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Background Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000–2006. Methods Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. Results Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%–75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. Conclusions The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

Factor H binding proteins (fHBP), are bacterial surface proteins currently undergoing human clinical trials as candidate serogroup B Neisseria meningitidis (MnB) vaccines. fHBP protein sequences segregate into two distinct subfamilies, designated A and B. Here, we report the specificity and vaccine potential of mono- or bivalent fHBP-containing vaccines. A bivalent fHBP vaccine composed of a member of each subfamily elicited substantially broader bactericidal activity against MnB strains expressing heterologous fHBP than did either of the monovalent vaccines. Bivalent rabbit immune sera tested in serum bactericidal antibody assays (SBAs) against a diverse panel of MnB clinical isolates killed 87 of the 100 isolates. Bivalent human immune sera killed 36 of 45 MnB isolates tested in SBAs. Factors such as fHBP protein variant, PorA subtype, or MLST were not predictive of whether the MnB strain could be killed by rabbit or human immune sera. Instead, the best predictor for killing in the SBA was the level of in vitro surface expression of fHBP. The bivalent fHBP vaccine candidate induced immune sera that killed MnB isolates representing the major MLST complexes, prevalent PorA subtypes, and fHBP variants that span the breadth of the fHBP phylogenetic tree. Importantly, epidemiologically prevalent fHBP variants from both subfamilies were killed.

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. 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 outer membrane protein LP2086, a human factor H binding protein, is undergoing clinical trials as a vaccine against invasive serogroup B meningococcal (MnB) disease. As LP2086 is a surface protein, expression of capsular polysaccharide could potentially limit accessibility of anti-LP2086 antibodies to LP2086 expressed on the surface of bacteria. To determine whether variability in expression levels of the serogroup B capsule (Cap B) might interfere with accessibility of anti-LP2086 antibody binding to LP2086, we evaluated the ability of anti-Cap B and anti-LP2086 antibodies to bind to the surface of 1263 invasive clinical MnB strains by flow cytometry. One of the anti-LP2086 monoclonal antibodies used recognizes virtually all LP2086 sequence variants. Our results show no correlation between the amount of Cap B expressed and the binding of anti-LP2086 antibodies. Furthermore, the susceptibility of MnB bacteria to lysis by anti-LP2086 immune sera was independent of the level of Cap B expressed. The data presented in this paper demonstrates that Cap B does not interfere with the binding of antibodies to LP2086 expressed on the outer membrane of MnB clinical isolates.

Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are common causative agents of human mucosal infections. To formulate a mucosal vaccine against these pathogens, recombinant lipidated P4 (rLP4) and P6 (rLP6) proteins of NTHi and ubiquitous cell surface protein A (UspA) of M. catarrhalis were used for active immunization experiments in a mouse nasal challenge model. BALB/c mice were immunized intranasally with these proteins formulated with a chemically synthesized adjuvant, RC529 in an aqueous formulation (RC529-AF). Three weeks after the last immunization, these animals were challenged intranasally with NTHi strain SR7332.P1 and nasal colonization measured 3 days later. To determine local and systemic immune responses, bronchoalveolar washes (BAW) and sera were collected prior to NTHi challenge. The serum and mucosal samples were analyzed by ELISA for rLP4, rLP6 and UspA2 protein-specific IgG, IgG subclass and IgA antibody titers and bactericidal titers were determined against the TTA24 and 430–345 strains of M. catarrhalis. Results of these experiments show that these proteins combined with RC529-AF administered intranasally to mice elicited (1) significantly increased rLP4/rLP6/UspA2 protein-specific circulating IgG and IgA antibody responses; (2) local rLP4/rLP6/UspA2-specific IgA responses in the respiratory tract; and (3) more than a two log reduction of nasal colonization of NTHi strain SR7332 from the nasal tissues of mice. The serum IgG subclass distribution was predominantly IgG2a, representing a Th1 response. The antiserum also exhibited bactericidal activities to several strains of M. catarrhalis. These data indicate that intranasal immunization with rLP4/rLP6/UspA2 proteins combined with RC529-AF may be able to provide a way for inducing local mucosal immunity and for prevention of otitis media in children.

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae ( H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4 + CT, a mixture of rP4 and rP6 + CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4 + CT or a mixture of rP4, rP6 + CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4 + CT and a mixture of rP4, rP6 + CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4 + CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.

