Optimization of the Process for Preparing Bivalent Polysaccharide Conjugates to Develop Multivalent Conjugate Vaccines against Streptococcus pneumoniae or Neisseria meningitidis and Comparison with the Corresponding Licensed Vaccines in Animal Models

Objective:This study aimed to describe,optimize and evaluate a method for preparing multivalent conjugate vaccines by simultaneous conjugation of two different bacterial capsular polysaccharides(CPs)with tetanus toxoid(TT)as bivalent conjugates.Methods:Different molecular weights(MWs)of polysaccharides,activating agents and capsular polysaccharide/protein(CP/Pro)ratio that may influence conjugation and immunogenicity were investigated and optimized to prepare the bivalent conjugate bulk.Using the described method and optimized parameters,a 20-valent pneumococcal conjugate vaccine and a bivalent meningococcal vaccine were developed and their effectiveness was compared to that of corresponding licensed vaccines in rabbit or mouse models.Results:The immunogenicity test revealed that polysaccharides with lower MWs were better for Pn1-TT-Pn3 and MenA-TT-MenC,while higher MWs were superior for Pn4-TT-Pn14,Pn6A-TT-Pn6B,Pn7F-TT-Pn23F and Pn8-TT-Pn11A.For activating polysaccharides,1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP)was superior to cyanogen bromide(CNBr),but for Pn1,Pn3 and MenC,N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDAC)was the most suitable option.For Pn6A-TT-Pn6B and Pn8-TT-Pn11A,rabbits immunized with bivalent conjugates with lower CP/Pro ratios showed significantly stronger CP-specific antibody responses,while for Pn4-TT-Pn14,higher CP/Pro ratio was better.Instead of interfering with the respective immunological activity,our bivalent conjugates usually induced higher IgG titers than their monovalent counterparts.Conclusion:The result indicated that the described conjugation technique was feasible and efficacious to prepare glycoconjugate vaccines,laying a solid foundation for developing extended-valent multivalent or combined conjugate vaccines without potentially decreased immune function.

Safety of a 13-Valent Pneumococcal Conjugate Vaccine in Elderly Adults Previously Immunized with a 23-Valent Pneumococcal Polysaccharide Vaccine: An Open-Label Trial

An open-label, multicenter study was conducted to describe the safety of the 13-valent pneumococcal conjugate vaccine (PCV13) in 1049 individuals aged ≥68 years, who had previously been immunized with the unconjugated 23-valent pneumococcal polysaccharide vaccine (PPSV23). In addition, the safety profile of PCV13 in this study was compared, in a post-hoc descriptive analysis, to that observed in other elderly populations, who had received PCV13 or PPSV23 as part of other completed studies. Local (56.6%) and systemic reactions (58.4%) were very common, but were mainly mild, and of short duration (mean: 1.3 - 4.6 days). There were no related serious adverse events (AEs) within 1 month after PCV13. 123 days after PCV13 and 94 days after a nonstudy influenza vaccine, a case of transient Guillain-Barré syndrome occurred, which the investigator assessed as possibly related to the vaccination. Reactogenicity observed in this study population was generally similar to that of other elderly study populations with PPSV23-preimmunized adults, and with PPSV23-naive adults. Reactogenicity was less common in this study than that observed in PPSV23-preimmunized adults who were revaccinated with PPSV23 rather than a subsequent dose of PCV13. There were no related serious AEs reported after PCV13 and PPSV23 in these comparator studies. Conclusion: PCV13 may be administered safely to older adults previously immunized with PPSV23. (ClinicalTrials. gov Identifier: NCT00500266)

Persistence of Pleural Effusions and Empyemas after Pneumococcal Conjugate Vaccine Implementation in Uruguay

In Uruguay a post pneumococcal conjugate vaccine implementation surveillance of hospitalized children with pneumonia showed an increase of complicated pneumonias, while uncomplicated pneumonias decreased. Out of 151 pleural effusions, 62 were empyemas requiring drainage, the rest of cases were treated with antibiotics with a favorable outcome. Patient’s vaccinated status varied. Pneumococcal etiology was poorly documented. The few identified sero-types were 1 and 3, a fact that urges PCV13 use for their control.

