Interaction of the Hepatitis B Core and the Innate Immune System Byung O. Lee, Amy Tucker, Lars Frelin, Matti Sallberg, Joyce Jones, Cory Peters, Janice Hughes, David Whitacre, This information is current as Bryan Darsow, Darrell L. Peterson and David R. Milich of September 24, 2021. J Immunol 2009; 182:6670-6681; ; doi: 10.4049/jimmunol.0803683 http://www.jimmunol.org/content/182/11/6670 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Interaction of the Hepatitis B Core Antigen and the Innate Immune System1

Byung O. Lee,* Amy Tucker,* Lars Frelin,* Matti Sallberg,† Joyce Jones,* Cory Peters,* Janice Hughes,* David Whitacre,*‡ Bryan Darsow,*‡ Darrell L. Peterson,§ and David R. Milich2*

Previous studies demonstrated that the primary APCs for the hepatitis B core Ag (HBcAg) were B cells and not dendritic cells DC). We now report that splenic B1a and B1b cells more efficiently present soluble HBcAg to naive CD4؉ T cells than splenic) B2 cells. This was demonstrated by direct HBcAg-biotin-binding studies and by HBcAg-specific activation in vitro in cultures of naive HBcAg-specific T cells and resting subpopulations. The inability of DC to function as APCs for exogenous HBcAg relates to lack of uptake of HBcAg, not to processing or presentation, because HBcAg/anti-HBc immune complexes can be efficiently presented by DC. Furthermore, HBcAg-specific CD4؉ and CD8؉ T cell priming with DNA encoding HBcAg does not Downloaded from

require B cell APCs. TLR activation, another innate immune response, was also examined. Full-length (HBcAg183), truncated ␬ (HBcAg149), and the nonparticulate HBeAg were screened for TLR stimulation via NF- B activation in HEK293 cells expressing human TLRs. None of the HBc/HBeAgs activated human TLRs. Therefore, the HBc/HBeAg proteins are not ligands for human

TLRs. However, the ssRNA contained within HBcAg183 does function as a TLR-7 ligand, as demonstrated at the T and B cell levels

in TLR-7 knockout mice. Bacterial, yeast, and mammalian ssRNA encapsidated within HBcAg183 all function as TLR-7 ligands. http://www.jimmunol.org/ These studies indicate that innate immune mechanisms bridge to and enhance the adaptive immune response to HBcAg and have important implications for the use of hepadnavirus core proteins as vaccine carrier platforms. The Journal of Immunology, 2009, 182: 6670–6681.

he hepatitis B virus (HBV)3 core gene encodes two tially primes Th1 cells, whereas HBeAg preferentially primes Th2 polypeptides. Initiation of translation at the first start cells (3); HBcAg-specific Th cells mediate anti-envelope as well as T codon (AUG) results in a 25-kDa precore protein that is anti-HBc Ab production (4); and HBcAg is an effective carrier secreted as HBeAg after removal of 19 residues of the leader se- platform for heterologous epitopes (5). These characteristics are

quence and 34 C-terminal amino acids. Initiation of translation at unique to and require the particulate structure of the HBcAg. A by guest on September 24, 2021 the second AUG leads to the synthesis of a 183-aa, 21-kDa protein number of hypotheses have been suggested to explain the en- that assembles to form 27-nm particles that comprise the virion hanced immunogenicity of HBcAg relative to HBeAg and to other nucleocapsid (hepatitis B core (HBc) Ag). Although HBeAg and particulate Ag. We have previously suggested that the orientation HBcAg are serologically distinct, these Ag are cross-reactive at the of the array of protein spikes distributed over the surface of the level of Th cell recognition because they are colinear throughout HBcAg shell may be optimal for cross-linking BCR and up-reg- most of their primary sequence. The HBcAg possesses unique im- ulation of costimulatory molecules (6), especially because the munologic features. For example, HBcAg can function as both a T dominant B cell epitopes are positioned at the tip of the spikes (7, cell-independent and a T cell-dependent Ag (1); HBcAg is ϳ1000- 8). Furthermore, we have previously reported that B cells rather fold more immunogenic than the HBeAg (2); HBcAg preferen- than non-B cell professional APCs such as dendritic cells or mac- rophages (DC/M␾) act as the primary APCs for HBcAg, which may explain its enhanced immunogenicity especially in terms of *Vaccine Research Institute of San Diego, San Diego, CA 92109; †Karolinska Insti- Ab production (6). Other investigators have suggested that the tutet, Clinical Microbiology, Karolinska University Hospital Huddinge, Stockholm, Sweden; ‡VLP Biotech, San Diego, CA 92109; and §Virginia Commonwealth Uni- binding of the protamine-like C terminus of HBcAg to cell mem- versity, Department of Biochemistry, Richmond, VA 23298 brane heparan sulfates (HS) or HS and chondroitin sulfates may Received for publication November 3, 2008. Accepted for publication March play a role in the enhanced immunogenicity of HBcAg (9, 10). An 14, 2009. interaction between HBcAg and TLR-2 has been proposed (9). It The costs of publication of this article were defrayed in part by the payment of page has also been suggested that contaminating TLR-4 and TLR-2 li- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. gands present in Escherichia coli-derived HBcAg may explain its 1 This work was supported by National Institutes of Health Grants R01 AI049730 and enhanced immunogenicity (11, 12). Lastly, the presence of encap- R01 AI20720 and grants from the Swedish Foundation. L.F. was supported by sidated ssRNA bound to the protamine-like domain within full- grants from Erik and Edith Fernstro¨ms Foundation and Gålo¨Foundation/Gemzeu´s length rHBcAg produced in E. coli or yeast expression systems has Foundation. been shown to enhance Th1-type immune responses (13). 2 Address correspondence and reprint requests to Dr. David R. Milich, Vaccine Re- search Institute of San Diego, 3030 Bunker Hill Street, Suite 300, San Diego, CA In an attempt to clarify or consolidate these disparate hypothe- 92109. ses, we examined Ag presentation of the HBcAg in vitro and in 3 Abbreviations used in this paper: HBV, hepatitis B virus; BMDC, bone marrow- vivo in detail to determine the ability of B cell subpopulations and derived ; DC, dendritic cell; HS, heparan sulfate; KO, knockout; M␾, DC/M␾ cells to function as primary APCs for either exogenous or ; Tg, transgenic. intracellular HBcAg particles. Furthermore, the possible role of Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 HBcAg-specific TLR-mediated activation of APCs was explored.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803683 The Journal of Immunology 6671

These studies reveal the involvement of the innate immune system Biotinylation of HBcAg150C in a rather unique APC pathway for exogenous HBcAg particles, rHBcAg150C (2 mg/ml) in 1.0 ml of PBS containing 1 mM EDTA was which is dependent on the mode of immunization. mixed with 100 ␮l of a biotin-N-(6-(biotinamido)hexyl)-3Ј-(2Ј-pyridyl- dithio)-propionamide stock solution (4 mM; Pierce) and vortexed, and the Materials and Methods reaction mixture was incubated for2hatroom temperature. Excess re- agents were removed by dialysis against PBS. Based on the absorbance Mice change at 343 nm, the biotin reacted with between 0.5 and 1 cysteine/ C57BL/10 (B10), B10.S, and 7/16-5 TCR-transgenic (Tg) mice were ob- monomer (240 monomers/particle). tained from the breeding colony of the Vaccine Research Institute of San Immunization protocols Diego. The B cell knockout (KO; ␮MT) mice originally produced by K. Rajewsky (14) were backcrossed onto B10, B10.S, and 7/16-5 TCR-Tg For protein immunization with HBcAg/WHcAg, groups of three to five backgrounds. The TLR-7KO mice were obtained from R. Flavell (Yale female mice were injected i.p. with 10–20 ␮g of protein either in saline or University, New Haven, CT) and bred at the Vaccine Research Institute of emulsified in CFA or IFA for both Ab production and T cell experiments. San Diego. The C3H.HeJ and C3H.HeSn mice and the CBA/J and CBA/N For DNA immunization, groups of 5–10 female mice were injected with (xid) mice were obtained from The Jackson Laboratory. All animal care 100 ␮g of plasmid DNA in PBS i.m. in the tibialis cranialis muscle. Mice was performed according to National Institutes of Health standards as set were boosted at 4 wk. For immunization with the RBL-5/C tumor cell line, forth in the Guide for the Care and Use of Laboratory Animals (1996). 5 ϫ 106 RBL-5/C tumor cells were given s.c. Recombinant proteins and synthetic peptide ELISA for detection of murine Abs

