Relative stability of meningococcal serogroup A and X polysaccharides

Prior to the introduction of the MenAfriVac™ serogroup A glycoconjugate vaccine in September 2010, serogroup A was the major epidemic disease-causing meningococcal serogroup in the African meningitis belt. However, recently serogroup X meningococcal (MenX) disease has received increased attention because of outbreaks recorded in this region, with increased endemic levels of MenX disease over the past 2 years. Whereas polysaccharide–protein conjugate vaccines against meningococcal serogroups A, C, W and Y (MenA, MenC, MenW, MenY) are on the market, a vaccine able to protect against MenX has never been achieved.     

Development and pre-clinical evaluation of a synthetic oligosaccharide-protein conjugate vaccine against Neisseria meningitidis serogroup C

Significant improvement has been made in the development of vaccines against Neisseria meningitidis infections since the introduction of polysaccharide-protein conjugate vaccines. Conventional bacterial capsular polysaccharide (PS) based conjugate vaccines require unique and expensive manufacturing facilities, complex production processes and extensive quality testing. Synthetic oligosaccharide (OS) based approach is one of the novel technologies that is being developed to simplify production of conjugate vaccines. OSs can be chemically synthesized to a desired length long enough to represent the antigenic epitopes which often present as a homogenous mixture. We prepared OSs corresponding to tetramer and octamer of N. meningitidis serogroup C (MenC) PS by organic synthesis. The MenC tetramer and octamer were further conjugated with tetanus toxoid to produce respective monovalent conjugates having the desired physico-chemical characteristics. The conjugates were evaluated in a mouse model for immunogenicity and compared with a licensed PS conjugate vaccine. Synthetic conjugates could induce anti-MenC PS IgG as well as serum bactericidal titers at levels comparable to those elicited by the licensed vaccine. The increase in length of synthetic oligomers from tetramer to octamer did not appear to increase immunogenicity. The results establish the pre-clinical proof of concept for a synthetic MenC oligosaccharide conjugate vaccine candidate.     

Characterization of size, structure and purity of serogroup X Neisseria meningitidis polysaccharide, and development of an assay for quantification of human antibodies

Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using (13)C, (1)H and (31)P NMR spectroscopy and total correlation spectroscopy (TOCSY) and (1)H-(13)C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC-SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule.     

Development of a bivalent conjugate vaccine candidate against rotaviral diarrhea and tuberculosis using polysaccharide from Mycobacterium tuberculosis conjugated to ΔVP8* protein from rotavirus

Conjugation of carbohydrate antigens with a carrier protein is a clinically proven strategy to overcome the poor immunogenicity of bacterial polysaccharide. In addition to its primary role, which is to help generate a T cell-mediate long-lasting immune response directed against the carbohydrate antigen, the carrier protein in a glycoconjugate vaccine can also play an important role as a protective antigen. Among carrier proteins currently used in licensed conjugate vaccines, non-typeable Haemophilus influenzae protein D has been used as an antigenically active carrier protein. Our previous studies also indicate that some carrier proteins provide B cell epitopes, along with T cell helper epitopes.Herein we investigated the dual role of truncated rotavirus spike protein ΔVP8* as a carrier and a protective antigen. Capsular polysaccharide lipoarabinomannan (LAM), purified from Mycobacterium tuberculosis (M.tb), was chemically conjugated with ΔVP8*. Mouse immunization experiments showed that the resultant conjugates elicited strong and specific immune responses against the polysaccharide antigen, and the responses were comparable to those induced by Diphtheria toxoid (DT)-based conjugates. The conjugate vaccine induced enhanced antibody titers and functional antibodies against ΔVP8* when compared to immunization with the unconjugated ΔVP8*. Thus, these results indicate that ΔVP8* can be a relevant carrier protein for glycoconjugate vaccine and the glycoconjugates consisting of ΔVP8* with LAM are effective bivalent vaccine candidates against rotavirus and tuberculosis.     