Potential carrier priming effect in Australian infants after 7-valent pneumococcal conjugate vaccine introduction

AIM:To investigate evidence of clinical protection in infants after one dose of 7-valent pneumococcal conjugate vaccine(7vPCV) owing to carrier priming.METHODS:Using Australian National Notifiable Diseases Surveillance System data,we conducted a descriptive analysis of cases of vaccine type invasive pneumococcal disease(VT-IPD) during "catch-up" years,when 7vPCV was carrier primed by prior administration of DTPa vaccine.We compared the number of VT-IPD cases occurring 2-9 wk after a single dose of 7vPCV(carrier primed),with those < 2 wk post vaccination,when no protection from 7vPCV was expected yet.Further comparison was conducted to compare the occurrence of VT-IPD cases vs non-VT-IPD cases after a single carrier-primed dose of 7vPCV.RESULTS:We found four VT-IPD cases occurring <2 wk after one carrier primed dose of 7vPCV while only one case occurred 2-9 wk later.Upon further comparison with the non-VT-IPD cases that occurred after one carrier primed dose of 7vPCV,two cases were detected within 2 wk,whereas seven occurred within2-9 wk later;suggesting a substantial level of protection from VT-IPD occurring from 2 wk after carrier-primed dose of 7vPCV.CONCLUSION:This data suggest that infants may benefit from just one dose of 7vPCV,likely through enhanced immunity from carrier priming effect.If this is proven,an adjusted 2-dose schedule(where the first dose of PCV is not given until after DTPa) may be sufficient and more cost-effective.

<i>Streptococcus pneumoniae</i>Upper Respiratory Carriage in Costa Rican Children with Otitis Media before the Introduction of the Heptavalent Conjugated Vaccine in the National Immunization Program

Objective: The aim of this study was to analyze the NP/OP S. pneumoniae serotype distribution and potential vaccine coverage in Costa Rican children with Otitis Media (OM) before the introduction of PCV-7 in the National Immunization Program (NIP). Methods: Between 2002 and 2006, NP and OP samples were obtained from 641 children from 6 to 79 months of age, at the time of OM diagnosis. S. pneumoniae serotyping and antimicrobial susceptibility were performed. Results: 386 S. pneumoniae isolates were recovered. The most common S. pneumoniae serotypes (ST) were: ST 6B, ST 14, ST 19F. Penicillin non-susceptibility was observed among 57% of the isolates obtained from children < 24 months of age. 15% strains were multidrug resistant. Potential vaccine coverage was: PCV-7: 60%;PCV-10: 62%;and PCV-13: 76% and against penicillin non-susceptible and multidrug resistant isolates was: PCV-7;59% and 83%, respectively;PCV-10: 60% and 85%, respectively and PCV-13: 74% and 96%, respectively. Conclusions: S. pneumoniae was isolated from the NP and/or OP in the majority (59%) of studied children with OM. At a statistical significant level, only serotype 3 was more frequently isolated among children >24 months of age. Antibiotic non-susceptibility and MDR were significantly higher in children <24 months of age. This study demonstrates that PCV-13 offers the highest potential vaccine coverage and serves to assess the impact of introduction of one of the conjugated vaccines in the NIP in Costa Rica.

Impact of Seven Valent Pneumococcal Conjugate Vaccine on Nasopharyngeal Carriage in Young Children in Okinawa, Japan

