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*(L)-ARGININE
The Journal of Immunology, 2002, 168: 4951-4959.
Copyright © 2002 by The American Association of Immunologists

Priming Th1 Immunity to Viral Core Particles Is Facilitated by Trace Amounts of RNA Bound to Its Arginine-Rich Domain1

Petra Riedl*, Detlef Stober*, Claude Oehninger*, Karl Melber{dagger}, Jörg Reimann* and Reinhold Schirmbeck2,*

* Institute of Medical Microbiology and Immunology, University of Ulm, Ulm, Germany; and {dagger} Rhein Biotech, Düsseldorf, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{gamma} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The p21 (21 kDa) core proteins (C proteins)3 of hepatitis B virus (HBV) self-assemble into immunogenic, icosahedral 30 nm particles known as hepatitis B core Ag (C protein) (HBcAg) (1). Core particles are permeable for small molecules, e.g., nucleotides. The C protein contains two domains, i.e., the N-terminal 149 aa domain is required for its oligomerization into capsids, and its C-terminal 34–36 aa arginine (Arg)-rich region (with similarity to protamines) nonspecifically binds nucleic acids (2). Core particles contain viral pregenomic RNA (reviewed in Ref. 3). The nucleic acid found in rHBcAg particles produced in bacteria is RNA between 30 and 3000 nucleotides in length (4).

Translation initiation at different AUG codons within the core gene region of HBV leads to expression of either the HBcAg, or the hepatitis B precore Ag (E protein) (HBeAg). The in vivo function of HBeAg in the HBV life cycle is unknown. HBeAg plays no role in HBV replication and assembly. High HBeAg serum levels correlate with high rates of virus replication, high levels of viremia, and high infectivity. The core AUG directs expression of the 183–185 residue particle-forming 21-kDa core protein (HBcAg) (Fig. 1GoA). HBcAg derived from the HBV subtype adw contains two additional residues at position 151 and 152. The 5' precore AUG directs production of the 25-kDa precore protein that is longer than the HBcAg protein by 29 N-terminal residues. A 19 aa signal sequence that targets the precore protein precursor to the secretory pathway is removed in the endoplasmic reticulum which generates a p22 intermediate from which the C-terminal 34 residues (aa 150–183) are cleaved. This gives rise to the secreted p17 protein known as HBeAg. Particle formation of HBeAg is prevented by a disulfide bridge between cysteines at position 7 (in the precore region) and 61 (in the core region) (5). The HBcAg sequences 1–149 or 1–144 can be efficiently expressed in bacteria. The resulting HBcAg-149 or -144 Ags form particles (6). Native HBcAg and mutant HBcAg-149 particles, as well as nonparticulate soluble HBeAg thus share a 149 aa sequence, but differ structurally and with respect to the C-terminal nucleic acid-binding domain (present in HBcAg but neither in mutant HBcAg-149 or -144, nor in HBeAg) (Fig. 1GoA). Most Ab responses against HBcAg and HBeAg are cross-reactive (1, 7). H-2 class II- (8, 9) and class I- (10, 11) restricted T cell responses, cross-reactive to HBcAg and HBeAg, have been identified in mice. HBcAg elicits Th1 immune responses while HBeAg elicits Th2 immune responses (8, 9, 12, 13, 14). It is unknown why these largely identical viral proteins, presented to the immune system either as a secreted 17-kDa protein, or as a 30-nm protein particle, stimulate strikingly different types of immune responses.



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  		FIGURE 1. A, Recombinant core and precore Ags of HBV. The HBV core gene sequences encoding aa 1–183 (HBcAg), aa -10–149 (HBeAg), aa 1–149 (HBcAg-149), or aa 1–144 (HBcAg-144) were cloned into bacterial expression vectors, and the respective Ags were produced. Purified Ags were boiled in buffer containing SDS and 2-ME and analyzed by SDS-PAGE. Coomassie blue staining revealed the expected protein bands. Purified bacterial HBcAg formed 28-nm particles, as revealed by electron microscopy. B, rHBcAg particles contain bacterial RNA. a, Purified HBcAg, HBcAg-144, HBcAg-149, or HBeAg were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The same gel was stained with Coomassie blue to demonstrate comigration of nucleic acids and HBcAg protein. b, HBcAg was treated with RNase, DNase, or Proteinase K before agarose gel analyses. c, The RNA content of HBcAg, HBeAg, HBcAg-144, and HBcAg-149 preparations was determined as described in Materials and Methods. The data shown are nanograms of RNA per 1 µg protein.

