Flagellin/Virus-like Particle Hybrid Platform with High Immunogenicity, Safety, and Versatility for Vaccine Development

Abstract

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.

CpG 1018 Is an Effective Adjuvant for Influenza Nucleoprotein

“…Mice were transferred to the animal biosafety level 2 (ABSL2) facility of URI for challenge. Mice were intranasally instilled with 8× LD50 of mouse-adapted influenza pandemic 2009 H1N1 (pdm09) viruses under light anesthesia as in our previous report [ 24 ]. Body weight and survival were monitored daily for 14 days.…”

Respiratory Viruses and Virus-like Particle Vaccine Development: How Far Have We Advanced?

“…Mice immunized with these modified insect-derived VLPs were better protected than those immunized with the flagellin-HBc M2e VLPs with unmodified D3 region. Replacing the D3 domain with the M2e antigens further enhanced the survival of immunized mice following challenge infection with the A/Puerto Rico/8/34 (H1N1), A/California/09/2009 (H1N1pdm09), and A/Philippines/2/82 (H3N2) viruses [ 48 ].…”

Effective adjuvantation of nanograms of influenza vaccine and induction of cross-protective immunity by physical radiofrequency adjuvant

“…Fifty percent lethal dose (LD50) in mice was determined following a well-established method 24 . Mouse-adapted pdm09 viruses and influenza A/ Puerto Rico/8/34 H1N1 (PR8) viruses were prepared as in our prior reports 20,25,26 . Mice were intranasally challenged with different doses of H3N2, pdm09, or PR8 viruses under anesthesia.…”