In Japan, the heptavalent pneumococcal conjugate vaccine (PCV7) became available in February 2010 and was subsidized by the national funding system from May 2011 in Okinawa, after which it was incorporated into the national immunization practice (NIP) in April 2013 using a 3 + 1 schedule for all infants. We conducted an annual survey in 2012 to determine the effect of PCV7 on nasopharyngeal colonization by pneumococcal serotypes and to analyze the risk factors for colonization in infants. Nasopharyngeal swabs for pneumococcal isolation and serotyping were obtained from infant 2 to 22 months of age before and after PCV7 immunization among 4 clinics in Okinawa, Japan. Between January 2012 and December 2012, nasopharyngeal swabs for bacterial cultures were obtained among 782 infants aged 2 to 22 months old and demographic data was obtained among 725 participant infants. Among the 725 evaluable infants, 193 pneumococcal strains were detected in 180 infants for an overall nasopharyngeal carriage of 24.8%. The main capsular serotypes isolated were 6C (16.1%), 19A (12.4%) and 15B (9.8%). Carriage of PCV7 serotypes accounted for 21.8% (42/193). The result of multivariate data analysis showed the pneumococcal carriage rate of non-PCV7 serotypes was significantly (P < 0.001) high in infant with siblings and daycare attendance. On the other hand, the result of multivariate data analysis showed that carriage rate of PCV7 serotype had only significantly high risk in infant with siblings and did not have a significant risk dependent on age and daycare attendance. Carriage PCV7 serotypes increased in the presence of other siblings, while PCV7 vaccination was shown to eliminate daycare attendance as a risk. The results of this study demonstrates that PCV7 vaccination decrease the overall nasopharyngeal carriage of PCV7 serotypes in vaccinated children including children at risk such as children attending day-care centers.

Effect of 7 and 13-Valent Pneumococcal Conjugate Vaccines Different Number of Doses for Pneumonia Control in 2008 and 2010 Birth Cohort Children

The 7-valent pneumococcal conjugate vaccine (PCV) was introduced in Uruguay in March 2008. In April 2010, it was replaced by PCV13. Surveillance of both vaccines was performed on hospitalized children with consolidated pneumonia. The effect of different number of vaccine doses was evaluated in 2008 and 2010 birth cohorts vaccinated with PCV7 and PCV13 respectively. The study aims to estimate the effects of PCV7 and PCV13 different number of doses on consolidated pneumonia, through the study of hospitalized children from 2008 and 2010 birth cohorts. Vaccination records of every child were available providing precise vaccination data;therefore a new approach was used to estimate PCVs effect. Incidence rate was calculated for each year of the study and for the different number of vaccine doses used each year. Exposure was calculated as person per year and rate ratio values determined the decrease of consolidated pneumonias. This decrease in percentage was estimated as the difference between the incidence with no vaccine and the incidence of every one of the doses. Incidence rate ratio revealed significant values for the three vaccine doses of PCVs for both cohorts. Upon comparing incidences, significant reduction percentages of consolidated pneumonia admissions were found. The reduction percentage of consolidated pneumonia for fully vaccinated (3 doses) patients was 69.3% and 84.6 % for PCV7 and PCV13, respectively. These results confirm that PCV7 and PCV13 are highly effective for reducing pediatric hospitalizations due to consolidated pneumonia, as reported by other national publications and demonstrated by international researchers.

Synthesis, Characterization, and Immunological Properties of LPS-Based Vaccines Composed of O-Polysaccharides Conjugated with Recombinant Exoprotein A from Pseudomonas aeruginosa

Pseudomonas aeruginosa remains one of the major pathogens affecting immunocompromised patients. LPS-based monovalent (MV) and polyvalent (PV) conjugate vaccines were prepared from the most prevalent strains of P. aeruginosa International Antigenic Typing Scheme (IATS) 6, 10, 11 and 20 to evaluate their immunogenicity and protective capacities from infection by the pathogens. Conjugation of the O-polysaccharide (O-PS) antigens of P. aeruginosa strains to the common immunogenic recombinant Exotoxin A (rEPA) supports the multi-antigenic approach for the development of a vaccine that provides cross protection against various strains of the pathogen. The O-PSs were indirectly conjugated through adipic acid dihydrazide (ADH) to rEPA by carbodiimidemediated condensation reaction. Mice were immunized with the conjugates emulsified with monophosphoryl lipid A (MPL) or Freund's adjuvant compared with conjugates without adjuvant, unconjugated mixture of rEPA and O-PS emulsified with MPL, and sterile saline. The MV and PV vaccines emulsified with MPL adjuvant elicited the highest anti-O-PS IgM and IgG antibodies. Immunization of mice with MV vaccines derived from IATS 10, 11, and 20, emulsified with MPL adjuvant provided a high level of protection against the homologous bacterial strain. Similarly, high protection was obtained when mice were immunized using PV and challenged separately with bacterial strains 10, 11, and 20, but lower protection against the IATS 6 strain. Also, high cross protection of MV vaccine derived from O-PS of IATS 10 and 20 was obtained against P. aeruginosa IATS 11 strain. The in vivo protection correlated with the level of anti-O-PS IgG in the mice serum.

