Utility of Hybrid Transferrin Binding Protein Antigens for Protection Against Pathogenic Neisseria Species

Abstract

The surface transferrin receptor proteins from Neisseria gonorrhoeae have been recognized as ideal vaccine targets due to their critical role in survival in the human male genitourinary tract. Recombinant forms of the surface lipoprotein component of the receptor, transferrin binding protein B (TbpB), can be readily produced at high levels in the Escherichia coli cytoplasm and is suitable for commercial vaccine production. In contrast, the integral outer membrane protein, transferrin binding protein A (TbpA), is produced at relatively low levels in the outer membrane and requires detergents for solubilization and stabilization, processes not favorable for commercial applications. Capitalizing on the core β-barrel structural feature common to the lipoprotein and integral outer membrane protein we engineered the lipoprotein as a scaffold for displaying conserved surface epitopes from TbpA. A stable version of the C-terminal domain of TbpB was prepared by replacing four larger exposed variable loops with short linking peptide regions. Four surface regions from the plug and barrel domains of Neisseria TbpA were transplanted onto this TbpB C-lobe scaffold, generating stable hybrid antigens. Antisera generated in mice and rabbits against the hybrid antigens recognized TbpA at the surface of Neisseria meningitidis and inhibited transferrin-dependent growth at levels comparable or better than antisera directed against the native TbpA protein. Two of the engineered hybrid antigens each elicited a TbpA-specific bactericidal antibody response comparable to that induced by TbpA. A hybrid antigen generated using a foreign scaffold (TbpB from the pig pathogen Haemophilus parasuis) displaying neisserial TbpA loop 10 was evaluated in a model of lower genital tract colonization by N. gonorrhoeae and a model of invasive infection by N. meningitidis. The loop 10 hybrid antigen was as effective as full length TbpA in eliminating N. gonorrhoeae from the lower genital tract of female mice and was protective against the low dose invasive infection by N. meningitidis. These results demonstrate that TbpB or its derivatives can serve as an effective scaffold for displaying surface epitopes of integral outer membrane antigens and these antigens can elicit protection against bacterial challenge.

Keywords: epitope; hybrid antigen; lipoprotein; outer membrane protein; protein engineering; scaffold.

Figure 1

(A) Cartoon diagram of the solved crystal structures of N. meningitidis TbpB C Lobe (PDB 3VE2) from strain M982. Loop 20 (26 amino acids; 413–439), loop 21 (30 amino acids; 444–474), loop 23 (21 amino acids; 499–520) and loop 31(21 amino acids; 657–676) are illustrated as dotted lines. (B) Surface regions of TbpA targeted for display on the hybrid antigens. Cartoon diagram of N. meningitidis M982 TbpA modeled with Phyre2 (27) using PDB 3V89 as a template, with the loop structures of interest shown in green (loop 3 helix), red (plug domain), pink (loop 11) and blue (loop 10). Structures on the apical side are surface facing while beta strands are within the outer membrane. (C) SDS-PAGE of the proteins produced from the genes encoding the wildtype C lobe, the LCL, and the four TbpA-LCL hybrid antigens that were cloned and purified. (D) Cartoon diagram of a Phyre2 model of a Neisseria TbpB (green) hosting TbpA loop 10 (red) as an example of a novel hybrid antigen displaying an exogenous loop.

Figure 2

Cartoon schematic of the insertion sites for exogenous loops on the C lobe of TbpB. Four insertion sites (loops 20, 21, 23, and 31) are shown in red, while the remaining loops and beta-sheets are annotated for both the barrel and handle domains of the C-lobe.

Figure 3

Phylogenetic analysis of Neisseria species TbpA (Left) and TbpB (Right) protein sequences. N. meningitidis sequences are shown in red, N. gonorrhoeae sequences are shown in blue, and any commensal Nesseria sequences or sequences lacking species annotation are shown in black, with all of the variants described in the (Figure 4) alignment denoted on the trees. The trees are shown at the same scale (0.4) in order to better compare the extent of diversity between the proteins. N. meningitidis isotype I TbpBs (including known isotype I containing strains B16B6 and 90/18311) are shown at the bottom with the corresponding TbpAs also shown at the bottom of the trees.

