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Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection.

ABSTRACT: To better characterize the vaccine potential of Neisseria meningitidis transferrin binding proteins (Tbps), we have overexpressed TbpA and TbpB from Neisseria meningitidis isolate K454 in Escherichia coli. The ability to bind human transferrin was retained by both recombinant proteins, enabling purification by affinity chromotography. The recombinant Tbps were evaluated individually and in combination in a mouse intraperitoneal-infection model to determine their ability to protect against meningococcal infection and to induce cross-reactive and bactericidal antibodies. For the first time, TbpA was found to afford protection against meningococcal challenge when administered as the sole immunogen. In contrast to the protection conferred by TbpB, this protection extended to a serogroup C isolate and strain B16B6, a serogroup B isolate with a lower-molecular-weight TbpB than that from strain K454. However, serum from a TbpB-immunized rabbit was found to be significantly more bactericidal than that from a TbpA-immunized animal. Our evidence demonstrates that TbpA used as a vaccine antigen may provide protection against a wider range of meningococcal strains than does TbpB alone. This protection appears not to be due to complement-mediated lysis and indicates that serum bactericidal activity may not always be the most appropriate predictor of efficacy for protein-based meningococcal vaccines.

SUBMITTER: West D

PROVIDER: S-EPMC98056 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection.

West D D Reddin K K Matheson M M Heath R R Funnell S S Hudson M M Robinson A A Gorringe A A

Infection and immunity 20010301 3

To better characterize the vaccine potential of Neisseria meningitidis transferrin binding proteins (Tbps), we have overexpressed TbpA and TbpB from Neisseria meningitidis isolate K454 in Escherichia coli. The ability to bind human transferrin was retained by both recombinant proteins, enabling purification by affinity chromotography. The recombinant Tbps were evaluated individually and in combination in a mouse intraperitoneal-infection model to determine their ability to protect against mening ...[more]

PMID: 11179327

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Project description:Neisseria meningitidis utilization of human serum transferrin (hTF)-bound iron is an important pathogenicity determinant. The efficiency of this system would clearly be increased through preferential binding of diferric hTF over the iron-free form. To characterize this process, functionally active meningococcal transferrin-binding protein A (TbpA) and TbpB have been purified from N. meningitidis using a novel purification procedure. The association of isolated Tbps and Tbps in the presence of hTF was investigated by gel filtration. Co-purified TbpA+B formed a complex of molecular mass 300 kDa which bound 1-2 molecules of hTF. Purified TbpA formed a complex of 200 kDa, indicating association as a dimer, whereas TbpB aggregated to form multimers of variable sizes. On recombining TbpA and TbpB, a stable complex of equivalent size to co-purified TbpA+B was formed. This complex may be composed of a single TbpA dimer and 1 molecule of TbpB. The technique of surface plasmon resonance (SPR) was used to demonstrate clearly that TbpB of either high (85 kDa) or low (68 kDa) molecular-mass preferentially bound diferric hTF in comparison with iron-free hTF. This selectivity was not observed with TbpA, but was found at low levels with co-purified TbpA+B. Individual TbpA and TbpB, recombined in a 1:1 molecular ratio, showed iron-mediated discriminatory binding at an intermediate level. SPR was also used to show that TbpA and TbpB bound to distinct regions of hTF, and that prior saturation with TbpB reduced subsequent TbpA binding. The results demonstrated that hTF bound more TbpA than TbpB, with an approximate ratio of 2:1. We have demonstrated that in vitro, TbpA+B exists as a receptor complex composed of a TbpA dimer and one molecule of TbpB, and that TbpB selectively binds diferric hTF. We propose that, in vivo, TbpA and TbpB also exist as a receptor complex, with TbpB selectively binding diferric hTF, bringing it close to TbpA, the transmembrane component, where the ferric iron can be transported to the periplasm.

Project description:BackgroundNeisseria meningitidis (Nm) is the cause of epidemic meningitis and fulminant meningococcal septicemia. The clinical presentations and outcome of meningococcal septic shock is closely related to the circulating levels of lipopolysaccharides (LPS) and of Neisseria meningitidis DNA (Nm DNA). We have previously explored the distribution of Nm DNA in tissues from large organs of patients dying of meningococcal septic shock and in a porcine meningococcal septic shock model.Objective1) To explore the feasibility of measuring LPS levels in tissues from the large organs in patients with meningococcal septic shock and in a porcine meningococcal septic shock model. 2) To evaluate the extent of contamination of non-specific LPS during the preparation of tissue samples.Patients and methodsPlasma, serum, and fresh frozen (FF) tissue samples from the large organs of three patients with lethal meningococcal septic shock and two patients with lethal pneumococcal disease. Samples from a porcine meningococcal septic shock model were included. Frozen tissue samples were thawed, homogenized, and prepared for quantification of LPS by Pyrochrome® Limulus Amoebocyte Lysate (LAL) assay.ResultsN. meningitidis DNA and LPS was detected in FF tissue samples from large organs in all patients with meningococcal septic shock. The lungs are the organs with the highest LPS and Nm DNA concentration followed by the heart in two of the three meningococcal shock patients. Nm DNA was not detected in any plasma or tissue sample from patients with lethal pneumococcal infection. LPS was detected at a low level in all FF tissues from the two patients with lethal pneumococcal disease. The experimental porcine meningococcal septic shock model indicates that also in porcinis the highest LPS and Nm DNA concentration are detected in lungs tissue samples. The quantification analysis showed that the highest concentration of both Nm DNA and LPS are in the organs and not in the circulation of patients with lethal meningococcal septic shock. This was also shown in the experimental porcine meningococcal septic shock model.ConclusionOur results suggest that LPS can be quantified in mammalian tissues by using the LAL assay.

Project description:BackgroundNeisseria meningitidis can cause severe infection in humans. Polymorphism of Complement Factor H (CFH) is associated with altered risk of invasive meningococcal disease (IMD). We aimed to find whether polymorphism of other complement genes altered risk and whether variation of N. meningitidis factor H binding protein (fHBP) affected the risk association.MethodsWe undertook a case-control study with 309 European cases and 5,200 1958 Birth Cohort and National Blood Service cohort controls. We used additive model logistic regression, accepting P<0.05 as significant after correction for multiple testing. The effects of fHBP subfamily on the age at infection and severity of disease was tested using the independent samples median test and Student's T test. The effect of CFH polymorphism on the N. meningitidis fHBP subfamily was investigated by logistic regression and Chi squared test.ResultsRs12085435 A in C8B was associated with odds ratio (OR) of IMD (0.35 [95% CI 0.19-0.67]; P = 0.03 after correction). A CFH haplotype tagged by rs3753396 G was associated with IMD (OR 0.56 [95% CI 0.42-0.76], P = 1.6x10⁻⁴). There was no bacterial load (CtrA cycle threshold) difference associated with carriage of this haplotype. Host CFH haplotype and meningococcal fHBP subfamily were not associated. Individuals infected with meningococci expressing subfamily A fHBP were younger than those with subfamily B fHBP meningococci (median 1 vs 2 years; P = 0.025).DiscussionThe protective CFH haplotype alters odds of IMD without affecting bacterial load for affected heterozygotes. CFH haplotype did not affect the likelihood of infecting meningococci having either fHBP subfamily. The association between C8B rs12085435 and IMD requires independent replication. The CFH association is of interest because it is independent of known functional polymorphisms in CFH. As fHBP-containing vaccines are now in use, relationships between CFH polymorphism and vaccine effectiveness and side-effects may become important.

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Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection.

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Recombinant Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection.

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