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 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.

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.

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.

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.

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.

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.

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.

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.

贵州省基于离散选择实验的13价肺炎球菌结合疫苗的选择偏好研究

目的了解儿童家长对13价肺炎球菌结合疫苗(13-valent pneumococcal conjugate vaccine,PCV13)的选择偏好,分析不同人群的偏好异质性,为疫苗的合理推广和使用提供建议。方法基于离散选择实验,在多阶段随机整群抽样的基础上,纳入2月龄至10周岁儿童的家长,使用电子问卷对儿童家长进行面对面调查,利用混合logit模型来评估受访者对PCV13的选择偏好。结果此次调查共收到有效问卷1476份,通过一致性检验的问卷1175份。所有疫苗属性对选择偏好均有统计学意义,有效性和保护时长是儿童家长更注重的疫苗属性。包含交互效应的混合logit模型显示,大专及以上学历的家长更偏好90%有效性以及低不良事件发生率的疫苗,家庭人均月收入大于3000元的家庭更偏好进口疫苗。在为儿童选择PCV13方面,儿童家长更愿意为疫苗高有效性和10年的保护时长支付更多的费用,普遍更偏好国产疫苗和全程接种费用低的疫苗。结论研究结果将为后续相关部门对于PCV13的管理决策提供数据支撑,并且为疫苗生产方提供参考,有助于合理推广PCV13并提高其接受度。

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.