Full-length rHBcAg183 of the ayw subtype was produced in E. coli,as Sera were collected by retro-orbital bleeding of isofluorane-anesthetized described previously (5). Yeast-derived HBcAg183 (ayw) was obtained mice and pooled. Abs were detected by an indirect solid-phase ELISA with Downloaded from commercially from Meridian Life. A truncated version of HBcAg (residues rHBcAg or rHBeAg or P. falciparum repeat peptides as the solid-phase 1–149) was produced in E. coli. This material is particulate, but expresses ligands, as described previously (2). Serial dilutions of both test and pre- both HBcAg and HBeAg antigenicity at the level of Ab recognition and is immunization sera were made, and the data were expressed as Ab end- designated HBcAg149 or HBcAg150C (additional C-terminal cysteine for point titers, in which the highest dilution of sera required to yield an OD biotinylation). A rHBeAg corresponding in sequence to serum-derived (492 nm) three times the preimmunization sera was considered positive. HBeAg encompassing the 10 precore amino acids remaining after cleavage IgG-, IgG1-, IgG2a-, IgG2b-, and IgG3-specific secondary Abs were pur- of the precursor and residues 1–149 of HBcAg was produced, as described chased from Southern Biotechnology Associates. http://www.jimmunol.org/ previously (15). The presence of the 10 precore amino acids prevents par- ϩ ticle assembly, and HBeAg is recognized efficiently by a HBeAg-specific Determination of CD4 cytokine production mAb, but displays little HBc antigenicity. Recombinant hybrid woodchuck Spleen cells from either primed wild-type or B cell KO mice were cultured hepatitis core Ag (WHcAg) particles containing a Plasmodium falciparum (5 ϫ 106 cells/ml) with various concentrations of a panel of Ag. Culture malaria circumsporozoite repeat sequence (NANPNVDPNANP3) inserted supernatants were harvested at 48 h for IL-2 determination and at 96 h for in the external loop were produced, as previously described (16). Anti-HBc IFN-␥ determination. Cytokines were measured using commercial ELISA mAbs 3105 and 3120 were purchased from the Immunology Institute. kits, according to the manufacturer’s protocol (eBiosciences). Purification of core Ag Detection of HBcAg-specific lytic CTLs The core proteins were precipitated from the bacterial lysate by the addi- Spleen cells derived from DNA plus tumor cell-immunized B10 mice were by guest on September 24, 2021 tion of solid ammonium sulfate to 45% saturation (277 g/liter). The pre- resuspended in complete RPMI 1640 medium, and in vitro stimulation was cipitates were collected by centrifugation, redissolved in a minimum of conducted for 5 days in the presence of 0.05 ␮M H-2Kb-restricted HBc- buffer (10 mM sodium phosphate buffer (pH 6.8)), and dialyzed exten- Ag93–100 peptide (sequence MGLKFRQL) (22). After 5 days of in vitro sively against the same buffer. The protein solutions were then applied to stimulation, effector cells were harvested and a 4-hour 51Cr assay was a Bio-Rad BioGel HTP hydroxyapatite column (5 cm ϫ 5–10 cm, depend- performed in 96-well V-bottom plates in a total volume of 200 ␮l. A total ing on amount of protein) and eluted with 50 mM sodium phosphate buffer of 1 ϫ 106 target cells (e.g., RMA-S) was pulsed with 50 ␮M H-2Kb- (pH 6.8). The core Ag pass through unretained. The proteins were then restricted HBcAg93–100 peptide for 90 min, and then subsequently labeled applied to a Sepharose CL-4B column (5 ϫ 100 cm). for 1 h with 25 ␮lof51Cr (5 mCi/ml; MP Biomedicals) and washed three Endotoxin was removed from the core preparations by a modification of times in PBS. Different numbers of effectors and 51Cr-labeled target cells a phase separation with Triton X-114 (17). A solution of the protein at a (5 ϫ 103 cells/well) were added to wells at E:T ratios of 100:1, 50:1, and concentration of 5 mg/ml was made with 1% Triton X-114 and incubated 25:1. The level of cytolytic activity was determined after incubation of at 4°C for 30 min with constant stirring. The solution was then incubated effectors and targets for 4 h. at 37°C for 10 min and then centrifuged at 20,000 ϫ g for 10 min. The protein solution was recovered from above the detergent. This procedure Fractionation of cell populations was repeated four times. Finally, the protein was precipitated by lowering ϩ the pH to 5. Residual detergent remained in solution. The protein was Five- to 10-wk-old 7/16-5 TCR-Tg mice were used as a CD4 T cell recovered by centrifugation and dissolved in endotoxin-free buffer. Before source. Single-cell suspensions of splenocytes were labeled with anti-CD4 ϳ Microbeads (Miltenyi Biotec) and passed through a LS column (Miltenyi Triton X-114 treatment, the core preparations contained 10 ng of endo- ϩ toxin/␮g HBcAg; after phase separation with Triton X-114, the endotoxin Biotec); the cells retained in the column were collected as CD4 T cells. content was between 0.01 ng/␮g HBcAg to an undetectable amount as For fractionation of B cells, splenocytes from B10 mice were labeled with determined by the QCL-1000 chromogenic Limulus amebocyte lysate end- anti-CD90.2 Microbeads to deplete T cells and then passed through the LS point assay (Cambrex). Yeast-derived HBcAg contained no measurable column; cells contained in the flow-through were termed enriched B lin- 183 eage cells. Enriched B cells were then labeled with anti-CD5 Microbeads endotoxin. Unless otherwise specified, only endotoxin-free HBcAg prep- ϩ arations were used. for positive selection of B1a cells. Similarly, CD5 B1a cells were col- Synthetic peptides derived from the HBcAg or WHcAg sequences or the lected from peritoneal exudate cells. Purity of B1a cells was subsequently analyzed by FACS. For isolation of the splenic B1b population (B220ϩ P. falciparum repeat sequence were synthesized by the simultaneous pep- Ϫ/low ϩ tide synthesis method, as previously described (18). CD21 CD11b ), flow through from the B1a cells was further labeled with CD11b Microbeads and positively selected as an enriched B1b cell Plasmid DNA and tumor cell line expressing HBcAg population. FACS analysis was performed to determine the purity and showed Ͼ75% were B220ϩ CD11bϩ. Flow through from this procedure A HBcAg gene fragment (552 nt) of the ayw subtype was amplified and was collected and used as B2 cells. Bone marrow-derived DC (BMDC) cloned into the eukaryotic expression vector pVAX1 (Invitrogen), as pre- were prepared by an established protocol. Briefly, bone marrow progenitor viously described (19). The HBcAg expression plasmid was designated cells were harvested from femur and tibia bones of B10 mice and nega- HBcAg-pVAX1. Plasmid DNA was grown and purified, as described (20). tively depleted of B cells using B220 Microbeads. Bone marrow progenitor The purified plasmid DNA was dissolved and diluted in sterile PBS to a cells were cultured in RPMI 1640 containing 10% FCS with 20 ng/ml concentration of 1 mg/ml. A Rauscher virus-induced T cell lymphoma mouse rGM-CSF. On day 2, supernatant and nonadherent cells were as- (RBL-5) cell line (H-2b) stably expressing HBcAg (RBL-5/C) (21) was pirated and replaced with fresh medium containing mouse rGM-CSF. Ad- provided by F. Chisari (The Scripps Research Institute, La Jolla, CA). herent immature DC were harvested by gentle pipetting on day 7. 6672 ROLE OF B1 CELLS AND TLR-7 IN HBcAg IMMUNE RESPONSES

