Molecular size distribution in pentavalent (A, C, Y, W, X) meningococcal polysaccharide conjugate vaccine by HPSEC-UV-MALS-RI method- a conceivable stability indicating parameter.

Molecular size distribution (MSD) of polysaccharides serves as a key parameter that directly correlates to the immunogenicity of vaccine. MSD at meningococcal polysaccharide (A, C, Y and W) or conjugate bulk level is well established under detailed pharmacopeial and WHO guidelines. We report here, a newly developed method for determination of molecular size distribution of pentavalent Meningococcal conjugate vaccine comprising of A, C, Y, W and X (MenFive). Although serogroup specific molecular size could not be estimated here; lot to lot consistency monitoring, molecular aggregates distribution in final lot, are key takeaways of this method. Determination of MSD in pentavalent fill finished product was quite challenging. Various columns/detectors combination, buffers, physico-chemical conditions (temperature, 2-8 °C, 25 °C, 40 °C and 60 °C; flow rate, 0.3 mL to 0.8 mL), liquid/lyophilized formulations, were explored. Polymer-based packed columns were explored for estimation for MSD by aqueous size exclusion chromatography, using combinations of- Shodex OHPAK SB 807 HQ, Shodex OHPAK SB 806 HQ, G6000 PWXL, coupled with guard Shodex OHPAK SB-G-6B. MenFive showed heterogenous distribution of molecules ranging from 200 to 19000 kDa, indicating its complex nature. However, 1000-8000 kDa was dominant range, comprising of ≥ 50 % distribution of molecules, in both liquid as well as lyophilized formulations, with average molecular weight around 6000-6500 kDa. The molar mass distribution after slicing would provide an insight to the conformation of molecules through its presentation as HMW, LMW, aggregates and subsequently, the presence of dominant population of molecules of a particular molecular weight and its total contribution in the sample.

Quantitation of residual sodium dodecyl sulfate in meningococcal polysaccharide by gas chromatography-mass spectrometry.

Sodium dodecyl sulfate (SDS) is a commonly used surfactant in protein solubilization and also during the polysaccharide purification. A GC-MS method has been developed to quantitate residual SDS in meningococcal polysaccharide serogroups A,C,W,Y and X circumventing the need of spectroscopic assays and HPLC based methods which are either unstable or requires the confirmation by MS. The developed method is based on quantitative conversion of SDS to 1-dodecanol at elevated temperature. Meningococcal polysaccharides and SDS standards were treated with methanolic-HCl and extracted in n-Hexane. The conversion of SDS to 1-dodecanol was confirmed by mass spectra and separation was achieved using a DB-5ms column. The mass spectral analysis of 1-dodecanol showed characteristic ions at m/z 168, 140 and 125. The GC-MS method validation performed on intermediate and purified meningococcal polysaccharides showed linearity with r2 > 0.99 over the concentration range of 2.5-200 µg/ml with LOD and LOQ of 1.27 and 3.85 respectively. The method was found to be precise, robust and accurate with spike recovery ranging 83-117%. The GC-MS method can be used in the quantitation of residual SDS during polysaccharide purification and provides valuable information about consistency of polysaccharide manufacturing process for development of pentavalent meningococcal conjugate vaccine.

A comparison of pyrogen detection tests in the quality control of meningococcal conjugate vaccines: The applicability of the Monocyte Activation Test.

