Silencing an immunodominant epitope of hepatitis B surface antigen reveals an alternative repertoire of CD8 T cell epitopes of this viral antigen

Vaccine 28 (2010) 114–119

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Silencing an immunodominant epitope of hepatitis B surface antigen reveals an alternative repertoire of CD8 T cell epitopes of this viral antigen Andreas Wieland 1 , Petra Riedl 1 , Jörg Reimann, Reinhold Schirmbeck ∗ Department of Internal Medicine I, University Hospital of Ulm, Ulm, Germany

a r t i c l e

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Article history: Received 9 June 2009 Received in revised form 28 August 2009 Accepted 23 September 2009 Available online 8 October 2009 Keywords: Immunodominance CD8 T cells Hepatitis B virus

a b s t r a c t Immunodominance hierarchies operating in immune responses to viral antigens limit the diversity of the elicited T cell responses. The Ld /S28–39 -restricted CD8 T cell response to the hepatitis B surface antigen (HBsAg or S) prevents copriming of Dd - and Kb -restricted CD8 T cell responses. We exchanged L to V at position S39 of HBsAg to construct mutant SL39V . Comparable levels of wild-type S and mutant SL39V were produced by transiently transfected cells, and mice immunized with the pCI/S and pCI/SL39V DNA vaccines showed comparable serum antibody responses to HBsAg. The pCI/S but not pCI/SL39V DNA vaccination induced Ld /S28–39 -specific CD8 T cell responses. However, the pCI/SL39V DNA vaccine efficiently primed CD8 T cell responses to the subdominant Dd - and Kb -restricted epitopes, confirming the immunosuppressive phenotype of the Ld /S28–39 -specific CD8 T cell response. A single point mutation within the HBsAg can hence completely silence a ‘dominant’ CD8 T cell response thereby facilitating priming of a multispecific repertoire of suppressed, ‘subdominant’ epitopes. The data have practical implications for understanding HBV-specific CD8 T cell responses and for the design of novel vaccination strategies. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction CD8 T cells recognize antigenic peptides (or epitopes) presented on the surface of antigen-presenting cells (APC) by major histocompatibility (MHC) class I molecules [1,2]. Because CD8 T cells recognize and eliminate cells infected by intracellular pathogens, the specific and efficient stimulation of CD8 T cells is a priority in many vaccine designs [3]. Priming of a broad repertoire of pathogen-specific CD8 T cell responses is expected to enhance the efficacy of their specific control of infection. However, the introduction of diverse antigenic information into a vaccine encounters the risk of establishing immunodominance hierarchies that can strikingly limit the immunogenicity of many codelivered, subdominant epitopes [4–6]. Immunodominance is affected by many different factors that govern antigen presentation and T cell activation, e.g., antigen processing, competition of antigenic peptides for MHC class I-binding, competition between responding T cells, competition of T cells for APC [7–16]. Immunodominance operates in many T cell responses to viral or tumour-specific antigens, e.g., during the different phases of acute and persistent hepatitis B virus (HBV) infection [17]. Mul-

∗ Corresponding author at: University of Ulm, Department of Internal Medicine I, Albert Einstein Allee 23, 89081 Ulm, Germany. Tel.: +49 0731 500 44685; fax: +49 0731 500 40294. E-mail address: [email protected] (R. Schirmbeck). 1 Theses authors contributed equally to this work. 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.09.096

tispecific CD8 T cell responses against HBV antigens have been successfully induced in different mouse strains [18,19]. Using Ld negative BALB/cdm2 and Ld -positive BALB/c mice, we previously showed that the immunodominant, Ld /S28–39 -specific CD8 T cell response to the 226-residue hepatitis B surface antigen (HBsAg, S) [20], elicited by a DNA-based vaccine, suppresses copriming of CD8 T cells specific to a Dd -restricted (S201–209 ) HBsAg epitope [21,22]. Moreover, the Ld /S28–39 -specific CD8 T cell response downmodulated Kb -restricted T cell responses to HBsAg in F1dxb (BALB/c × B6) mice [22]. This dominant CD8 T cell response hence strikingly limits the diversity of HBsAg-specific CD8 T cell immunity. Here, we used a simple DNA vaccination approach [23] to study the induction of multispecific CD8 T cell responses to HBsAg in mice. We generated a mutant HBsAg (SL39V ) with a single amino acid exchange (L to V) in the anchor position S39 of the Ld /S28–39 epitope [24], and compared the induction of CD8 T cell responses by pCI/S (encoding wild-type HBsAg) and pCI/SL39V (encoding mutant HBsAg) in mouse strains that express (BALB/c; BALB/c × B6) or do not express (BALB/cdm2 ; B6) the Ld -molecule. 2. Materials and methods 2.1. Mice C57BL/6 (B6; expressing MHC class I Kb - and Db -molecules) and BALB/c mice (expressing MHC class I Ld -, Kd - and Dd molecules) were obtained from Janvier (Le Genets-St-Isle; France).