FIGURE 1. Naive B cells function as APCs for naive, HBcAg-specific CD4ϩ T cells in vitro. A, Fraction- ated, HBcAg-specific CD4ϩ T cells (1 ϫ 106) derived from three unprimed, TCR-Tg mice (7/16-5) were cocultured for 4 days with either purified, resting splenic B cells (1 ϫ 106) or splenic DC/M␾ (1 ϫ 106)de- rived from three wild-type mice, plus the indicated concentrations of HBc-

Ag183 or the HBcAg-derived peptide (p120–140). CD4ϩ T cell activation was measured by CD25 expression, as Downloaded from determined by FACS analysis. B, Unprimed spleen cells pooled from either three wild-type (WT) 7/16-5 TCR-Tg mice or three 7/16-5 TCR-Tg mice bred on a B cell KO (BKO) background were cultured (5 ϫ 5 10 ) with the indicated concentrations http://www.jimmunol.org/ ␥ of HBcAg183, and IL-2 and IFN- production was determined by ELISA at day 2 or 4, respectively. Values are given as pg/ml. This experiment was performed on two occasions, and the results are representative. by guest on September 24, 2021

Flow cytometry analysis Results To analyze T cell activation, Ag-specific T cells from 7/16-5 TCR-Tg mice Resting B cells, but not DC/M␾, can efficiently present HBcAg (V␤11ϩ CD4ϩ T cell) were cultured with HBcAg or p120–140 peptide for and prime naive T cells 4 days. At day 4, cultured cells were stained for CD25. For surface stain- ϩ ␾ ing, cells were incubated in 3% FBS in PBS containing 10 ␮g/ml 2.4G2 to Fractionated B cells (B220 ) or DC/M cells representing the block FcR binding, followed by the addition of fluorochrome-conjugated APC source were cocultured with naive HBcAg-specific, splenic Abs. All fluorochrome-conjugated Abs were obtained from eBioscience. CD4ϩ T cells derived from 7/16-5 TCR-Tg mice (Fig. 1A). Both Flow cytometry was performed on a LSR flow cytometer (BD Biosciences) APC populations are capable of presenting the HBcAg-derived at the Salk Institute. Data were analyzed using FlowJo software (Tree Star). ϩ peptidic p120–140 Ag to naive 7/16-5 CD4 T cells, resulting in TLR ligand screening T cell activation, as demonstrated by up-regulation of cell surface CD25 (Fig. 1A) or T cell proliferation (Fig. 3A). However, only TLR stimulation was tested by assessing NF-␬B activation in HEK293 cells ectopically expressing seven different human TLRs, including the splenic B cells efficiently presented HBcAg183 to naive 7/16-5 ϩ following: TLR 2, 3, 4, 5, 7, 8, and 9, and a reporter plasmid, pNiFty, CD4 T cells, resulting in HBcAg dose-dependent T cell activa- carrying the human secreted embryonic alkaline phosphatase gene con- tion (Fig. 1A). In a similar experiment, naive 7/16-5 TCR-Tg ␬ trolled by an NF- B-inducible ELAM-1 composite promoter (InvivoGen). spleen cells or naive spleen cells derived from 7/16-5 TCR-Tg Four HBc/HBeAg preparations were incubated with the TLR-expressing ϫ KO HEK293 cells at a concentration of 2 ␮g/ml. The four HBc/HBeAg prep- mice bred onto a B cell KO background (7/16-5 B ) were arations varied in the amounts of contaminating LPS ranging from 10 cultured with HBcAg183, and HBcAg-specific presentation and T ng/␮g to undetectable. cell activation were measured by IL-2 and IFN-␥ cytokine pro- duction. Efficient HBcAg-specific IL-2 and IFN-␥ production re- Core protein-binding assay quired the presence of B cells (Fig. 1B). Note that in both exper- ϩ B1a and B2 cells were prepared as described above. Biotinylated HBc150c iments the HBcAg-specific CD4 T cells and the B cell APCs ϫ 6 was added to 1 10 B cells and incubated at 4°C for 1 h. Cells were were resting and unprimed (naive), an advantage of using TCR-Tg collected and stained for B220 and streptavidin-PE for further FACS ϩ analysis. mice because the high frequency of HBcAg-specific CD4 T cells precludes the necessity for immunization. Statistical analysis To determine whether the preferential B cell presentation of ϩ Data were analyzed using the Student’s t test. SPSS software was used for HBcAg was unique to 7/16-5 CD4 T cells and/or relevant in all statistical analysis. vivo, B10.S/ϩ/ϩ mice or B10.S/BKO mice were immunized with The Journal of Immunology 6673

FIGURE 2. Efficient CD4ϩ T cell priming in vivo with soluble HBcAg requires B cells. Groups of three B10.S/ϩ/ϩ or B10.S/BKO mice were immunized (i.p.) ␮ with HBcAg183 (10 g) emulsified in CFA. Ten days after immunization, spleen cells were harvested, pooled, and cultured with the indicated concentrations of a

panel of recall Ag (HBcAg183, HBeAg, and p120–140). Culture supernatants were collected at days 2 and 4 for ␥ the determination of IL-2 and IFN- , respectively, by ELISA. Groups of three mice each were also immu- nized with the HBsAg (20 ␮g, CFA), and spleen cells were cultured with varying concentrations of HBsAg in vitro (inset). This experiment was performed on at least three occasions, and the results are representative. Downloaded from

HBcAg183 either emulsified in CFA, IFA, or saline (only the re- nonparticulate HBeAg, and the dominant peptidic T cell site, sults of HBcAg/CFA immunization are shown in Fig. 2). In B10.S/ p120–140). The HBcAg Ag was the superior recall Ag in vitro. In ϩ/ϩ mice, HBcAg efficiently primed T cells measured by IL-2 contrast, HBcAg-specific T cells were not efficiently primed in and IFN-␥ production in response to a panel of recall Ag (HBcAg, B10.S/BKO mice (Fig. 2). This defect was specific for the HBcAg http://www.jimmunol.org/

FIGURE 3. Preferential Ag pre- sentation of HBcAg by B1a and B1b cells. A, Fractionated, splenic B cells 5 5 (4 ϫ 10 ) or BMDC (4 ϫ 10 ) were by guest on September 24, 2021 cocultured with unprimed, CFSE-la- beled 7/16-5 TCR-Tg CD4ϩ T cells (1 ϫ 105) in the presence of 2 ␮g/ml

