抗鼠疫耶尔森菌F1抗原单克隆抗体对小鼠的被动免疫保护作用

目的研究抗鼠疫耶尔森菌F1抗原单克隆抗体(单抗)被动免疫BALB/c小鼠后的抗鼠疫保护效果,确认鼠疫抗体治疗依据,探索鼠疫免疫学治疗的评价手段。方法将BALB/c小鼠随机分组,用不同剂量的抗鼠疫耶尔森菌F1抗原单抗4C6和2D2分别进行免疫,再分别用不同剂量的鼠疫强毒菌攻击,通过小鼠的存活率和存活时间,判定F1单抗免疫小鼠被动保护其抵抗鼠疫强毒菌攻击的有效性和剂量相关性。结果在14 d的观察期内,在100.0 MLD鼠疫强毒菌攻击下,4C6单抗400.0μg组、200.0μg组、100.0μg组、50.0μg组和25.0μg组小鼠的存活率分别为100%、83%、50%、50%和0%,平均存活时间分别为15.00 d、14.67 d、13.00 d、13.50 d和9.67 d;2D2单抗400.0μg组、200.0μg组、100.0μg组、50.0μg组、25.0μg组小鼠的存活率分别为83%、50%、50%、33%和0%,平均存活时间分别为14.83 d、13.33 d、12.67 d、12.33 d和8.00 d。在10.0 MLD鼠疫强毒菌攻击下,4C6单抗50.0μg组、25.0μg组和12.5μg组小鼠的存活率分别为83%、83%和17%,平均存活时间分别为14.17 d、14.50 d、9.83 d;2D2单抗50.0μg组、25.0μg组、12.5μg组小鼠的存活率分别为100%、33%和33%,平均存活时间分别为15.00 d、12.33d和11.67 d。2.0 MLD和1.0 MLD攻击的未免疫对照组小鼠全部死亡,平均存活时间分别为6.1 d和6.7 d。F1抗原单抗的免疫剂量与小鼠的平均存活时间和存活率有比较明显的相关性(P<0.05)。结论用抗鼠疫耶尔森菌F1抗原单抗被动免疫小鼠,在受到鼠疫强毒菌攻击时,对小鼠具有免疫保护作用。     

鼠疫F1-V重组融合蛋白抗原的制备及其免疫保护效果的研究

为制备鼠疫耶尔森氏菌F1-V重组融合蛋白抗原,观察其免疫原性和免疫保护效果,通过疏水层析、阴离子交换层析、凝胶过滤层析纯化鼠疫F1-V重组融合蛋白抗原.用氢氧化铝凝胶吸附制备试验性鼠疫F1-V重组融合蛋白抗原,皮下接种健康BALB/c小鼠,ELISA检测血清F1-V抗体效价、MTT法测定淋巴细胞增殖能力,进一步用400LD50鼠疫耶尔森氏菌141标准毒株皮下攻毒,观察动物的存活情况.通过三步柱层析纯化获得的鼠疫F1-V重组融合蛋白抗原纯度达到90%以上.氢氧化铝凝胶吸附的鼠疫F1-V重组融合蛋白抗原免疫BALB/c小鼠三次,血清抗F1-V抗体效价为1∶(51200±800),对耶尔森氏菌141强毒株攻击的保护率是90%.上述结果表明,制备的鼠疫F1-V重组融合蛋白抗原具有良好的免疫原性和免疫保护效果,为研制鼠疫F1-V重组融合蛋白疫苗奠定了基础.     

鼠疫F1抗原和V抗原双组分候选疫苗的免疫学评价

目的通过由提取的鼠疫F1抗原和重组鼠疫V(rV)抗原组成的鼠疫候选疫苗免疫豚鼠,对其免疫效果进行评价。方法将豚鼠随机分成5个试验组,在免疫后不同时间点采血进行抗体检测、MTT法淋巴细胞增殖试验以及皮肤迟发型超敏反应(DTH)检测。结果抗体检测结果显示,双组分鼠疫候选疫苗能诱导较强的体液免疫应答;MTT细胞增殖结果显示,脾脏淋巴细胞特异性增殖不明显;中剂量组、高剂量组针对F1和rV抗原DTH阳转率均为100%。结论双组分鼠疫候选疫苗能诱导豚鼠体液免疫和细胞免疫应答,该疫苗有希望成为我国新一代鼠疫疫苗。     

