A novel live attenuated vaccine candidate protects chickens against subtype B avian metapneumovirus

Avian metapneumovirus(aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses. Subtype B aMPV has recently become the dominant epidemic strain in China. We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate. The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation.The LN16-A strain did not revert to a virulent strain after five serial passages in chickens. The genomic sequence of LN16-A differed from that of the parent wild-type LN16(wtLN16) strain and had nine amino acid mutations. In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4^(+) T lymphocytes, and the Th1(IFN-γ) and Th2(IL-4 and IL-6)cytokines. We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation. Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

A Natural Vaccine Candidate Strain Against Cholera

E1 Tor Vibrio cholerae (EVC) strains may be classifled into two kinds-epidemigenic (EEVC) strains and non-epidemigenic (NEEVC) strains-based on a phage-biotyping system. A large number of EEVC strains have been screened for toxigenic and putative colonization attributes. One such naturally occurring strain (designated IEM 101) has been found which is devoid of genes encoding cholera toxin (CT), accessory cholera enterotoxin (ACE), zonula occludens toxin (ZOT), but possesses RS1 sequences and toxin-coregulated pilus A gene (tcpA) although tcpA is poorly expressed. It expresses type B pili but does not posses type C pili. It is an E1 Tor Ogawa strain and does not cause fluid accumulation in rabbit ileal loop tests. Active immunization of rabbits with strain IEM 101 elicited good protection against challenge with virulent strains of V cholerae O1. Oral administrationcaused no side effects in 15 human volunteers, colonized the gut for four to ten days and elicited good immune responses

Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

Evaluation of the Protective Efficacy of a Fused OmpK/Omp22 Protein Vaccine Candidate against Acinetobacter baumannii Infection in Mice

Acinetobocter baumannfi （A. Baumannii） is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A. boumonnii. The bacterial load in the blood, pathological changes in the lung tissue and survival rates after challenge were evaluated. Mice immunized with OmpK/Omp22 fused protein provided significantly greater protection against A. boumonnfi challenge than those immunized with either of the two proteins individually. The results provide novel clues for future design of vaccines against A. boumonnii.

Clinical development update: 1st dengue vaccine candidate

A candidate tetravalent dengue vaccine is being assessed in three clinical trials involving more than 35,000 children between the ages of 2 and 16 years in the Asia-Pacific and Latin American countries.We report the results of long-term follow-up interim analyses and integrated efficacy analyses.We are assessing the incidence of hospitalization for virologically confirmed dengues a surrogate safety end point during follow-up in years 3–6 of two phase 3 trials,CYD14 and CYD15,and a phase 2b trial,CYD23/57.We estimated vaccine efficacy using pooled data from the first 25 months of CYD14 and CYD15.

SARS-CoV-2: Vaccines in the pandemic era

Coronavirus disease 2019(COVID-19),caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),has caused millions of infections and deaths worldwide since its emergence in December 2019.As there is little or no natural immunity in the human population or specific anti-COVID-19 drugs,researchers from the government,academia and industry are developing vaccines at an unprecedented speed to halt the pandemic.In this review,the results of animal experiments and clinical trials on several vaccine technical platforms are summarized,and several challenges are also discussed to further promote the development,evaluation and application of vaccines during the challenging situation of the global pandemic.

Vaccine development for leptospirosis:A systematic review

Objective:To assess the efficacy of various types of vaccines developed for leptospirosis.Methods:A comprehensive search was conducted in three databases:PubMed,Scopus,and Cochrane Library.Two authors(YS and MN)selected the articles based on manual screening.The study eligibility criteria are all Leptospira species regardless of any cluster(pathogenic,intermediate and non-pathogenic).This study recorded articles with positive and negative results and showed a comparison among various membrane proteins as vaccine candidates.The studies on the effectiveness of outer membrane protein as vaccine candidates were also included.The articles obtained in the databases were imported into the WPS spreadsheet,and duplicate documents were removed manually.Results:A total of 24 studies were included in the review,which evaluated various types of leptospirosis vaccines.Multiple vaccines were developed and tested;however,the heterogeneity of Leptospira species pose a challenge.As an effective approach,an epitope based vaccine shows quite a promising result.However,sufficient validation,testing and clinical trials are required.Conclusions:Developing an effective vaccine for leptospirosis remains a global health priority.While significant progress has been made in recent years,there is a need for further research to optimize vaccine development and to ensure that vaccines are accessible and effective for high-risk populations.

In the era of rapid mRNA-based vaccines:Why is there no effective hepatitis C virus vaccine yet?

Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections,there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility.Indeed,the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected.To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must.The coronavirus disease 19(COVID-19)pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2(SARSCoV-2)virus,which has renewed interest on fighting HCV epidemic with vaccination.The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications.We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus,together with some key aspects of HCV immunology which have,so far,ham-pered the progress in this area.The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches,some of which have been recently and successfully employed for SARS-CoV-2 vaccines.Finally,some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.

The Efficacy of a Live Attenuated TW I-Type Infectious Bronchitis Virus Vaccine Candidate

Infectious bronchitis(IB) is a highly contagious avian disease caused by infection with infectious bronchitis virus(IBV),which seriously affects the development of the global poultry industry. The distribution of TW I-type IBV in China has increased in recent years, becoming a widespread genotype. We previously isolated a TW I-type IBV strain termed CK/CH/GD/GZ14 in 2014, but its pathogenicity and possibility for vaccine development were not explored. Therefore, this research aimed to develop a live-attenuated virus vaccine based on the CK/CH/GD/GZ14 strain. The wild type IBV CK/CH/GD/GZ14 strain was serially passaged in SPF embryos for 145 generations. The morbidity and mortality rate of wildtype strain in 14 day-old chickens is 100% and 80% respectively, while the morbidity rate in the attenuated strain was 20%in the 95 th and 105 th generations and there was no death. Histopathological observations showed that the pathogenicity of the 95th and 105th generations in chickens was significantly weakened. Further challenge experiments confirmed that the attenuated CK/CH/GD/GZ14 strain in the 95th and 105 th generations could resist CK/CH/GD/GZ14(5th generation)infection and the protection rate was 80%. Tracheal cilia stagnation, virus shedding, and viral load experiments confirmed that the 95 th and 105th generations provide good immune protection in chickens, and the immunogenicity of the 105th generation is better than that of the 95th generation. These data suggest that the attenuated CK/CH/GD/GZ14 strain in the105th generation may be applied as a vaccine candidate against TW I-type IBV.

RR1 and RR2 gene deletion affects the immunogenicity of a live attenuated pseudorabies virus vaccine candidate in natural pig host

As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pigs has not been adequately investigated. In this study, we deleted RR1 and RR2 genes based on a TK/g E/g I triple gene-deleted pseudorabies virus and tested its efficacy in pigs as a vaccine candidate. The rescued virus showed similar growth properties and plaque size in vitro as its parent strain. In an animal study, the virus could elicit humoral immune responses shown by generation of g B-specific antibodies and virus neutralizing antibodies.However, vaccination could not provide protection against virulent pseudorabies virus challenge since vaccinated pigs showed clinical pseudorabies-specific syndromes. The deficiency in protection may due to the generation of late and low levels of gB antibodies and virus neutralizing antibodies.

Neospora caninum immune mapped protein 1(NcIMP1) is a novel vaccine candidate against neosporosis

The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.

利用大蜡螟幼虫和小鼠感染模型筛选猪链球菌血清2、3和9型三价灭活疫苗候选菌株

旨在评估临床分离的猪链球菌血清2、3、9型强毒株制备的三价灭活疫苗对小鼠的免疫保护效果。使用大蜡螟幼虫感染模型和小鼠感染模型,从临床分离的猪链球菌中筛选出血清2、3和9型强毒株各1株(SS1803、SS1803024、SS1696),检测其生长曲线和毒力因子。将菌株分别灭活及混合灭活后与佐剂混合,用4×10^(7)CFU、8×10^(7)CFU SS1803、2×10^(8)CFU、5×10^(8)CFU SS1803024、1×10^(8)CFU、5×10^(8)CFU SS1696及三价灭活苗(2×10^(7)CFU SS1803^(+)1.5×10^(8)CFU SS1803024^(+)3×10^(7)CFU SS1696)分别对BALB/c小鼠进行两轮免疫,采集血清检测特异性抗体水平,分别用3种菌株致死剂量攻毒后观察小鼠免疫保护率,同时用亚致死剂量攻毒三价苗免疫组,测定小鼠血、脑、肺、脾的组织载菌量和细胞因子IL-6、IL-12水平,并观察脑、肺、脾、肾中的组织病理变化。结果显示:3株疫苗候选菌株的毒力基因鉴定分别为SS1803:gapdh^(+)/sly^(+)/fbps^(+)/orf2^(+)/mrp^(-)/89K^(-)/gdh^(+)/epf^(+),SS1803024:gapdh^(+)/sly^(+)/fbps^(+)/orf2^(+)/mrp^(-)/89K^(-)/gdh^(+)/epf^(-),SS1696:gapdh^(+)/sly-/fbps^(+)/orf2^(+)/mrp^(-)/89K^(-)/gdh^(+)/epf^(-)。单价和三价疫苗免疫组均能产生对应免疫血清型的IgG抗体,以产生IgG1型抗体为主。三价疫苗免疫组能对2、3、9型3个菌株攻毒分别提供83.3%、66.7%和66.7%的保护率,能显著降低感染后小鼠组织中的载菌量,降低血清炎性因子水平和组织病变程度。本研究筛选临床流行率高的2、3、9型强毒株,联合制备三价灭活疫苗,能够对小鼠提供良好的免疫保护力,为猪链球菌多价疫苗的研发提供新的材料。