The meningococcal C conjugate vaccine (MenCC) is an interesting model with which to test the efficacy of the Monocyte Activation Test (MAT) as an alternative method of pyrogen testing in the quality control of vaccines. The MenCC that has been produced by Bio-Manguinhos in Brazil is in the final development stage, and, as recommended in the guidelines for MenCC production, its pyrogen content must be determined by using the Limulus Amoebocyte Lysate (LAL) assay and the Rabbit Pyrogen Test (RPT). This represents an ideal opportunity to compare LAL and RPT data with data obtained by using a MAT system with cryopreserved whole blood and IL-6/IL-1ß as marker readouts. In order to assess the compatibility of the MAT with MenCC, endotoxin and non-endotoxin pyrogen content was quantified by using MenCC samples spiked with lipopolysaccharide (LPS), lipoteichoic acid or zymosan standards. The presence of the aluminium-based adjuvant interfered with the MAT, increasing the readout of IL-1ß in LPS-spiked MenCC batches. This infringed the product-specific validation criteria of the test, and led to IL-6 being chosen as the more suitable marker readout. No pyrogenic contaminants were identified in the MenCC batches tested, demonstrating consistency among the different systems (MAT, RPT and the LAL assay). In conclusion, the introduction of the MAT during MenCC development could contribute to the elimination of animal tests post-licensing, ensuring human protection based on an effective non-animal based method of quality control.

Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA.

Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease.

Trumenba (bivalent rLP2086) is a vaccine licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B (NmB) in individuals 10-25 years of age in the USA. The vaccine is composed of two factor H binding protein (fHbp) variants that were recombinantly expressed in Escherichia coli as native lipoproteins: rLP2086-A05 and rLP2086-B01. The vaccine was shown to induce potent bactericidal antibodies against a broad range of NmB isolates expressing fHbp that were different in sequence from the fHbp vaccine antigens. Here, we describe the characterization of the vaccine antigens including the elucidation of their structure which is characterized by two distinct motifs, the polypeptide domain and the N-terminal lipid moiety. In the vaccine formulation, the lipoproteins self-associate to form micelles driven by the hydrophobicity of the lipids and limited by the size of the folded polypeptides. The micelles help to increase the structural stability of the lipoproteins in the absence of bacterial cell walls. Analysis of the lipoproteins in Toll-like receptor (TLR) activation assays revealed their TLR2 agonist activity. This activity was lost with removal of the O-linked fatty acids, similar to removal of all lipids, demonstrating that this moiety plays an adjuvant role in immune activation. The thorough understanding of the structure and function of each moiety of the lipoproteins, as well as their relationship, lays the foundation for identifying critical parameters to guide vaccine development and manufacture.

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.

Interchangeability of meningococcal group C conjugate vaccines with different carrier proteins in the United Kingdom infant immunisation schedule.

An open, non-randomised study was undertaken in England during 2011-12 to evaluate vaccine antibody responses in infants after completion of the routine primary infant immunisation schedule, which included two doses of meningococcal group C (MenC) conjugate (MCC) vaccine at 3 and 4 months. Any of the three licensed MCC vaccines could be used for either dose, depending on local availability. Healthy term infants registered at participating general practices (GPs) in Hertfordshire and Gloucestershire, UK, were recruited prospectively to provide a single blood sample four weeks after primary immunisation, which was administered by the GP surgery. Vaccination history was obtained at blood sampling. MenC serum bactericidal antibody (SBA) and IgG antibodies against Haemophilus influenzae b (Hib), pertussis toxin (PT), diphtheria toxoid (DT), tetanus toxoid (TT) and thirteen pneumococcal serotypes were analysed according to MCC vaccines received. MenC SBA responses differed significantly (P<0.001) according to MCC vaccine schedule as follows: MenC SBA geometric mean titres (GMTs) were significantly lower in infants receiving a diphtheria cross-reacting material-conjugated MCC (MCC-CRM) vaccine followed by TT-conjugated MCC (MCC-TT) vaccine (82.0; 95% CI, 39-173; n=14) compared to those receiving two MCC-CRM (418; 95% CI, 325-537; n=82), two MCC-TT (277; 95% CI, 223-344; n=79) or MCC-TT followed by MCC-CRM (553; 95% CI, 322-949; n=18). The same group also had the lowest Hib geometric mean concentrations (0.60 µg/mL, 0.27-1.34) compared to 1.85 µg/mL (1.23-2.78), 2.86 µg/mL (2.02-4.05) and 4.26 µg/mL (1.94-9.36), respectively. Our results indicate that MCC vaccines with different carrier proteins are not interchangeable. When several MCC vaccines are available, children requiring more than one dose should receive MCC vaccines with the same carrier protein or, alternatively, receive MCC-TT first wherever possible.

