Control Strategy Approach for a Well-Characterized Vaccine Drug Product.

Trumenba (MenB-FHbp; bivalent rLP2086), the first meningococcal serogroup B vaccine approved in the United States and subsequently approved in Europe, Canada, and Australia, is well-characterized. Pfizer devised a control strategy approach by using a simplified control strategy wheel for Trumenba based on International Council for Harmonisation (ICH) Q8 (R2), Q9, Q10, and Q11 guidelines, which provide complementary guidance on pharmaceutical development, quality risk management, quality systems, and development and manufacture of drug substances, respectively. These guidelines ensure product quality using a scientific and risk-based approach. Trumenba contains two factor H binding proteins (FHbps), one from each of the two FHbp subfamilies (A and B), adsorbed onto aluminum phosphate. Trumenba manufacturing processes are complicated by the recombinant protein expression of Subfamily A and B proteins and the nature of the drug product (suspension in syringes); the latter also introduces challenges in controlling product critical quality attributes during the development process. In such complex systems, the control strategy is critical to ensuring consistent desired product quality; it also supports the regulatory requirement of continued improvement through continuous process verification and aids regulatory filing. This article describes Pfizer's approach toward robust control strategy development, built on product and process understanding, and links control strategy to regulatory document sections and flow of controls. Specifically, an approach is presented on product quality attribute criticality determination based on safety and efficacy and on an understanding of process parameter criticality. This was achieved by studying the impact of the approach on product quality attributes to define process parameter and in-process controls. This approach is further explained through Trumenba case studies, highlighting specific quality attributes and the associated controls implemented, and provides a holistic view of controls employed for both drug substance and drug product.

Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency.

Vaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi-component meningococcal serogroup B (MenB)-4C. MenB-4C is a multi-component, protein-based vaccine against MenB consisting of factor H-binding protein, Neisserial heparin-binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine-mediated protection by a whole cell enzyme-linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine-induced antibodies and capacity to activate complement-mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement-mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB-4C vaccination still induced effective complement-dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement-deficient children against MenB.

A homopolymeric adenosine tract in the promoter region of nspA influences factor H-mediated serum resistance in Neisseria meningitidis.

Although usually asymptomatically colonizing the human nasopharynx, the Gram-negative bacterium Neisseria meningitidis (meningococcus) can spread to the blood stream and cause invasive disease. For survival in blood, N. meningitidis evades the complement system by expression of a polysaccharide capsule and surface proteins sequestering the complement regulator factor H (fH). Meningococcal strains belonging to the sequence type (ST-) 41/44 clonal complex (cc41/44) cause a major proportion of serogroup B meningococcal disease worldwide, but they are also common in asymptomatic carriers. Proteome analysis comparing cc41/44 isolates from invasive disease versus carriage revealed differential expression levels of the outer membrane protein NspA, which binds fH. Deletion of nspA reduced serum resistance and NspA expression correlated with fH sequestration. Expression levels of NspA depended on the length of a homopolymeric tract in the nspA promoter: A 5-adenosine tract dictated low NspA expression, whereas a 6-adenosine motif guided high NspA expression. Screening German cc41/44 strain collections revealed the 6-adenosine motif in 39% of disease isolates, but only in 3.4% of carriage isolates. Thus, high NspA expression is associated with disease, but not strictly required. The 6-adenosine nspA promoter is most common to the cc41/44, but is also found in other hypervirulent clonal complexes.

Predicting the Susceptibility of Meningococcal Serogroup B Isolates to Bactericidal Antibodies Elicited by Bivalent rLP2086, a Novel Prophylactic Vaccine.

Bivalent rLP2086 (Trumenba), a vaccine for prevention of Neisseria meningitidis serogroup B (NmB) disease, was licensed for use in adolescents and young adults after it was demonstrated that it elicits antibodies that initiate complement-mediated killing of invasive NmB isolates in a serum bactericidal assay with human complement (hSBA). The vaccine consists of two factor H binding proteins (fHBPs) representing divergent subfamilies to ensure broad coverage. Although it is the surrogate of efficacy, an hSBA is not suitable for testing large numbers of strains in local laboratories. Previously, an association between the in vitro fHBP surface expression level and the susceptibility of NmB isolates to killing was observed. Therefore, a flow cytometric meningococcal antigen surface expression (MEASURE) assay was developed and validated by using an antibody that binds to all fHBP variants from both fHBP subfamilies and accurately quantitates the level of fHBP expressed on the cell surface of NmB isolates with mean fluorescence intensity as the readout. Two collections of invasive NmB isolates (n = 1,814, n = 109) were evaluated in the assay, with the smaller set also tested in hSBAs using individual and pooled human serum samples from young adults vaccinated with bivalent rLP2086. From these data, an analysis based on fHBP variant prevalence in the larger 1,814-isolate set showed that >91% of all meningococcal serogroup B isolates expressed sufficient levels of fHBP to be susceptible to bactericidal killing by vaccine-induced antibodies.IMPORTANCE Bivalent rLP2086 (Trumenba) vaccine, composed of two factor H binding proteins (fHBPs), was recently licensed for the prevention of N. meningitidis serogroup B (NmB) disease in individuals 10 to 25 years old in the United States. This study evaluated a large collection of NmB isolates from the United States and Europe by using a flow cytometric MEASURE assay to quantitate the surface expression of the vaccine antigen fHBP. We find that expression levels and the proportion of strains above the level associated with susceptibility in an hSBA are generally consistent across these geographic regions. Thus, the assay can be used to predict which NmB isolates are susceptible to killing in the hSBA and therefore is able to demonstrate an fHBP vaccine-induced bactericidal response. This work significantly advances our understanding of the potential for bivalent rLP2086 to provide broad coverage against diverse invasive-disease-causing NmB isolates.