HBcAg149 or p120–140 peptide. Af- ter 4 days, dilution of CFSE as a mea- sure of CD4ϩ T cell proliferation was determined by FACS analysis. B, The indicated fractionated cell populations (4 ϫ 105) (BMDC, splenic B1a, splenic B1b, and splenic B2 cells) de- rived from unprimed B10 mice were ␮ cultured with HBcAg149 (2.0 g/ml) and CD4ϩ T cells (1 ϫ 105) derived from 7/16-5 TCR-Tg mice. After 4 days of in vitro culture, CD4ϩ T cell activation was measured by CD25 ex- pression determined by FACS analy- sis. C, Unprimed, B10, total spleen cells, fractionated splenic B2 cells, or fractionated, splenic B1a cells (4 ϫ 105) were cocultured with unprimed, 7/16-5 TCR-Tg CD4ϩ T cells (1 ϫ 5 ␮ 10 ) and HBcAg149 (0.04–5.0 g/ml) and CD4ϩ T cell activation was mea- sured by IL-2 (day 2) and IFN-␥ (day 4) production, as determined by ELISA. The results are representative of experiments performed on two sep- arate occasions. 6674 ROLE OF B1 CELLS AND TLR-7 IN HBcAg IMMUNE RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 5. Anti-HBc Ab production in vivo is influenced by B1a cells. A, Groups of five wild-type CBA/J or CBA/N (xid) mice were immunized ␮ ␮ (1°) with HBcAg149 (20 g in saline) and boosted (2°) (i.p.) with 10 gof HBcAg149 in saline, and serum anti-HBc Ab levels were determined by ELISA at the indicated times (E-E, ●-●). Serum anti-HBc IgG Ab was quantitated by endpoint dilution of sera (titer). Groups of three mice each were also immunized (1°) with HBsAg (20 ␮g; IFA), and anti-HBs Ab was determined by ELISA (Ⅺ-Ⅺ, f-f). B, Groups of three CBA/J or CBA/N ␮ by guest on September 24, 2021 (xid) mice were immunized (i.p.) with HBcAg149 (20 g of insaline), and 4 wk later serum IgM and IgG isotype-specific anti-HBc Abs were deter- mined by ELISA. Pre-M, pre-existing IgM anti-HBc (before immuniza- tion). Results represent the mean values (ϮSD) of individual mouse sera .(p ϭ 0.002 ,ءءء ;p ϭ 0.005 ,ءء ;p ϭ 0.02 ,ء) FIGURE 4. Splenic B1a cells preferentially bind biotinylated ϫ 6 HBcAg150C. Enriched B2 cells or B1a cells (1 10 ) were incubated at 4°C for 1 h with the indicated concentrations of biotinylated HBcAg150C. BMDC were unable to present HBcAg149 efficiently to naive HBc- Cells were collected and stained for the B cell marker, B220, and for ϩ Ag-specific CD4 T cells, as measured by CD25 up-regulation on streptavidin binding, and analyzed by FACS. CD4ϩ T cells (Fig. 3B) and IL-2 and IFN-␥ cytokine production in vitro (Fig. 3C).

Furthermore, direct binding studies of biotinylated-HBcAg150C because the particulate envelope protein of HBV, HBsAg, primed particles to separated BMDC, splenic B2 cells, or splenic B1a cells ϩ ϩ KO HBsAg-specific T cells equally in / and B mice (Fig. 2, were performed. Fig. 4 demonstrates that biotinylated HBcAg150C inset). It was also notable that the low number of T cells primed in binds preferentially to an enriched B1a cell population in a dose- B10.S/BKO mice by HBcAg was more efficiently activated in vitro dependent manner and not to a B2 cell population. BMDC do not

by HBeAg and p120–140 rather than the HBcAg, demonstrating bind biotinylated HBcAg150C (data not shown). Furthermore, to that HBeAg and p120–140 are not as dependent on B cells for illustrate the consequences of B1a cell APC function in vivo, we APC function as the particulate HBcAg. immunized CBA/N (xid) mice, which lack B1a cells, with HBc-

ϩ Ag149 in saline and compared the response with CBA/J control B cell subsets preferentially present HBcAg to naive CD4 mice (Fig. 5). Significantly lower and slower developing anti-HBc T cells Ab responses were apparent in CBA/N (xid) mice. Note, however, We next examined whether a specific B cell subset was responsible that CBA/N (xid) mice did demonstrate a moderate secondary anti- for the APC function. Fractionated, naive HBcAg-specific CD4ϩ HBc Ab response. Therefore, it appears that B1a cells may be T cells (from 7/16-5 TCR-Tg mice) were labeled with CFSE and responsible for the early and/or initial anti-HBc Ab response. As a cocultured with fractionated splenic B cells or BMDC as APCs further control, these two strains were also immunized with the (Fig. 3A). Only CD4ϩ T cells cocultured with splenic B cells pro- HBsAg, and no significant differences were observed in the anti-

liferated efficiently in the presence of HBcAg149. Note that both B HBs Ab responses. We also measured IgM and the various anti- cells and BMDC were efficient APCs for the HBcAg-derived pep- HBc IgG isotype responses in CBA vs CBA/N mice (Fig. 5B). tide, p120–140. Importantly, splenic B1a cells and splenic B1b Note that CBA/J mice possess IgM anti-HBc Abs before immu- cells were superior APCs as compared with splenic B2 cells, and nization (pre-M), whereas CBA/N mice do not. All murine strains The Journal of Immunology 6675

FIGURE 6. Plasmid DNA immu- nization bypasses the requirement for B cell APCs to prime HBcAg-specific CD4ϩ and CD8ϩ T cells. A, Groups of three B10/ϩ/ϩ or B10/BKO mice were immunized i.m. with 100 ␮gof HBcAg-pVAX1 or unimmunized. Four weeks after immunization, spleen cells were harvested, pooled, and cultured for 2 days (IL-2) or for 4 days (IFN-␥) with the indicated con- centrations of HBcAg183, HBeAg, or p120–140 as recall Ag, and T cell priming was determined by measur- ing IL-2 and IFN-␥ production in cul- ture supernatants by ELISA. For un- immunized groups, only the response to HBcAg183 is shown. Values are

given as pg/ml. B, Groups of six B10/ Downloaded from ϩ/ϩ or B10/BKO mice were immu- nized i.m. twice with 100 ␮g of HBc- Ag-pVAX1 or the empty pVAX1 vector (four mice/group). All groups were boosted with RBL-5/C (5 ϫ 106) tumor cells given s.c. Six weeks after the tumor cell boost, spleens were http://www.jimmunol.org/ harvested and restimulated for 5 days in the presence of the HBcAg93–100 H-2Kb-restricted peptide, and thereaf- ter used in a standard 4-h Cr51 release assay at the indicated E:T ratios. Each line represents an individual mouse. by guest on September 24, 2021 previously tested possess IgM anti-HBc Abs spontaneously, which APCs can process and present HBcAg when it is complexed to most likely represents natural Ab produced by the innate immune anti-HBc mAbs (mAbs 3105 ϩ 3120), but not free HBcAg. In system; in mice at least, this appears to be produced by B1a cells contrast, B cell APCs can present free HBcAg or immune-com- because it is absent in CBA/N mice. However, upon immunization, plexed HBcAg, as shown in Fig. 7. Therefore, it appears that CBA/N mice did produce IgM anti-HBc Abs in levels equal to DC/M␾ can process and present HBcAg to naive CD4ϩ and CD8ϩ CBA/J mice (Fig. 5B). Therefore, pre-existing IgM anti-HBc is pro- T cells, but do not efficiently bind and internalize free exogenous duced by B1a cells, and IgM anti-HBc produced in response to im- munization is produced by another B cell subset, most likely B1b cells or marginal zone B2 cells. CBA/J mice produced significantly more IgG anti-HBc Abs of all IgG isotypes than CBA/N mice.

The requirement for B cell APCs does not apply to immune-complexed HBcAg or HBcAg expressed intracellularly The preceding experiments were performed using protein rHBcAg as Ag and immunogen and demonstrated that DC/M␾ cells do not serve as efficient APCs for exogenous HBcAg. However, a single immunization with a DNA plasmid encoding the HBcAg (HBcAg- pVAX1) did not require B cells as APCs for efficient CD4ϩ T cell priming (Fig. 6A). Both wild-type B10 mice and B10/BKO mice immunized with HBcAg-pVAX1 demonstrated efficient HBcAg-, HBeAg-, and p120–140-specific T cell cytokine responses in splenic in vitro Ag recall cultures (Fig. 6A). We have previously shown that a prime/boost immunization strategy using HBcAg- FIGURE 7. Non-B cell APCs can process and present immune-com- plexed HBcAg to naive, CD4ϩ T cells, but not free HBcAg. Either free pVAX1 and a boost with a HBcAg-expressing tumor cell line ␮ ϩ HBcAg183 (1 g/ml) or HBcAg183 complexed to HBcAg-specific mAbs (RBL-5/C) elicits an efficient CD8 CTL response to the HBcAg ϩ ␮ ϩ (3105 3120) (2 g/ml) were added to 7/16-5 TCR-Tg spleen cells (B (23). The induction of an efficient CD8 CTL response to intra- cellϩ) or 7/16-5/BKO spleen cells (B cellϪ), and CD4ϩ T cell activation cellular HBcAg also does not require B cells as APCs (Fig. 6B). It was determined by IL-2 (day 2) and IFN-␥ (day 4) cytokine production in appears that DC/M␾ APCs can cross-present intracellular HBcAg culture supernatants measured by ELISA. Values are expressed as pg/ml. derived from transfected or tumor cells. Furthermore, non-B cell The results are representative of three separate experiments. 6676 ROLE OF B1 CELLS AND TLR-7 IN HBcAg IMMUNE RESPONSES

