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The adhesin complex protein (ACP) of Neisseria meningitidis is a new adhesin with vaccine potential.

Hung MC, Heckels JE, Christodoulides M - MBio (2013)

Bottom Line: 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.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.We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP.

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton, Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT

Unlabelled: 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.

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(A and B)  Role of ACP in meningococcal invasion of human cells. Chang, Hep2, HUVEC, and meningioma cell monolayers were challenged with MC58 ¢18 and MC58 ¢18 ΔACP strains (MOI, 100 to 200), and the numbers of both associated and internalized bacteria were quantified. Results of representative experiments for each cell line are shown, as reported by Virji et al. (15). Experiments were repeated 3 times, and the differences in association and/or invasion between the strains were reproducible between experiments, regardless of any quantitative variations between experiments. (C) Effect of rabbit anti-rACP serum on internalization of MC58 ¢18 strain into Chang epithelial cells and HUVECs. Cell monolayers (n = 3 experiments for each cell line) were infected for 4 h with strain MC58 ¢18 in the presence of 10% (vol/vol) preimmune and postimmune rabbit anti-rACP serum and the numbers of internalized bacteria quantified. For all figures, the columns represent the numbers of associated and/or internalized bacteria and the error bars the standard deviations of the results determined with triplicate wells. *, P < 0.05.
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fig8: (A and B)  Role of ACP in meningococcal invasion of human cells. Chang, Hep2, HUVEC, and meningioma cell monolayers were challenged with MC58 ¢18 and MC58 ¢18 ΔACP strains (MOI, 100 to 200), and the numbers of both associated and internalized bacteria were quantified. Results of representative experiments for each cell line are shown, as reported by Virji et al. (15). Experiments were repeated 3 times, and the differences in association and/or invasion between the strains were reproducible between experiments, regardless of any quantitative variations between experiments. (C) Effect of rabbit anti-rACP serum on internalization of MC58 ¢18 strain into Chang epithelial cells and HUVECs. Cell monolayers (n = 3 experiments for each cell line) were infected for 4 h with strain MC58 ¢18 in the presence of 10% (vol/vol) preimmune and postimmune rabbit anti-rACP serum and the numbers of internalized bacteria quantified. For all figures, the columns represent the numbers of associated and/or internalized bacteria and the error bars the standard deviations of the results determined with triplicate wells. *, P < 0.05.

Mentions: A potential role for ACP in cellular invasion was investigated using the gentamicin assay (15). Initially, internalization of the wild-type capsulated (Cap+) MC58 and MC58 ∆ACP strains by Chang, Hep2, HUVECs, and meningioma cells was examined, but bacterial recovery after gentamicin treatment was very low and differences between the variants were not significant (P > 0.05). These data showed that ACP did not play a role in invasion of encapsulated strains, so we compared the interactions of noncapsulated (Cap−) ACP-expressing and MC58 ΔACP strains. Moreover, to avoid the potentially masking effects of the important meningococcal host binding factors of pili and Opa and Opc proteins (16), invasion of a noncapsular strain lacking their expression, i.e., MC58 ¢18 (Cap− Pil− Opa− Opc− ACP+), was compared with that of its ACP knockout mutant, MC58 ¢18 ∆ACP (Cap− Pil− Opa− Opc− ACP−). Compared to the MC58 ¢18 strain, absence of ACP reduced association of the mutant strain to all the cell lines by ~50% to 75% (P < 0.05) (Fig. 8A). Internalization of MC58 ¢18 by human cells was observed, but the numbers of bacteria recovered per monolayer after gentamicin treatment were low (Fig. 8B). Nevertheless, there was a significant reduction in the numbers of internalized MC58 ¢18 ∆ACP in Chang and Hep2 epithelial cells compared to the strain expressing ACP (P < 0.05) and also after taking into account the differences in association between the ACP+ and ACP− strains. In contrast, although there was an apparent reduction in the numbers of internalized MC58 ¢18 ∆ACP bacteria in HUVECs, this was not genuine, since the percentages of reduction in association and internalization of MC58 ¢18 ∆ACP were similar (P > 0.05) to those seen with MC58 ¢18. Very low numbers of MC58 ¢18 bacteria were recovered from meningioma cell monolayers, and no significant difference was observed with the MC58 ¢18 ∆ACP strain (P = 0.181); the inability of meningococci to invade these cells is consistent with our previous findings (17).