重组鼠疫菌V抗原的纯化及其豚鼠免疫保护力初探

采用Ni^2 亲和层析方法,对用工程化大肠杆菌BL21(DE3)表达的重组鼠疫菌v抗原进行纯化,目标蛋白纯度达到90％以上。以氢氧化铝凝胶配制吸附疫苗,经二针次肌内注射免疫实验豚鼠后,对皮下注射400个致死剂量(MLD)强毒鼠疫菌攻击有一定保护效力,存活率为20％。结果表明,重组鼠疫菌V抗原有望作为改进的F1 V亚单位疫苗的主要成分。     

鼠疫菌F1抗体/抗原酶联免疫诊断试剂盒的应用评价

目的对兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒进行临床应用评价。方法采用双抗原/抗体夹心酶联免疫吸附试验(ELISA)、间接血球凝集试验(IHA)、胶体金免疫层析试验(GICA)3种方法的诊断试剂对比检测云南省地方病防治所中心实验室保藏的和现场采集的血清样品和脏器样品,对血清样品做鼠疫菌F1抗体检测,对脏器样品做鼠疫菌F1抗原检测。结果在358份血清样品中,ELISA试剂检出F1抗体阳性52份(14.52%),IHA试剂检出阳性37份(10.34%),GICA试剂检出阳性45份(12.57%)。ELISA与IHA试剂的符合率为95.23%,与GICA试剂的符合率为96.92%。经统计学χ2检验,ELISA试剂检出F1抗体阳性率高于IHA试剂(χ2=11.53,P=0.000 7),与GICA试剂检出的差异无统计学意义(χ2=3.27,P=0.070 4)。进一步分析滴度差值频数,ELISA试剂检测人血清的敏感性高于IHA试剂的样品占87.5%。在117份脏器样品中,3种试剂均检出F1抗原阳性15份(12.82%),符合率100%。滴度差值频数比较,ELISA试剂检测敏感性高于反向间接血球凝集试验(RIHA)试剂的样品为78.57%。结论兰州生物制品研究所有限责任公司研制的鼠疫菌F1抗体酶联免疫诊断试剂盒和鼠疫菌F1抗原酶联免疫诊断试剂盒性质特异,其敏感性优于IHA试剂盒和GICA试剂条,值得在鼠疫的监测和快速诊断中推广应用。     

中试规模纯化重组鼠疫rF1-V融合蛋白及其免疫原性分析

重组F1-V融合蛋白(rF1-V)是目前在进行临床研究的鼠疫亚单位疫苗的主要成分。本研究摸索了rF1-V的可溶表达条件,并对条件进行了优化和放大,确定的中试发酵工艺为:在重组菌对数生长期中期加入50μmol/LIPTG,25℃诱导表达5h。通过硫酸铵分级沉淀、离子交换、疏水相互作用层析和凝胶过滤四步纯化,最终得到纯度为99%、回收率大于20%且各项检测指标合格的蛋白。在此基础上,将蛋白使用氢氧化铝佐剂进行吸附,在小鼠体内进行了免疫原性研究。ELISA测定两次皮下免疫后血清的抗体滴度。比较融合蛋白免疫组(rF1-V)与单一抗原免疫组(rF1、rV)以及联合抗原免疫组(rF1+rV)之间体液免疫反应的差异。结果显示:20μgrF1-V免疫剂量组诱导的抗F1抗体滴度明显高于其他组,抗V抗体滴度与其他组相比没有显著差异。表明本工艺制备的rF1-V抗原有望作为鼠疫亚单位疫苗的主要组分。     

F1-V重组亚单位疫苗对鼠疫耶尔森氏菌141强毒株皮下攻毒保护性的研究

在此实验中, 设计了一种包含2种成分的重组融合蛋白作为疫苗成分来防护鼠疫耶尔森氏菌(Yersinia)可能产生的生物威胁. 重组F1-V蛋白与铝佐剂结合, 分别以10, 20, 50 mg剂量免疫BALB/C小鼠, 周期为2个月. 检测小鼠血清抗体水平和T辅助细胞的亚型. 免疫后小鼠以25~600 LD₅₀剂量的鼠疫耶尔森氏菌141强毒株进行皮下攻毒实验. 结果证明, F1-V重组蛋白在 小鼠体内诱导产生足够保护的免疫应答. 血清IgG水平是产生最终保护力的一个重要因素. 20 μg的免疫剂量可以诱导血清抗体效价高达51200, 使小鼠对400 LD50的鼠疫耶尔森氏菌产生100%的保护. F1-V重组蛋白引发的抗体亚型主要为IgG1类, 说明抗体反应趋向Th2型反应. 流式细胞分析表明, 铝佐剂主要帮助F1-V重组融合蛋白诱导强烈的体液免疫而不是CTL细胞免疫应答.表明F1-V重组亚单位疫苗株有希望成为一种新型的鼠疫疫苗候选株.     