FIGURE 9. Endotoxin contamination of HBcAg does not enhance anti- HBc Ab production in vivo. Groups of three LPS-sensitive (C3H.HeSnJ) or LPS-insensitive (C3H.HeJ) mice were immunized with either a highly ϩ FIGURE 8. The HBcAg protein is not a TLR ligand. Four HBc/HBeAg (LPS ) HBcAg149 preparation (10 ng/ml) or a HBcAg149 preparation free Ϫ preparations were screened for TLR ligand activity on a panel of human of LPS contamination (LPS ). Mice were immunized i.p. with 10 ␮gof

TLR-expressing HEK293 cells. TLR ligand activity was determined by HBcAg149 in saline, and 5 wk later serum anti-HBc IgG isotypes were Downloaded from NF-␬B activation, as described in Materials and Methods. The two full- measured by ELISA. length HBcAg183 preparations contained no endotoxin, the HBeAg prep- aration contained low level endotoxin (1.0 ng/ml), and the truncated

HBcAg149 preparation was contaminated with 10 ng/ml endotoxin. Role of TLR-7

Full-length HBcAg183, which contains a protamine-like C termi- nus, encapsidates pregenomic RNA during natural infection, and rHBcAg encapsidates ssRNA when produced in prokaryotic or http://www.jimmunol.org/ HBcAg even though it is a particulate Ag. This most likely reflects eukaryotic expression systems. Furthermore, the enhanced immu- the absence of any ligands for DC/M␾ endocytic receptors on nogenicity of full-length vs truncated HBcAg is well known and HBcAg (i.e., no carbohydrate or lipid). has been attributed to the encapsidated ssRNA (13). However, for- mal proof that the encapsidated ssRNA acts as a TLR-7 ligand is KO Potential role of TLRs in Ag presentation of the HBcAg lacking. Therefore, B10/ϩ/ϩ and B10/TLR-7 mice were im- munized with either truncated HBcAg149 or full-length HBcAg183, Various preparations of HBcAg and the nonparticulate HBeAg and anti-HBc Ab production was then determined. As shown in were examined for the ability to signal through a number of human by guest on September 24, 2021 TLRs (Fig. 8). Only a truncated HBcAg149 preparation, which con- tained E. coli-derived LPS as a contaminant (10 ng/␮g HBcAg), signaled through TLR-2 and TLR-4, whereas the cleanest HBc-

Ag183 preparation, which was produced in yeast, demonstrated zero TLR signaling. Therefore, it appears that the HBcAg/HBeAg proteins do not possess TLR-binding capability, but the LPS that can copurify with rHBcAg produced in E. coli can bind and signal through TLRs 2 and 4. Recently, a number of publications have suggested that this contaminating LPS may explain the enhance immunogenicity of HBcAg in mice and that researchers have been unaware of this problem (11, 12). Although we disagree that re- searchers have not been aware of this issue and, in fact, have rig- orously controlled for the presence of LPS, it was important to address the possible effects that contaminating LPS may have on HBcAg immunogenicity studies in mice. Therefore, LPS-sensitive (C3H.HeSn) and congenic LPS-insensitive (C3H.HeJ) mice, which possess a mutation preventing TLR-4 signaling, were im- munized with either a highly LPS-contaminated (10 ng/␮g) HBc-

Ag149 preparation or a LPS-negative HBcAg149 preparation, and anti-HBc Ab IgG isotype distribution was then determined. As shown in Fig. 9, in LPS-sensitive C3H.HeSn mice, the LPS-neg- ative HBcAg149 preparation elicited equal or greater anti-HBc Abs FIGURE 10. Enhanced immunogenicity of full-length HBcAg183 is me- diated by TLR-7 signaling. Groups of three wild-type B10/ϩ/ϩ or B10/ of all IgG isotypes than the LPS-positive HBcAg149 preparation. KO Reciprocally, in LPS-insensitive mice, the LPS-negative and LPS- TLR-7 mice were immunized i.p. with 10 ␮g in saline of either full- length HBcAg (ssRNAϩ) or HBcAg (ssRNAϪ). At 1, 2, 4, and 6 wk positive HBcAg preparations elicited equal anti-HBc IgG iso- 183 149 149 after immunization, IgG anti-HBc Abs in pooled sera were measured by type responses, with the exception of a higher IgG3 anti-HBc re- ELISA. Groups of three wild-type B10/ϩ/ϩ mice or B10/TLR-7KO mice sponse elicited by the LPS-negative HBcAg149 (Fig. 9). Therefore, ␮ were immunized i.p. with 10 g in saline of full-length HBcAg183 (ss- the level of LPS contamination likely to occur in E. coli-derived RNAϩ). Two weeks after immunization, anti-HBc Abs of the indicated HBcAg preparations is not determinative in terms of the humoral IgG isotypes were measured in pooled sera by ELISA. The results depicted anti-HBc response in vivo and cannot explain the enhanced im- represent mean values (ϮSD) of experiments performed using pooled se- .(p ϭ 0.005 ,ءء ;p ϭ 0.05 ,ء) munogenicity of HBcAg in mice. rum samples on two separate occasions The Journal of Immunology 6677

␮ Fig. 10, immunization (10 g in saline) with full-length HBcAg183 in B10/ϩ/ϩ mice elicited significantly higher titer (ϳ25-fold) anti-

HBc Ab at every time point compared with truncated HBcAg149.

Full-length HBcAg183 also elicited significantly higher titer anti- HBc Ab in B10/ϩ/ϩ mice as compared with B10/TLR-7KO mice. ϩ ϩ Truncated HBcAg149 was equally immunogenic in B10/ / and B10/TLR-7KO mice. Also note that the humoral response to full- KO length HBcAg183 in B10/TLR-7 mice was identical with the ϩ ϩ response to the truncated HBcAg149 in either B10/ / mice or B10/TLR-7KO mice. Furthermore, the anti-HBc IgG isotype dis- tribution was dramatically influenced by the absence of TLR-7 signaling (Fig. 10). Anti-HBc IgG1 was enhanced in TLR-7KO mice, IgG2a anti-HBc Abs were totally absent in TLR-7KO mice, and IgG2b anti-HBc Abs were significantly reduced in TLR-7KO mice as compared with wild-type mice. Anti-HBc IgG3 Abs were not significantly different in wild-type vs TLR-7KO mice. These results indicate that the ssRNA of bacterial origin that is encapsi- FIGURE 11. HBcAg-specific T cell cytokine production is regulated by KO dated within full-length HBcAg183 acts as a TLR-7 ligand and TLR-7 signaling. Groups of three B10/ϩ/ϩ and B10/TLR-7 mice were Downloaded from ␮ ϩ enhances anti-HBc Ab production while shifting IgG isotype dis- immunized i.p. with 10 g of full-length HBcAg183 (ssRNA ) in saline, and 4 wk later in vitro splenic recall cultures were performed in the pres- tribution via signaling through endosomal TLR-7 within B cells or other APCs. These results also indicate that the difference in ence of the indicated concentration of HBcAg183. HBcAg-specific IL-2 (day 2) and IFN-␥ (day 4) production was measured by ELISA and ex- immunogenicity between full-length HBcAg183 and truncated

