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Using structure-based design to engineer chimeric protein vaccines

Related publication: Hollingshead S., Jongerius I., Exley R. M., Johnson S., Lea S. M. & Tang C. M. Structure-based design of chimeric antigens for multivalent protein vaccines. Nat. Commun. 9, 1051 (2018). DOI: 10.1038/s41467-018-03146-7

Publication keywords: Meningococcal; Vaccine; Serogroup B; fHbp; PorA

A major World Health Organisation objective is to develop vaccines against pathogenic bacteria. However, vaccine development is often hindered by variation in the molecules at the surface of the bacteria, and the manufacturing challenges inherent to working with such molecules. Researchers conducted a proof-of-principle study to assess the feasibility of taking portions of intractable molecules and creating chimeras by splicing them into well-behaved molecules, with the ultimate goal of producing vaccines that raised immune responses against both types of molecule.

They used Macromolecular Crystallography beamlines I02 and I04-1 to carry out structural investigations to confirm that this chimera-generating approach doesn’t compromise key structural features of the molecules. This is especially important when it comes to molecules with the potential for use in therapeutic drugs. The study demonstrated that the approach is valid. Structures of the chimeric molecules revealed that both molecules retained the structure observed in their native context, or in immune-relevant complexes. Immunisation studies revealed that the chimeric molecules were able to elicit immune responses against each component.

: Figure 1: (a) Schematic of N. meningitidis cell surface, shown are the major antigens fHbp (in a complex with human complement factor H domains 6-7) and PorA. (b) Structural alignment of fHbp
scaffolds from V1 fHbp peptides with a PorA loop inserted at residue 151 (pink, PDB: 5NQP), 294 (green, PDB: 5NQX) or 309 (orange, PDB: 5NQY). (c) Structural alignment of the PorA VR2 epitopes in the
fHbp scaffold (151, pink; 294, green; 309, orange) with a linear peptide of PorA VR2 P1.16 (black) in a complex with a bactericidal Fab fragment (grey) from monoclonal antibody MN12H2 (PDB: 2MPA).

To combat the rise in multi-drug resistant bacteria, the World Health Organisation is advocating the development of vaccines against bacterial pathogens. Historically, vaccines were based on protein toxoids, polysaccharide capsules, or outer membrane vesicles. However, these approaches are not feasible for many pathogens, and immune evasion mechanisms, such as antigenic diversity, further limit the strategies available to develop successful vaccines. In addition, many vaccine candidates are integral membrane proteins which can present manufacturing problems, and issues such as generating immune responses against epitopes that are masked in the whole organism. To circumvent these issues, we used structure-based design to engineer chimeric antigens (ChAs), and developed a next generation vaccine for prevention of meningococcal disease.

The meningococcal ChAs produced for our study are composed of two major N. meningitidis antigens, factor H binding protein (fHbp), a surface exposed lipoprotein¹, and PorA, an integral outer membrane porin² (Fig. 1a). In the ChAs, fHbp is exploited as a molecular scaffold to display an immunogenic surface exposed loop from PorA, known as the variable region 2 (VR2, Fig. 1a). Both fHbp and the VR2 loop exhibit antigenic diversity, with fHbp peptides falling into three variant groups or two subfamilies depending on the classification system: V1 (subfamily B), V2, and V3 (both subfamily A). In general, immunisation with a particular fHbp induces cross-protection against strains that express an fHbp belonging to the same, but not a different, variant group, although there can be cross-protection between fHbp variant groups V2 and V3 (subfamily A). Unlike fHbp, the PorA VR2 offers limited or no cross-protection against strains expressing a PorA peptide with a different VR2. Therefore to optimise coverage of the vaccine, the fHbp and PorA VR2 peptides used must be carefully selected. This was achieved using the comprehensive epidemiology data available in the meningococcal genome library³.