用提呈载体构建鼠疫肽疫苗

鼠疫菌是肺、腺鼠疫病原因子。由于当前注册的疫苗有局限性,需要更合理有效的亚单位疫苗战胜各型鼠疫。结合佐剂的新方法使形成新疫苗有了新方案。为了开发新一代鼠疫疫苗,我们选择了一个免疫主基,鼠疫菌荚膜蛋白F1。已知此肽具有B(3个序列B1,     

鼠疫耶尔森菌F1抗原的性质研究

鼠疫菌F1抗原是鼠疫亚单位新疫苗最重要的候选抗原,对其性质的充分认识,将有助于抗原制造工艺和新疫苗的开发。F1抗原的性质研究包括:微观结构,一级核苷酸、氨基酸序列,二级结构,高分子聚集形态,以及F1抗原的理化性质。     

不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平分析

目的研究伤寒Vi多糖蛋白结合疫苗免疫效果及不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平,以确定合适的免疫剂量。方法将150只清洁级NIH雌性小鼠随机分为5组,分别为A组(0.625μg结合疫苗组)、B组(1.250μg结合疫苗组)、C组(2.500μg结合疫苗组)、D组(2.500μg多糖组)及阴性组(10mmol/L PBS),每组30只;另领取10只为空白对照(不接种)。A、B、C、D组及阴性组小鼠经腹股沟皮下注射,剂量0.1 m L/只,每隔2周免疫1次,共免疫3次,每次免疫后第7天采血。采用ELISA检测小鼠血清抗体效价,同时对不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体效价进行分析比较。结果与D组相比,A、B、C三组诱导的抗体水平与之均有统计学意义(P<0.05);A组与B组、A组与C组之间的抗体水平也具有统计学意义(P<0.05),而B组与C组之间的抗体水平无统计学意义(P>0.05)。说明与多糖疫苗相比,伤寒Vi多糖蛋白结合疫苗能够诱导更高的抗体水平,且具有明显的剂次加强效应。同时证明1.250μg的伤寒Vi多糖蛋白结合疫苗可诱导与2.500μg伤寒Vi多糖蛋白结合疫苗相同的抗体水平。结论伤寒Vi多糖蛋白结合疫苗的两种免疫剂量在小鼠体内可诱导相同的抗体水平,在选择接种剂量时,可致免疫应答的无统计学意义的低剂量可能是较为经济和安全的选择。     

Protection against lethal subcutaneous challenge of virulent Y. pestis strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V pro-tein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25― 600 LD50. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD50 virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.     

基于rV270抗原的鼠疫多孔微球疫苗的制备及其免疫保护作用评价

目的：评价生物可降解高分子材料多孔微球作为鼠疫亚单位疫苗佐剂的可行性。方法：制备可生物降解的高分子材料多孔微球,将rV270抗原蛋白吸附到多孔微球中制备微球疫苗,肌肉注射免疫BALB/c小鼠,初次免疫后21d加强免疫1次,于初次免疫后第10周用600LD50鼠疫耶尔森氏菌攻毒,攻毒后观察14d。结果：攻毒后,微球疫苗免疫的小鼠全部存活,且健康状况良好,对照组小鼠几乎全部死亡。结论：生物可降解多孔微球可作为免疫佐剂用于鼠疫亚单位疫苗研制。     

