The cytochemical marking of lymphocyte subpopulations in determining the immunoreactivity status of children vaccinated with a live measles vaccine

In 54 children aged 1.5-2 years, immunized with the same batch of live measles vaccine prepared from strain, the relative and absolute numbers of different lymphocyte subpopulations were determined in parallel by means of two cytochemical reactions: for acid alpha-naphthyl acetate esterase and acid phosphatase in their dynamics. According to the data obtained in these reactions, the determination of lymphocyte subpopulations by two cytochemical methods in the same blood sample yielded similar results indicating that immunization with attenuated measles virus strain induced a rise in the number or zero lymphocytes and was accompanied by the decrease of the immunoregulatory index (T-helper/T-suppressor ratio), most pronounced in children with a high level of antibody formation.     

Mumps vaccine L-Zagreb, prepared in chick fibroblasts. I. Production and field trials

Leningrad-L3 Mumps Vaccine virus has been further attenuated by adaptation and passage on SPF chick embryo fibroblast cell cultures. This new mumps strain has been designated L-Zagreb and has been used to prepare mumps vaccines which meet the WHO requirements. Observations during both the field trial period prior to registration and during the later use of the vaccine showed that the few reactions observed were mild and that seroconversion was obtained in 88-98% of vaccines. The morbidity of mumps in Croatia declined more than tenfold after the introduction of the new vaccine. During a mumps epidemic, vaccine efficiency was calculated to be 97-100%.     

Inactivation of SAM-Methyltransferase is the Mechanism of Attenuation of a Historic Louse Borne Typhus Vaccine Strain

Louse borne typhus (also called epidemic typhus) was one of man''s major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine.     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

Induction of mucosal protection against primary, heterologous simian immunodeficiency virus by a DNA vaccine

An effective vaccine against human immunodeficiency virus (HIV) should protect against mucosal transmission of genetically divergent isolates. As a safe alternative to live attenuated vaccines, the immunogenicity and protective efficacy of a DNA vaccine containing simian immunodeficiency virus (SIV) strain 17E-Fr (SIV/17E-Fr) gag-pol-env was analyzed in rhesus macaques. Significant levels of cytotoxic T lymphocytes (CTL), but low to undetectable serum antibody responses, were observed following multiple immunizations. SIV-specific mucosal antibodies and CTL were also detected in rectal washes and gut-associated lymphoid tissues, respectively. Vaccinated and naive control monkeys were challenged intrarectally with SIV strain DeltaB670 (SIV/DeltaB670), a primary isolate whose env is 15% dissimilar to that of the vaccine strain. Four of seven vaccinees were protected from infection as determined by the inability to identify viral RNA or DNA sequences in the peripheral blood and the absence of anamnestic antibody responses postchallenge. This is the first report of mucosal protection against a primary pathogenic, heterologous isolate of SIV by using a commercially viable vaccine approach. These results support further development of a DNA vaccine for protection against HIV.     

表达大肠杆菌LT-B/ST融合抗原的减毒鼠伤寒沙门氏菌株的构建

编码LT-B/ST融合抗原的基因插入pYA248载体中,构建了重组质粒pXZL66。该重组质粒转入无毒鼠伤寒沙门氏菌SR-11,ΔCya,Δcrp,Δasd菌株X4072。此无抗药性的杂合菌株X4072(pXZL66)表达的LT-B/ST融合抗原具有LT和ST抗原性而没有生物毒性,可望成为预防ETEC腹泻和相应的沙门氏菌病双价口服活疫苗候选株。     

Rubella Vaccine Trial in Children

A study with RA 27/3 attenuated rubella virus vaccine (Plotkin strain) showed that this produced a significant antibody response in all of twenty-one vaccinated non-immune children without any appreciable marked clinical reactions. Serological examination of 53 non-immune and 29 immune siblings living in the same households failed to show any evidence of transmission of infection.     

Development of a live attenuated varicella vaccine.

The Oka strain of varicella virus, isolated in our labolatory, was serially cultivated in guinea-pig embryo cultures (GPEC), and a considerable amount of cell-free virus was obtained from infected cell. GPEC passage virus at the 6th passage level was used in a small scale field trial. Susceptible children of 1 to 10 years old were injected subcutaneously with 100 to 1,000 PFU of virus. No clinical reactions due to the vaccination were observed in any children, and a high rate of antibody response was obtained with viral doses of more than 200 PFU. Attenuated virus obtained by passage in GPEC was propagated in human diploid (WI-38) cells, and it was also effective in inducing an immune response without clinical reactions. The results show that the Oka strain of varicella virus passaged in GPEC and human diploid (WI-38) cells may be used safely and effectively as a live attenuated vaccine.     