Figure 2: Serum bactericidal assay titres. (a) α-PorA SBA titres generated using ChA/alum antisera and serogroup B N. meningitidis isolates with mismatched fHbp variants. (b) α-fHbp SBA titres generated using ChA/alum antisera and serogroup B N. meningitidis isolate H44/76 ΔporA.

A proof of principle study was designed to ascertain: i) the introduction of a PorA VR2 loop did not alter the overall architecture of fHbp, ii) functional immune responses could be elicited against both fHbp and PorA antigens, iii) ChA composition can be manipulated to adapt to circulating fHbp and PorA antigens.

Structures were determined of ChAs with PorA VR2 P1.16 inserted into position 151, 294, or 309 of a V1 fHbp (Fig. 1b). These structures were solved using molecular replacement to resolutions of 2.9 Å, 3.7 Å, and 2.6 Å, respectively. All ChA scaffolds align with good agreement to native fHbp (Fig. 1b), demonstrating that the structure of fHbp is not perturbed by insertion of VR2 loop P1.16. In each structure, the VR2 loop adopts a β-turn conformation that extends away from the fHbp scaffold, and mimics an identical free VR2 peptide in a complex with a bactericidal Fab fragment (Fig. 1c).

The adaptability of the ChA approach was tested by generating ChAs composed from different combinations of fHbp and PorA VR2. The comprehensive meningococcal genome data available for strains isolated in the UK enable design of ChAs that have exact sequence matches to the most common antigens in a given region³. Prevalent PorA VR2s in circulating serogroup B N. meningitidis isolates are P1.4, P1.9, P1.14, P1.16 and P1.10_1, and prevalent fHbp peptides are V1.4 and V3.45. Five different ChAs were constructed, in which a VR2 peptide was inserted into position 151 in V1.4 or V3.45. The ability of these ChAs to elicit immune responses was examined by immunising groups of CD1 mice with each ChA, followed by the use of a serum bactericidal assay (SBA), to assess the ability of the immune sera to initiate complement-mediated lysis of N. meningitidis. This assay demonstrated PorA specific SBA titres ranging between ≥20 and ≥1280 (Fig. 2a) (above the ≥8 threshold for an accepted correlate of protective immunity against N. meningitidis). Furthermore, fHbp specific SBA titres, obtained from ChAs composed of fHbp V1.1 and VR2 P1.16, range between ≥1024 and ≥4096 (Fig. 2b).

Previous studies had found that linear PorA VR2 peptides failed to elicit antibodies that recognised native PorA, while cyclic VR2 peptides, with identical residues fixed in a β-turn, elicited PorA specific bactericidal antibodies. In the ChAs generated in this study, the VR2 N- and C- termini are effectively locked into a “biological” β-turn peptide mimetic by neighbouring fHbp β-strands, as confirmed by our atomic structures.

This study provides proof of principle that ChAs can be used to display selected antigens from a soluble protein, which forms a scaffold, and an epitope from an integral membrane protein. The structural data conclusively demonstrates that fHbp is not perturbed by insertion of a PorA VR2, and that the VR2 loop P1.16 adopts a conformation known to induce bactericidal antibody responses. The immune response data demonstrates that correlates of protection are elicited by both antigens in the ChA, and that ChA composition can be adapted to provide protection against relevant circulating N. meningitidis strains.

References:

Pizza M. et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science (80-. ). 287, 1816–1820 (2000). DOI: 10.1126/science.287.5459.1816
van der Ley P. et al. Topology of outer membrane porins in pathogenic Neisseria spp. Infect. Immun. 59, 2963 LP-2971 (1991)
Meningitis Research Foundation Meningococcus Genome Library (https:// pubmlst.org/bigsdb?db=pubmlst_neisseria_mrfgenomes)

Funding acknowledgement:

Action Medical Research (Award number GN2205).

Corresponding author:

Prof Susan Lea, University of Oxford, [email protected]

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Using structure-based design to engineer chimeric protein vaccines