Petra Riedl

Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5–20 ng RNA/μg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-γ release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20–100 μg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 μg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 μg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Priming of immune responses to hepatitis B surface antigen with minimal DNA expression constructs modified with a nuclear localization signal peptide

Abstract Nuclear localization signal (NLS) peptides conjugated to DNA increase transfection efficiency in vitro. We tested in mice whether conjugation of NLS peptides to DNA vaccines enhances their immunogenicity after intramuscular injection or gene gun mediated intradermal delivery. We constructed the plasmid pMOK-HBsAY that contains a transcription unit encoding hepatitis B surface antigen (HBsAg) and bacterial sequences for amplification of plasmid DNA. From this plasmid we derived the minimal expression construct pMOK-HBsAY-MIDGE, a covalently closed linear DNA that contains only the HBsAg transcription unit. Both constructs stimulated similar (predominantly IgG1) antibody response to HBsAg after gene gun immunization. In contrast, pMOK-HBsAY plasmid DNA was more efficient than pMOK-HBsAY-MIDGE DNA in priming predominantly IgG2a antibody responses to HBsAg after intramuscular injection. Both constructs efficiently primed cytotoxic T lymphocyte responses after intramuscular immunization. When a NLS peptide was coupled to the pMOK-HBsAY-MIDGE DNA, HBsAg transfection efficiency in vitro and priming of antibody responses to HBsAg after intramuscular (but not gene gun mediated) injection was enhanced 10- to 15-fold. These data show: (a) MIDGE constructs can be used as DNA vaccines indicating that bacterial sequences are not essential cofactors; and (b) in intramuscular (but not gene gun mediated) delivery the immunogenicity of a MIDGE-based vaccine is enhanced by coupling NLS peptides to the vector DNA.

The immunodominant, Ld-restricted T cell response to hepatitis B surface antigen (HBsAg) efficiently suppresses T cell priming to multiple Dd-, Kd-, and Kb-restricted HBsAg epitopes.

MHC-I-restricted CTL responses of H-2d (Ld+ or Ld−) and F1 H-2dxb mice to hepatitis B surface Ag (HBsAg) are primed by either DNA vaccines or HBsAg particles. The Dd/S201–209 and Kd/S199–208 epitopes are generated by processing endogenous HBsAg; the Kb/S208–215 epitope is generated by processing exogenous HBsAg; and the Ld/S28–39 epitope is generated by exogenous as well as endogenous processing of HBsAg. DNA vaccination primed high numbers of CTL specific for the Ld/S28–39 HBsAg epitope, low numbers of CTL specific for the Dd/S201–209 or Kd/S199–208 HBsAg epitopes in BALB/c mice, and high numbers of Dd/S201–209- and Kd/S199–208-specific CTL in congenic H-2d/Ld− dm2 mice. In F1dxb mice, the Kd-, Dd-, and Kb-restricted CTL responses to HBsAg were strikingly suppressed in the presence but efficiently elicited in the absence of Ld/S28–39-specific CTL. Once primed, the Kd- and Dd-restricted CTL responses to HBsAg were resistant to suppression by immunodominant Ld/S28–39-specific CTL. The Ld-restricted immunodominant CTL reactivity to HBsAg can thus suppress priming to multiple alternative epitopes of HBsAg, independent of the processing pathway that generates the epitope, of the background of the mouse strain used, and of the presence/absence of different allelic variants of the K and D MHC class I molecules.

Antigenic Epitopes Fused to Cationic Peptide Bound to Oligonucleotides Facilitate Toll-Like Receptor 9-Dependent, but CD4+ T Cell Help-Independent, Priming of CD8+ T Cells

A priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4+ T cell help-independent CD8+ T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8+ T cell responses to proteins or peptides. We show that the adjuvant effect of ISS+ ODN on CD8+ T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8+ T cell priming. Fusing antigenic epitopes to cationic (8- to 10-mer) peptides bound to immune-stimulating ISS+ ODN or nonstimulating NSS+ ODN (without CpG-containing sequences) generated immunogens that efficiently primed long-lasting, specific CD8+ T cell immunity of high magnitude. Different MHC class I-binding epitopes fused to short cationic peptides of different origins showed this adjuvant activity. Quantitative ODN binding to cationic peptides strikingly reduced the toxicity of the latter, suggesting that it improves the safety profile of the adjuvant. CD8+ T cell priming supported by this adjuvant was Toll-like receptor 9 dependent, but required no CD4+ T cell help. ODN (with or without CpG-containing sequences) are thus potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of Ag delivery prevailing in many viral nucleocapsids.

Noncovalent Association with Stress Protein Facilitates Cross-Priming of CD8+ T Cells to Tumor Cell Antigens by Dendritic Cells

A viral oncogene carrying well-defined Kb/Db-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the Kb/Db-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-γ-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, Db/Kb-restricted IFN-γ-producing CD8+ T cells were primed during the growth of transfected H-2d Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F1(b × d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.

