Lipid-Based Nanoparticles Loaded with Hepatitis C Virus Glycoproteins for Induction of Potent Immune Responses

Immune Responses J. Bazzill, Y. Fan, E. Giang, S. Castillo, M. Law, J. J. Moon University of Michigan, Scripps Research Institute Purpose Subunit vaccines are highly attractive for vaccination against virulent pathogens due to their ease of manufacturing and proven safety. However, conventional subunit antigens are susceptible to degradation in vivo, and they lack the ability to activate B-cells in a multivalent or controlled manner. Our goal in this study is to design nanoparticles that can protect subunit antigens from degradation, display antigens in a multivalent fashion, and simultaneously deliver adjuvant molecules to elicit potent immune responses. Here, we produced interbilayer-crosslinked multilamellar vesicles (ICMVs) designed for multivalent display of hepatitis C virus (HCV) envelope glycoprotein E2 in two variants, E2 truncated to amino acid 661 (E2.661) and E2 truncated to amino acid 717 with a C terminal hexa-histidine tag (E2-his). ICMVs carrying E2 variants were evaluated for their antigen loading, display, and immunogenicity in mice. Methods ICMVs carrying E2.661 and E2-his were synthesized as described previously [1,2]. To utilize the selective interaction between hexahistidine tags and (Ni) nitrilotriacetic acid (NTA), phospholipids functionalized with NTA were added during ICMV synthesis (NTA ICMVs) when formulated with E2-his. Antigen loading of ICMVs was assessed by SDS gel electrophoresis, while particle size, polydispersity, and zeta potential were analyzed by dynamic light scattering. Direct immunofluorescence staining of ICMVs was used to determine preservation of antigen configuration and display on ICMVs. Female C57BL/6 mice (n = 5) were subcutaneously injected at the tail base five times every three weeks with a prime dose of 10 μg antigen plus 1.0 μg monophosphryl lipid A (MPLA, Toll-like receptor-4 agonist) and booster doses of 5 μg antigen and 0.5 μg MPLA. Control groups were E2.661 formulated with alum, MPLA, or MPLA plus alum. Sera were collected on days -3, 18, 39, 60, 81, 102, and 144 to determine antigen-specific IgG titers. Sera from the final two time-points were assessed for neutralizing activity against HCV pseudovirus particles. Results ICMVs and NTA ICMVs were loaded with E2.661 or E2-his, respectively, with an average efficiency of 46% and 75%, respectively. Antigen-loaded ICMVs showed a diameter of 210 nm on average with a polydispersity index of 0.27 and zeta potentials of -24 mV. Direct binding of broadly neutralizing antibodies (bNAbs) AR3A and HCV1 were markedly increased, compared to isotype control, with 5 and 10-fold higher binding for E2.661 ICMVs, and 25 and 31-fold higher binding for E2-his NTA ICMVs, respectively. Recognition by the conformational epitope recognizing AR3A bNAb indicated preservation of the tertiary glycoprotein structure of E2 variants on the surfaces of ICMVs. After prime immunization in C57BL/6 mice, E2-specific serum antibody titers were >10-fold higher for both ICMV groups, compared with the control groups containing antigen admixed with either MPLA, alum, or MPLA and alum, and strong antibody titers were maintained up to 60 days after the final vaccination. Only the immune sera from ICMVs groups neutralized autologous pseudovirus of 1a H77 strain in vitro. Modest neutralization was observed in 3 out of 5 mice immunized with E2.661 ICMVs, while high neutralization (86%) was observed in 1 out of 5 mice immunized with E2-his NTA ICMVs. At 60 days after the final immunization, 2 of the 3 sera from the E2.661 ICMV and the E2-his NTA ICMV serum retained their neutralizing capacity. Conclusion Eliciting broadly neutralizing antibodies against HCV is a daunting task due to its high genetic diversity. ICMVs loaded with recombinant E2 glycoprotein are dense immunogenic particles with the potential to display antigens in a configurational manner that maintains native structure of the glycoprotein. In vivo immunizations with E2.661and E2-his-loaded ICMVs induced substantial increases in antigen-specific serum antibody titers with modest capability for neutralization in vitro. It remains to be seen whether ICMV vaccine can be further optimized and immune responses amplified in order to achieve potent protection against heterologous virus isolates.