Miao-Chiu Hung

The Neisseria meningitidis Macrophage Infectivity Potentiator Protein Induces Cross-Strain Serum Bactericidal Activity and Is a Potential Serogroup B Vaccine Candidate

ABSTRACT A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)3. Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.

The Adhesin Complex Protein (ACP) of Neisseria meningitidis Is a New Adhesin with Vaccine Potential

ABSTRACT The acp gene encoding the 13-kDa adhesin complex protein (ACP) from Neisseria meningitidis serogroup B strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant ACP (rACP) was used for immunization studies. Analysis of the ACP amino acid sequences from 13 meningococcal strains, isolated from patients and colonized individuals, and 178 strains in the Bacterial Isolate Genome Sequence (BIGS) database showed the presence of only three distinct sequence types (I, II, and III) with high similarity (>98%). Immunization of mice with type I rACP in detergent micelles and liposomes and in saline solution alone induced high levels of serum bactericidal activity (SBA; titers of 1/512) against the homologous strain MC58 and killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/512). Levels of expression of type I, II, or III ACP by different meningococcal strains were similar. ACP functioned as an adhesin, as demonstrated by reduced adherence of acp knockout (MC58 ΔACP) meningococci to human cells in vitro and the direct surface binding of rACP and by the ability of anti-rACP sera to inhibit adherence of wild-type bacteria. ACP also mediated the invasion of noncapsular meningococci into human epithelial cells, but it was not a particularly impressive invasin, as the internalized bacterial numbers were low. In summary, the newly identified ACP protein is an adhesin that induces cross-strain bactericidal activity and is therefore an attractive target antigen for incorporation into the next generation of serogroup B meningococcal vaccines. IMPORTANCE Infections caused by Neisseria meningitidis serogroup B are still significant causes of mortality and morbidity worldwide, and broadly protective vaccines of defined antigen composition are not yet licensed. Here, we describe the properties of the adhesin complex protein (ACP), which we demonstrate is a newly recognized molecule that is highly conserved and expressed to similar levels in meningococci and facilitates meningococcal interactions with human cells. We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP. Taken together, these properties demonstrate that ACP merits serious consideration as a component of a broadly protective vaccine against serogroup B meningococci. Infections caused by Neisseria meningitidis serogroup B are still significant causes of mortality and morbidity worldwide, and broadly protective vaccines of defined antigen composition are not yet licensed. Here, we describe the properties of the adhesin complex protein (ACP), which we demonstrate is a newly recognized molecule that is highly conserved and expressed to similar levels in meningococci and facilitates meningococcal interactions with human cells. We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP. Taken together, these properties demonstrate that ACP merits serious consideration as a component of a broadly protective vaccine against serogroup B meningococci.

Recombinant Protein Truncation Strategy for Inducing Bactericidal Antibodies to the Macrophage Infectivity Potentiator Protein of Neisseria meningitidis and Circumventing Potential Cross-Reactivity with Human FK506-Binding Proteins

ABSTRACT A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (−LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.

The Biology of Neisseria Adhesins

Members of the genus Neisseria include pathogens causing important human diseases such as meningitis, septicaemia, gonorrhoea and pelvic inflammatory disease syndrome. Neisseriae are found on the exposed epithelia of the upper respiratory tract and the urogenital tract. Colonisation of these exposed epithelia is dependent on a repertoire of diverse bacterial molecules, extending not only from the surface of the bacteria but also found within the outer membrane. During invasive disease, pathogenic Neisseriae also interact with immune effector cells, vascular endothelia and the meninges. Neisseria adhesion involves the interplay of these multiple surface factors and in this review we discuss the structure and function of these important molecules and the nature of the host cell receptors and mechanisms involved in their recognition. We also describe the current status for recently identified Neisseria adhesins. Understanding the biology of Neisseria adhesins has an impact not only on the development of new vaccines but also in revealing fundamental knowledge about human biology.

A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins

The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p>0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32-64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)3. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.

Vaccine potential of bacterial macrophage infectivity potentiator (MIP)-like peptidyl prolyl cis/trans isomerase (PPIase) proteins

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

Potential of the adhesin complex protein of Neisseria meningitidis for next-generation meningococcal vaccines

Author for correspondence: Neisseria Research, Molecular Microbiology, Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, SO166YD, UK M.Christodoulides@soton.ac.uk “...Neisseria ACP is the first member of a class of small Mr OM antigens for which a role in human host cell interactions has been demonstrated. Moreover, Neisseria ACP fulfils the selection criteria for a ‘universal’ meningococcal vaccine antigen and in the future, ACP-like proteins identified in other bacterial pathogens may become components of both human and veterinary vaccines.”