Development of a non-biased, high-throughput ELISA for the rapid evaluation of immunogenicity and cross-reactivity.

Traditional ELISA-based protein analysis has been predicated on the assumption that proteins bind randomly to the solid surface of the ELISA plate polymer (polystyrene or polyvinyl chloride). Random adherence to the plate ensures equal access to all faces of the protein, an important consideration when evaluating immunogenicity of polyclonal serum samples as well as when examining the cross-reactivity of immune serum against different antigenic variants of a protein. In this study we demonstrate that the soluble form of the surface lipoprotein transferrin binding protein B (TbpB) from three different bacterial pathogens (Neisseria meningitidis, Actinobacillus pleuropneumoniae, and Mannheimia haemolytica) bind the ELISA plate in a manner that consistently obscures the transferrin binding face of the proteins' N-lobe. In order to develop a non-biased ELISA where all faces of the protein are accessible, the strong interaction between biotin and avidin has been exploited by adding a biotin tag to these proteins during Escherichia coli-based cytoplasmic expression and utilizing streptavidin or neutravidin coated ELISA plates for protein capture and display. The use of avidin coated ELISA plates also allows for rapid purification of biotin-tagged proteins from crude E. coli lysates, removing the requirement of prior affinity purification of each protein to be included in the ELISA-based analyses. In proof of concept experiments we demonstrate the utility of this approach for evaluating immunogenicity and cross-reactivity of serum from mice and pigs immunized with TbpBs from human and porcine pathogens.

Proof of Concept for Prevention of Natural Colonization by Oral Needle-Free Administration of a Microparticle Vaccine.

There has been substantial interest in the development of needle-free vaccine administration that has led to a variety of approaches for delivery through the skin for induction of a systemic immune response. The mucosal administration of vaccines has inherently been needle-free, but the simple application of vaccines on the mucosal surface by itself does not lead to mucosal immunity. Since many important bacterial infections develop after initial colonization of the upper respiratory tract of the host, prevention of colonization could not only prevent infection but also eliminate the reservoir of pathogens that reside exclusively in that ecologic niche. This study was designed to provide proof of concept for a needle-free immunization approach that would reduce or eliminate colonization and prevent infection. In order to accomplish this a microparticle vaccine preparation was delivered just below the oral mucosal epithelial cell layer where it would lead to a robust immune response. A vaccine antigen (mutant transferrin binding protein B) shown to be capable of preventing infection in pigs was incorporated into a polyphosphazene microparticle preparation and delivered by a needle-free device to the oral sub-epithelial space of pigs. This vaccination regimen not only provided complete protection from infection after intranasal challenge by Glaesserella parasuis but also eliminated natural colonization by this bacterium. Notably, the complete prevention of natural colonization was dependent upon delivery of the microparticle preparation below the epithelial layer in the oral mucosa as intradermal or intramuscular delivery was not as effective at preventing natural colonization. This study also demonstrated that a primary immunization in the presence of maternal antibody limited the resulting antibody response but a robust antibody response after the second immunization indicated that maternal antibody did not prevent induction of B-cell memory.

Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species.

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core ß-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.

Photo-Cross-Linking Mass Spectrometry and Integrative Modeling Enables Rapid Screening of Antigen Interactions Involving Bacterial Transferrin Receptors.

Structure-based approaches to the delineation of immunogens for vaccine development have a throughput requirement that is difficult to meet in practice with conventional methods of structure determination. Here we present a strategy for rapid and accurate structure generation in support of antigen engineering programs. The approach is developed around the modeling of interactions between host transferrin (Tf) and the bacterial vaccine target transferrin binding protein B (TbpB) from Gram-negative pathogens such as Neisseria meningitidis. Using an approach based solely on cross-linking mass spectrometry (XL-MS) data, monomeric structural models, and the Integrative Modeling Platform (IMP), we demonstrate that converged representations of the Tf:TbpB interactions can be returned that accurately reflect the binding interface and the relative orientation of the monomeric units, with the capacity to scale to the analysis of interactions from any number of additional strains. We show that a key element to accurate modeling involves the application of hetero-bifunctional cross-linkers incorporating fast-acting photoactivatable diazirines coupled with conventional amine-targeting N-hydroxysuccinimide esters, and we demonstrate that conventional homo-bifunctional reagents used in cross-linking kinetically trap dynamic states in the ensemble. Therefore, the application of both classes of cross-linker provides an opportunity to empirically detect protein dynamics during integrative structural modeling.