 
Oligodeoxynucleotides (ODN) with immunostimulating sequences are potent activators of the innate and specific immune system (reviewed in Refs. 15 , 16 , and 17). Synthetic ODN with CpG-containing immunostimulating sequences mixed with protein Ags prime Th1 immunity including CTL responses (18, 19, 20, 21, 22, 23, 24, 25, 26). The dose range required for the optimal adjuvant effect of ODN in mice is 10–50 µg ODN/vaccine. As rHBcAg particles bind RNA to the C-terminal Arg-rich domain, we studied whether RNA associated with HBcAg particles was critical for priming Th1 immunity. The data described show that nanogram amounts of RNA, bound to native HBcAg particles through its C-terminal Arg-rich domain, facilitate priming of Th1 immunity. This reveals that prokaryotic and eukaryotic RNA encapsulated into viral nucleocapsids are potent adjuvants.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/cJ (H-2d) mice were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). Breeding pairs of these mice were obtained from Bomholtgard Breeding and Research Center (Ry, Denmark). Female mice were used at 10–16 wk of age.

rAg preparations

rHBcAg particles were produced in Escherichia coli (NF-1) using the plasmid pLEH2 kindly provided by Dr. M. Nassal (University of Freiburg, Freiburg, Germany). This construct expresses HBcAg under control of the bacteriophage {lambda} pL promoter. Upon expression in bacteria, HBcAg proteins self-assemble into core particles. Expression and purification of bacterial HBcAg particles has been described (4). Bacterial HBeAg was obtained from American Research Products (catalog no. 12-3068; Belmont, MA). rHBcAg-149 particles were produced in E. coli (GI-698) using the plasmid pPLC/c1–149 kindly provided by Dr. M. Nassal (Freiburg, Germany). Expression and purification of bacterial HBcAg-149 particles have been described (4, 27). rHBcAg-144 particles were obtained from American Research Products (catalog no. 12-3169-1). Where indicated, HBcAg particles were disrupted by incubation for 30 min at 56°C in a buffer containing 0.5% SDS and 2% 2-ME. Disrupted HBcAg was injected immediately after treatment. Nucleic acids bound to the recombinant proteins were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Where indicated, HBcAg particles were incubated either with 50 µg proteinase K (catalog no. 19133; Qiagen, Hilden, Germany) for 90 min at 37°C, with 2 U DNase I (catalog no. 776785; Roche, Mannheim, Germany) in 10 mM MgCl2 for 60 min at 37°C, or with 2.5 µg RNase (catalog no. 1119915; Roche) for 60 min at room temperature. The RNA content of the Ag preparations was determined by spectrophotometer measurement after proteinase K digestion of the Ag and removal of the proteins using an RNeasy Mini kit (catalog no. 74104; Qiagen). In addition, the OD of the bound RNA was measured after agarose gel electrophoresis. These ODs were then compared with defined nucleic acid standards using ImageMaster VDS software (Amersham Biosciences, Piscataway, NJ).

Determination of LPS

LPS contamination of the Ags produced in E. coli was determined using the E-Toxate test (catalog no. 210-C1; Sigma-Aldrich, Taufkirchen, Germany) following the instructions of the manufacturer (technical bulletin no. 210). Different HBcAg preparations produced in the E. coli strains NF-1, JM101, or W3110 contained 1–4 ng LPS/µg protein. LPS levels were determined using different E. coli-derived LPS preparations as standards including the LPS (catalog no. L2143; Sigma-Aldrich) that we used as adjuvant in the immunization experiments. HBcAg-144 and -149 particle preparations produced in a bacterial expression system contained 0.01- 0.03 ng LPS/µg protein.

HBV core-encoding expression vectors used for nucleic acid immunization

The construction of the plasmids pCI/C (encoding the HBcAg 1–183 aa) and pCI/E (encoding the HBeAg/HBcAg -29–183 aa) has been described (10, 11, 28). The HBcAg-149 core Ag was cloned into pCI by amplifying the desired fragment and introducing a forward KpnI site and a reverse stop codon following residue 149 (followed by a SalI site). Expression of the different HBV core Ags from plasmid DNA was tested in transient transfection assays. Chicken hepatoma cells (LMH) were transfected with plasmid DNA using the CaPO4-method. Two days later, cells were labeled with [35S]methionine (catalog no. SJ1015; Amersham Biosciences) for 16 h and extracted with lysis buffer. Cell supernatants and lysates were immunoprecipitated for HBcAgs with a polyclonal rabbit anti-HBV core serum (a generous gift of Dr. H.-J. Schlicht; University of Ulm, Ulm, Germany) and protein A Sepharose. Samples were processed for SDS-PAGE followed by fluorography as described (10).

HBcAg-expressing transfectants

The generation of stable P815 transfectants expressing HBcAg (P815/C) or HBeAg (P815/E) has been described (10, 11). RNA bound to eukaryotic HBcAg and HBeAg was determined. Briefly, P815/C, P815/E, or control P815/BMG cells (transfected with the expression vector without insert) were labeled for 16 h with 200 µCi [3H]uracil (catalog no. TRK 408; Amersham Biosciences) and extracted with lysis buffer (100 mM NaCl, 1% aprotinin, 1 µg/ml leupeptin, 0.2% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, 100 mM Tris-HCl (pH 8.0)). HBcAg was immunoprecipitated from cell supernatants and lysates with a polyclonal rabbit anti-HBcAg serum (kindly provided by Dr. H.-J. Schlicht) and protein A-Sepharose. Immunoprecipitates were washed repeatedly and 3H counts were determined.