用反向遗传技术致弱基因VIId型鹅源新城疫病毒ZJI株

将新城疫病毒ZJI株基因组cDNA全长分成7个片段,依次连接并克隆至TVT7R转录载体中,构建了含ZJI株全基因组cDNA的转录载体(pNDV/ZJI),pNDV/ZJI与3个辅助表达质粒pCI-NP、pCI-P和pCI-L共转染BSR-T7/5细胞,成功拯救出了具有感染性的新城疫病毒粒子。设计两对引物,经overlapPCR方法将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸后,替换pNDV/ZJI上的对应序列,构建了转录载体pNDV/ZJIFM,将pNDV/ZJIFM与3个辅助表达质粒共转染BSR-T7/5细胞,成功拯救出了致弱的基因VIId型鹅源新城疫病毒NDV/ZJIFM,获救病毒的鸡胚最小致死剂量平均死亡时间(MDT)大于120h,同时该病毒的脑内接种致病指数(ICPI)为0.16,上述结果表明,获救病毒的毒力已被致弱,是一个较为理想的疫苗候选株。     

Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8(+) T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4(+) T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.     

表达ApxIA的血清7型胸膜肺炎放线杆菌弱毒菌株的构建及特性分析

猪传染性胸膜肺炎是由胸膜肺炎放线杆菌引起的一种高度接触传染疾病,严重阻碍着全球养猪业的发展,疫苗接种是控制该病的有效措施。为提高胸膜肺炎放线杆菌弱毒疫苗的免疫效力,以及探索胸膜肺炎放线杆菌弱毒疫苗作为呼吸系统病原疫苗载体的可行性,通过穿梭质粒pJFF224-XN将完整的apxIA基因导入apxIIC基因缺失突变株HB04C-中,构建了含有apxIA和apxIIA基因的弱毒疫苗菌株HB04C2(apxIIC-/apxIIA+/apxIA+)。通过对HB04C2的生物学特性分析发现,穿梭质粒可稳定传代,并表达ApxIA,其生长特性未受穿梭质粒的影响。将HB04C2以气管接种方式免疫仔猪,可产生针对ApxIA和ApxIIA的抗体。二免后2周以高致病性的血清1型胸膜肺炎放线杆菌攻毒,该弱毒疫苗可提供良好的免疫保护效果。     

Expression of foreign proteins in a <Emphasis Type="Italic">Vibrio cholerae</Emphasis> vaccine strain using the stationary phase hemagglutinin/protease promoter

The use of the hemagglutinin(HA)/protease promoter and secretion signals to drive expression and secretion of a foreign antigen in a live genetically attenuated cholera vaccine candidate is demonstrated. A Vibrio cholerae vaccine strain, containing a HA/protease-tetanus toxin C fragment (TCF) fusion, produced soluble-and cell-associated TCF. The fraction of TCF secreted to the culture medium was degraded unless expressed in a HA/protease-defective vaccine strain. Comparison of the hapA promoter with the strong Tac promoter using quantitative real time PCR revealed that at least five times more TCF mRNA was produced when expressed from the hapA promoter.     

Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations

The viability of BCG vaccine has traditionally been monitored using a colony-forming unit (CFU) assay. Despite its widespread use, results from the CFU assay can be highly variable because of the characteristic clumping of mycobacteria, their requirement for complex growth media, and the three week incubation period needed to cultivate slow-growing mycobacteria. In this study, we evaluated whether an ATP luminescence assay (which measures intracellular ATP content) could be used to rapidly estimate the viability of lyophilized and/or frozen preparations of six different BCG vaccine preparations - Danish, Tokyo, Russia, Brazil, Tice, and Pasteur - and two live attenuated mycobacterial vaccine candidates - a ΔlysAΔpanCD M. tuberculosis strain and a ΔmmaA4 BCG vaccine mutant. For every vaccine tested, a significant correlation was observed between intracellular ATP concentrations and the number of viable attenuated bacilli. However, the extractable intracellular ATP levels detected per cell among the different live vaccines varied suggesting that validated ATP luminescence assays with specific appropriate standards must be developed for each individual live attenuated vaccine preparation. Overall, these data indicate that the ATP luminescence assay is a rapid, sensitive, and reliable alternative method for quantifying the viability of varying live attenuated mycobacterial vaccine preparations.     