Elimination of Immunodominant Epitopes from Multispecific DNA-Based Vaccines Allows Induction of CD8 T Cells That Have a Striking Antiviral Potential

Immunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C), and polymerase (Pol) proteins and/or the OVA Ag as stress protein-capturing fusion proteins. Priming of mono- or multispecific, HLA-A*0201- or Kb-restricted CD8 T cell responses by these DNA vaccines differed. Kb/OVA257–264- and Kb/S190–197-specific CD8 T cell responses did not allow priming of a Kb/C93–100-specific CD8 T cell response in mice immunized with multidomain vaccines. Tolerance to the S- Ag in transgenic Alb/HBs mice (that express large amounts of transgene-encoded S- Ag in the liver) facilitated priming of subdominant, Kb/C93–100-specific CD8 T cell immunity by multidomain Ags. The “weak” (i.e., easily suppressed) Kb/C93–100-specific CD8 T cell response was efficiently elicited by a HBV core Ag-encoding vector in 1.4HBV-Smut tg mice (that harbor a replicating HBV genome that produces HBV surface, core, and precore Ag in the liver). Kb/C93–100-specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-Smut transgenic mice where they suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific antiviral effects to HBV-expressing hepatocytes.

Translation from Cryptic Reading Frames of DNA Vaccines Generates an Extended Repertoire of Immunogenic, MHC Class I-Restricted Epitopes

To test whether simple expression units used in DNA vaccines can generate immunogenic, MHC class I-binding epitopes by translating other than the primary open reading frame (ORF), we constructed a vector (pCI/SX) that encodes the small hepatitis B surface Ag in the primary ORF, and a C-terminal fragment (residue 344–832) of the polymerase (Pol) in an alternative (out-of-frame) reading frame. pCI/SX efficiently primed multispecific, HLA-A2-restricted CD8+ T cell responses to epitopes of hepatitis B surface Ag and of Pol (Pol3, Pol803–811). Pol3-containing products generated from pCI/SX were detected only by T cell assays, but not by biochemical assays. Priming Pol-specific T cell responses to epitopes generated from alternative ORFs depended on promoter sequences that drive transcription in the DNA vaccine (human CMV-derived promoter sequences being more efficient than SV40-derived promoter sequences). Human CMV promoter-driven Pol constructs encoding different Pol fragments in primary or alternative reading frames elicited comparable levels of Pol3-specific T cell responses. We confirmed efficient T cell priming to epitopes from alternative ORFs by constructing DNA vaccines that encode an SV40-derived cT1–272 protein fused either in frame or out of frame with an immunogenic OVA fragment (OVA18–385). Similar OVA-specific CD8+ T cell responses were primed by both alternative vaccine constructs. Hence, DNA vaccine-stimulated T cell responses to epitopes generated from alternative ORFs seem to be a regular event, although its biological role and risks are largely unexplored.

Binding immune‐stimulating oligonucleotides to cationic peptides from viral core antigen enhances their potency as adjuvants

Priming specific Th1 immunity by recombinant hepatitis B core antigen (HBcAg) depends on its arginine (Arg)‐rich, 34–36‐residue‐long C terminus, and nucleotides bound to it. This adjuvant activity intrinsic to HBcAg facilitates priming of Th1 immunity to co‐delivered, unrelated antigens, such as hepatitis B surface antigen (HBsAg), or ovalbumin (OVA) that prime specific Th2 immunity when injected without adjuvants. We loaded immune‐stimulating, CpG‐containing oligodeoxynucleotides (ODN) to the HBcAg‐derived Arg‐rich peptides C‐1 (HBcAg150–157, RRRDRGRS) or C‐2 (HBcAg164–179, SPRRRRSQSPRRRRSQ). When these peptide/nucleotide complexes were co‐injected into mice with HBsAg, hepatitis B precore antigen (HBeAg) or OVA, the animals developed strikingly higher serum IgG2 antibody titers and cytotoxic T lymphocyte responses than animals co‐injected with these antigens and ‘free’ (not peptide‐bound) ODN. Potent Th1‐promoting adjuvants can thus be synthesized that mimic priming of anti‐viral immunity by natural nucleocapsid particles.

Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain

The N‐terminal domain of large tumor antigens (T‐Ag) of polyomaviruses forms a DnaJ‐like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild‐type) SV40 T‐Ag show stable, ATP‐dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady‐state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73capturing, chimeric antigens containing an N‐terminal, T‐Ag‐derived domain, and different C‐terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N‐terminal 77 residues representing the J domain of T‐Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73‐bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a “natural adjuvant” for enhanced immunogenicity for T and B cells.

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8+ T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8+ T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.