Immunization of mice

Mice were immunized i.m. (into both tibialis anterior muscles) or s.c. into the base of the tail with the indicated doses of recombinant proteins. rHBeAg protein or HBcAg-149 particles were mixed with either poly I/C (catalog no. P-1530; Sigma-Aldrich), or murine rIL-12 (a generous gift of Dr. Gubler; Roche, Nutley, NJ), bacterial RNA isolated from E. coli (NF-1) using the RNeasy Maxi kit (catalog no. 75162; Qiagen), or 50–1000 ng LPS (catalog no. L2143; Sigma-Aldrich). Ag-encoding plasmid DNA was injected intradermally (0.1–1 µg) with the Helios Gene Gun system (catalog no. 165-2431, 2432; Bio-Rad, Munich, Germany) as described (29).

Determination of serum Ab levels

Serum samples were repeatedly obtained from individual immunized mice by tail bleedings. Ag-specific IgG, IgG1, and IgG2a serum Abs were determined by end-point dilution ELISA as described (30, 31). Briefly, microELISA plates (Nunc-Maxisorp, Wiesbaden, Germany) were coated with 150 ng rAgs/well in 50 µl 0.1 M sodium carbonate buffer (pH 9.5) at 4°C. Serial dilutions of the sera in loading buffer (PBS supplemented with 3% BSA and 2% Tween 20) were added to the Ag-coated wells. Serum Abs were incubated for 2 h at 37°C followed by four washes with PBS supplemented with 0.05% Tween 20. Bound serum Abs were detected using HRP-conjugated anti-mouse IgG Abs (catalog no. 02067E; BD PharMingen, Hamburg, Germany) at a dilution of 1/2000 followed by incubation with o-phenylenediamine x 2 HCl (catalog no. 6172-24; Abbott Laboratories, Abbott Park, IL) in PBS (pH 6.0). The reaction was stopped by 1 M H2SO4 and the extinction was determined at 492 nm. End-point titers were defined as the highest serum dilution that resulted in an absorbance value three times greater than that of negative control sera (derived form nonimmunized mice).

Generation of dendritic cells (DC) from bone marrow (BM)

The in vitro generation of DC from murine BM has been described (32). Briefly, BM cells prepared from femurs were depleted of CD4+CD8+B220+ lymphocytes and MHC-class-II+ cells (Abs, catalog no. 492-01, 494-01, 495-01, 524-01; Miltenyi Biotec, Bergisch-Gladbach, Germany) by MACS (following the manufacturer’s instructions) and cultured at a density of 106 cells/ml in UltraCulture medium (catalog no. 12-725F; BioWhittaker, Walkersville, MD) supplemented with 5 ng/ml GM-CSF (catalog no. 315-03; PeproTech, Rocky Hill, NJ), 2 mM glutamine, and antibiotics. On days 3 and 5, cells were fed by medium exchange. On days 7–8 of culture, nonadherent cells were harvested and CD11c+ cells were purified by MACS (catalog no. 130-052-001; Miltenyi Biotec). CD11c+ DC were pulsed with Ag for 2 h, washed, and cultured for 24 h after which IL-12 p70 release was determined. In addition, unfractionated spleen cells from normal B6 mice were pulsed with Ag for 2 h, washed, and cultured for 24 h after which IFN-{gamma} release was determined.

Splenic CD4+ T cells were purified from nonimmune mice, from HBcAg, HBcAg-149, or HBeAg immune mice, and restimulated in vitro with HBcAg-149-pulsed DC. Their specific IFN-{gamma} release in 48 h cocultures was determined.

Cytokine detection by ELISA

IFN-{gamma} and IL-12 p70 were measured in cell culture supernatants by conventional double-sandwich ELISA using the mAb R4–6A2 (catalog no. 18181D; BD PharMingen) and biotinylated mAb XMG1.2 (catalog no. 18112D; BD PharMingen) for IFN-{gamma} detection, and using the mAb RedT/G297-289 (catalog no. 20011D; BD PharMingen) and mAb C17.8 (catalog no. 18482D) for IL-12 p70 detection. Extinction was analyzed at 405/490 nm on a TECAN microplate-ELISA reader (Tecan, Crailsheim, Germany) using EasyWin software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of HBeAg, HBcAg, and HBcAg-149 in bacteria

Native or truncated HBcAg particles, or nonparticulate HBeAg protein (Fig. 1GoA), were expressed in bacteria. When lysates of transformed bacteria were analyzed by SDS-PAGE, the expected 21-kDa HBcAg protein, the truncated 17 kDa HBcAg-149, and the 17-kDa HBeAg protein were revealed (Fig. 1GoA). Electron microscopy of the purified rHBcAg, HBcAg-144, and HBcAg-149 revealed their well-characterized particle structure (Fig. 1GoA; data not shown). Native HBcAg particles, but not HBeAg protein, contain nucleic acids (Fig. 1GoBa). Nucleic acids and HBcAg protein comigrated in the native agarose gels suggesting that nucleic acids tightly associate with the particulate structure (Fig. 1GoBa) (2, 6). Truncated HBcAg-144 or -149 core proteins form particles, but lack the Arg-rich C terminus and exhibit drastically reduced RNA binding activity (Fig. 1GoBa). The >98% reduction in RNA content of mutant vs wild-type HBcAg confirms published data (2, 4). HBcAg-bound nucleic acids were completely eliminated by RNase treatment but remained readily detectable after DNase treatment (Fig. 1GoBb). HBcAg particles completely digested by proteinase K released RNA that varied in length from 30 to 3000 nucleotides (Fig. 1GoBb). Proteinase K-released nucleic acids were eliminated by RNase treatment but remained readily detectable after DNase treatment (data not shown). Hence, RNA, but not DNA, is bound by native HBcAg particles. Five to 20 nanograms bacterial RNA are bound per microgram HBcAg protein (Fig. 1GoBc). We found no evidence that core-specific RNA is associated with rHBcAg particles. Hybridization analyses with core specific probes did not reveal detectable levels of core-specific RNA in the RNA recovered from HBcAg (data not shown). Bacterial RNA of various lengths is thus associated with particulate HBcAg but no, or only trace, amounts of RNA are detectable in particulate HBcAg-144 or -149, and in nonparticulate HBeAg protein (that share a common 144 or 149 residue core sequence).