纳米递送技术用于幽门螺杆菌疫苗研发的进展

幽门螺杆菌感染与一系列胃肠道疾病的发生息息相关,而目前用于抗幽门螺杆菌治疗的抗生素耐药性极大增加了感染复发率,导致其根治困难。疫苗是防治感染性疾病最有效的策略之一,然而,至今仍未有幽门螺杆菌疫苗上市。运用纳米技术提高疫苗稳定性和有效性已在实验室研究中初见成效,因此,本文对幽门螺杆菌疫苗的研发现状进行了总结,并归纳和讨论了幽门螺杆菌疫苗纳米递送系统的研究进展,包括黏液惰性纳米粒、耐酸性纳米粒、纳米囊泡等,以期为后续开发有效、安全的幽门螺杆菌疫苗提供参考。

H9N2亚型禽流感流行株灭活疫苗种毒的筛选

为筛选出具有良好免疫原性的禽流感病毒(AIV)H9N2亚型灭活流行株种毒,选择2008年中国大陆8个省份15株H9N2亚型AIV分离株进行抗原性分析,选取代表流行株进行鉴定并制备灭活疫苗,进行免疫效力评估。实验结果显示,2008年分离株之间抗原性比较接近,与2000年前分离株的抗原性相差较大;2008年分离的8株病毒HA基因核苷酸同源率在93.2%～98.6%之间,而CK/SH/10/01毒株与这8株病毒的同源率仅在91.9%～93.5%之间;CK/ZJ/17/08、CK/SD/2CZ/08、DK/FJ/560/08、CK/FJ/521/08、CK/HuN/174/08和CK/HuN/33/08候选株病毒在SPF鸡胚上连续传15代HA价及致病性等均未改变,SPF鸡鼻腔感染106EID50各毒株后均无任何症状和死亡出现;候选株灭活疫苗免疫SPF鸡3周时,产生针对疫苗株抗原检测的HI抗体介于10.35log2～11.811log2,以106EID50剂量鼻腔感染途径攻毒后,只有CK/HuN/174/08和CK/HuN/33/08株灭活疫苗免疫鸡不仅可以对同源毒株的攻击提供良好的免疫保护,而且对2008年分离的异源毒株的攻击也能提供比较理想的免疫保护,可作为适合我国大部分地区应用的H9N2亚型禽流感灭活疫苗种毒株。

日本血吸虫(中国大陆株)基因工程疫苗的研究

本文报道应用PCR技术或RT－PCR技术从日本血吸虫成虫cDNA文库或成虫mRNA中扩增到Sj28GST、C－Sjparamyosin、H-Sj23、Sj14、Sj22．6和Sj．TPI6个编码日本血吸虫中国大陆株抗原的基因，并把它们克隆入适合载体中，在大肠杆菌系统和蚕核型多角体病毒（BmNPV）系统中进行表达．免疫学检测证明这6种基因重组抗原都具有较好的抗原性。我们进一步应用4种比较成熟的基因重组抗原Sj26GST、Sj28GST、C－Sj．paramyosin和H－Sj23进行3批绵羊试验和1批黄、水牛田间试验，评估这几种重组抗原在绵羊和黄、水牛中的免疫保护效果，rSj26GST和rSj28GST分别获得62．1％和50.4％－68.5％的显著保护；n－paramyosin获得42.9％－55．3％、r－C－paramyosin获得44．2％－47．1％的显著保护，r－H－Sj23为51．2％－66．1％的显著保护．各组免疫后的粪便虫卵数和组织虫卵数也有不同程度的减少．用r－C－Sjparamyosin、r－Sj28GST和r－H－Sj23免疫典、水牛在血吸虫重疫区田间试验的结果表明，减虫率水牛高于黄牛，显示上述试验的3种日本血吸虫重组分子抗原有希望成为家畜日本血吸虫病的候选疫苗抗原．