pressed as pg/ml. The results are representative of two separate HBcAg149 resides solely in ssRNA signaling through TLR-7 be- experiments. cause HBcAg183 and HBcAg149 are equivalently immunogenic in http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 12. Ab production to hybrid-WHc- Ag particles is influenced by TLR-7 signaling. A, Groups of three B10/ϩ/ϩ or B10/TLR-7KO mice were immunized i.p. with 20 ␮g of either truncated hybrid-WHcAg (149-Mal-78-UTC) (ssRNAϪ) or full-length hybrid-WHcAg (FLW- Mal-78-UTC) (ssRNAϩ) particles emulsified in IFA. Six weeks after immunization, anti-WHc (carrier), anti-NANP (insert), and anti-NVDP (insert) Abs were measured in pooled sera by ELISA. B, Groups of three B10 wild-type mice were immunized i.p. with 20 ␮g of either trun- cated hybrid-WHcAg (149-Mal-78-UTC) (ss- RNAϪ) or full-length hybrid-WHcAg (FLW- Mal-78-UTC) (ssRNAϩ) particles emulsified in IFA. Six-week sera were collected and pooled, and IgG1 and IgG2a anti-WHc, anti-NANP, and

anti-NVDP Abs were measured by ELISA. G1/ G2a ratios are also shown. The results represent mean values (ϮSD) of experiments performed using pooled serum samples on two separate oc- .(p ϭ 0.005 ,ءء ;p ϭ 0.05 ,ء) casions 6678 ROLE OF B1 CELLS AND TLR-7 IN HBcAg IMMUNE RESPONSES

KO TLR-7 mice. rHBcAg183 expressed in E. coli or yeast behaves similarly; therefore, both prokaryotic and eukaryotic ssRNA within HBcAg can function as TLR-7 ligands. Immunization with a pVAX1 DNA vector encoding full-length HBcAg183 elicits greater anti-HBc Ab production than a pVAX1 vector encoding truncated HBcAg149, suggesting that mammalian ssRNA can also function as a TLR-7 ligand (data not shown). The ssRNA-TLR-7 pathway predictably affected HBcAg-specific CD4ϩ T cell prim- ing as well as in vivo anti-HBc Ab production, as shown in Fig. 11. Splenic T cells derived from B10/TLR-7KO mice immunized with ϩ HBcAg183 (i.e., ssRNA ) produced significantly less IL-2 and IFN-␥ than T cells derived from B10/ϩ/ϩ-immunized mice in HBcAg recall spleen cultures. Because the woodchuck hepadnavirus core protein (WHcAg) has been developed as a vaccine carrier platform (16), it was of interest to determine whether TLR-7 signaling was also relevant to FIGURE 13. TLR-7 signaling occurs in B cells. Two groups of six immunization with hybrid-WHcAg particles. For this purpose, B10/BKO mice served as recipients for the adoptive transfer of 30 ϫ 106 ϩ ϩ ϩ full-length WHcAg187 (ssRNA ) carrying two P. falciparum ma- fractionated splenic B cells derived from either B10/ / mice or B10/ KO laria-neutralizing B cell epitopes (i.e., NANP and NVDP) in the TLR-7 mice. Successful B cell grafting was confirmed by the pres- Downloaded from loop region at position 78 and a truncated WHcAg (ssRNAϪ) ence of serum IgG 3 days after adoptive transfer. At 7 days after adop- 149 ϩ particle carrying the identical malaria B cell inserts were used to tive transfer, mice were immunized i.p. with HBcAg183 (ssRNA ) (20 KO ␮g in saline) and boosted with 10 ␮g of HBcAg in saline. Sera were immunize B10/ϩ/ϩ and B10/TLR-7 mice; anti-WHc and anti- 183 collected, pooled, and assayed for IgM anti-HBc Abs at the indicated insert Ab responses were then compared. As shown in Fig. 12A, time points by ELISA. the anti-WHc, anti-NANP, and anti-NVDP Ab responses were sig-

nificantly higher (13- to 25-fold) in wild-type (ϩ/ϩ) mice immu- http://www.jimmunol.org/ nized with the ssRNAϩ hybrid-WHcAg particle as compared with the ssRNAϪ particle. Furthermore, the ssRNAϩ hybrid-WHcAg ϩ TCR-Tg CD4 T cells in culture, and HBcAg149 as well as HBc particle elicited 25-fold higher anti-insert Ab responses in B10/ Ag183 can activate T cells. However, for production of IgM anti- KO ϩ/ϩ mice as compared with B10/TLR-7 mice, demonstrating HBc Ab to occur in this primary in vitro culture system, the HBc the positive effect of signaling through TLR-7. Note also that the Ag must be associated with a TLR signal (Fig. 14). Naive, HBcAg- ϩ Ϫ immunogenicity of ssRNA and ssRNA hybrid-WHcAg parti- specific 7/16-5 spleen cells cultured with HBcAg in vitro for 4–5 KO cles was virtually identical in TLR-7 mice, again illustrating days produced IgM anti-HBc Abs in the supernatant only if the that only TLR-7 signaling distinguished between the full-length HBcAg possessed ssRNA as a TLR-7 ligand or if HBcAg was and truncated hybrid-WHcAg particles. Furthermore, immuniza- associated with LPS as a contaminant, which is a TLR-4 ligand by guest on September 24, 2021 ϩ tion of B10/ϩ/ϩ mice with ssRNA hybrid-WHcAg particles (Fig. 14). The LPS does not have to be physically associated with elicited IgG2a-predominant anti-WHc, anti-NANP, and anti- the HBcAg because HBcAg plus soluble LPS will elicit IgM anti- Ϫ NVDP Abs, whereas ssRNA hybrid-WHcAg particles elicited HBc Ab production as well (data not shown). It appears that pri- IgG1-predominant Ab responses (Fig. 12B). mary in vitro anti-HBc Ab production requires three signals, in- cluding the following: BCR cross-linking, cognate T cell help, and a TLR signal. Evidence that early TLR-7 signaling occurs in B cells The preceding experiments indicate that B cells, and preferentially B1a and B1b cells, act as the primary APCs for exogenous HBc- Ag, and that TLR-7 signaling enhances anti-HBc Ab production and HBcAg-specific T cell cytokine production. This suggests, but does not prove that TLR-7 signaling occurs, at least initially, in B cells. To determine whether TLR-7 signaling occurs in HBcAg- specific B cells and is relevant to in vivo anti-HBc Ab production, a B cell transfer experiment was used. Recipient, B cell KO mice were adoptively transferred with purified, splenic B cells (30 ϫ 106) derived either from wild-type (ϩ/ϩ) mice or from TLR-7KO mice, in which case only B cells lacked TLR-7 expression and non-B cell APC populations were wild type with respect to TLR-7. Three days after the B cell transfer, mice were immunized (20 ␮g) ␮ ϩ and boosted (10 g) with HBcAg183 (ssRNA ) in saline, and IgM anti-HBc Abs were measured by ELISA (using this protocol IgG anti-HBc is not efficiently produced). As shown in Fig. 13, TLR-7 expression in B cells was required for an efficient primary IgM FIGURE 14. In vitro IgM anti-HBc Ab production requires three sig- nals. TCR-Tg 7/16-5 spleen cells were cultured with varying concentra- anti-HBc response in vivo, indicating the relevance of TLR-7 sig- ␮ tions of HBcAg preparations, which contained LPS (10 ng/ g) HBcAg149 naling to this response. After the boost, IgM anti-HBc Ab produc- Ϫ ϩ ϩ Ϫ RNA /LPS ; ssRNA, HBcAg183 RNA /LPS ; neither LPS or ssRNA, tion was less dependent on B cell TLR-7 expression. An in vitro Ϫ Ϫ HBcAg149 RNA /LPS , or LPS without HBcAg particles. After 5 days of correlate assay also indicated the importance of B cell TLR sig- culture, supernatants (SN) were collected and IgM anti-HBc Abs were naling for IgM anti-HBc Ab production. As noted in Fig. 1, measured by ELISA. This experiment was performed on three separate unprimed B cells present HBcAg to and activate naive, 7/16-5 occasions, and the results are representative. The Journal of Immunology 6679