Figure 4

Sequence alignments of the loops of TbpA used in the hybrid antigens across 11 strains including six N. meningitidis isotype 2 strains (M982, H44/76, 020, 010, BZ163 and MC58), two N. meningitidis isotype 1 strains (B16B6 and 90/18311) and three N. gonorrhoeae strains (MS11, FA19 and FA1090). Clustal Omega amino acid alignments displayed in JalView of the four regions of TbpA of interest include (A) Loop 10; (B) Loop 11; (C) Helix 3 and (D) the Plug Domain.

Figure 5

In vitro characterization of the LCL scaffold and the LCL-TbpA hybrid antigens. (A) Mouse sera were evaluated in a whole cell ELISA against both wild-type N. meningitidis M982 (Black) and M982 with TbpB knocked out (M982ΔtbpB; Gray). Sera were assayed in triplicate from individual mice (4 to 5 mice per group) and averaged and displayed as mean +/- SEM. Significance was determined as a significant increase in titer compared with mice that received adjuvant alone by two-way ANOVA with Sidak's multiple comparison test for both wildtype and knockout data sets. **p ≤ 0.01, ****p ≤ 0.0001. (B,C) Iron starved N. meningitidis M982ΔtbpB was grown with antisera from rabbits immunized with the antigens of interest. Iron-loaded hTf was added and bacteria were grown for 2.5 h and enumerated. Bacteria supplemented with LCL antiserum grew at the same rate as those supplemented with pre-challenge rabbit sera (B), as compared by an unpaired t-test. Growth was significantly decreased in bacteria supplemented with whole TbpA antisera, as well as antisera from rabbits immunized with LCL + Loop 10, Loop 11 and the plug domain (C). Graph represents data generated from three independent experiments plated in duplicate. Significance was determined as a significant decrease in final CFU/ml compared with bacteria grown with LCL alone by ordinary one-way ANOVA with post-hoc analysis by Holm-Sidak's multiple comparison test. *p ≤ 0.05, **p ≤ 0.01. (D) Serum bactericidal activity of mouse sera. Sera from each mouse was assayed against N. meningitidis strain M982 lacking TbpB. Data shown are averages of 4 to 5 mice per group ± SEM. A bactericidal titer of ¼ is the limit of detection of bacterial killing. Some values fall below the cut off due to some animals within the group showing killing at the 1:4 dilution while others produced no bacterial killing.

Figure 6

Whole-cell serum IgG ELISA titres of mice immunized with alum, TbpA or Hp7-L10 when captured by (A) heat-killed N. gonorrhoeae strain MS11 or (B) heat-killed N. meningitidis strain M982. Female mice were used in the lower genital tract gonococcal colonization, while male mice were challenged systemically with N. meningitidis. Significance was determined as a significant increase in titer compared with mice that received alum alone by ordinary one-way ANOVA with post-hoc analysis by Dunnett's multiple comparisons test. ****p < 0.0001.

Figure 7

Lower genital tract gonococcal colonization in immunized female mice. Mice were immunized with one of TbpA (17 mice, with 2 excluded due to non-infection; blue), H. parasuis TbpB hosting the meningococcal TbpA loop 10 (17 mice, with 5 excluded due to non-infection; purple) or alum alone (14 mice, with 5 excluded due to non-infection; black) and challenged with 10⁷ CFU of N. gonorrhoeae intra-vaginally. Mice were immunized and challenged in two cohorts and are shown as combined data. Duration of colonization is shown as percent (A) as well as by median with interquartile range (B). The recovered CFU from vaginal washes from each mouse is shown (C) broken down by each immunization group as well as averages are shown. Duration of gonococcal colonization was compared by a log rank [Mantel-Cox] test, however no significance was observed.

Figure 8

Survival of immunized mice in a model of invasive meningococcal infection of mice immunized with either the low dose [(A); 2.5 × 10⁷ CFU per mouse] or the high dose [(B); 7.5 × 10⁷ CFU/mouse]. Mice were immunized with one of TbpA (blue), H. parasuis TbpB hosting the meningococcal TbpA loop 10 (Hp7-L10; purple) or alum alone (black) and challenged with a lethal dose of N. meningitidis strain M982 systemically. Mouse survival is shown at the top, with clinical scores and bacterial burden in the blood shown for individual mice for each immunization group (descending from the top): alum, TbpA, and H. parasuis TbpB hosting loop 10.