表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性

摘要:【目的】 利用平衡致死系统构建表达产类志贺氏毒素大肠杆菌(Shiga-like toxin Escherichia coli , SLTEC)保护性抗原的减毒猪霍乱沙门氏菌。【方法】 构建表达SLT-IIeB－FedF的重组质粒 ,再将其电转入终宿主菌减毒猪霍乱沙门氏菌ΔasdC500株中构建成口服活疫苗株 ,经聚丙烯酰胺凝胶电泳检测SLT-IIeB－FedF融合蛋白的表达情况,并观察重组菌体外培养的稳定性。【结果】 　利用宿主-载体平衡致死系统构建了表达SLTEC保护性抗原的重组减毒猪霍乱沙门氏菌     

高致病性猪繁殖与呼吸综合征病毒TJ株致弱过程中遗传变异和致病性分析

为了研制高致病性猪繁殖与呼吸综合征(HP-PRRS)弱毒疫苗,将高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)TJ株进行了致弱驯化,在Marc-145细胞上对其进行了连续传代,每5～10代进行噬斑克隆纯化病毒。对致弱过程中不同代次病毒进行遗传变异及致病性分析。结果表明,TJ株在致弱过程中各基因均存在不同程度的变异,至第140代,共有58个氨基酸发生突变,同时在非结构蛋白nsp2区域,在不连续的30个氨基酸缺失(481位和533～561位)之后又出现连续120个氨基酸的缺失,与VR-2332相比,该缺失位点位于推定氨基酸序列的628～747位。动物接种试验结果表明,TJ株经Marc-145细胞传至第20代时,病毒对猪的致病性明显减弱,推测TJ株在这一传代过程中非结构蛋白nsp2-nsp5、nsp7和结构蛋白GP5所发生的遗传变异对病毒毒力致弱起到一定作用。     

表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。     

马传染性贫血弱毒疫苗株核衣壳蛋白基因的表达与鉴定

从感染驴白细胞的马传染性贫血弱毒疫苗株前病毒DNA中克隆了编码核衣壳蛋白 (pll)的基因 ,在大肠杆菌中得到了表达 ,而表达的蛋白是一种可溶性的融合蛋白 ,其氨基端带有 6个组氨酸的标签 ,因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。经间接ELISA和免疫印迹试验检测 ,这种表达的融合蛋白可与马传贫阳性血清样品发生反应 ,而与健康马血清无任何反应 ,显示其具有良好的抗原性和特异性 ,可用于马传贫弱毒疫苗株在体内外复制及在接种马体内免疫应答的研究。     

Immunogenicity and protective effect of hemolysis mutants of Mycoplasma pneumoniae

Two attenuated strains of Mycoplasma pneumoniae, P24-S1 and P24-S11, were tested for their ability as a live vaccine to confer on hamsters immune resistance against challenge infection with a virulent strain of M. pneumoniae, FH-P24. Fifty percent protection was obtained by vaccination with the P24-S1 strain administered once or twice. In contrast, only 10% of the animals were protected by the P24-S11 vaccine even when it was given three times. Vaccination with the P24-S1 strain resulted in higher humoral and cellular immune responses than the P24-S11 did. These results suggest that the P24-S1 strain has the primary qualities a vaccine which may be used for protection against human mycoplasmal infection.     

Induction of interferon in man by vaccines.

The purpose of this study was to extend the spectrum of vaccines with interferon-inducing potential in man. The vaccines selected for study were the commercially available attenuated poliomyelitis vaccine type 2 (Sabin strain) and the new live attenuated influenza A/England/42/72 (H3N2) vaccine ("Alice" strain). Five subjects, two of whom had low or undetectable polio type 2 neutralizing antibody levels were given the type 2 vaccine (10-4.7 TCID50) in the standard manner orally. Even though the two individuals with low titers experienced a fourfold or greater antibody rise and one of them shed the virus in his stool, neither they nor the remaining three volunteers developed detectable levels of interferon in their sera obtained at very closely spaced intervals from day 0 to day 25 following immunization. Fifteen subjects were given approximately 10-7.5 TCID50 of influenza A/England/42/72 (H3N2) by nasal drops. Specimens consisting of sera and nasal washings were obtained at closely timed intervals for 23 days, starting with day 3 following immunization. Interferon could be detected in three of nine (33.3%) subjects who had fourfold or greater HI antibody rises. No interferon was detected in nasal washings, however. It is concluded that poliomyelitis is not a good interferon inducers in man. Live attenuated influenza vaccine does induce an interferon response in subjects with low initial serum antibody titers. This response is at best modest. The latter finding also suggests that the attenuation of the Alice strain of influenza A vaccine is not dependent on its interferon inducing potential.     

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.