Murine immune responses to rHBcAgs

BALB/c mice (H-2d) were immunized i.m. by a single injection of 3 µg rHBcAg, mutant HBcAg-144, mutant HBcAg-149, or HBeAg (in PBS without adjuvants). Serum IgG Ab titers were determined 4–16 wk postvaccination. Core and precore Ags contain cross-reactive (e1 and e2) Ab-binding epitopes (1). This was confirmed when we used rHBcAg particles, HBcAg-149 particles, or nonparticulate HBeAg as detection Ags in the ELISA. The injection of HBcAg particles induced high titers of serum IgG that bound HBcAg, HBcAg-149, and HBeAg (Fig. 2GoA). Similarly, Ab responses primed by injection of HBcAg-149 and -144 particles are detected with all three Ags tested (Fig. 2GoA) indicating that mutant HBcAg-144 or -149 particles contain intact Ab-binding epitopes (1). Nonarticulate HBeAg induced 10–20 fold lower Ab titers, irrespective of the detection Ag used (Fig. 2GoA).



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  		FIGURE 2. Immunogenicity of rHBcAg and HBeAg. BALB/c mice were noninjected (0) or injected i.m. with two different batches of HBcAg (1, 2), HBeAg (3), HBcAg-149 (4), or HBcAg-144 (5) without adjuvants (3 µg/mouse). A, Serum IgG Abs obtained 8 wk postimmunization were tested in ELISA for specific binding to HBcAg, HBcAg-149, and HBeAg. Mean specific Ab titers in sera of three to six mice per group are shown. B, HBcAg-149-specific IgG1 and IgG2a Abs were determined and the IgG1-IgG2a ratios were calculated. C, Immune spleen cells were obtained from primed mice 10 days after a boost injection. Controls (0) were from nonimmunized BALB/c mice. Splenic CD4+ T cells were purified and cocultured with syngeneic DC pulsed with rHBcAg-149. After 48 h, their IFN-{gamma} release was determined.

 
The isotype profile of the serum Ab response to HBcAg particles indicated that predominantly IgG2a Abs were induced, although low levels of specific IgG1 Abs were also found. The IgG1/IgG2a ratios of Ab responses to HBcAg were <0.3, indicating preferential priming of anti-HBcAg Th1 immunity (Fig. 2GoB). Different HBcAg preparations produced in different E. coli strains induced similar Th1-biased immunity (Fig. 2GoB; data not shown). In contrast, specific IgG1 Abs predominate in the Ab response to mutant HBcAg-149 or -144 particles, or HBeAg which is evident by IgG1-IgG2a ratios of >20 (Fig. 2GoB). This confirms and extends previous reports (14). The HBcAg-specific Th1 (IgG2a)-biased immune response and the Th2 (IgG1)-biased response induced by mutant HBcAg-149 or -144 particles and HBeAg were observed in immunization experiments using a dose range of 0.1–10 µg of the respective Ags (data not shown). The isotype profiles of Abs elicited by the different Ags were identical when HBcAg, HBcAg-149, or HBeAg was used as detection Ag (Fig. 2GoB; data not shown).

The polarization of the HBcAg- and HBcAg-149-specific immune response was confirmed by the specific cytokine release of T cells primed by either HBcAg, or HBcAg-149. Splenic CD4+ T cells were purified from nonimmune mice, or from HBcAg and HBcAg-149 immune mice, and restimulated in vitro with HBcAg-149 pulsed dendritic cells (DC). Their specific IFN-{gamma} release in 48 h cocultures was determined by ELISA. IFN-{gamma} release was detected only in T cell populations immune to HBcAg, not in T cell populations immune to HBeAg or HBcAg-149, and not in nonimmune control populations (Fig. 2GoC). The specific cytokine release data thus confirm that HBcAg preferentially induces Th1 responses.