Safety Observation Study on Haemophilus Influenza Type B Conjugate Vaccines Injected at Different Sites in Chinese Infants

In the present study, the safety of Hoemophilus influenza type b conjugate vaccines inoculated in the upper arm deltoid and vastus lateralis muscle was evaluated. 680 infants aged 2-5 months and 6-12 months were selected to be the research subjects in whom the Hib conjugate vaccines were inoculated by injection in the upper arm deltoid and vastus lateralis muscle, respectively. The safety analysis indicated that there were no statistic differences in the incidence rates of adverse reactions when the Hib conjugate vaccines were inoculated at different sites. So we concluded that the safety of inoculation injection of Hib conjugate vaccines in vastus lateralis muscle was the same as that inoculated in the upper arm deltoid.

Immunogenicity and protective immunity against otitis media caused by pneumococcus in mice of Hib conjugate vaccine with PsaA protein carrier

This study evaluated the immunogenicity and protective immunity of a Hemophilus influenzae b （Hib） polysaccharide conjugate vaccine with the pneumococcal surface adhesin A （PsaA） protein carrier in young mice. The Hib polysaccharide was conjugated with the rPsaA protein carrier, which was produced using recombinant DNA technology. A total of 15 young mice aged 3 weeks to 5 weeks were immunized with the conjugate vaccine, and another 15 young mice of the same age were immunized with the licensed Hib-tetanus toxoid （TT） vaccine. Furthermore, the third group of 15 young mice was inoculated with phosphate buffer saline as control. The immunized mice were inoculated with pneumococcus in the middle ear. Results showed that IgG antibody responses against both the PsaA protein and Hib polysaccharide were observed in the Hib-PsaA group. However, no statistical difference was observed in the titer of ｜gG against the Hib polysaccharide between Hib-PsaA and Hib-TT groups. The elimination rate of pneumococcus and the inflammation of the middle ear showed the effectiveness of protective immunity against otitis media caused by pneumococcus. Our results suggest that the Hib polysaccharide can be successfully conjugated with rPsaA via amide condensation. This new Hib-PsaA conjugate vaccine can induce both anti-PsaA and anti-Hib immune responses in young mice and elicit effective protection against acute otitis media caused by pneumococcus.

13-Valent pneumococcal conjugate vaccines vaccination innovative strategy in Weifang City,China:a case study

The World Health Organization(WHO)prioritizes pneumococcal disease as a vaccine-preventable disease and recommends the inclusion of pneumococcal conjugate vaccines(PCV)in national immunization programs worldwide.However,PCV is not included in the National Immunization Program in China and has low vaccination coverage due to its high cost.To address this,Weifang City implemented an innovative strategy for a 13-valent PCV(PCV13)on June 1,2021.This strategy aimed to provide one dose of PCV13 free of charge for children aged 6 months to 2 years in registered households and to adopt a commercial insurance model with one dose of PCV13 free of charge in 2023 for children over 2 years old.The Health Commission of Weifang and other departments conducted a comprehensive investigation and considered various factors,such as vaccine efectiveness,safety,accessibility,vaccine price,and immunization schedules,for eligible children(under 5 years old).Stakeholder opinions were also solicited before implementing the policy.The Commission negotiated with various vaccine manufacturers to maximize its negotiating power and reduce vaccine prices.The implementation plan was introduced under the Healthy Weifang Strategy.Following the implementation of this strategy,the full course of vaccination coverage increased signifcantly from 0.67 to 6.59%.However,vaccination coverage is still lower than that in developed countries.Weifang’s PCV13 vaccination innovative strategy is the frst of its kind in Chinese mainland and is an active pilot of non-immunization program vaccination strategies.To further promote PCV13 vaccination,Weifang City should continue to implement this strategy and explore appropriate fnancing channels.Regions with higher levels of economic development can innovate the implementation of vaccine programs,broaden fnancing channels,improve accessibility to vaccination services,and advocate for more localities to incorporate PCV13 into locally expanded immunization programs or people-benefting projects.A monitoring and evaluation system should also be established to evaluate implementation efects.

