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, 12 (7), e0181508
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Contribution of Factor H-Binding Protein Sequence to the Cross-Reactivity of Meningococcal Native Outer Membrane Vesicle Vaccines With Over-Expressed fHbp Variant Group 1

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Contribution of Factor H-Binding Protein Sequence to the Cross-Reactivity of Meningococcal Native Outer Membrane Vesicle Vaccines With Over-Expressed fHbp Variant Group 1

Arianna Marini et al. PLoS One.

Abstract

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV.

Conflict of interest statement

Competing Interests: This work was sponsored by the University of Birmingham. AM, AC, IH, and CM were beneficiaries of grant funding from European Union’s European Industrial Doctorate Programme, VADER (Vaccine Design and Immune Responses) [FP7/2007-2013]. ID, OK, CM, FM, SR, and AS were employees of Novartis Vaccines and Diagnostics Srl at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March, 2015, MA, ID, OK, FM, SR, AS are now employees of the GSK group of companies. AM was PhD Student at the University of Birmingham, SG at the University of Bologna, OR and MA at the University of Naples at the time of the studies, and supervised by Novartis Vaccines and Diagnostics. AS and ID report ownership of GSK shares and/or restricted GSK shares. SG and ID are listed as inventors on patents owned by the GSK group of companies. GSK has filed patent applications in Europe (EP 13703562.2) and the United States (US 14/375222) entitled “Promoters For Increased Protein Expression in Meningococcus”. The patent applications have claims directed to specific promoter sequences for increasing expression of outer membrane proteins, including fHbp. Material including modified bacterial strains and NOMV over-expressing fHbp disclosed in our manuscript entitled “Contribution of Factor H-Binding Protein Sequence to the Cross- Reactivity of Meningococcal Native Outer Membrane Vesicles (NOMV) with Over- Expressed fHbp v.1 Peptides” can be obtained and used without restriction for research purposes by implementing the teachings of the published patent specifications and/or the manuscript. Data and materials described in the manuscript may be provided to third parties for research purposes subject to GSK’s policies and guidelines, which are available upon request. Unauthorized use of bacterial strains incorporating GSK’s proprietary promoter sequences for commercial purposes would constitute patent infringement. Licenses for commercial use of patented promoter sequences may be granted by GSK, subject to negotiation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Section of matrix for pairwise comparison of fH-contact residues on fHbp v.1 IDs.
Each cellmn contains the number of different amino acids in the fH-binding site between IDm and IDn. The darkness of shading of each intersection corresponds to the number of differences, with white = 0 differences. In the table below the matrix, for each IDm the number of fHbp v.1 peptides differing for an increasing number of amino acids is given, together with the average number of different residues between IDm and all other v.1 peptides. IDs belonging to v.1 between 12 and 60, and between 80 and 677 are not shown (full matrix available in S2 Table).
Fig 2
Fig 2. NOMV characterisation.
(A) SDS-PAGE and Coomassie staining of NOMV. Samples: NOMV from MenW wild type; NOMV OE ID 1; NOMV OE ID 5; NOMV OE ID 9; NOMV OE ID 74; NOMV fHbp KO. 5 μg of each NOMV were loaded. The location of different proteins is indicated by the arrows. (B) Western blot to detect fHbp v.1 in NOMV from different mutants. As controls, NOMV fHbp KO and NOMV from MenA wild type (expressing fHbp ID 5) were also loaded. 0.2 μg of NOMV were loaded per lane. (C) Western blot for comparison of fHbp presence in NOMV from MenA wild type (expressing fHbp ID 5) and in NOMV OE ID 9. 0.05 to 0.5 μg of NOMV were loaded.
Fig 3
Fig 3. Anti-fHbp IgG antibody levels in sera from mice immunised with NOMV or recombinant fHbp.
Anti-fHbp IgG antibody levels were measured in all immunisation groups against each of the 4 fHbp IDs. 1 μg/mL of recombinant fHbp was used for ELISA plate coating. Mice were immunised twice, four weeks apart, with 5 μg NOMV fHbp KO (A), or 1 μg NOMV OE one of the four different fHbp IDs (B-E), or with 20 μg recombinant fHbp (F-I). Serum samples analysed were obtained 2 weeks after the second dose. Each symbol represents a serum sample from an individual mouse; bars represent the geometric mean of each group. Mann-Whitney test 2-tailed was performed to compare pairs of groups: * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4
Fig 4. Comparison of IgG antibody levels against different fHbp ID within the same immunisation group.
Comparison of IgG antibody levels against different recombinant fHbp IDs, in individual mice. Each symbol represents an individual mouse, identified by a number; 1 μg/mL of recombinant fHbp was used for ELISA plate coating. A) Mice immunised with NOMV OE ID 74 show linear correlation between anti-fHbp ID 74 IgG ELISA units (X axis) and anti-fHbp ID 5 IgG ELISA units (Y axis), by the Pearson Correlation test. B) Mice immunised with NOMV OE ID 5 do not show linear correlation between anti-fHbp ID 5 IgG ELISA units (X axis) and anti-fHbp ID 74 IgG ELISA units (Y axis).
Fig 5
Fig 5. Serum bactericidal responses induced in individual mice immunised with NOMV or recombinant fHbp.
Mice were immunised twice with NOMV (A-E), or with recombinant fHbp (F-I), four weeks apart, as described in the materials and methods. Serum samples obtained two weeks after the second dose were tested against a set of seven strains, expressing the four different fHbp ID types: ID 1 (), ID 5 (), ID 9 (), or ID 74 (). Each symbol represents the reciprocal titre of an individual mouse; horizontal bars represent geometric mean titres of the group.
Fig 6
Fig 6. Correlation of IgG antibody levels against recombinant ID 74 and SBA titres against MenX strains.
Each symbol represents a single mouse immunised with recombinant fHbp ID 74 and identified by a number. No linear correlation was evident according to the Pearson Correlation test.
Fig 7
Fig 7. Correlation of SBA titres against different strains, in mice immunised with NOMV OE ID 1.
Each symbol represents the reciprocal SBA titre of a mouse, identified by a number. According to the Pearson Correlation test, linear correlation is present between SBA titres against strains carrying the same fHbp ID, but not between SBA titres against strains carrying different fHbp ID.

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Grant support

The research leading to this publication has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7/2007- 2013/ under REA grant agreement n° 316940, European Industrial Doctorate Programme VADER (Vaccine Design and Immune Responses), whose beneficiaries were GSK Vaccines Institute for Global Health (former Novartis Vaccine Institute for Global Health) and University of Birmingham. GSK Vaccines Institute for Global Health and University of Birmingham also contributed. ID, OK, CM, FM, SR, and AS were employees of Novartis Vaccines and Diagnostics Srl at the time of the study. Following the acquisition of Novartis Vaccines by the GSK group of companies in March, 2015, MA, ID, OK, FM, SR, AS are now employees of the GSK group of companies. The funder provided support in the form of salaries for authors [AM, MA, ID, OK, FM, CM, SR, AS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
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