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The Ld- BALB/cdm2 mouse line (expressing MHC class I Kd - and Dd -molecules) was obtained from the Jackson Laboratories (Bar Harbor, Maine). Where indicated we used F1dxb (BALB/c × B6) and Ld- F1dxb (BALB/cdm2 × B6) mice in our vaccination studies. Mice were bred and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). Male and female mice were used at 6–8 weeks of age. All studies were conducted after institutional board approval in accordance with the German Federal Animal Protection Law. 2.2. Plasmids The construction of the pCI/S vector has been described previously [25]. We exchanged the sequence encoding the L at position S39 of HBsAg to V by PCR. 2.3. Characterization of antigen expression HEK cells were transiently transfected with the indicated plasmid DNAs using the calcium phosphate method. Cells were labelled with 35 S-methionine between 36 and 48 h post transfection and cell culture supernatants were collected, diluted with pH 8.0 lysis buffer (100 mM NaCl, 0.5% NP40 and 50 mM Tris–hydrochloride) supplemented with the protease inhibitors, leupeptin and aprotinin, and precipitated with a polyclonal rabbit anti-HBsAg serum and protein A-sepharose. Precipitates were washed and recovered from protein A-sepharose with pH 6.8 elution buffer (1.5% SDS, 5% mercaptoethanol, and 7 mM Tris–hydrochloride). The samples were then processed for SDS-PAGE and subsequent gel fluorography. 2.4. DNA immunization Plasmids were produced in E. coli and purified by Plasmid Factory GmbH (Bielefeld, Germany). Mice were injected with 100 ␮g plasmid DNA into tibialis anterior muscles. 2.5. Determination of specific CD8 T cells by flow cytometry (FCM) Spleen cells (5 × 105 /100 ␮l) were incubated for 5 h in Ultra Culture medium with 5 ␮g/ml of the indicated peptides in the presence of brefeldin A (5 ␮g/ml) (cat. no. 15870; Sigma, Taufkirchen, Germany). Cells were harvested, washed, and surface stained with PE-conjugated anti-CD8 antibody (cat. no. 553033; Biosciences, Heidelberg, Germany). Surface stained cells were fixed with 2% paraformaldehyde in PBS. Fixed cells were resuspended in permeabilization buffer (HBSS, 0.5% BSA, 0.5% saponin, 0.05% sodium azide), stained with FITC-conjugated anti-IFN␥ antibody (cat. no. 554411; BD Biosciences, Heidelberg, Germany) for 30 min at room temperature, and washed twice in permeabilization buffer. Stained cells were resuspended in PBS-BSA buffer (PBS supplemented with 0.3% (w/v) BSA and 0.1% (w/v) sodium azide). Frequencies of IFN␥+ CD8 T cells were determined by FCM analyses. For some of the analyzed epitopes specific tetramers or pentamers are commercially available: Ld /S28–39 -specific pentamers (Proimmune, Oxford, UK) and Kb /S190–197 -specific tetramers (Beckman-Coulter, Marseille, France). Determination of CD8 T cell responses with these reagents has been described [26]. 2.6. Statistics The data were analyzed with GraphPad PRISM software, version 4.0 (GraphPad Software, San Diego, CA). The statistical significance of differences in the mean CD8 T cell frequencies between groups was determined with the unpaired Student’s t-test. A value of (*) P < 0.05 was considered significant.