New insights about functional and cross-reactive properties of antibodies generated against recombinant TbpBs of Haemophilus parasuis.

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund's adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.

Identification of a Large Family of Slam-Dependent Surface Lipoproteins in Gram-Negative Bacteria.

The surfaces of many Gram-negative bacteria are decorated with soluble proteins anchored to the outer membrane via an acylated N-terminus; these proteins are referred to as surface lipoproteins or SLPs. In Neisseria meningitidis, SLPs such as transferrin-binding protein B (TbpB) and factor-H binding protein (fHbp) are essential for host colonization and infection because of their essential roles in iron acquisition and immune evasion, respectively. Recently, we identified a family of outer membrane proteins called Slam (Surface lipoprotein assembly modulator) that are essential for surface display of neisserial SLPs. In the present study, we performed a bioinformatics analysis to identify 832 Slam related sequences in 638 Gram-negative bacterial species. The list included several known human pathogens, many of which were not previously reported to possess SLPs. Hypothesizing that genes encoding SLP substrates of Slams may be present in the same gene cluster as the Slam genes, we manually curated neighboring genes for 353 putative Slam homologs. From our analysis, we found that 185 (~52%) of the 353 putative Slam homologs are located adjacent to genes that encode a protein with an N-terminal lipobox motif. This list included genes encoding previously reported SLPs in Haemophilus influenzae and Moraxella catarrhalis, for which we were able to show that the neighboring Slams are necessary and sufficient to display these lipoproteins on the surface of Escherichia coli. To further verify the authenticity of the list of predicted SLPs, we tested the surface display of one such Slam-adjacent protein from Pasteurella multocida, a zoonotic pathogen. A robust Slam-dependent display of the P. multocida protein was observed in the E. coli translocation assay indicating that the protein is a Slam-dependent SLP. Based on multiple sequence alignments and domain annotations, we found that an eight-stranded beta-barrel domain is common to all the predicted Slam-dependent SLPs. These findings suggest that SLPs with a TbpB-like fold are found widely in Proteobacteria where they exist with their interaction partner Slam. In the future, SLPs found in pathogenic bacteria can be investigated for their role in virulence and may also serve as candidates for vaccine development.

A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response.

Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs.

Sequence and structural diversity of transferrin receptors in Gram-negative porcine pathogens.

Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a challenge to future vaccine development, our results suggest a rationally designed TbpB-based vaccine may provide protection against all three pathogens.

Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

Improvement of immunogenicity of meningococcal lipooligosaccharide by coformulation with lipidated transferrin-binding protein B in liposomes: implications for vaccine development.

Among various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group B Neisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluated in vitro using the Limulus Amebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, an in vivo study demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

Gonococcal transferrin binding protein chimeras induce bactericidal and growth inhibitory antibodies in mice.

We have previously demonstrated the full-length gonococcal transferrin binding proteins (TbpA and TbpB) to be promising antigens in the development of a protective vaccine against Neisseria gonorrhoeae. In the current study we employed a genetic chimera approach fusing domains from TbpA and TbpB to the A2 domain of cholera toxin, which naturally binds in a non-covalent fashion to the B subunit of cholera toxin during assembly. For one construct, the N-terminal half of TbpB (NB) was fused to the A2 subunit of cholera toxin. In a second construct, the loop 2 region (L2) of TbpA was genetically fused between the NB domain and the A2 domain, generating a double chimera. Both chimeras were immunogenic and induced serum bactericidal and vaginal growth-inhibiting antibodies. This study highlights the potential of using protective epitopes instead of full-length proteins in the development of an efficacious gonococcal vaccine.

Stimulation of mucosal and systemic antibody responses against recombinant transferrin-binding protein B of Actinobacillus pleuropneumoniae with chitosan after tracheal administration in piglets.

This study evaluated the suitability of using a chitosan formulation as an adjuvant to enhance both the mucosal and systemic immune responses against recombinant transferrin-binding protein B (rTbp B) of Actinobacillus pleuropneumoniae via direct tracheal administration. The chitosan formulation was found to enhance mucosal immune response, as measured by the secretory IgA level in lung lavage fluid and lung homogenate extracts, and systemic immune response, as measured by the serum IgG level.

Vaccination of mice with gonococcal TbpB expressed in vivo from Venezuelan equine encephalitis viral replicon particles.

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.

Intranasal administration of recombinant Neisseria gonorrhoeae transferrin binding proteins A and B conjugated to the cholera toxin B subunit induces systemic and vaginal antibodies in mice.

The transferrin binding proteins (TbpA and TbpB) comprise the gonococcal transferrin receptor and are considered potential antigens for inclusion in a vaccine against Neisseria gonorrhoeae. Intranasal (IN) immunization has shown promise in development of immunity against sexually transmitted disease pathogens, in part due to the induction of antigen-specific genital tract immunoglobulin A (IgA) and IgG. Conjugation of antigens to the highly immunogenic cholera toxin B subunit (Ctb) enhances antibody responses in the serum and mucosal secretions following IN vaccination. In the current study, we characterized the anti-Tbp immune responses following immunization of mice IN with recombinant transferrin binding proteins (rTbpA and rTbpB) conjugated to rCtb. We found that both rTbpA-Ctb and rTbpB-Ctb conjugates administered IN induced antibody responses in the serum and genital tract. IN immunization resulted in both IgA and IgG in the genital tract; however, subcutaneous immunization mainly generated IgG. Surprisingly, rTbpA alone was immunogenic and induced serum and mucosal antibody responses similar to those elicited against the rTbpA-Ctb conjugate. Overall, rTbpB was much more immunogenic than rTbpA, generating serum IgG levels that were greater than those elicited against rTbpA. Bactericidal assays conducted with sera collected from mice immunized IN with TbpA and/or TbpB indicated that both antigens generated antibodies with bactericidal activity. Anti-TbpA antibodies were cross-bactericidal against heterologous gonococcal strains, whereas TbpB-specific antibodies were less cross-reactive. By contrast, antibodies elicited via subcutaneous immunization were not cross-bactericidal against heterologous strains, indicating that IN vaccination could be the preferred route for elicitation of biologically functional antibodies.

The gonococcal Fur-regulated tbpA and tbpB genes are expressed during natural mucosal gonococcal infection.

Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

Role of transferrin receptor from a Neisseria meningitidis tbpB isotype II strain in human transferrin binding and virulence.

Neisseria meningitidis acquires iron through the action of the transferrin (Tf) receptor, which is composed of the Tf-binding proteins A and B (TbpA and TbpB). Meningococci can be classified into isotype I and II strains depending on whether they harbor a type I or II form of TbpB. Both types of TbpB have been shown to differ in their genomic, biochemical, and antigenic properties. Here we present a comparative study of isogenic mutants deficient in either or both Tbps from the isotype I strain B16B6 and isotype II strain M982. We show that TbpA is essential in both strains for iron uptake and growth with iron-loaded human Tf as a sole iron source. No growth has also been observed for the TbpB- mutant of strain B16B6, as shown previously, whereas the growth of the analogous mutant in M982 was similar to that in the wild type. This indicates that TbpB in the latter strain plays a facilitating but not essential role in iron uptake, which has been observed previously in similar studies of other bacteria. These data are discussed in relation to the fact that isotype II strains represent more than 80% of serogroup B meningococcal strains. The contribution of both subunits in the bacterial virulence of strain M982 has been assessed in a murine model of bacteremia. Both the TbpB- TbpA- mutant and the TbpA- mutant are shown to be nonvirulent in mice, whereas the virulence of the TbpB- mutant is similar to that of the wild type.

Determination of surface-exposed, functional domains of gonococcal transferrin-binding protein A.

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and beta strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative beta strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA.

Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis.

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

Immunogenicity of gonococcal transferrin binding proteins during natural infections.

In this study, we examined the immune response during gonococcal infection to the individual transferrin binding proteins by using a quantitative enzyme-linked immunosorbent assay (ELISA). Recombinant transferrin binding protein A (rTbpA) and rTbpB were purified under nondenaturing conditions for use as ELISA antigens. Sera and secretions from culture-positive individuals were analyzed for antibodies to rTbpA and rTbpB and compared to samples from individuals with no history of gonococcal infection. Although antibodies to both rTbpA and rTbpB were detected in serum, in most cases the antibody levels were not significantly different from those measured in the control population. Also, previous history of gonococcal infection did not increase antibody levels in serum, suggesting the lack of an anamnestic response. Analysis of secretion samples revealed antibody levels that were generally below the limits of detection in our assay. Overall, this study demonstrated a paucity of systemic and local antibody responses to rTbps as a result of natural infection and represents a baseline over which a protective antibody response will have to be generated in order to develop an efficacious gonococcal vaccine.