Accelerating Pneumococcal Conjugate Vaccine introductions in Indonesia:key learnings from 2017 to 2022

Despite high pneumococcal disease and economic burden in Indonesia and interest to introduce pneumococcal conjugate vaccine (PCV), there were challenges in establishing a comprehensive strategy to accelerate and enable the introduction in country in the early 2010s. Starting in 2017, Clinton Health Access Initiative and partners supported the government of Indonesia with evidence-based decision-making and implementation support for introducing PCV into the routine immunization program. Indonesia has since accelerated PCV roll out, with nationwide reach achieved in 2022. On the path to PCV introduction, several challenges were observed that impacted decision making on whether and on how to optimally roll out PCV, resulting in significant introduction delays;including (1) a complex country context with a devolved government structure, fragmented domestic funding streams, and an imminent transition out of major immunization donor (Gavi) support;(2) strong preference to use domestically sourced products, with limited experience accessing global pooled procurement mechanism including for vaccines;and (3) concerns around programmatic feasibility and sustainability. This case study documents key insights into the challenges experienced and how those were systematically addressed to accelerate new vaccine introduction in Indonesia, with support from local and global stakeholders over time. The learnings would be beneficial for other countries yet to introduce critical new vaccines, in particular those with similar archetype as Indonesia e.g., middle-income countries with domestic manufacturing capacity and/or countries recently transitioning out of Gavi support.

Novel polysaccharide-protein conjugates provide an immunogenic 13-valent pneumococcal conjugate vaccine for S.pneumoniae

Pneumonia remains the single leading cause of childhood death worldwide.Despite the commercial availability of multiple pneumococcal conjugate vaccines(PCVs),high dosage cost and supply shortages prevent PCV delivery to much of the developing world.The current work presents high-yield pneumococcal conjugates that are immunogenic in animals and suitable for use in human vaccine development.The 13-valent pneumococcal conjugate vaccine(PCV-13)investigated in this research incorporated serotypes 1,3,4,5,6A,6B,7F,9V,14,18C,19A,19F,and 23F.Pneumococcal polysaccharides(PnPSs)and CRM197 carrier protein were produced and purified in-house,and used to prepare PnPS-CRM conjugates using unique,cyanide-free,in vacuo glycation conjugation methods.In vitro characterization confirmed the generation of higher molecular weight PnPS-CRM conjugates low in free protein.In vivo animal studies were performed to compare PnuVax's PCV-13 to the commercially available PCV-13,Prevnar®13(Pfizer,USA).A boost dose was provided to all groups post-dose 1 at t?14 days.Post-dose 2 results at t?28 days showed that all 13 serotypes in PnuVax's PCV-13 were boostable.Per serotype IgG GMCs demonstrated that PnuVax's PCV-13 is immunogenic for all 13 serotypes,with 10 of the 13 serotypes statistically the same or higher than Prevnar®13 post-dose 2.As a result,the novel polysaccharideprotein conjugates developed in this work are highly promising for use in human PCV development.The in vacuo conjugation technique applied in this work could also be readily adapted to develop many other conjugate vaccines.

Apoferritin nanoparticle based dual-antigen influenza conjugate vaccine with potential cross-protective efficacy against heterosubtypic influenza virus

Ferritin nanoparticles with self-assembling properties have been widely explored as vaccine carrier by displaying foreign antigens through genetic fusion strategy.In the present work,an apoferritin(AFt)nanoparticle was tested as influenza vaccine carrier by chemically conjugating a matrix protein 2 ectodomain(M2e)antigen peptide or/and the full-length hemagglutinin(HA)antigen on the outer surface of the AFt,with heterobifunctional sSMCC or SM(PEG)_(24) containing PEG chain as linkers.To each AFt nanoparticle,about 30-32 M2e or 1.8 HA antigen could be coupled.The AFt-(PEG)24-M2e,in which the M2e was coupled through SM(PEG)_(24) containing PEG chain,conferred higher protective efficacy in immunized mice than AFt-M2e did,but was less effective than AFt-(PEG)_(24)-HA.When both M2e and HA were coupled,the synthesized dual-antigen vaccine candidate AFt-(PEG)_(24)-M2e/HA elicited high level of M2e and HA antigen-specific antibodies and conferred 100%protection against lethal infection of homologous PR8 HI N1 virus strain and 70%protection against a heterologous A/FM/1/47(FM1,H1N1)strain,which was more effective than the M2e or HA single antigen vaccine candidates.The potential cross-protective effect of the dual-antigen vaccine was further demonstrated by significant specific hemagglutination inhibition(HAI)titers in serum of the immunized mice against three other heterologous viral strains including A/Singapore/GPl908/2015(IVR-180)H1N1,A/Anhui/1/2005 H5N1,and A/Hong Kong H3N2.

