Protective Immunity Against Ocular Herpes Simplex Virus Infection by Immunization With Glycoprotein D Epitopes Extended by N–Epsilon–Palmitoyl–Lysine Moiety that Stimulate Maturation of Dendritic Cells

Abstract

Abstract: : We have recently identified four immunodominant CD4(+) T cell peptide epitopes from the HSV–1 gD that induce Th₁ immune responses and confer ocular protection when delivered in mice with the experimental adjuvant Montanide ISA–720. Purpose: Methods: In the present study, to render the immunization clinically feasible, these T cell peptide epitopes were covalently linked with a single palmitic acid moiety (lipopeptides), using the newly described chemoselective ligation method, and delivered without exogenous adjuvants. Results: The physicochemical properties of each lipopeptide were compatible with multi–dimensional analysis, using RP–HPLC, Edman sequencing, electrospray mass spectrometry, and 2D–NMR. The influence of the lipid content on the immuno–stimulatory capacity of lipopeptides was also investigated following covalent attachment to the gD peptide backbone of either an N–epsilon–palmitoyl–lysine (palmitoyl–LP) or cholesterol–lysine (cholesterol–LP). Immunization of mice with the palmitoyl–LP, but not with its cholesterol–LP analog, induced a strong T–cell dependent protective immunity against lethal ocular HSV–1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1–type immune response was stimulated by the palmitoyl–LP, as opposed to a Th2 response generated by its cholesterol–LP analog. The palmitoyl–LP was efficiently taken up in vitro by immature dendritic cells (DCs) in a time and dose–dependent manner. Moreover, palmitoyl–LP induced MHC and costimulatory molecule expression and production of pro–inflammatory IL–12 and TNF–a cytokines by immature DCs. Finally, DCs stimulated with palmitoyl–LP induced Ag–specific T–cell responses through the toll like receptor–2 (TLR–2) pathway. Conclusions: The purity of these totally synthetic lipopeptides, which enables manufacturing of defined immunogenic molecules under GMP conditions, together with the strength of their T cell immunogenicity and protective efficacy in mice provides a safe and powerful immunogenic formulation that can be tested in humans.

Keywords: herpes simplex virus • immunomodulation/immunoregulation • cytokines/chemokines