domain of NS4A (amino acids 21 - 34).     

苦瓜蛋白对柯萨奇B3病毒性心肌炎小鼠半胱天冬酶3活性及凋亡的抑制作用

为研究苦瓜蛋白对凋亡的关键酶半胱天冬酶 3及凋亡的调节作用 ,用已经建立起来的方法从苦瓜果肉中提取苦瓜蛋白。将BALB/C小鼠分为 4组 :病毒对照组、正常对照组、药物对照组和药物治疗组 ,分别在第 0天、第 3天、第 7天和第 14天各处死 5只小鼠 ,第 2 1天全部处死动物 ,心肌组织半胱天冬酶 3活性测定按照CAL BIOCHEM公司的试剂说明书进行 ,并稍加改正 ,凋亡鉴定按照Oncogene公司的TdT DNA裂解片断末端原位标记试剂说明书进行。结果发现 ,①病毒对照组第 7天开始出现半胱天冬酶 3活性 (0 .6 3± 0 .2 1pmol/min ,n =5 ) ,第 14天的活性 (10 .9± 1.5pmol/min ,n =5 )显著高于第 7天 ,第 2 1天的活性 (12 .6± 1.3pmol/min ,n =5 )又高于第 14天。②药物治疗组 ,只有一个第 2 1天的标本有半胱天冬酶 3活性 (0 .4 1pmol/min) ,其它两组均未检测到此酶的活性。③DNA裂解片断末端原位标记法发现 ,病毒对照组在第 7天心肌中有少数细胞凋亡 ,第 14天、第 2 1天凋亡细胞明显增多 ,在非病变区域可发现单个凋亡的心肌细胞。治疗组未发现凋亡细胞 ,其它两组也未发现凋亡细胞。结果提示 ,CVB3病毒性心肌炎中发现有明显的凋亡现象 ,凋亡和半胱天冬酶 3发现于第 7天 ,且随着时间增加而逐渐加重(增强 ) ;苦瓜蛋白可显     

The Bluetongue Disabled Infectious Single Animal (DISA) Vaccine Platform Based on Deletion NS3/NS3a Protein Is Safe and Protective in Cattle and Enables DIVA

The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.