Preclinical immunogenicity study of trivalent meningococcal AWX-OMV vaccines for the African meningitis belt

In the recent decade, epidemic meningitis in the African meningitis belt has mostly been caused by Neisseria meningitidis of serogroups A, W and X (MenA, MenW and MenX, respectively). There is at present no licensed vaccine available to prevent MenX meningococcal disease. To explore a trivalent MenAWX vaccine concept, we have studied the immunogenicity in mice of MenX outer membrane vesicles (X-OMV) or MenX polysaccharide (X-PS) when combined with a bivalent A-OMV and W-OMV (AW-OMV) vaccine previously shown to be highly immunogenic in mice. The vaccine antigens were produced from three representative wild type strains of MenA (ST-7), MenW (ST-11) and MenX (ST-751) isolated from patients in the African meningitis belt. Groups of mice were immunized with two doses of X-OMV or X-PS combined with the AW-OMV vaccine or as individual components. All vaccine preparations were adsorbed to Al(OH)₃. Sera from immunized mice were tested by ELISA and immunoblotting. Functional antibody responses were measured as serum bactericidal activity (SBA) and opsonophagocytic activity (OPA). Immunization of mice with X-OMV, alone or in combination with AW-OMV induced high levels of anti-X OMV IgG. Moreover, X-OMV alone or in combination with the AW-OMV vaccine induced high SBA and OPA titers against the MenX target strain. X-PS alone was not immunogenic in mice; however, addition of the AW-OMV vaccine to X-PS increased the immunogenicity of X-PS. Both AWX vaccine formulations induced high levels of IgG against A- and W-OMV and high SBA titers against the MenA and MenW vaccine strains. These results suggest that a trivalent AWX vaccine, either as a combination of OMV or OMV with X-PS, could potentially prevent the majority of meningococcal disease in the meningitis belt.     

Quality,immunogenicity and stability of meningococcal serogroup ACWY-CRM197, DT and TT glycoconjugate vaccines

A physicochemical and immunological study of the stability of three different meningococcal (Men) ACWY conjugate vaccines was performed to evaluate any patterns of serogroup oligo- or polysaccharide-specific or carrier protein-specific stability that would affect immunogenicity. Critical quality and stability-indicating characteristics were measured, with the study supporting the suitability of both HPLC-SEC and HPAEC-PAD methods to detect changes following inappropriate vaccine storage. All three final products, ACWY-CRM₁₉₇, -DT and -TT conjugate vaccines had expected quality indicator values and similar immunogenicity in a mouse model (anti-PS IgG and rSBA) when stored at +2–8 °C. When stored at ≥+37 °C, all conjugated carrier proteins and serogroup saccharides were affected. Direct correlations were observed between the depolymerization of the MenA saccharide as evidenced by a size-reduction in the MenA conjugates (CRM₁₉₇, DT and TT) and their immunogenicity. MenA was the most labile serogroup, followed by MenC; then MenW and Y, which were similar. At high temperatures, the conjugated carrier proteins were prone to unfolding and/or aggregation. The anti-MenC IgG responses of the multivalent conjugate vaccines in mice were equivalent to those observed in monovalent MenC conjugate vaccines, and were independent of the carrier protein. For any newly developing MenACWY saccharide-protein conjugate vaccines, a key recommendation would be to consider the lyophilization of final product to prevent deleterious degradation that would affect immunogenicity.     

Natural immunity against capsular group X N. meningitidis following an outbreak in Togo, 2007

BackgroundCapsular group X N. meningitidis (MenX) has emerged as a cause of localized disease outbreaks in sub-Saharan Africa, but the human immune response following exposure to MenX antigens is poorly described. We therefore assessed the natural immunity against MenX in individuals who were living in an area affected by a MenX outbreak during 2007 in Togo, West Africa. During 2009, 300 healthy individuals (100 aged 3–5 years, 100 aged 13–19 years and 100 aged 20–25 years) were included in the study, and serum responses were compared with sera from age-matched controls from the U.K. and Burkina Faso.MethodsMenX serum bactericidal antibody (SBA) was measured using rabbit complement, and antibodies against MenX polysaccharide (XPS) and outer membrane vesicles (XOMVs) were quantified by ELISA.ResultsThe proportion of Togolese individuals with an SBA titer of ≥8 against the MenX strain was 29% (95% confidence interval (CI) 18–41) among those aged 3–5 years, 34% (95% CI 9–60) among those aged 13–19 years and 32% (95% CI 24–40) among those aged 20–25 years. These were significantly higher than observed in the control populations from the U.K (range 13–16%) and Burkina Faso (range 2–6%).ConclusionIn Togolese individuals, the concentration of serum IgG against XPS was higher among the two older age groups as compared to the youngest age group. Antibody concentrations against MenX PS correlated significantly with SBA titers. This supports further development of a MenX PS based conjugate vaccine. Further studies are needed to verify the ability of MenX PS to induce SBA in humans.     