Native HBcAg particles (but neither mutant HBcAg-149 or -144 particles, nor HBeAg protein) stimulate IL-12 p70 and IFN-{gamma} release by nonimmune cells

Although bacterial DNA or synthetic CpG-containing ODN are potent adjuvants that facilitate priming of Th1 immunity (19) and trigger DC maturation (19, 20, 33, 34, 35, 36, 37, 38, 39, 40), little is known about the adjuvants effect of RNA. Therefore, we tested whether RNA-containing HBcAg particles induce release of IL-12 p70 or IFN-{gamma} by cells from nonimmune mice. When spleen cells from nonimmune mice were pulsed in vitro with 2 or 20 µg/ml native HBcAg particles, they released high amounts of IFN-{gamma} (Fig. 3GoA). IFN-{gamma} release was not detected when spleen cells were pulsed with 20 µg/ml mutant HBcAg-149 or -144 particles, or HBeAg protein (Fig. 3GoA). Furthermore, we measured IL-12 p70 release of purified DC pulsed with 20 µg/ml of either native HBcAg particles, mutant HBcAg-149 particles, or native HBeAg protein. Native HBcAg particles, but not mutant HBcAg-149 particles or HBeAg protein, stimulated release of bioactive IL-12 p70 from DC (Fig. 3GoB). Hence, priming of Th1 immunity by native HBcAg particles may be initiated by IL-12-dependent activation of innate immunity and may depend on particulate HBcAg-containing RNA.



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  		FIGURE 3. Innate immune response to native or mutant HBcAg or HBeAg. A, Unfractionated spleen cells from normal BALB/c mice were pulsed with 2 or 20 µg/ml HBcAg, or 20 µg/ml HBcAg-149, HBcAg-144, or HBeAg for 2 h, washed, and cultured for 24 h. IFN-{gamma} release was determined by ELISA. B, Myeloid DC generated in vitro from BALB/c BM precursors were pulsed with 20 µg/ml HBcAg, HBcAg-149, or HBeAg, washed, and cultured for 24 h. IL-12 p70 release was determined by ELISA.

 
Bacterial RNA facilitates priming of Th1 immunity to HBeAg

Mice injected i.m. with either 1 µg native HBcAg particles (containing 10 ng of bound RNA) or 5 µg HBeAg protein (in PBS without adjuvants) generated specific serum Ab responses with isotype profiles indicating preferential priming of either Th1 or Th2 immunity (Fig. 4Go, groups 1 and 2). We tested whether bacterial RNA mixed to HBeAg can enhance and/or modulate the immune response it elicits. Compared with the injection of HBeAg without adjuvants, the codelivery of 50 µg purified bacterial RNA with HBeAg elicited strikingly enhanced serum IgG titers with a Th1-biased isotype profile (Fig. 4Go, groups 2 and 3). These data were confirmed by analyses of the cytokine release profile of splenic CD4+ T cells primed by either native HBeAg or HBeAg mixed with bacterial RNA (data not shown). This adjuvant effect was as potent as codelivering 200 µg double-stranded poly I/C with HBeAg (Fig. 4Go, group 4). Codelivery of low amounts (<20 µg) of bacterial RNA or poly I/C with HBeAg had no detectable adjuvants effect (Fig. 4Go, group 5; data not shown). Hence, when RNA is mixed with HBeAg, ~1000-fold higher doses had to be codelivered to prime a Th1-biased immunity comparable to that induced by native HBcAg particles associated with RNA (Fig. 4Go, groups 1, 3, and 4). Codelivery of 100 ng murine rIL-12 p70 with 5 µg HBeAg enhanced the specific serum Ab response and shifted its polarization toward a Th1-biased pattern (Fig. 4Go, group 7). This suggests that IL-12 is a mediator in the RNA-mediated adjuvant effect as IL-12 p70 release by DC is stimulated by RNA-containing HBcAg particles (Fig. 3GoB).



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  		FIGURE 4. Codelivery of RNA can change the polarization profile of the murine immune response to HBeAg. BALB/c mice were immunized i.m. with (1) 1 µg HBcAg particles (containing 10 ng RNA), (2) 5 µg HBeAg, (3) 5 µg HBeAg mixed with 50 µg bacterial RNA, (4) 5 µg HBeAg mixed with 200 µg poly I/C, (5) 5 µg HBeAg mixed with 20 µg poly I/C, (6) 5 µg HBeAg mixed with 50 ng LPS, or (7) 5 µg HBeAg mixed with 100 ng IL-12. Mean titers of anti-HBeAg serum IgG, IgG1, or IgG2a Abs were determined 10 wk postimmunization by ELISA using HBeAg as detection Ag. Mean serum Ab titers of four mice per group, and their mean IgG1-IgG2a ratios are shown.

 
Codelivery of different batches and different doses of E. coli-derived LPS (50–1000 ng/injection) with HBeAg or HBcAg-149 did not enhance or modulate priming of Ag-specific Th1 immunity (Fig. 4Go, group 6; data not shown) while HBcAg preparations (expressed in E. coli and containing 1–4 ng LPS/µg protein) efficiently induced Th1 immune responses. HBcAg particles boiled for 30 min (a treatment that destroys RNA and the conformation of the HBcAg particles but leaves LPS intact) induce Th2 immunity (data not shown). These findings indicate that bacterial RNA, but not LPS, mediate the Th1-inducing adjuvant effect.