The role of anti-NHba antibody in bactericidal activity elicited by the meningococcal serogroup B vaccine, MenB-4C.

BACKGROUND: MenB-4C (Bexsero®) is a multicomponent serogroup B meningococcal vaccine. For vaccine licensure, efficacy was inferred from serum bactericidal antibody (SBA) against three antigen-specific indicator strains. The bactericidal role of antibody to the fourth vaccine antigen, Neisserial Heparin binding antigen (NHba), is incompletely understood. METHODS: We identified nine adults immunized with two or three doses of MenB-4C who had sufficient volumes of sera and >3-fold increases in SBA titer against a strain with high NHba expression, which was mismatched with the other three MenB-4C antigens that elicit SBA. Using 1month-post-immunization sera we measured the effect of depletion of anti-NHba and/or anti-Factor H binding protein (FHbp) antibodies on SBA. RESULTS: Against three strains matched with the vaccine only for NHba, depletion of anti-NHba decreased SBA titers by an average of 43-79% compared to mock-adsorbed sera (P<0.05). Despite expression of sub-family A FHbp (mismatched with the sub-family B vaccine antigen), depletion of anti-FHbp antibodies also decreased SBA by 45-64% (P<0.05). Depletion of both antibodies decreased SBA by 84-100%. Against a strain with sub-family B FHbp and expression of NHba with 100% identity to the vaccine antigen, depletion of anti-NHba decreased SBA by an average of 26%, compared to mock-adsorbed sera (P<0.0001), and depletion of anti-FHbp antibody decreased SBA by 92% (P<0.0001). CONCLUSIONS: Anti-NHba antibody can contribute to SBA elicited by MenB-4C, particularly in concert with anti-FHbp antibody. However, some high NHba-expressing strains are resistant, even with an exact match between the amino acid sequence of the vaccine and strain antigens.

Neisseria meningitidis subverts the polarized organization and intracellular trafficking of host cells to cross the epithelial barrier.

Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular microtubule-dependent pathway and represents a crucial step in its pathogenesis. Despite this fact, the interaction of invasive meningococci with host subcellular compartments and the resulting impact on their organization and function have not been investigated. The influence of serogroup B strain MC58 on host cell polarity and intracellular trafficking system was assessed by confocal microscopy visualization of different plasma membrane-associated components (such as E-cadherin, ZO-1 and transferrin receptor) and evaluation of the transferrin uptake and recycling in infected Calu-3 monolayers. Additionally, the association of N. meningitidis with different endosomal compartments was evaluated through the concomitant staining of bacteria and markers specific for Rab11, Rab22a, Rab25 and Rab3 followed by confocal microscopy imaging. Subversion of the host cell architecture and intracellular trafficking system, denoted by mis-targeting of cell plasma membrane components and perturbations of transferrin transport, was shown to occur in response to N. meningitidis infection. Notably, the appearance of all of these events seems to positively correlate with the efficiency of N. meningitidis to cross the epithelial barrier. Our data reveal for the first time that N. meningitidis is able to modulate the host cell architecture and function, which might serve as a strategy of this pathogen for overcoming the nasopharyngeal barrier without affecting the monolayer integrity.

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.

Meningococcal biofilm growth on an abiotic surface - a model for epithelial colonization?

Neisseria meningitidis colonizes the human nasopharynx asymptomatically, often for prolonged periods, but occasionally invades from this site to cause life-threatening infection. In the nasopharynx aggregated organisms are closely attached to the epithelial surface, in a state in which the expression of components of the bacterial envelope differs significantly from that found in organisms multiplying exponentially in liquid phase culture or in the blood. We and others have hypothesized that here they are in the biofilm state, and to explore this we have investigated biofilm formation by the serogroup B strain MC58 on an abiotic surface, in a sorbarod system. Transcriptional changes were analysed, focusing on alteration in gene expression relevant to polysaccharide capsulation, lipooligosaccharide and outer-membrane protein synthesis - all phenotypes of importance in epithelial colonization. We report downregulation of genes controlling capsulation and the production of core oligosaccharide, and upregulation of genes encoding a range of outer-membrane components, reflecting phenotypic changes that have been established to occur in the colonizing state. A limited comparison with organisms recovered from an extended period of co-cultivation with epithelial cells suggests that this model system may better mirror natural colonization than do short-term meningococcal/epithelial cell co-cultivation systems. Modelling prolonged meningococcal colonization with a sorbarod system offers insight into gene expression during this important, but experimentally relatively inaccessible, phase of human infection.