Higher mass meningococcal group C-tetanus toxoid vaccines conjugated with carbodiimide correlate with greater immunogenicity

To examine the link between meningococcal C (MenC) vaccine size and immunogenic response, a panel of MenC glycoconjugate vaccines were prepared differing in chain length, molar mass and hydrodynamic volume. The preparations consisted of different lengths of MenC polysaccharide (PS) covalently linked to monomeric purified tetanus toxoid (TT) carrier protein using the coupling reagent ethylcarbodiimide hydrochloride (EDC). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) and viscometry analysis confirmed that the panel of MenC-TT conjugates spanned masses of 191,500 to 2,348,000 g/mol, and hydrodynamic radii ranging from 12.1 to 47.9 nm. The two largest conjugates were elliptical in shape, whereas the two smallest conjugates were more spherical. The larger conjugates appeared to fit a model described by multiple TTs with cross-linked PS, typical of lattice-like networks described previously for TT conjugates, while the smaller conjugates were found to fit a monomeric or dimeric TT configuration. The effect of vaccine conjugate size on immune responses was determined using a two-dose murine immunization. The two larger panel vaccine conjugates produced higher anti-MenC IgG1 and IgG2b titres after the second dose. Larger vaccine conjugate size also stimulated greater T-cell proliferative responses in an in vitro recall assay, although cytokines indicative of a T-helper response were not measurable. In conclusion, larger MenC-TT conjugates up to 2,348,000 g/mol produced by EDC chemistry correlate with greater humoral and cellular murine immune responses. These observations suggest that conjugate size can be an important modulator of immune response.     

Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines

An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the H_C domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8 × 10⁶ g/mol to larger than 20 × 10⁶ g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the H_C domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the H_C domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.     

Molecular shape and immunogenicity of meningococcal polysaccharide group A conjugate vaccine

Neisseria meningitidis is a leading cause of severe bacterial infections in infants and young children. As a major virulence factor, meningococcal capsular polysaccharide (PS) is poorly immunogenic and generally does not induce immunological memory. Conjugation of PS with a carrier protein can significantly increase the PS-specific immunogenicity and induce immunological memory. It is well known that the molecular shape/size of the conjugate vaccine is important for its immunogenicity. However, little is known about the molecular shape/size of the meningococcal conjugate vaccine. A meningococcal PS–ovalbumin (OVA) conjugate vaccine was prepared using cystamine as linker. Four components (P1–P4) with different molecular size were fractionated from the conjugate. Small angle X-ray scattering (SAXS) analysis revealed that the conjugate vaccine exhibited a rod-like shape similar to virus-like particles. PS-specific immunogenicity of the conjugate vaccine was related to its molecular shape and increased as a function of its molecular size. Thus, the present study provides a three-dimensional shape of the conjugate vaccine and helps to identify optimal design of a potent meningococcal conjugate vaccine.     

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM₁₉₇ have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM₁₉₇, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM₁₉₇ showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM₁₉₇ before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM₁₉₇. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins.     

Could the multicomponent meningococcal serogroup B vaccine (4CMenB) control Neisseria meningitidis capsular group X outbreaks in Africa?