Release of RNA from HBcAg particles eliminates its ability to prime Th1 immunity

The described data indicate that low amounts of RNA delivered in a particle-associated form are potent adjuvants that facilitate priming of Th1 immunity. Treatment of HBcAg particles with SDS and 2-ME (or SDS alone) disrupts the HBcAg particles into its subunits and releases all RNA (Fig. 5GoA). This treatment does not destroy HBcAg immunogenicity as many of the Ab specificities are directed against linear epitopes (1). Injection of native or denatured HBcAg (in PBS without adjuvants) into mice induced comparable specific serum IgG titers when read against either native HBcAg (Fig. 5GoB, upper panel) or denatured linearized HBcAg (Fig. 5GoB, lower panel). The polarization of the immune responses induced by injecting these different formulations of the same Ags differed strikingly: while specific IgG2a Abs predominated in mice vaccinated with HBcAg particles (IgG1-IgG2a ratio, <0.3), injection of denatured HBcAg (with released "free" RNA) preferentially primed specific IgG1 Ab responses (IgG1-IgG2a ratio, >10) (Fig. 5GoB). Similar isotype profiles were revealed in Ab populations binding either native particles or denatured HBcAg protein (Fig. 5GoB). This supports the interpretation that low amounts of RNA incorporated into native HBcAg particles are required for priming Th1 immunity.



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  		FIGURE 5. Immunogenicity of denatured (linearized) HBcAg devoid of RNA. A, HBcAg particles (6 µg) were either not treated (lane 1), treated for 30 min at 56°C with SDS (lane 2), or treated for 30 min at 56°C with SDS plus 2-ME (lane 3). The nucleotide content was analyzed using agarose gel electrophoresis followed by ethidium bromide staining. B, BALB/c mice were immunized i.m. with 2 µg (1) native, (2) SDS-treated, or (3) SDS/2-ME-treated (linearized) HBcAg. Mean titers of specific IgG, IgG1, or IgG2a serum Abs were determined 8 wk postimmunization using as detection Ags either native HBcAg particles (upper panel), or SDS/2-ME-treated denatured HBcAg (lower panel). Mean serum Ab titers and their mean IgG1-IgG2a ratios of three mice per group are shown.

 
Priming HBcAg- and HBeAg-specific immune responses by DNA-based vaccination

DNA vaccines were constructed that expressed either the native HBcAg (pCI/C), the truncated HBcAg-149 (pCI/C-149), or the native HBeAg (pCI/E) (Fig. 6GoA). Transient transfection assays showed intracellular expression of the p21 HBcAg protein, the p25 and p22 precore intermediates, and the p17 HBcAg-149 protein by these vectors in which expression is driven by CMV promoter/enhancer sequences (Fig. 6GoB). The secreted p17 HBeAg was detected in the cell culture supernatant (Fig. 6GoB).



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  		FIGURE 6. DNA vaccines encoding native or truncated HBcAg, or HBeAg. A, A 5' precore AUG directs production of precore (preC) protein precursor consisting of 29 aa precore and 183 aa core Ag sequence. A 19 aa signal sequence is removed from the precore sequence and truncation of the C-terminal 34 aa of the core sequence generates the secreted 17-kDa HBeAg protein. The second AUG directs synthesis of the 1–183 aa sequence encoding the 21-kDa HBcAg protein. Sequences encoding the 29 aa precore and the 183 aa of the core Ag (pCI/E), the 183 aa core Ag (pCI/C), or the N-terminal 149 aa of the core Ag (pCI/C-149) were cloned into the pCI expression vector in which expression of the Ags is driven by human CMV promoter/enhancer sequences. B, Expression of Ags from these vectors. LMH cells were transiently transfected with pCI/C, pCI/E, or pCI/C-149 vector DNA using the CaPO4 method, cultured for 36 h, labeled with [35S]methionine, washed, lysed, and HBcAg/HBeAg-reactive material was immunoprecipitated (lane a). In addition, HBcAg/HBeAg-reactive material was immunoprecipitated from the supernatant (lane b). Precipitates were analyzed by SDS-PAGE followed by fluorography. The p21 HBcAg, the intracellular p25 and p22 HBeAg-intermediates and the secreted p17 HBeAg, and the 17-kDa HBcAg-149 are indicated. C, RNA binds to HBcAg expressed in mouse cells. HBcAg- (P815/C) or HBeAg- (P815/E) expressing transfectants, or control P815 cells were labeled with [3H]uracil, extracted, and immunoprecipitated for HBcAgs using polyclonal rabbit anti HBcAg serum. The amount of 3H radiolabel incorporated into RNA coprecipitated with protein was determined.

 
Binding of RNA to HBcAg particles in eukaryotic cells was demonstrated by labeling stable murine transfectants expressing HBcAg (P815/C) or HBeAg (P815/E) with [3H]uracil. Cells were lysed, the core Ag was immunoprecipitated using a polyclonal rabbit antiserum, and the immunoprecipitates were washed and analyzed for [3H] content. Radiolabeled RNA was associated with HBcAg particles but not with intracellular or secreted HBeAg (Fig. 6GoC). The nonparticulate p22 and p25 intermediates of HBeAg (that contain the Arg-rich C terminus) were not associated with RNA. This finding indicates that the particulate structure of HBcAg is an essential prerequisite for RNA binding. Similar results were obtained using different HBcAg-expressing cell lines (data not shown). Similar to the prokaryotic expression data in bacteria (Fig. 1GoB), the eukaryotic expression of HBcAg (but not HBeAg) leads to encapsulation of eukaryotic RNA into HBV core particles.

