Background
Persistent chronic HBV infection is associated with impaired functional T-cell specific responses and a lack of memory T-cells against core (C), polymerase (P), and envelope proteins (S). Therapeutic vaccination is aimed to relieve exhausted T-cells and to stimulate recall memory T-cells. We have developed an antigen-presenting cell (APC)-targeting approach allowing the delivery of specific antigens through a mAb targeting the CD40 receptor.
Methods
HBV C, P, and S proteins (genotype Dayw) were screened in silico to identify immunogenic and conserved epitopes. Six selected peptides were fused to a humanized anti-CD40 IgG4 mAb. The immunogenicity of the resulting CD40.HBV vaccines was evaluated in hCD40 transgenic mice in a prime/boost regimen (10mcg/mouse with 50mcg of Poly-ICLC). IgG binding to S and C was measured via Luminex assay, while T-cell immunity was assessed using IFN-γ ELISpot. Additionally, PBMCs from NUC-treated chronically infected donors (CHB, n=19) were stimulated in vitro with 1nM to 10nM of the vaccine, and the expansion of functional CD4+ and CD8+ memory T-cells was analyzed by FACS.
Results
Two peptides from S and 2 from C were selected and successfully conjugated with the CD40 mAb (CD40.HBV(S/C)), or the non-targeting IgG4 control mAb (IgG4.HBV(S/C)) while 2 peptides from P were associated with a second vaccine (CD40.HBV(P)). The co-injection of CD40.HBV(S/C) and CD40.HBV(P) induced significant serum HBs and HBc IgG responses (P<0.0001 and P<0.01, respectively), and S/P-specific IFN-γ ELISpot responses (p<0.01, p<0.0001, respectively) compared to mock injection. We confirmed in vivothe advantage of the CD40 targeting in eliciting specific T-cell responses by comparing the CD40.HBV(S/C) with IgG4.HBV(S/C). Both vaccines activated human polyfunctional (IFN-g±IL-2±TNF) CD4+ T-cells specific to S, C, and P in vitro, with significantly enhanced responses compared to equivalent amounts of non-targeted antigens (3 ng/mL of peptide pools). CD8+ T-cell responses against HBc and HBV P were amplified in vitro in 40% of NUC-treated CHB patients.
Conclusions
Two CD40-targeting vaccine candidates, incorporating 6 highly immunogenic HBV peptides, were produced with high quality and demonstrated immunogenicity in vivo, and were able to recall memory T-cell responses from PBMCs of NUC-treated CHB patients. These vaccines are promising candidates for further testing in humanized mice and for assessing their therapeutic potential.
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