A new vaccine, 4CMenB, is composed of surface proteins of Neisseria meningitidis and is aimed to target serogroup B (MenB) isolates. The vaccine components are present in meningococcal isolates of other serogroups allowing potential use against meningococcal isolates belonging to non-B serogroups. Isolates of serogroup X (MenX) have been emerged in countries of the African meningitis belt. 4CMenB may offer a vaccine strategy against these isolates as there is no available capsule-based vaccine against MenX. We used the Meningococcal Antigen Typing System (MATS) to determine presence, diversity and levels of expression of 4CMenB antigens among 9 MenX isolates from several African countries in order to estimate the potential coverage of MenX by the 4CMenB vaccine. We performed bactericidal assays against these isolates, using pooled sera from 4CMenB-vaccinated infants, adolescents and adults. The African MenX isolates belonged to the same genotype but showed variation in the vaccine antigens. MATS data and bactericidal assays suggest coverage of the 9 African MenX isolates by 4CMenB but not of two unrelated MenX isolates from France. 4CMenB vaccine can be considered for further investigation to control MenX outbreaks in Africa.     

Thermostable formulations of a hepatitis B vaccine and a meningitis A polysaccharide conjugate vaccine produced by a spray drying method

Thermostable vaccines promise to simplify the logistics of vaccine distribution and expand the immunization coverage. In this study, a pilot-scale spray drying process was developed and used to produce glassy state formulations of a recombinant hepatitis B (HepB) vaccine containing aluminum adjuvant and Neisseria meningitidis A (MenA) protein–polysaccharide conjugate vaccine, representing two common types of subunit vaccines in use today: the spray-dried HepB vaccine formulations were stable for at least 24 months at 37 °C while several MenA vaccine formulations exhibited complete stability at temperatures up to 60 °C. This study demonstrates the feasibility of producing thermostable vaccines with advanced processing and formulation technologies.     

Carrier priming with CRM197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: Biophysical and immunochemical interpretation

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM₁₉₇), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM₁₉₇ or DT does not suppress the response to the carbohydrate moiety of CRM₁₉₇ meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM₁₉₇ structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM₁₉₇ were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM₁₉₇ induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM₁₉₇ and related conjugates. The low intrinsic immunogenicity of CRM₁₉₇ as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM₁₉₇ and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM₁₉₇ and DT in a carrier priming context.     

Evaluation of the non-toxic mutant of the diphtheria toxin K51E/E148K as carrier protein for meningococcal vaccines

Diphtheria toxin mutant CRM₁₉₇ is a common carrier protein for glycoconjugate vaccines, which has been proven an effective protein vector for, among others, meningococcal carbohydrates. The wide-range use of this protein in massive vaccine production requires constant increase of production yields and adaptability to an ever-growing market. Here we compare CRM₁₉₇ with the alternative diphtheria non-toxic variant DT-K51E/E148K, an inactive mutant that can be produced in the periplasm of Escherichia coli. Biophysical characterization of DT-K51E/E148K suggested high similarity with CRM₁₉₇, with main differences in their alpha-helical content, and a suitable purity for conjugation and vaccine preparation. Meningococcal serogroup A (MenA) glycoconjugates were synthesized using CRM₁₉₇ and DT-K51E/E148K as carrier proteins, obtaining the same conjugation yields and comparable biophysical profiles. Mice were then immunized with these CRM₁₉₇ and DT-K51E/E148K conjugates, and essentially identical immunogenic and protective effects were observed. Overall, our data indicate that DT-K51E/E148K is a readily produced protein that now allows the added flexibility of E. coli production in vaccine development and that can be effectively used as protein carrier for a meningococcal conjugate vaccine.     

Characterization of invasive Neisseria meningitidis isolates recovered from children in Turkey during a period of increased serogroup B disease, 2013–2017