2岁以下儿童肺炎球菌疫苗接种情况及应用效果分析

目的分析2岁以下儿童肺炎球菌疫苗接种率及其应用效果。方法采用多阶段随机抽样法,调查2岁以下儿童13价肺炎球菌疫苗(PCV13)接种率,了解呼吸道疾病的发生率和住院率,分析疫苗应用效果。结果PCV13全程接种率为56.26%。非全程接种组有43.45%存在因病就医,高于完成PCV13全程免疫的儿童(28.18%),其住院率(13.69%)高于完成PCV13全程免疫的儿童(3.64%),差异有统计学意义(P<0.05)。结论儿童PCV13全程接种率偏低,需采取针对性措施提高儿童PCV13接种率。

衢州市单中心儿童肺炎链球菌感染流行及血清型分布特征研究

目的探讨衢州市儿童肺炎链球菌感染流行及血清型分布特征,为指导该地区的疫苗应用和研发提供依据。方法收集2020年1月至2022年6月衢州市人民医院肺炎链球菌感染患儿的临床资料及不同来源标本中分离的肺炎链球菌,进行系列菌株鉴定实验,并通过乳胶凝集试验、荚膜肿胀试验对所有菌株进行血清型鉴定,分析相关感染性疾病和血清型分布的流行特征。结果共收集到107株肺炎链球菌,除2株不能明确分型外共鉴定出21种血清型,排名前5位的血清型为19F、6B、14、19A和3。肺炎链球菌多糖结合疫苗(PCV)7、PCV13和PCV20的血清型覆盖率分别为56.1%(60/107)、71.0%(76/107)、78.5%(84/107)。肺炎链球菌引起儿童支气管肺炎是主要感染类型,不同PCV覆盖血清型肺炎链球菌引起支气管肺炎病例数的比较表明,PCV7覆盖血清型与非PCV7血清型无明显差异;但PCV13(P=0.008)和PCV20(P=0.002)覆盖血清型明显高于非该类疫苗覆盖血清型引起肺炎病例数。结论引起衢州市儿童肺炎链球菌疾病的主要流行血清型为19F、6B、14、19A和3,其中支气管肺炎为最主要感染类型;PCV13和PCV20对该地区儿童感染肺炎链球菌支气管肺炎有保护力,建立肺炎球菌疾病发病状况的监测系统,针对肺炎链球菌血清型分布和感染性疾病进行长期流行病学监测是非常必要的。

合成生物学在多糖结合疫苗研发中的应用

由于合成生物学的快速发展,目前已经实现了人工设计DNA和蛋白质的合成,对具有重要生物学功能、结构也更为复杂的糖类物质进行精准设计合成,也将是合成生物学未来发展的重要方向。近年来,一种基于细菌寡糖转移酶体系的蛋白多糖偶联技术发展迅速,已被广泛应用于病原细菌多糖结合疫苗的生物合成制备。本文综述了该技术体系中的寡糖转移酶元件、载体蛋白元件、异源多糖抗原合成线路以及工程菌株改造等核心关键模块的最新研究进展。使用生物活体系统,发酵生产多糖结合疫苗,具有产物均一性好、步骤简便、绿色环保等优势,是一种亟待发展的新兴技术,同时也存在一些技术细节需要完善。未来,寡糖转移酶的定向进化、纳米颗粒型蛋白载体的应用、多糖合成基因的组合重排、工程菌株的代谢途径优化,将有望进一步促进多糖结合疫苗的生物合成研究。