Factor H binding protein (fHBP) is currently under investigation as a potential vaccine antigen for protection against meningococcal serogroup B (MenB) disease. This study describes the distribution of genotypes among all (n=58) MenB, and a total of 80 representative non-MenB (serogroups A, C, Y and W135) isolates causing invasive disease in South Africa in 2005 using fHBP sequence analysis, PorA, FetA and multilocus sequence typing. There was less fHBP diversity among non-MenB isolates compared to MenB isolates. fHBP subfamily variant A32 was the most common fHBP variant among MenB isolates and was represented by 17% (10/58) of the isolates, while fHBP variant B16 was the most prevalent variant among non-MenB strains and was represented by 40% (32/80) of isolates. Overall, subfamily B domain N6 (modular group I) was most prevalent (57%, 79/138). Twenty PorA and 16 FetA types were identified among MenB isolates whereas non-MenB serogroups were largely associated with specific serosubtypes. The most common MenB clonal complex (ST-41/44/lineage 3) was represented by 29% (17/58) of the MenB isolates, while each of the non-MenB serogroups had a major clone represented by at least 75% of the isolates within the serorogroup. Our data highlight that non-MenB meningococcal isolates also harbor fHBP.

A family of outer membrane lipoproteins of Neisseria meningitidis, LP2086, has been shown to induce serum bactericidal activity against a broad variety of meningococcal strains. Two sub-families of serologically distinct LP2086 proteins (A and B) have been identified. In the present study, we have shown that polyclonal anti-serum against rLP2086 is protective in vivo in an infant rat passive-protection model. Additionally, the LP2086 protein is displayed on the surface of 91% meningococcal strains as measured in a whole cell ELISA using polyclonal anti-sera raised against these proteins. We also demonstrate based on the reactivity of anti-rLP2086 antibody with recombinantly expressed C- and N-terminal fragments of rLP2086 in a Western blot assay that the C-terminal fragment of LP2086 dictates sub-family specificity and the N-terminal fragment determines the family specificity. A formulation containing family A and B of LP2086 potentially would provide broad protection against a majority of Neisseria meningitidis strains.

Neisseria meningitidis is a major cause of bacterial meningitis in the human population, especially among young children. There is a need to develop a non-capsular vaccine to prevent meningococcal B infections due to the inadequate immune response elicited against the capsular polysaccharide of these strains. Previously, we developed a Swiss Webster adult mouse intranasal challenge model for group B N. meningitidis and evaluated several potential vaccine candidates including a meningococcal outer membrane protein, P2086, through parenteral immunization. Since N. meningitidis is a respiratory pathogen, a mucosal immune response may play an important role in the defense against meningococcal infections. Thus, intranasal immunization may be more effective than traditional parenteral immunization. In this study, mice were immunized intranasally with purified recombinant lipidated P2086 protein (rLP2086) adjuvanted with either CT-E29H, a genetically modified cholera toxin that is significantly reduced in enzymatic activity and toxicity or RC529-AF, a synthetic immunostimulant molecule in aqueous formulation. rLP2086-specific serum and mucosal IgG and IgA antibodies were induced. IgG antibodies reacted with whole cells of multiple strains of group B N.meningitidis. The antibodies have functional activity against N. meningitidis as demonstrated by bactericidal assays. Moreover, immunized mice exhibited reduced nasal colonization of group B meningococcal strains in the intranasal challenge model. These results demonstrate that an intranasal immunization with rLP2086 protein formulated with a detoxified cholera toxin or RC529-AF could prevent the initial colonization of group B meningococcus and become an effective immunization strategy against group B N. meningitidis.

Neisseria meningitidisis a major cause of bacterial meningitis in the human population, especially among young children. There is a need to develop a non-capsular vaccine to prevent meningococcal B infections due to the inadequate immune response elicited against the capsular polysaccharide of these strains. Previously, we developed a Swiss Webster adult mouse intranasal challenge model for group BN. meningitidisand evaluated several potential vaccine candidates including a meningococcal outer membrane protein, P2086, through parenteral immunization. SinceN. meningitidisis a respiratory pathogen, a mucosal immune response may play an important role in the defense against meningococcal infections. Thus, intranasal immunization may be more effective than traditional parenteral immunization. In this study, mice were immunized intranasally with purified recombinant lipidated P2086 protein (rLP2086) adjuvanted with either CT-E29H, a genetically modified cholera toxin that is significantly reduced in enzymatic activity and toxicity or RC529-AF, a synthetic immunostimulant molecule in aqueous formulation. rLP2086-specific serum and mucosal IgG and IgA antibodies were induced. IgG antibodies reacted with whole cells of multiple strains of group BN.meningitidis. The antibodies have functional activity againstN. meningitidisas demonstrated by bactericidal assays. Moreover, immunized mice exhibited reduced nasal colonization of group B meningococcal strains in the intranasal challenge model. These results demonstrate that an intranasal immunization with rLP2086 protein formulated with a detoxified cholera toxin or RC529-AF could prevent the initial colonization of group B meningococcus and become an effective immunization strategy against group BN. meningitidis.