Discussion it appears that HBcAg particles are ignored by DC/M␾ APCs at Although previous studies indicated that HBcAg-specific B cells least at concentrations of 1 ␮g/ml or less. The finding that DC/M␾ were the primary APCs for HBcAg, the readout system consisted cells were efficient APCs for HBcAg immune complexed to of presentation to HBcAg-specific T cell hybridomas, which do not HBcAg-specific mAbs established that once the HBcAg is inter- require costimulatory signals for T cell activation (6, 24). In the nalized, presumably through FcR-mediated uptake, processing of HBcAg by DC/M␾ APCs and subsequent presentation to naive current study, it was demonstrated that naive HBcAg-specific ϩ CD4ϩ T cells, derived from unprimed 7/16-5 TCR-Tg mice, were CD4 T cells occur efficiently. Furthermore, immunization with a DNA plasmid encoding HBcAg elicited very efficient CD4ϩ T cell activated by HBcAg presented by resting, naive B cells in vitro, ϩ and furthermore, that DC/M␾ APCs could not serve as efficient and CD8 CTL responses in B cell KO mice. This indicates that APCs for exogenous HBcAg. The ability of resting B cells to B cells are not required as APCs for intracellular HBcAg, which ϩ can apparently be cross-presented by DC/M␾ to both HBcAg-spe- prime naive CD4 T cells remains a controversial subject, with ϩ ϩ opinions ranging from B cells being essential as APCs (25–27); B cific CD4 and CD8 T cells. Because HBcAg exists as an ex- cells being unable to function as APCs (28, 29); and B cells in- ogenous Ag as well as an intracellular Ag within hepatocytes dur- ducing anergy in naive T cells (30). This lack of consensus may ing a natural infection, B cell presentation of exogenous HBcAg to CD4ϩ T cells and DC/M␾ cross-presentation of intracellular HBc- relate to the nature of the Ag under study. For example, the HBcAg ϩ ϩ is a particulate, multivalent protein Ag capable of cross-linking Ag to CD4 and CD8 T cells are both relevant APC pathways BCRs sufficiently to induce B7.2 and B7.1 costimulatory mole- during HBV infection. Predictably, B cell presentation of exoge- nous HBcAg leads to significantly greater anti-HBc Ab production cules on resting, HBcAg-specific B cells (6). Furthermore, in the as compared with DC/M␾ presentation of intracellular HBcAg; Downloaded from present study, it was demonstrated that B1a cells are superior and, reciprocally, HBcAg-specific CD8ϩ T cells and possibly APCs for the HBcAg as compared with B2 cells and a relatively CD4ϩ T cells are more efficiently primed by DC/M␾ presentation high frequency of B1a cells bind biotinylated HBcAg . Fur- 150C of intracellular HBcAg. thermore, in mice lacking B1a cells, the in vivo anti-HBc response The HBcAg and WHcAg have been proposed as vaccine carrier was delayed as compared with wild-type mice. B1a cells are mem- platforms for heterologous T and B cell epitopes (16, 39). Vacci- bers of the innate immune system that produce IgM natural Abs

nation with hybrid-HBcAg/WHcAg particles would at least ini- http://www.jimmunol.org/ and play a role in defense against bacterial infection (31). The tially rely on B cells for primary APC function; however, once specificities characteristic of B1a cells are encoded by distinctive anti-HBc and anti-insert Abs are produced, immune-complexed germline genes, possibly with preferential Ig H chain-L chain com- hybrid-HBcAg/WHcAg particles would be recognized by DC/M␾ binations, resulting in a restricted or hard-wired Ab repertoire (32). APCs as well. In this regard, it is interesting that a linear motif that binds HBcAg Historically, naive B cell activation, maturation, and Ab pro- has been identified in the framework region 1-CDR1 junction of an duction have been assumed to be dependent on the sequential in- IgM anti-HBc hybridoma (9C8). This motif is present in H chains tegration of two signals, as follows: BCR cross-linking by Ag, of the mouse V 1 family and human V 1 and V 7 families (33). H H H followed by cognate interaction with Th cells through an immu- A cryo-electron microscopic reconstruction of an immune com- nological synapse involving CD40-CD40L interaction and cyto- by guest on September 24, 2021 plex between HBcAg and a Fab of 9C8 indicates binding via the kines (40, 41). More recent studies suggest a three-signal pathway VL chain rather than the VH chain; however, the VL chain frame- for naive B cell activation that includes TLR ligands acting di- work region 1-CDR1 junction region contains a motif with 73% rectly on B cells, resulting in coligation of the BCR and TLRs (42, similarity to the VH1 consensus motif (34). These studies suggest 43). Although the absolute requirement for TLR signaling in naive interaction between HBcAg and Igs derived from restricted V re- B cell activation has been questioned (44), there is no disagree- gion germline genes, which is consistent with preferential recog- ment that coligation of BCRs and TLRs augments Ab responses. nition of HBcAg by B1 cells. Because WHcAg as well as HBcAg There has been some debate as to whether HBcAg can function is preferentially presented by B cell APCs (data not shown) and directly as a TLR ligand or whether LPS or some other contami- these hepadnavirus core proteins are not cross-reactive at the Ab nant indirectly activates TLR-2/4 (45). The current study did not level, we suggest that structural features of the spikes on the cap- find any evidence that the HBcAg protein possessed TLR-activat- sids surface rather than specific amino acids per se may serve as ing activity, which was restricted to HBcAg particles that were recognition sites for B1 cell Ig receptors. Although not extensively contaminated with LPS (i.e., TLR-2/4 activation). However, the studied, B1a cells have been reported to be very efficient APCs ssRNA encapsidated in either E. coli-derived or yeast-derived full- (35). length HBcAg stimulated significant TLR-7 activation. For exam- As interesting as the HBcAg-B1 cell APC pathway may be, the ϩ ple, full-length HBcAg183 (ssRNA ) elicited 25-fold higher anti- more surprising observation is the inability of non-B cell profes- HBc responses in wild-type mice as compared with TLR-7KO sional APCs (i.e., DC/M␾) to efficiently present exogenous HBc- mice. Furthermore, in the absence of TLR-7 (TLR-7KO mice), full- Ag to CD4ϩ T cells in vitro or in vivo. DC/M␾ cells are known to ϩ Ϫ length HBcAg183 (RNA ) and truncated HBcAg149 (ssRNA ) be highly efficient APCs due to constitutive expression of costimu- were equally immunogenic. Therefore, the significantly greater latory molecules, and are also known to be able to cross-present immunogenicity of HBcAg183 as compared with HBcAg149 in virus-like particles and other large particulate Ag (36–38). Nev- wild-type mice is mediated entirely through ssRNA-TLR-7 signal- ertheless, immunization of B cell KO mice even with HBcAg ing within B cell APCs, as demonstrated by adoptive transfer of emulsified in CFA, a very strong APC activator, was unable to TLR-7-negative B cells into B cell KO mice immunized with ϩ efficiently prime CD4 T cells (Fig. 2). The fact that immunization HBcAg183 (Fig. 13). Predictably, the ssRNA encapsidated within ϩ in CFA was unable to prime HBcAg-specific CD4 T cells in B HBcAg183 also enhanced HBcAg-specific T cell cytokine produc- cell KO mice indicated that the defect in APC function was not at tion. Furthermore, ssRNA-TLR-7 signaling skewed the anti-HBc the level of DC/M␾ activation, and suggested that the uptake of Ab isotype distribution toward IgG2a,IgG2b and away from IgG1 free HBcAg particles by DC/M␾ was the limiting factor. Because indicative of a Th1-type response. the HBcAg is nonglycosylated and contains no lipid and appar- Because of our interest in using the WHcAg as a vaccine carrier ently no other ligands recognized by DC/M␾ endocytic receptors, platform for heterologous B and T cell epitopes, hybrid-WHcAg 6680 ROLE OF B1 CELLS AND TLR-7 IN HBcAg IMMUNE RESPONSES

carrying P. falciparum malaria-protective B cell epitopes (NANP, lack efficient B1a cell function, pre-existing IgM anti-HBc Abs are NVDP) were used to immunize wild-type and TLR-7KO mice. Hy- not present in serum, and the induced IgG anti-HBc Ab response brid-WHcAg particles containing ssRNA were significantly more is lower and delayed as compared with wild-type CBA mice. Nev- immunogenic and elicited IgG2a/2b-predominant anti-WHc and ertheless, a secondary IgG anti-HBc Ab response occurs after anti-NANP and anti-NVDP Ab responses as compared with ss- boosting in CBA/N (xid) mice, although less efficient than the