Antibodies Targeting a Conserved Surface Polysaccharide Are Protective Against a Wide Range of Microbial Pathogens Producing β-1-6-Linked Poly-N-Acetylglucosamine(PNAG)

The b-1-6-linked poly-N-acetylglucosamine(PNAG)polymer is a conserved surface polysaccharide produced by many bacteria,fungi,and protozoan(and even filarial)parasites.This wide-ranging expression makes PNAG an attractive target for vaccine development,as it potentially encompasses a broad range of microorganisms.Significant progress has been made in discovering important properties of the biology of PNAG expression in recent years.The molecular characterization and regulation of operons for the production of PNAG biosynthetic proteins and enzymes have been studied in many bacteria.In addition,the physiological function of PNAG has been further elucidated.PNAG-based vaccines and PNAG-targeting antibodies have shown great efficacy in preclinical research.Furthermore,clinical tests for both vaccines and antibodies have been carried out in humans and economically important animals,and the results are promising.Although it is not destined to be a smooth road,we are optimistic about new vaccines and immunotherapeutics targeting PNAG becoming validated and eventually licensed for clinical use against multiple infectious agents.

冻干b型流感嗜血杆菌结合物疫苗中磷含量测定方法建立及验证

目的:建立冻干b型流感嗜血杆菌(Hib)结合疫苗磷含量检测的超滤法联合紫外分光光度法,并对该方法进行验证及初步应用。方法:用去离子水将样品溶解后超滤,收集超滤管中Hib多糖,采用等体积比硫酸高氯酸消解样品,使用钼酸铵在酸性条件下与磷形成磷钼酸,与维生素C生成蓝色化合物,在825 nm下有最大吸收。对该方法进行线性、精密度、稳定性、准确度验证。用建立的方法检测3批次商业化冻干Hib结合疫苗磷含量。结果:标准磷在质量浓度1~10μg·mL^(-1)内线性关系良好(r=1.000),线性方程为Y=8.878×10^(-2)X+2.402×10^(-3),方法在低、中、高浓度水平下回收率均值分别为在97.35%、96.65%、97.42%(n=3),RSD分别为2.55%、0.95%、0.95%;重现性试验的RSD为2.81%;中间精密度RSD 2.14%;磷标准品衍生物溶液室温放置1 h稳定,RSD为0.39%。结论:该方法结果准确、可靠,可用于冻干b型流感嗜血杆菌结合疫苗磷含量检测,同时也为其他多糖蛋白结合疫苗磷含量测定提供了思路。

Site-Specific Conjugation of Cell Wall Polyrhamnose to Protein SpyAD Envisioning a Safe Universal Group A Streptococcal Vaccine

Development of an effective vaccine against the leading human bacterial pathogen group A Streptococcus(GAS)is a public health priority.The species defining group A cell wall carbohydrate(GAC,Lancefield antigen)can be engineered to remove its immunodominant N-acetylglucosamine(GlcNAc)side chain,implicated in provoking autoimmune cross-reactivity in rheumatic heart disease,leaving its polyrhamnose core(GACPR).Here we generate a novel protein conjugate of the GACPR and test the utility of this conjugate antigen in active immunization.Instead of conjugation to a standard carrier protein,we selected SpyAD,a highly conserved GAS surface protein containing both B-cell and T-cell epitopes relevant to the bacterium that itself shows promise as a vaccine antigen.SpyAD was synthesized using the XpressTM cell-free protein expression system,incorporating a non-natural amino acid to which GACpr was conjugated by site-specific click chemistry to yield high molecular mass SpyAD-GACPR conjugates and avoid disruption of important T-cell and B-cell immunological epitopes.The conjugated SpyAD-GACPR elicited antibodies that bound the surface of multiple GAS strains of diverse M types and promoted opsonophagocytic killing by human neutrophils.Active immunization of mice with a multivalent vaccine consisting of SpyAD-GACPR,together with candidate vaccine antigens streptolysin O and C5a peptidase,protected against GAS challenge in a systemic infection model and localized skin infection model,without evidence of cross reactivity to human heart or brain tissue epitopes.This general approach may allow GAC to be safely and effectively included in future GAS subunit vaccine formulations with the goal of broad protection without autoreactivity.