人tPA信号肽和Yersinia Pestis保护性抗原F1-V融合基因的构建及其免疫效果观察

为获得含有鼠疫F1和V抗原编码基因以及人tPA信号肽基因的重组质粒tPA-pVAX1/F1-V,并测定其诱导特异性免疫应答的能力, 用PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序,构建pVAX1/F1-V融合重组质粒.PCR扩增tPA信号肽片段并将其插入到F1-V的上游,构建tPA-pVAX1/F1-V融合重组质粒;转染COS-7细胞,Western blot法鉴定目的蛋白的表达.重组质粒tPA-pVAX1/F1-V加GM-CSF佐剂免疫BALB/c小鼠,观察免疫效果.400个LD50强毒鼠疫菌皮下攻毒观察保护率.结果表明,tPA-pVAX1/F1-V在COS-7细胞中表达;免疫鼠体内产生特异性抗体;抗体亚型分析、细胞因子等指标的测定表明,所构建DNA疫苗以诱发Th1型免疫为主;&#61472;攻毒保护率达90%.结果提示,已成功构建F1-V融合蛋白真核表达载体tPA-pVAX1/F1-V,且具有诱导特异性细胞免疫和体液免疫应答的能力, 对强毒鼠疫菌皮下攻毒有一定的保护效力,为鼠疫菌新型疫苗研制奠定了基础.     

鼠疫耶尔森氏菌rV270抗原的克隆、表达及其免疫保护作用评价

目的：为研制鼠疫亚单位疫苗,克隆、表达并纯化去除产生免疫抑制作用序列后的鼠疫耶尔森氏菌LcrV抗原（rV270）。方法：依据已知的LcrV的核苷酸序列,避开其产生免疫抑制作用的区段设计引物,扩增rV270基因并克隆到pET24a载体中,在大肠杆菌BL21中表达His-rV270融合蛋白：表达产物先后经Co^2＋亲和层析和Sephacryl S-200HR凝胶柱纯化,并在纯化过程中应用凝血酶切除His标塔;氢氧化铝佐剂吸附重组抗原后免疫BALB／c小鼠,初次免疫后第21天加强免疫1次,第5周使用104CFU鼠疫菌141强毒株攻毒,测定其免疫保护作用。结果：rV270以可溶性方式表达;应用Co^2＋亲和层析柱和Sephacryl S-200HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度的重组蛋白;攻毒实验中实验组小鼠全部存活,而对照组全部死亡。结论：获得了具有良好免疫保护作用的rV270蛋白,可用于鼠疫亚单位疫苗的研究。     

Protection against lethal subcutaneous challenge of virulent <Emphasis Type="Italic">Y. pestis</Emphasis> strain 141 using an F1-V subunit vaccine

In this study, we designed and engineered a two-component recombinant fusion protein antigen as a vaccine candidate against the possible biological threat of Yersinia pestis. The recombinant F1-V protein was formulated with Alhydrogel. A four-time injection with a dosage of 10, 20 and 50 μg/mouse in about two months was adopted for vaccination. Serum antibodies and subclass of T helper cells were measured and analyzed. After the final vaccination, the mice were challenged by 141 strain with 25–600 LD₅₀. In conclusion, the recombinant vaccine was capable of inducing protective immunity against subcutaneous challenge. The level of serum IgG was supposed to be a main factor that affected the final protection of challenge. 20 μg recombinant protein could induce an endpoint titre of serum IgG as high as 51200, which was enough to afford 100% protection against 400 LD₅₀ virulent 141 challenge. The antibody isotype analysis showed that the vaccine induced predominantly an IgG1 rather than IgG2a response. Flow cytometric analysis revealed that Alhydrogel significantly helped induce a stronger humoral immunity instead of CTL cellular response. These findings suggested that the plague F1-V subunit vaccine is promising for the next plague vaccine.     

A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen

We investigated the relative immunogenicity and protective efficacy of recombinant X85MF1 and X85V strains of DeltacyaDeltacrpDeltaasd-attenuated Salmonella Typhimurium expressing, respectively, secreted Yersinia pestis F1 and V antigens, following intranasal (i.n.) or i.n. combined with oral immunization for a mouse model. A single i.n. dose of 10(8) CFU of X85MF1 or X85V induced appreciable serum F1- or V-specific IgG titres, although oral immunization did not. Mice i.n. immunized three times (i.n. x 3) with Salmonella achieved the most substantial F1/V-specific IgG titres, as compared with corresponding titres for an oral-primed, i.n.-boosted (twice; oral-i.n. x 2) immunization regimen. The level of V-specific IgG was significantly greater than that of F1-specific IgG (P<0.001). Analysis of the IgG antibodies subclasses revealed comparable levels of V-specific Th-2-type IgG1 and Th-1-type IgG2a, and a predominance of F1-specific Th-1-type IgG2a antibodies. In mice immunized intranasally, X85V stimulated a greater IL-10-secreting-cell response in the lungs than did X85MF1, but impaired the induction of gamma-interferon-secreting cells. A program of i.n. x 3 and/or oral-i.n. x 2 immunization with X85V provided levels of protection against a subsequent lethal challenge with Y. pestis, of, respectively, 60% and 20%, whereas 80% protection was provided following the same immunization but with X85MF1.     

