Cationic domains in particle-forming and assembly-deficient HBV core antigens capture mammalian RNA that stimulates Th1-biased antibody responses by DNA vaccination

Jana Krieger,Reinhold Schirmbeck,Katja Stifter,Petra Riedl

doi:10.1038/s41598-018-32971-5

Abstract

The HBV core protein self-assembles into particles and encapsidates immune-stimulatory bacterial RNA through a cationic COOH-terminal (C150–183) domain. To investigate if different cationic domains have an impact on the endogenous RNA-binding of HBV-C antigens in mammalian cells, we developed a strep-tag (st) based expression/purification system for HBV-C/RNA antigens in vector-transfected HEK-293 cells. We showed that HBV-stC but not HBV-stC149 particles (lacking the cationic domain) capture low amounts of mammalian RNA. Prevention of specific phosphorylation in cationic domains, either by exchanging the serine residues S155, S162 and S170 with alanines (HBV-stCAAA) or by exchanging the entire cationic domain with a HIV-tat48–57-like sequence (HBV-stC149tat) enhanced the encapsidation of RNA into mutant core particles. Particle-bound mammalian RNA functioned as TLR-7 ligand and induced a Th1-biased humoral immunity in B6 but not in TLR-7−/− mice by exogenous (protein) and endogenous (DNA) vaccines. Compared to core particles, binding of mammalian RNA to freely exposed cationic domains in assembly-deficient antigens was enhanced. However, RNA bound to non-particulate antigens unleash its Th1-stimulating adjuvant activity by DNA- but not protein-based vaccination. Mammalian RNAs targeted by an endogenously expressed antigen thus function as a natural adjuvant in the host that facilitates priming of Th1-biased immune responses by DNA-based immunization.

Highlights

The innate immune system has evolved endo/lysosomal and cytoplasmic pattern recognition receptors (PRRs) for the detection of pathogen-associated molecular patterns (PAMPs) like foreign nucleic acids or conserved molecules and structures of an invading pathogen[5]
We investigated the non-specific binding of mammalian RNA to different cationic domains at the COOH terminus of HBV core (HBV-C)
To elucidate the adjuvant activity of mammalian RNA captured by HBV-C antigens, we vaccinated B6 and Toll-like receptors (TLRs)-deficient mice with recombinant antigens or antigen-expressing vector DNA and determined priming of Th1/Th2-biased core-specific antibody responses

Summary

Introduction

The innate immune system has evolved endo/lysosomal and cytoplasmic pattern recognition receptors (PRRs) for the detection of pathogen-associated molecular patterns (PAMPs) like foreign nucleic acids or conserved molecules and structures of an invading pathogen[5]. Heterologous bacterial RNA bound to recombinant HBV-C particles stimulates the innate immune system via the TLR-7 and induces a vigorous Th1-biased serum antibody response in mice[16,17]. There is evidence that RNA bound to the cationic C150–183 domain of endogenously expressed HBV-C particles in mammalian cells exert a specific adjuvant activity: HBV-C, but not HBV-C149 and HBV-E antigens (lacking the cationic domain) bound [3H]-uracil-labelled cellular RNA in vector-transfected cells and stimulated a Th1-biased core-specific humoral immunity in mice by DNA vaccination with the gene gun[16]. To elucidate the adjuvant activity of mammalian RNA captured by HBV-C antigens, we vaccinated B6 and TLR-deficient mice with recombinant antigens (exogenous antigens) or antigen-expressing vector DNA (endogenous antigens) and determined priming of Th1/Th2-biased core-specific antibody responses

Methods

Results

Conclusion