Induction of death receptor CD95 and co-stimulatory molecules CD80 and CD86 by meningococcal capsular polysaccharide-loaded vaccine nanoparticles.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis, and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. We formulated a novel nanovaccine containing meningococcal CPS as an antigen encapsulated in albumin-based nanoparticles (NPs) that does not require chemical conjugation to a protein carrier. These nanoparticles are taken up by antigen-presenting cells and act as antigen depot by slowly releasing the antigen. In this study, we determined the ability of CPS-loaded vaccine nanoparticles to induce co-stimulatory molecules, namely CD80, CD86, and CD95 that impact effective antigen presentation. Co-stimulatory molecule gene induction and surface expression on macrophages and dendritic cells pulsed with meningococcal CPS-loaded nanoparticles were investigated using gene array and flow cytometry methods. Meningococcal CPS-loaded NP significantly induced the surface protein expression of CD80 and CD86, markers of dendritic cell maturation, in human THP-1 macrophages and in murine dendritic cells DC2.4 in a dose-dependent manner. The massive upregulation was also observed at the gene expression. However, high dose of CPS-loaded NP, but not empty NP, induced the expression of death receptor CD95 (Fas) leading to reduced TNF-α release and reduction in cell viability. The data suggest that high expression of CD95 may lead to death of antigen-presenting cells and consequently suboptimal immune responses to vaccine. The CPS-loaded NP induces the expression of co-stimulatory molecules and acts as antigen depot and can spare antigen dose, highly desirable criteria for vaccine formulations.

Distribution of aluminum in hair of Brazilian infants and correlation to aluminum-adjuvanted vaccine exposure.

BACKGROUND: Nursing children are exposed to dietary aluminum (in breast milk and/or infant formulas) and through aluminum-adjuvanted vaccines (AAVs). We studied total hair-Al concentrations of nursing children that had been immunized with hepatitis B, DTP, and meningococcal vaccines. METHODS: We studied the hair of 37 young children (aged 26 to 824 days) who were exposed to cumulative doses of Al ranging from 0.63 to 6.88 mg from AAVs. Graphite furnace atomic absorption spectrometry was used to reliably measure total Al concentrations in hair samples. RESULTS: The analytical method proved sensitive enough to quantify Al in the hair of nursing children. At current levels of exposure it is possible to determine total Al in hair sample of 1.65 mg. Cumulative doses of AAV in children ranged from 0.63 to 6.88 mg Al. Median hair-Al was 47.7 µg g⁻¹ (ranging from 12.2 to 221.9 µg g⁻¹). There was no statistically significant correlation between hair-Al concentration and age of child (r=-0.049; p=0.774), total exposure from vaccine (r=-0.078; p=0.643), or the time elapsed after the last AAVs (r=0.015; p=0.931). CONCLUSION: Aluminum in children's hair can be reliably measured but we are still uncertain how representative it can be of the Al body burden.

Cysteine depletion causes oxidative stress and triggers outer membrane vesicle release by Neisseria meningitidis; implications for vaccine development.

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading.