The adhesin complex protein (ACP) of Neisseria meningitidis is a new adhesin with vaccine potential.

Hung MC, Heckels JE, Christodoulides M - MBio (2013)

(A and B)  Role of ACP in meningococcal invasion of human cells. Chang, Hep2, HUVEC, and meningioma cell monolayers were challenged with MC58 ¢18 and MC58 ¢18 ΔACP strains (MOI, 100 to 200), and the numbers of both associated and internalized bacteria were quantified. Results of representative experiments for each cell line are shown, as reported by Virji et al. (15). Experiments were repeated 3 times, and the differences in association and/or invasion between the strains were reproducible between experiments, regardless of any quantitative variations between experiments. (C) Effect of rabbit anti-rACP serum on internalization of MC58 ¢18 strain into Chang epithelial cells and HUVECs. Cell monolayers (n = 3 experiments for each cell line) were infected for 4 h with strain MC58 ¢18 in the presence of 10% (vol/vol) preimmune and postimmune rabbit anti-rACP serum and the numbers of internalized bacteria quantified. For all figures, the columns represent the numbers of associated and/or internalized bacteria and the error bars the standard deviations of the results determined with triplicate wells. *, P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585444&req=5

fig8: (A and B)  Role of ACP in meningococcal invasion of human cells. Chang, Hep2, HUVEC, and meningioma cell monolayers were challenged with MC58 ¢18 and MC58 ¢18 ΔACP strains (MOI, 100 to 200), and the numbers of both associated and internalized bacteria were quantified. Results of representative experiments for each cell line are shown, as reported by Virji et al. (15). Experiments were repeated 3 times, and the differences in association and/or invasion between the strains were reproducible between experiments, regardless of any quantitative variations between experiments. (C) Effect of rabbit anti-rACP serum on internalization of MC58 ¢18 strain into Chang epithelial cells and HUVECs. Cell monolayers (n = 3 experiments for each cell line) were infected for 4 h with strain MC58 ¢18 in the presence of 10% (vol/vol) preimmune and postimmune rabbit anti-rACP serum and the numbers of internalized bacteria quantified. For all figures, the columns represent the numbers of associated and/or internalized bacteria and the error bars the standard deviations of the results determined with triplicate wells. *, P < 0.05.
Mentions: A potential role for ACP in cellular invasion was investigated using the gentamicin assay (15). Initially, internalization of the wild-type capsulated (Cap+) MC58 and MC58 ∆ACP strains by Chang, Hep2, HUVECs, and meningioma cells was examined, but bacterial recovery after gentamicin treatment was very low and differences between the variants were not significant (P > 0.05). These data showed that ACP did not play a role in invasion of encapsulated strains, so we compared the interactions of noncapsulated (Cap−) ACP-expressing and MC58 ΔACP strains. Moreover, to avoid the potentially masking effects of the important meningococcal host binding factors of pili and Opa and Opc proteins (16), invasion of a noncapsular strain lacking their expression, i.e., MC58 ¢18 (Cap− Pil− Opa− Opc− ACP+), was compared with that of its ACP knockout mutant, MC58 ¢18 ∆ACP (Cap− Pil− Opa− Opc− ACP−). Compared to the MC58 ¢18 strain, absence of ACP reduced association of the mutant strain to all the cell lines by ~50% to 75% (P < 0.05) (Fig. 8A). Internalization of MC58 ¢18 by human cells was observed, but the numbers of bacteria recovered per monolayer after gentamicin treatment were low (Fig. 8B). Nevertheless, there was a significant reduction in the numbers of internalized MC58 ¢18 ∆ACP in Chang and Hep2 epithelial cells compared to the strain expressing ACP (P < 0.05) and also after taking into account the differences in association between the ACP+ and ACP− strains. In contrast, although there was an apparent reduction in the numbers of internalized MC58 ¢18 ∆ACP bacteria in HUVECs, this was not genuine, since the percentages of reduction in association and internalization of MC58 ¢18 ∆ACP were similar (P > 0.05) to those seen with MC58 ¢18. Very low numbers of MC58 ¢18 bacteria were recovered from meningioma cell monolayers, and no significant difference was observed with the MC58 ¢18 ∆ACP strain (P = 0.181); the inability of meningococci to invade these cells is consistent with our previous findings (17).

Bottom Line: 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.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.We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP.

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton, Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus