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

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.

Endogenously Expressed Antigens Bind Mammalian RNA via Cationic Domains that Enhance Priming of Effector CD8 T Cells by DNA Vaccination

“…It has been shown that non-specific “exogenous” bacterial RNAs function as a Toll-like receptor 7 (TLR-7) ligand and stimulated Th1-biased immune responses in mice, when co-delivered with recombinant antigens or when directly bound by particulate or non-particulate antigens. 1 , 2 , 3 , 4 , 5 , 6 Antigen-bound bacterial RNA has an >1,000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a recombinant antigen. 7 Under certain conditions, mammalian self-RNAs also stimulated TLR-7- or TLR-3-mediated autoreactive B cell responses.…”

“…When selectively expressed in bacterial, yeast, or mammalian expression systems, HBV-C protein self-assembled into particles that non-specifically bound heterologous RNAs. 1 , 2 , 4 , 6 , 16 , 17 The 34-residue COOH-terminal cationic domain of HBV-C (C150–183) is crucial for the non-specific RNA-binding of HBV-C particles, whereas HBV-C149 particles (lacking the cationic domain) did not bind RNA. 1 , 2 , 4 , 6 , 16 , 17 Non-phosphorylated HBV-C particles encapsidate high amounts of bacterial RNA but low amounts of mammalian RNA.…”

“… 1 , 2 , 4 , 6 , 16 , 17 The 34-residue COOH-terminal cationic domain of HBV-C (C150–183) is crucial for the non-specific RNA-binding of HBV-C particles, whereas HBV-C149 particles (lacking the cationic domain) did not bind RNA. 1 , 2 , 4 , 6 , 16 , 17 Non-phosphorylated HBV-C particles encapsidate high amounts of bacterial RNA but low amounts of mammalian RNA. 6 , 17 Prevention of specific phosphorylation in the cationic C150–183 domain by exchanging serine residues S155, S162, and S170 with alanine 6 , 17 , 18 , 19 or by exchanging the cationic C150–183 domain with a heterologous 14-residue HIV-tat 48–57 -like cationic domain (HBV-C149tat), lacking any phosphorylation sites, significantly enhanced the RNA-binding of these mutant core particles.…”

“… 1 , 2 , 4 , 6 , 16 , 17 Non-phosphorylated HBV-C particles encapsidate high amounts of bacterial RNA but low amounts of mammalian RNA. 6 , 17 Prevention of specific phosphorylation in the cationic C150–183 domain by exchanging serine residues S155, S162, and S170 with alanine 6 , 17 , 18 , 19 or by exchanging the cationic C150–183 domain with a heterologous 14-residue HIV-tat 48–57 -like cationic domain (HBV-C149tat), lacking any phosphorylation sites, significantly enhanced the RNA-binding of these mutant core particles. 6 Similarly, mammalian RNA efficiently bound to freely exposed cationic domains in assembly-deficient core antigens, indicating that stable RNA-binding primarily depends on interactions between positively charged cationic domains and negatively charged nucleic acids.…”

“… 6 , 17 Prevention of specific phosphorylation in the cationic C150–183 domain by exchanging serine residues S155, S162, and S170 with alanine 6 , 17 , 18 , 19 or by exchanging the cationic C150–183 domain with a heterologous 14-residue HIV-tat 48–57 -like cationic domain (HBV-C149tat), lacking any phosphorylation sites, significantly enhanced the RNA-binding of these mutant core particles. 6 Similarly, mammalian RNA efficiently bound to freely exposed cationic domains in assembly-deficient core antigens, indicating that stable RNA-binding primarily depends on interactions between positively charged cationic domains and negatively charged nucleic acids. 6 , 7 Both bacterial and mammalian RNAs bound to recombinant core particles (exogenous protein vaccines) or cellular RNAs bound to endogenously expressed core particles (endogenous DNA vaccines) function as TLR-7, but not TLR-3, ligands and induced a Th1-biased humoral immunity in C57Bl6/J (B6) and TLR-3 −/− , but not in TLR-7 −/− , mice.…”