Mice were immunized with 1 µg pCI/C, pCI/E, and pCI/C-149 expression plasmid DNA using the gene gun (Fig. 7Go). Serum IgG Ab titers were measured using HBcAg-149 particles as detection Ag. DNA immunization with pCI/C-primed IgG1 and IgG2a Abs, with an IgG1-IgG2a ratio of 0.5:1 (Fig. 7Go, group 1). In contrast, the serum Ab response of mice vaccinated with the pCI/E or pCI/C-149 showed preferential development of IgG1 titers, with IgG1-IgG2a ratios of >20 (Fig. 7Go, groups 2 and 3). Higher IgG1 titers were apparent in mice immunized with pCI/E plasmid DNA (IgG1-IgG2a ratio, 50:100) than in those immunized with pCI/C-149 plasmid DNA (IgG1-IgG2a ratio, 20:30). The IFN-{gamma} release of spleen cells from primed mice confirmed this polarization of the elicited anti-viral immune response (data not shown). The comparison of Figs. 2Go and 7Go show a similar polarization of HBcAg/HBeAg-specific Ab responses elicited by either gene gun-mediated DNA vaccination, or injection of recombinant proteins (without adjuvant). HBcAg (but not HBcAg-149 or HBeAg) expressed by mouse cells transiently transfected in vivo after DNA-based immunization and HBcAg particles (but not HBcAg-149 or HBeAg protein) produced in bacteria contain (prokaryotic or eukaryotic) RNA and efficiently prime Th1 immunity.



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  		FIGURE 7. The polarization of the immune response to native and mutant HBcAg, and HBeAg delivered by intradermal DNA vaccination. BALB/c mice were intradermally immunized with 1 µg particle-coated pCI/C (1), pCI/E (2), or pCI/C-149 (3) plasmid DNA using the gene gun. The specific serum Ab response, its isotype profile, and its IgG1-IgG2a ratio were determined 8 wk postimmunization. Mean Ab titers in sera of five mice per group are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The precore/core region of the HBV genome encodes two structurally different proteins with an identical reading frame. The p21 core Ag (HBcAg) self-assembles into nucleocapsid HBcAg particles. An alternative 5'-translation initiation leads to the synthesis of the secreted p17 precore protein HBeAg. Despite their extensive structural differences, HBcAg and HBeAg proteins share a common region of 149 aa (Fig. 1GoA). This region contains most T and B cell epitopes. This viral Ag system is thus an attractive model to study the humoral and cellular immune responses against similar epitopes primed by two different, natural variants of an Ag. As measured by the isotype profile of serum Abs and the cytokine release profile of primed spleen cells, the immune responses of H-2d (BALB/c) and H-2b (C57BL/6) mice to HBcAg particles were Th1-biased while the responses to HBeAg were Th2-biased. The described experiments were designed to identify the structural feature(s) that determine the different immunogenicity of these viral proteins.

HBcAg binds nucleic acids by its C-terminal Arg-rich region that is present in HBcAg but not HBeAg. Bacterial or insect DNA as well as synthetic ODN with immune-stimulating CpG sequences are potent activators of the innate and specific immune system that facilitate priming of Th1-biased responses (reviewed in Ref. 15 , 16 , and 17). Therefore, we tested whether the association of HBcAg with RNA is critical for its immunogenicity. Truncating the Arg-rich C terminus of HBcAg generated mutant HBcAg-149 or HBcAg-144 particles in which nucleic acid binding activity was drastically reduced (>98% compared with wild-type HBcAg) but not completely eliminated (Fig. 1Go) (2, 6). Residual nucleic acid binding by HBcAg-149 particles may be mediated by conformational determinants in the 149 aa region that are present in particulate (HBcAg-like), but not soluble (HBeAg-like), proteins. Alternatively, RNA may be nonspecifically captured during particle assembly. The difference in the RNA content of native HBcAg particles and truncated HBcAg-149 or HBcAg-144 particles correlated well with the difference in the type of immunogenicity that these variants of particulate viral Ags displayed. Mice responded predominantly with specific IgG2a Ab production to native HBcAg vaccination (IgG1-IgG2a ratios, <0.3), and with specific IgG1 Ab production to mutant HBcAg-149 or -144 particle vaccination (IgG1-IgG2a ratios, 10:20). Cytokine release profiles by in vivo primed and in vitro restimulated spleen cells confirmed these polarization patterns. Disruption of the HBcAg particles destroyed the intrinsic Th1-biased immunogenicity of this rAg (Fig. 5Go). A similar treatment of the HBcAg particles has been reported to reveal enhanced serological anti-HBeAg cross reactivity (41). Disruption of HBcAg particles released and/or inactivated the incorporated RNA, leading to a similar immunogenicity of linearized HBcAg and soluble HBeAg. These patterns were reproducibly detectable when we injected the rAg in the dose range of 0.1–10 µg/mouse without adjuvants by the i.m. or s.c. route.