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.     

H7N9灭活疫苗联合MF59佐剂在小鼠体内诱导的长效体液免疫应答

以BALB/c小鼠为模型,探讨H7N9流感病毒灭活疫苗免疫小鼠后所诱导的长效体液免疫应答的动态变化。不同剂量的流感H7N9全病毒灭活疫苗单独或辅以MF59佐剂肌肉注射免疫小鼠一次。连续采集免疫后小鼠15个月的血清,用ELISA方法检测特异性IgG抗体水平,血凝抑制(hemagglutination inhibition,HI)试验和微量中和(microneutralization,MN)试验检测第15个月时的HI抗体和中和抗体效价。实验结果发现,小鼠血清中的特异性IgG抗体水平随时间变化持续缓慢上升,第5个月时达到顶峰,随后略有下降但一直持续平稳状态;IgG抗体滴度与疫苗剂量成正相关,且添加佐剂能提高抗体滴度。HI及MN抗体检测表明,免疫后第15个月产生的抗体能有效中和病毒,且抗体跟疫苗剂量成正比。以上研究表明,H7N9流感病毒灭活疫苗免疫小鼠一次诱导产生的特异性抗体能在较长期内保持比较平稳的抗体滴度,为小鼠提供免疫保护;增加抗原剂量和添加MF59佐剂能增加疫苗特异性抗体水平。该研究为H7N9流感疫苗产生的长期保护效应提供了一定的数据积累和参考。     

Technical note: a rapid diagnostic test detects plague in ancient human remains: an example of the interaction between archeological and biological approaches (southeastern France, 16th-18th centuries)

A rapid diagnostic test (RDT) that detects Yersinia pestis F1 antigen was applied to 28 putative plague victims exhumed from seven burial sites in southeastern France dating to the 16th-18th centuries. Yersinia pestis F1 antigen was detected in 19 of the 28 (67.9%) samples. The 27 samples used as negative controls yielded negative results. Soil samples taken from archeological sites related to both positive and negative samples tested negative for F1 antigen. The detection threshold of the RDT for plague (0.5 ng/ml) is sufficient for a preliminary retrospective diagnosis of Y. pestis infection in human remains. The high specificity and sensitivity of the assay were confirmed. For two sites positive to F1 antigen (Lambesc and Marseille), Y. pestis-specific DNA (pla gene) had been identified previously by PCR-sequence based analyses. Specifically, the positive results for two samples, from the Lambesc cemetery and the Marseille pit burial, matched those previously reported using PCR. Independent analyses in Italy and France of different samples taken from the same burial sites (Draguignan and Martigues) led to the identification of both Y. pestis F1 antigen and Y. pestis pla and gplD genes. These data are clear evidence of the presence of Y. pestis in the ancient human remains examined in this study.     

Immune responses of mice to influenza subunit vaccine in combination with CIA07 as an adjuvant

CIA07 is an immunostimulatory agent composed of E. coli DNA fragments and modified LPS lacking the lipid A moiety. In this study, we investigated whether CIA07 promotes immune responses as an adjuvant to the influenza subunit vaccine. Balb/c mice were immunized intramuscularly once or twice at a 4-week interval with the trivalent influenza subunit vaccine antigen alone or in combination with CIA07 as adjuvant. Antigen-specific serum antibody titers and hemagglutination-inhibition (HI) antibody titers were assessed. At 4 weeks after each immunization, the antigen-specific total serum IgG antibody titer in mice receiving CIA07 was 2 to 3 times higher than that in animals administered antigen alone (P<0.05). The CIA07-treated group additionally displayed higher HI antibody titers against each of the 3 vaccine strains, compared to the antigen group. Animals receiving antigen alone displayed barely detectable antigen-specific serum IgG2a antibody titers. In contrast, coadministration of CIA07 with antigen led to significantly enhanced IgG2a antibody responses, suggesting that CIA07 stimulates a Th1-type immune response. Moreover, the CIA07-treated group displayed a marked increase in the number of interferon gamma-producing CD8(+) T cells in splenocytes. These data collectively demonstrate that CIA07 has the ability to induce both Th1-type cellular and Th2-type humoral immune responses to the influenza subunit vaccine, and support its potential as an effective adjuvant to the influenza vaccine.