Diverse Neisseria meningitidis strains belonging to various serogroups and clonal complexes cause epidemic and endemic life-threatening disease worldwide. This study aimed to investigate the genetic diversity of recent invasive meningococci in Turkey with respect to multilocus sequence type (MLST) and also meningococcal serogroup B (MenB) vaccine antigens to enable assessment of potential MenB strain coverage using the genetic Meningococcal Antigen Typing System (gMATS). Fifty-four isolates, representing 37.5% of all pediatric (ages 0–18 years) invasive meningococcal disease cases in Turkey from January 2013 to December 2017, underwent genome sequence analysis. Thirty-six (66.7%) isolates were MenB, 10 (18.5%) were serogroup W (MenW), 4 (7.4%) were serogroup A (MenA), 3 (5.6%) were serogroup Y (MenY) and 1 (1.8%) was serogroup X (MenX). The MenB isolates were diverse with cc35 (19.4%), cc41/44 (19.4%) and cc32 (13.8%) as the most prevalent clonal complexes. The MenW isolates (n = 10) comprised cc11 (n = 5), ST-2754 (cc-unassigned; n = 4) and cc22 (n = 1). gMATS was indicative of high 4CMenB coverage (72.2–79.1%) of Turkish invasive MenB strains from pediatric patients. Strain coverage of several clonal complexes differed from that seen elsewhere in Europe highlighting the importance of performing local assessments and also the use of phenotypic methods, i.e. MATS, where possible. All of the isolates possessed in-frame fhbp alleles and so were potentially covered by MenB-fHbp. Continued surveillance is essential to guide recommendations for current and future vaccines as well as understanding changes in epidemiology.     

Immunogenicity in mice of pneumococcal glycoconjugate vaccines using pneumococcal protein carriers

Antibody response and protective immunity were evaluated in mice immunized with pneumococcal glycoconjugate vaccines using two pneumococcal protein carriers. Mice injected with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) produced high levels of IgG and IgM antibodies to both the PS and the protein carrier. Higher PS antibody titers to the pneumococcal PS conjugates were measured by ELISA using PS-Ply or PS-tetanus toxoid (TT) conjugate as a coating antigen compared with PS mixed with methylated human serum albumin. Type 9V PS (10 microg) inhibited most of the 9V IgM and IgG antibody binding to the 9V-TT coated plate. In contrast, absorption with 19F PS did not inhibit 9V antibody binding. The avidity index of IgG antibodies in the 9V PS-Ply serum was 55.5 +/- 0.9, compared with 47.8 +/- 1.4 for 9V PS-Aly serum. Thus, high avidity of serum antibodies in conjugate-immunized mice can provide more effective functional activity for protection against pneumococcal infection. Mice immunized with these glycoconjugates exhibited rapid bacterial clearance from blood and provided cross-protection against challenge with heterologous serotypes of virulent pneumococci. These results reveal that conjugates using pneumococcal protein carriers can induce opsonophagocytic activity to destroy homologous and heterologous pneumococci, indicating that such conjugates can confer broader protective immunity than conjugates using non-pneumococcal proteins.     

Carrier priming effect of CRM197 is related to an enhanced B and T cell activation in meningococcal serogroup A conjugate vaccination. Immunological comparison between CRM197 and diphtheria toxoid

Glycoconjugate vaccines are composed of capsular polysaccharides (CPSs) of a pathogenic bacteria covalently linked to carrier proteins. Pre-exposure to the carrier is known to influence the efficacy of the glycoconjugate, by inducing enhanced or suppressed anti-CPS response. Following our previous work on the immunogenicity of diphtheria toxin mutant CRM₁₉₇ and formaldehyde-treated diphtheria toxoid (DT) as carriers for meningococcal A (MenA) conjugates in mouse model, we further investigated the role of the carrier on the immunological response to glycoconjugate vaccines. We previously showed that high dosage DT priming could result in carrier-induced epitopic suppression (CIES), an event that did not occur for CRM₁₉₇ priming, and we observed that anti-DT IgGs could cross-react with DT based conjugates in vitro. Here, we confirmed the cross-reactivity of anti-carrier IgGs with DT conjugates in vivo. Furthermore, we analyzed the splenocytes of animals primed with the carrier and subsequently immunized with the MenA conjugate. Pre-exposure to the carrier protein, both CRM₁₉₇ and DT, resulted in increased carrier-specific plasma and memory B cell response. However, only for CRM₁₉₇ priming an enhanced carbohydrate-specific plasma cell response was observed. Analysis of circulating IgGs confirmed these observations. Memory to the CPS resulted to be non-influenced by carrier priming. Analysis of T helper response showed an enhancement effect for CRM₁₉₇ priming, while DT priming resulted in constrained T cell activation. Stimulation with CRM₁₉₇, which does not require formaldehyde detoxification, of splenocytes from animal immunized with DT suggested that the formaldehyde treatment used to produce DT might be the cause of limited presentation of the antigen to the T cells. We concluded that the dominant carrier-specific B cell response in case of limited T cell recruitment might explain the previously observed CIES phenomenon in case of DT priming.     