RNA-negative hybrid-WHcAg particles in wild-type mice and wild-type response (Fig. 5). Furthermore, truncated HBcAg149, equivalent Ab responses in TLR-7KO mice. The finding that anti- which lacks encapsidated ssRNA, is still a relatively strong im- insert as well as anti-WHc Ab titers and IgG isotype distribution munogen in the absence of TLR-7 signaling, and TLR-7KO mice were affected by the presence of ssRNA within hybrid-WHcAg are still capable of producing anti-HBc Abs (Fig. 10). particles indicates that all three B cell specificities are regulated by The suggestions that HBcAg protein binding to TLR-2 (9) or

TLR-7 signaling. It appears that hybrid-WHcAg particles are ex- full-length HBcAg183 binding to cell membrane HS (9, 10), or the cellent delivery vehicles for nucleic acid TLR ligands because the activity of contaminating LPS may contribute to the enhanced im- ssRNA is protected from nucleases within the WHcAg capsid and munogenicity of the HBcAg in vivo (11, 12) could not be sub- is delivered intracellularly to the APC, in this case, Ag-specific B stantiated in this study (see Figs. 8 and 9). These previous sug- cells. The ssRNA present in hybrid-WHcAg can also be removed gestions were based solely on in vitro systems, whereas the present by RNase treatment and replaced with other TLR ligands, such as study relied heavily on evaluation of in vivo immune responses to CpG, a TLR-9 ligand (46). The adverse systemic effects reported the HBcAg. For example, in vitro IgM, anti-HBc production re- for TLR ligands such as CpG mixed with Ag as an adjuvant should quires three signals, as follows: BCR, cognate Th, and TLR-7; and

not be problematic because TLR ligands encapsidated within hy- LPS signaling through TLR-4 can substitute for the TLR-7 signal. Downloaded from brid-WHcAg particles are present in relatively low amounts and However, the amount of LPS present even in a highly contami- are delivered intracellularly to Ag-specific B cells or other APCs. nated HBcAg preparation had no significant effect on anti-HBc Ab Given the spatial segregation between the BCR on the plasma production in vivo (Fig. 9). Similarly, we observed nonspecific ␾ membrane and both TLR-7 and TLR-9, which are endosomal re- activation of DC/M in the presence of full-length HBcAg183 in ceptors, it has not been clear whether enhanced B cell responses to vitro at relatively high concentrations (Ն1.0 ␮g/ml), which may be

ssRNA- or dsDNA-containing Ag are achieved by synergistic mediated by HBcAg C-terminal binding to cell membranes (9, 52) http://www.jimmunol.org/ BCR-TLR coligation and/or whether coligation occurred in the (data not shown). However, the in vivo relevance is not apparent ␾ same cellular compartment. However, a recent study clarified this because DC/M do not efficiently present exogenous HBcAg183 to point by demonstrating that as internalized BCR-Ag traffics to au- resting or primed CD4ϩ T cells; and, in the absence of TLR-7

tophagosomes, it continues to signal through a phospholipase D- signaling, full-length HBcAg183 and truncated HBcAg149 are dependent pathway to recruit TLR-9-containing endosomes to au- equally immunogenic. Therefore, the particulate structure of the tophagosomes, where they colocalize with BCR-Ag complexes HBcAg characterized by the optimal spacing of the antigenic loops (47). The recruitment of TLR-9 to BCR-containing compartments at the tip of the capsid spikes, which are capable of cross-linking permits TLR-9 to sample BCR-bound Ag for the presence of its BCRs, together with efficient CD4ϩ T cell recognition, explains ligand (47). Although not experimentally demonstrated, it is likely the efficient adaptive humoral response to HBcAg, which can cer- by guest on September 24, 2021 that the synergistic interaction between BCRs and TLR-9 also oc- tainly be enhanced by the innate immune mechanisms described in curs between BCRs and TLR-7 because coligation of BCR and this study. TLR-7 within B cells has been described (48) and TLR-7 and TLR-9 are both localized in endosomes. Acknowledgments Although the relevance of TLR ligands encapsidated within hy- We gratefully acknowledge Francis V. Chisari (Scripps Research Institute, brid-HBcAg/WHcAg particles used for vaccine purposes is clear, La Jolla, CA) and members of his laboratory, especially Alana Althage, the relevance of the viral RNA/DNA incorporated in HBcAg to Masanori Isogawa, and Holly Maier, for scientific discussions, excellent TLR signaling in a natural HBV infection is less obvious. The help, and technical assistance. We also thank Melissa Grace for preparation of the manuscript. HBV has been characterized as a stealth virus in terms of innate immunity because in the liver of acutely HBV-infected chimpan- Disclosures zees, HBV does not induce an intrahepatic innate immune re- The authors have no financial conflict of interest. sponse during the lag phase of infection or the log phase of viral spread (49). It was suggested that HBV RNA/DNA species may be References shielded from recognition by TLR or other nucleic acid sensors 1. Milich, D. R., and A. McLachlan. 1986. The nucleocapsid of hepatitis B virus is because HBV replication occurs within HBcAg particles (49). both a T-cell-independent and a T-cell-dependent antigen. Science 234: 1398–1401. However, this would not preclude TLR signaling in APCs during 2. Milich, D. R., A. McLachlan, S. Stahl, P. Wingfield, G. B. Thornton, processing of HBcAg, which may enhance both innate and adap- J. L. Hughes, and J. E. Jones. 1988. Comparative immunogenicity of hepatitis B tive immune responses to the HBcAg during a natural infection. virus core and E . J. Immunol. 141: 3617–3624. 3. Milich, D. R., D. L. Peterson, F. Schodel, J. E. Jones, and J. L. Hughes. 1995. Furthermore, recent studies of very early acute HBV infections in Preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells humans indicate early development of NK and NT cell responses, is epitope and major histocompatibility complex dependent. J. Virol. 69: 2776–2785. suggesting that the innate immune system is able to sense HBV 4. Milich, D. R., A. McLachlan, G. B. Thornton, and J. L. Hughes. 1987. Antibody infection (50, 51). production to the nucleocapsid and envelope of the hepatitis B virus primed by The ability of the HBcAg to interact with the innate immune a single synthetic T cell site. Nature 329: 547–549. 5. Schodel, F., A. M. Moriarty, D. L. Peterson, J. A. Zheng, J. L. Hughes, H. Will, system was demonstrated by the preferential binding and presen- D. J. Leturcq, J. S. McGee, and D. R. Milich. 1992. The position of heterologous ϩ tation of HBcAg to CD4 T cells by B1a and B1b APCs and epitopes inserted in hepatitis B virus core particles determines their immunoge- TLR-7 signaling within B cell APCs. These interactions serve to nicity. J. Virol. 66: 106–114. 6. Milich, D. R., M. Chen, F. Schodel, D. L. Peterson, J. E. Jones, and J. L. Hughes. bridge the innate and adaptive immune responses to the HBcAg. 1997. Role of B cells in antigen presentation of the hepatitis B core. Proc. Natl. However, it is important to emphasize that these innate immune Acad. Sci. USA 94: 14648–14653. 7. Belnap, D. M., N. R. Watts, J. F. Conway, N. Cheng, S. J. Stahl, P. T. Wingfield, interactions enhance, but are not necessary for an adaptive immune and A. C. Steven. 2003. Diversity of core antigen epitopes of hepatitis B virus. response to the HBcAg. For example, in CBA/N (xid) mice, which Proc. Natl. Acad. Sci. USA 100: 10884–10889. The Journal of Immunology 6681

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