In previous studies protective antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with glycoconjugates prepared from O-deacylated lipopolysaccharide (LPS-OH) via direct reductive amination between the reducing end of the oligosaccharide molecule, created by treatment with alkaline phosphatase, and amino functionalities on the CRM(197) carrier protein. These glycoconjugates proved difficult to prepare because the presence of amide linked fatty-acyl groups results in glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare glycoconjugates in order to identify a robust, consistently reproducible strategy that produces glycoconjugates with a high loading of LPS derived oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the carbohydrate antigen following removal of the glycosidic phosphate to amino groups on CRM(197), however carbohydrate loading on the carrier protein was low. Glycoconjugates were then produced utilising amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked fatty acids from the lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-OH was treated with Dd amidase, the reducing glycosidic phosphate removed, and using a cystamine linker strategy, conjugated to the carrier protein. Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy, targeting lysine residues on the carrier protein did not result in high loading of the carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of carbohydrate antigens per CRM(197), which can be controlled by variation in the amount of activated carbohydrate utilised in the conjugation reaction.

La técnica de Western Blot como criterio de identidad para la vacuna antimeningocócica Men B / Western Blot technique as an identity criterion for Men B antimeningococcal vaccine

Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica Men B producida en el Instituto Finlay con el objetivo de demostrar un criterio de identidad. En el estudio de las proteínas antigénicas de la vacuna, P1.15 y P1.4 en vesícula de membrana externa,monograneles y producto final se emplearon en la identificación anticuerpos monoclonales específicos para estas proteínas. Los parámetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. El método cumplió con los parámetros señalados, por lo que se consideró validado.

Western Blot technique was developed and validated, applied to Men B meningococcal vaccine produced in Carlos J, Finlay Institute to demonstrate an identity criterion. In study of antigenic proteins of the vaccine, we used P1.15 y P1.4 in vesicle of external membrane, monogranels, and end product to identify the monoclonal antibodies specific of these proteins. Parameters developed in technique validation included: specificity, detection limit, repetition, average accuracy, reproduction, and strength. Method fulfilled with specified parameters, thus considering its validation.

Vaccines containing de-N-acetyl sialic acid elicit antibodies protective against neisseria meningitidis groups B and C.

Murine mAbs that were produced by immunization with a vaccine containing the N-propionyl derivative of Neisseria meningitidis group B (MenB) capsular polysaccharide (NPr MBPS) mediate protective responses against MenB but were not reactive with unmodified MBPS or chemically identical human polysialic acid (PSA). Recently, we showed that some of the mAbs were reactive with MBPS derivatives that contain de-N-acetyl sialic acid residues. In this study we evaluated the immunogenicity of de-N-acetyl sialic acid-containing derivatives of PSA (de-N-acetyl PSA) in mice. Four de-N-acetyl PSA Ags were prepared and conjugated to tetanus toxoid, including completely de-N-acetylated PSA. All of the vaccines elicited anti-de-N-acetyl PSA responses (titers >/=1/10,000), but only vaccines enriched for nonreducing end de-N-acetyl residues by treatment with exoneuraminidase or complete de-N-acetylation elicited high titers against the homologous Ag. Also, nonreducing end de-N-acetyl residue-enriched vaccines elicited IgM and IgG Abs of all subclasses that could bind to MenB. The results suggest that the zwitterionic characteristic of neuraminic acid, particularly at the nonreducing end, may be important for processing and presentation mechanisms that stimulate T cells. Abs elicited by all four vaccines were able to activate deposition of human complement proteins and passively protect against challenge by MenB in the infant rat model of meningococcal bacteremia. Some vaccine antisera mediated bactericidal activity against a N. meningitidis group C strain with human complement. Thus, de-N-acetyl PSA Ags are immunogenic and elicit Abs that can be protective against MenB and N. meningitidis group C strains.

[Perspectives for development of polyvalent meningococcal vaccine].

Experimental whole-culture oral polyvalent meningococcal vaccines against serogroups A, B and C consisting of three monovalent components in different proportions have been developed and evaluated. Kinetics of IgG response to meningococcal antigens (outer membrane proteins, polysaccharide, lypooligosaccharide of these serogroups) in sera of rabbits immunized orally with these preparations was measured. Sharp rise of IgG levels (on 2 - 3 orders) compared to baseline has been detected as well as persistence of high titers during the observation period (322 days).

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design.

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.