At least part of the adjuvant activity of RNA encapsulated in viral nucleocapsids seems derived from its ability to stimulate the innate immune system. Native (but not truncated) HBcAg particles stimulated IL-12 release by DC and IFN-{gamma} release by nonimmune spleen cells. Activation of DC by CpG-containing ODN has been shown to depend on stress kinase activity and is preceded by nonspecific endocytosis and endosomal maturation (20, 21, 34, 35, 42, 43). We reproduced the same effects with low amounts of nucleocapsid-bound RNA, demonstrating the immunostimulatory potency of nucleotides encapsulated in viral nucleocapsids. There is no evidence that the type or sequence of nucleic acid incorporated into HBcAg particles is critical. We used HBcAg particles purified from a bacterial expression source in the described experiments. These particles contain a heterogeneous population of bacterial RNA in the 30- to 3000-bp size range (Fig. 1GoB). It is difficult to characterize the RNA subsets (single- vs double-stranded) in this heterologous RNA. In contrast to previous reports (4), we found no HBcAg-specific message in this RNA pool. Labeling studies with [3H]uracil in mammalian cells indicated that HBcAg particles, but not HBeAg, incorporated eukaryotic RNA. In the absence of genomic RNA and reverse transcripts from HBV, host cell RNA seems to nonspecifically bind to the C-terminal domain of HBcAg. We tested the immunogenicity of HBcAg particles expressed in mouse cells in DNA-based immunization experiments using the gene gun. Intradermal injection of 0.1–1 µg DNA coated to gold particles usually primes Th2-biased responses (reviewed in Ref. 44). We have confirmed this in studies using expression vector DNA encoding different intracellular and/or secreted Ags (e.g., HBsAg, LHBsAg, HBeAg, or OVA) and intradermal DNA delivery with the gene gun in BALB/c and C57BL/6 mice (45, 46, 47). The only exception we have observed up to now is the priming of specific Th1 responses by intradermal delivery with the gene gun of particle-bound plasmid DNA encoding HBcAg (Fig. 7Go). These findings suggest that HBcAg particles capture nonspecifically low amounts of mouse cell RNA during biogenesis. This excludes bacterial contaminants or some specific features of bacterial RNA as the factors that mediate the immune-enhancing and -modulating effect of native HBcAg particles. Trace amounts of eukaryotic RNA associated with nucleocapsids thus have potent enhancing and modulating effects on the immune response. These data support our interpretation that this viral capsid is an exceptionally potent vehicle to deliver RNA adjuvants.

It seems that the particle-associated delivery is a key factor for the potency of nucleocapsid-associated RNA as an intrinsic adjuvant. The RNA within the particle is protected from degradation by nucleases during its extracellular phase and during uptake by cells. The HBcAg particles are taken up by endocytosis or macropinocytosis. For disruption of the particle and proteolytic degradation of the core protein, the particles have to reach an acid late endosomal or early lysosomal compartment. Uptake of each particle delivers 100–200 copies of the Ag to a single vesicle within the Ag-processing and -presenting cell. Together with the protein Ag, RNA oligonucleotides are delivered to the same acid vesicles. ODN with CpG motifs require uptake into an acid compartment to deliver their immunostimulatory effect (48). Triggering the immunostimulating effect of ODN involves the Toll-like receptor 9 (49) and signal transduction by MyD88, MAP kinases, and NF-{kappa}B (50, 51, 52). The amount of RNA within a single core particle released into a single vesicle may efficiently trigger the signal-transducing cascade. Hence, the protection from degradation and the targeted delivery of a critical amount of RNA to vesicles may contribute to the potency of this intrinsic adjuvant. These data may help to rationally design nontoxic adjuvants for anti-viral vaccines.


   Acknowledgments
 
We thank Tanja Guentert for excellent technical assistance. The generous gifts of HBeAg and HBcAg preparations from Dr. H.-J. Schlicht (Ulm, Germany) and Drs. V. Bruus and Heermann (Göttingen, Germany) are gratefully acknowledged. Drs. H.-J. Schlicht and M. Nassal kindly provided plasmids and bacteria for HBcAg and mutant HBcAg-149 expression. Dr. U. Gubler (Roche, Nutley, NJ) provided rIL-12.


   Footnotes
 
1 This work was supported by grants from the German Federal Ministry for Science and Technology (01GE 9907) and the Deutsche Forschungsgemeinschaft (DFG Schi 505/2-1) to R.S. and J.R. Back

2 Address correspondence and reprint requests to Dr. Reinhold Schirmbeck, Institute for Medical Microbiology and Immunology, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Germany. E-mail address: reinhold.schirmbeck{at}medizin.uni-ulm.de Back

3 Abbreviations used in this paper: C protein, core protein; HBV, hepatitis B virus; HBcAg, hepatitis B core Ag (C protein); HBeAg, hepatitis B precore Ag (E protein); ODN, oligodeoxynucleotide; DC, dendritic cell; BM, bone marrow. Back

Received for publication November 7, 2001. Accepted for publication March 12, 2002.


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