H9N2亚型禽流感病毒疫苗候选株的构建

为研制H9N2亚型禽流感病毒疫苗,本研究以具有良好抗原性的代表病毒株A/chicken/Hunan/S933/2008(H9N2)(HuN33)为HA和NA基因的供体,以A/Puerto Rico/8/34(H1N1)(PR8)的6个内部基因为骨架,利用反向遗传操作技术,救获了一株重组H9N2亚型禽流感病毒疫苗候选株Re-9。该疫苗候选株既具有很好的抗原性又具有较高的鸡胚增殖特性,从而为有效防控H9N2亚型禽流感的流行提供了技术储备。

胸膜肺炎放线杆菌血清型特异的疫苗候选基因的筛选及痤疮丙酸杆菌异源免疫

目的筛选猪胸膜肺炎放线杆菌(APP)1型与5型特异的疫苗候选基因,并研究痤疮丙酸杆菌对猪传染性胸膜肺炎的异源免疫。方法采用cDNA代表性差异分析、大肠杆菌核糖体展示、免疫筛选和RT-PCR技术筛选APP1型和5型血清型特异的疫苗候选基因,进行克隆、测序及同源序列分析。并用与APP疫苗候选基因具有同源序列的痤疮丙酸杆菌(PA)免疫小鼠,研究其对APP感染的异源免疫。结果获得血清1型APP6条疫苗候选基因,2条为未知基因,2条与PA同源性为98%,2条与人cDNA有同源性。获得血清5型APP7条疫苗候选基因,其中2条为未知基因,2条与PA同源性分别为93%和100%。用PA全菌免疫小鼠,产生的抗血清可与1型和5型APP发生强的免疫反应,ELISA检测效价达1∶3200;脾淋巴细胞检测结果显示,免疫组CD3+、CD4+T细胞的百分率及CD4+/CD8+比值与对照组相比均升高,两组CD3+T细胞百分率差异有显著意义。以10倍LD501型和5型APP分别感染PA免疫小鼠,小鼠存活率分别为95%和90%,并且在攻毒后第15天体内APP可全部被清除。结论1型与5型APP存在不同的疫苗候选基因,痤疮丙酸杆菌与之存在共同抗原,能够产生交叉免疫应答,对血清1型和5型APP的感染具有预防作用。

7.2分支H5N1亚型禽流感病毒反向遗传疫苗候选株的构建

为应对clade7.2 H5N1亚型禽流感病毒(AIV)持续发生的抗原性改变及其引发的潜在AIV的地方性流行,需要根据流行监测的抗原分析结果挑选合适的供体株制备疫苗候选株。本研究采用反向遗传操作系统,以A/Puerto Rico/8/34(PR8)的内部基因为骨架,以clade7.2 H5N1 AIV A/Chicken/Liao Ning/S4092/2011(CK/LN/S4092)基因组为模板经RT-PCR扩增其HA及NA基因,并对HA基因进行分子修饰,去除与H5亚型AIV致病力有关的HA蛋白裂解位点处的多个连续碱性氨基酸,使其获得低致病性AIV的分子特征(即将-PQIEGRRRKR-突变为-PQRETR-),从而构建出clade7.2 H5N1亚型AIV疫苗候选株rPR8-LN/S4092,为动物免疫试验奠定了基础。

2型猪链球菌层粘连蛋白结合蛋白的原核表达及免疫原性检测

目的表达2型猪链球菌(Streptococcus suis2,S.suis2)层粘连蛋白结合蛋白(Lmb)并检测其免疫原性。方法 PCR检测lmb基因在不同血清型S.suis中的分布。将S.suis2中国强毒株05ZYH33的lmb基因克隆至表达载体pET32a,转化大肠杆菌E.coliBL21,IPTG诱导表达,His亲和层析柱纯化重组蛋白。Western blot检测Lmb的免疫原性。结果 lmb基因存在于大多数S.suis血清型中。诱导表达并纯化后获得较高纯度的重组蛋白。重组Lmb能够和感染05ZYH33全菌的猪恢复期血清反应。结论 lmb基因在S.suis不同血清型中广泛分布,Lmb在细菌感染宿主过程中表达,可以作为疫苗开发的候选分子。

长角血蜱的免疫研究进展

以长角血蜱疫苗候选抗原为例,从长角血蜱蛋白水解酶、蜱生殖和发育相关分子及RNA干扰技术、IgG结合蛋白技术几方面概述了近年来国内外有关蜱疫苗的研究现状,并展望了蜱疫苗的发展趋势。