Non-cognate ligands of hepatitis C virus envelope broadly neutralizing antibodies induce virus-neutralizing sera in mice

Abstract

Introduction: The persistent rise in new Hepatitis C virus (HCV) infections threatens WHO efforts to eliminate HCV infection by 2030. Although direct-acting antiviral (DAA) drugs are efficacious, access remains limited, reinfections occur, and perinatal infections continue to pose long-term complications. Therefore, an effective anti-HCV vaccine is urgently needed.

Methods: We employed a highly complex combinatorial Myomedin-loop scaffold library to identify variants binding to paratopes of HCV E2-specific broadly neutralizing antibodies (bNAbs) HC-1AM and HC84.26.WH.5DL. The selected binders, named SHB and WIN, respectively, represent non-cognate mimotopes of the aforementioned bNAbs. These binders were subsequently used as immunogens in experimental mice to elicit serum antibodies capable of binding to HCV E2 and neutralize HCV pseudotyped viruses.

Results and discussion: The non-cognate mimotopes SHB and WIN competed with the E2 glycoprotein for bNAbs binding and, after immunizing experimental mice, elicited E2- and HCV-pseudovirus-specific antibodies. WIN- and SHB-immunized mice exhibited neutralization against 15 HCV pseudoviruses with varying neutralization sensitivities. The most potent binders WIN028 and WIN047, were modified with a C-terminal His-tag, allowing the generation of WIN proteoliposome and subsequent use in experimental mice immunizations. Hyperimmune sera exhibited improved binding to HCV E2 and neutralized 60% of the tested HCV pseudoviruses. The broad neutralization of HCV pseudoviruses achieved by hypperimmune sera from SHB- and WIN-immunized mice highlights the potential of this approach in the HCV vaccine design.

Keywords: broadly neutralizing antibodies; hepatitis C; mimotope; myomedins; protein mimicry; protein scaffolds; vaccine.

Figure 1

Scheme of Myomedin loop variant library assembly using multistep PCR showing the forward and reverse primers.

Figure 2

ELISA screening of bacterial lysates for binding to (a) HC-1AM (SHB variants) and (b) HC84 (WIN variants) antibodies (green color). As a negative control, isotype R04 antibody (blue color) was used. Specific binding was detected using anti-V5-HRP conjugated antibody. Variants selected for further analysis are indicated in red color.

Figure 3

Binding and competition ELISA of SHB variants to the HC-1AM antibody: (a) Purified SHB variants were incubated with either the HC-1AM bNAb or the isotype control (human IgG1 λ or anti-HCMV R04 antibody) coated onto an ELISA plate. Specific binding was detected using an anti-V5 HRP-conjugated antibody. The colored lines show the specific binding of SHB variants to HC-1AM and the gray colored lines show the binding of SHB variants to the isotype control. (b) In the competition ELISA, serial dilutions of E2 protein were incubated with a constant concentration of SHB variants. The binding of the variants to the specific target antibody was detected using an anti-V5 tag antibody.

Figure 4

Binding and competition ELISA of WIN variants to the HC84 antibody: (a) Purified WIN variants were incubated with either the HC84 bNAb or the isotype control (human IgG1 λ or anti-HCMV R04 antibody) coated onto an ELISA plate. Specific binding was detected using an anti-V5 HRP-conjugated antibody. The colored lines show the specific binding of WIN variants to HC84 and the gray colored lines show the binding of WIN variants to the isotype control. (b) In the competition ELISA, serial dilutions of E2 protein were incubated with a constant concentration of WIN variants. The binding of the variants to the specific target antibody was detected using an anti-V5 tag antibody.

Figure 5

Cross-binding of selected SHB variants with (a) CBH-2, (b) HC33, and (c) HC84 HCV antibodies. Purified SHB variants were serially diluted and added into wells coated with HC-1AM antibody and antibodies selected for cross-binding. Specific binding was detected using an anti-V5 HRP-conjugated antibody. The color lines show specific binding of SHB variants to HC-1AM and gray color lines show the binding of SHB variants to antibodies considered for cross-binding.

Figure 6

Cross-binding of selected WIN variants with (a) CBH-2, (b) HC33, and (c) HC-1AM HCV antibodies. Purified WIN variants were serially diluted and added into wells coated with HC84 antibody and antibodies selected for cross-binding. Specific binding was detected using an anti-V5 HRP-conjugated antibody. The color lines show the specific binding of WIN variants to HC84 and gray color lines show the binding of WIN variants to antibodies considered for cross-binding.

Figure 7

Analysis of binding antibodies from mouse hyper-immune sera. Experimental mice were immunized intradermally with (a) SHB, WIN proteins and (b) with proteoliposomal formulation of WIN028 and WIN047 with His-tag on N- (WIN028-L, WIN047-L) or C-terminus (WIN028C-L, WIN047C-L). Sera from (a) soluble SHB-immunized, (b) soluble WIN-immunized, and (c) WIN-proteoliposome immunized mice were collected two weeks after last immunization and the serum IgG reactivities with (a, b) HCV E2 protein and (c) UK Nottingham Panel (23) HCV pseudotyped viruses 1.21.2 (Genotype 1), 2.3.1 (Genotype 2), and 4.2.1 (Genotype 4) immobilized on 96-well plates were determined by ELISA. Statistical significance was calculated using the ANOVA Kruskal-Wallis model with Dunn’s post-test (* p < 0.05, ** p < 0.01). (d) Linear regression analysis of WIN-proteoliposomes immunized murine serum IgGs binding and the neutralization breadth toward 1.21.2., 2.3.1, and 4.2.1. pseudoviruses.

Figure 8

Neutralization capacity of hyper-immune sera of mice immunized with SHB and WIN. Sera from mice immunized with (a) soluble WIN and SHB proteins and (b) select WIN proteoliposomes were tested against a panel of 15 HCV-pseudotyped HIV viruses. 50% virus neutralization was reached by reciprocal serum dilution as shown by the color-coded values. The relative mean neutralization dilution reflects the average titers of the pseudoviruses used. The sum of the neutralized pseudoviruses for each Myomedin-induced sera variant is represented as the number of neutralized PVs and is calculated at reciprocal titers higher than 30. (c) Shows the comparison between the neutralization of bNAbs HC-1AM and HC84. The pseudoviruses were selected to include representatives from the most to the least neutralization sensitive HCV strains from the Nottingham Panel (23).