Analytical technology development to monitor the stability of Polysaccharide-Protein conjugate vaccines

The covalent attachment of a bacterial-derived capsular polysaccharide to protein is of critical importance in transforming the polysaccharide from an antigen with limited immunogenicity in infants and older adults to an antigen that can prevent potentially fatal disease. For a polysaccharide-protein conjugate vaccine (PCV) candidate to be successful, it must be sufficiently stable. Chemical breakage of carbohydrate bonds in the polysaccharide may result in the reduction of “conjugate dose” and could negatively impact immunogenicity and the ability of the vaccine to prime for memory responses. Therefore, development of analytical tools to monitor the integrity of a polysaccharide-protein conjugate (glycoconjugate) vaccine is of practical significance. In this work, reducing SDS-PAGE, Intrinsic Protein Fluorescence Spectroscopy (IPFS), Differential Scanning Fluorimetry (DSF) were evaluated methods to study the impact of time, temperature, and formulation composition on the stability of a glycoconjugate vaccine prepared by multisite coupling of polysaccharide to a carrier protein. In addition, an automated capillary Western system was also evaluated to study the impact of storage on glycoconjugate vaccine stability. Two streptococcus pneumoniae polysaccharide-protein conjugates (serotype 3 and serotype 19A) were chosen to examine their physicochemical stability when formulated as a single antigen vaccine. While all methods require only a small amount of test article and can test multiple samples per assay run, automated capillary Western has the additional advantage of being highly sensitive even at low concentrations in complex vaccine formulations that contain aluminum adjuvant and multiple antigens. Results suggest that automated capillary Western is stability-indicating and may be an effective analytical technology tool for the formulation development of a multivalent glycoconjugate vaccine.     

A new fully liquid presentation of MenACWY-CRM conjugate vaccine: Results from a multicentre,randomised, controlled,observer-blind study

BackgroundThe currently licensed quadrivalent MenACWY-CRM conjugate vaccine presentation consists of two vials (lyophilised MenA and liquid MenCWY) to be reconstituted before injection. A new fully liquid formulation in a single vial has been developed to further improve the vaccine presentation. Since the MenA structure is subject to hydrolytic degradation, this study was conducted to compare the immunogenicity and safety of the investigational MenACWY-CRM liquid vaccine with the licensed vaccine.MethodsIn this multicentre, randomised, controlled, observer-blind, phase 2b study, 979 healthy adults were administered a single dose of MenACWY-CRM liquid presentation or the currently licensed MenACWY-CRM vaccine. MenA free saccharide generation was accelerated to approximately 30% in the liquid presentation and MenA polysaccharide O-acetylation was reduced to approximately 40%, according to a controlled procedure. Immunological non-inferiority of the MenACWY-CRM liquid to the licensed vaccine, as measured by human serum bactericidal assay (hSBA) geometric mean titres (GMTs) against MenA 1 month post-vaccination, was the primary study objective. Safety assessment was among the secondary objectives.ResultsImmune responses against each serogroup were similar between the two vaccine groups and was non-inferior for MenA. Adjusted hSBA GMTs for MenA were 185.16 and 211.33 for the MenACWY-CRM liquid presentation and currently licensed vaccine presentation, respectively. The between-group ratio of hSBA GMTs for MenA was 0.88, with a two-sided 95% confidence interval lower limit of 0.64, greater than the prespecified non-inferiority margin of 0.5, thus meeting the primary study objective. Both vaccines were well tolerated. No serious adverse events were considered related to vaccination.ConclusionsThe levels of MenA free saccharide and polysaccharide O-acetylation did not affect the immunogenicity of the fully liquid presentation, which was demonstrated to be non-inferior to the immunogenicity of the currently licensed MenACWY-CRM vaccine against MenA. The immunogenicity, reactogenicity and safety profiles of the two vaccine presentations were similar.