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B.

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.

Vaccine-induced opsonophagocytic immunity to Neisseria meningitidis group B.

The role of opsonophagocytosis (OP) in protection against meningococcal disease is controversial because patients with deficiencies in terminal complement proteins whose sera support OP but not bactericidal activity (BA) are at greatly increased risk of disease. We assayed complement-mediated BA and OP bactericidal activity in sera from 32 adults immunized with an outer membrane vesicle vaccine given alone or combined with an investigational recombinant protein, genome-derived neisserial antigen (GNA2132). The sera were heat inactivated to remove internal complement activity, and BA was measured with exogenous nonimmune human serum as a complement source. OP was measured with human polymorphonuclear cells (PMNs) and C6-depleted complement, which without PMNs did not support BA. Before immunization, 9 to 19% of sera from subjects in both vaccine groups combined had BA titers of >or=1:4, which increased to 41 to 72% after immunization (P < 0.01 against each of three test strains). The percentages of sera with OP titers of >or=1:5 were 3 to 16%, which increased to 55 to 72% (P < 0.001 for each strain). Most postimmunization BA-positive sera were OP positive, but 10 to 37% of BA-negative sera also were OP positive. Comparing the two vaccine groups, there were no significant differences in the percentages of sera with BA or OP activity except for a higher percentage of OP against one strain in postimmunization sera from subjects in the combination vaccine group (P

Prevalence of factor H-binding protein variants and NadA among meningococcal group B isolates from the United States: implications for the development of a multicomponent group B vaccine.

BACKGROUND: Two promising recombinant meningococcal protein vaccines are in development. One contains factor H-binding protein (fHBP) variants (v.) 1 and 2, whereas the other contains v.1 and 4 other antigens discovered by genome mining (5 component [5C]). Antibodies against fHBP are bactericidal against strains within a variant group. There are limited data on the prevalence of strains expressing different fHBP variants in the United States. METHODS: A total of 143 group B isolates from patients hospitalized in the United States were tested for fHBP variant by quantitative polymerase chain reaction, for reactivity with 6 anti-fHBP monoclonal antibodies (MAb) by dot immunoblotting, and for susceptibility to bactericidal activity of mouse antisera. RESULTS: fHBP v.1 isolates predominated in California (83%), whereas isolates expressing v.1 (53%) or v.2 (42%) were common in 9 other states. Isolates representative of 5 anti-fHBP MAb-binding phenotypes (70% of isolates) were highly susceptible to anti-fHBP v.1 or v.2 bactericidal activity, whereas 3 phenotypes were approximately 50% susceptible. Collectively, antibodies against the fHBP v.1 and v.2 vaccine and the 5C vaccine killed 76% and 83% of isolates, respectively. CONCLUSIONS: Susceptibility to bactericidal activity can be predicted, in part, on the basis of fHBP phenotypes. Both vaccines have the potential to prevent most group B disease in the United States.

Carbohydrate composition of meningococcal lipopolysaccharide modulates the interaction of Neisseria meningitidis with human dendritic cells.

Meningococcal lipopolysaccharide (LPS) is of crucial importance for the pathogenesis of invasive infection. We show that sialylation and elongation of the alpha-chain effectively shields viable unencapsulated Neisseria meningitidis from recognition by human dendritic cells (DC). In contrast, beta- and gamma- chain of the LPS carbohydrate moiety play only a minor role in the interaction with DC. The protective function of the LPS for the bacteria can be counteracted in vivo by phase variation of the lgtA gene encoding LPS glycosyltransferase A. Capsule expression protects N. meningitidis efficiently from recognition and phagocytosis by DC independent of the LPS structure. Despite the significant impact of LPS composition on the adhesion and phagocytosis of N. meningitidis no differences were found in terms of cytokine levels secreted by DC for IL1-beta, IL-6, IL-8, TNF-alpha, IFN-gamma and GM-CSF. However, significantly lower levels of the regulatory mediator IL-10 were induced by encapsulated strains in comparison to isogenic unencapsulated derivatives. IL-10 secretion was shown to depend on phagocytosis because poly alpha-2,8 sialic acid did not influence IL-10 secretion. The use of truncated LPS isoforms in vaccine preparations can therefore not only result in attenuation but also in more efficient targeting of DC.