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3. Results 3.1. Construction and expression of HBsAg with the SL39V mutation The small, 226-residue HBsAg contains four (Ld -, Dd - and Kb restricted) epitopes specifically recognized by CD8 T cells from H-2d and H-2b mice listed in Fig. 1A [20,21,27,28]. The 12-residue Ld /S28–39 epitope (IPQSLDSWWTSL) contains the Ld -binding motif [20,21], i.e., a P at the anchor position P2 and a L at the COOHterminal anchor position [24]. Similarly, long CD8 T cell epitopes have been described [29]. We introduced the single, conservative amino acid exchange L to V into the COOH-terminal anchor position S39 to generate the HBsAg mutant SL39V (Fig. 1A). This sequence was inserted into the pCI expression vector to generate the pCI/SL39V DNA vaccine. Transient transfection of HEK cells revealed that comparable levels of secreted small HBsAg particles were produced from the pCI/S and pCI/SL39V expression constructs (Fig. 1B). BALB/c mice immunized with these constructs developed comparable levels of conformation-specific, anti-HBsAg serum antibodies (Fig. 1C). The single amino acid exchange thus did not alter expression, stability, conformation or immunogenicity (for B cells) of HBsAg. 3.2. Vaccination with pCI/SL39V DNA does not prime a Ld /S28–39 -specific CD8 T cell response A single intramuscular injection of the pCI/S plasmid DNA into BALB/c mice efficiently induced a Ld /S28–39 -specific CD8 T cell response readily detectable by either staining with Ld /S28–39 pentamers or by an IFN␥ response specifically inducible by a 5 h ex vivo restimulation with antigenic S28–39 peptide (Fig. 2A and B). Pentamer+ CD8 T cell numbers were usually 2–3-fold higher than the number of CD8 T cells showing specifically inducible IFN␥+ expression because only a subset of the specific CD8 T cells could be specifically induced to produce this cytokine during the short ex vivo restimulation with antigenic peptide. Ld /S28–39 -specific CD8 T cell responses peaked at day 12 post vaccination (Fig. 2B). In contrast, vaccination with pCI/SL39V plasmid DNA did not prime Ld /S28–39 -specific CD8 T cells (Fig. 2A and C). Neither pentamer staining, nor specific ex vivo restimulation of primed splenocytes with the S28–39(L39V) peptide revealed evidence for priming of a Ld /S28–39 -specific CD8 T cell response to the mutant SL39V antigen (Fig. 2A and C). Cross-reactive recognition of the mutant S28–39(L39V) peptide by Ld /S28–39 -specific CD8 T cells (primed by wt S antigen) was not or only barely detectable (Fig. 2C). The L39V mutation of HBsAg thus destroyed the antigenicity of its Ld -binding S28–39 epitope. 3.3. The Ld /S28–39 -specific CD8 T cell response to HBsAg is dominant We tested if the deletion of the Ld /S28–39 -specific CD8 T cell response impairs or enhances CD8 T cell responses to the other HBsAg epitopes primed by the same vaccine. BALB/c mice were immunized with either the pCI/S or the pCI/SL39V DNA vaccines and their specific responses to the Ld /S28–39 or Dd /S201–209 epitopes of HBsAg were read out 12 days post priming (Fig. 3A). The pCI/S vaccine efficiently primed Ld /S28–39 -specific CD8 T cells but did not (or very inefficiently) prime responses to the Dd -binding S201–209 epitope (Fig. 3A, group 2). In contrast, high numbers of Dd /S201–209 - but no Ld /S28–39 -specific CD8 T cells were found in pCI/SL39V -vaccinated mice (Fig. 3A, group 3). 8–12-Fold higher numbers of Dd /S201–209 -specific CD8 T cells were found in pCI/SL39V as compared to pCI/S vaccinated mice (Fig. 3A, groups 2 and 3). Simi-

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Fig. 1. Expression of a mutant HBsAg. (A) Schematic presentation of the 226-residue HBsAg and the four well-defined MHC class I-restricted epitopes. A pCI/SL39V vector was generated by PCR technique. The mutant SL39V contains a single acid exchange (L to V) at position S39. Plasmids were produced in E. coli and purified by PlasmidFactory GmbH (Bielefeld, Germany). (B) HEK293 cells were transiently transfected with pCI/S (lane 1) and pCI/SL39V (lane 2). Cells were labeled with 35 S-methionine and cell supernatants were immunoprecipitated for HBsAg. Samples were analyzed by SDS-PAGE and fluorography of the gels. The positions of the glycosylated (gp27) and non-glycosylated (p24) HBsAg bands are indicated. (C) BALB/c mice (five mice per group) were immunized with 100 ␮g with pCI/S (group 1) and pCI/SL39V DNA (group 2) (injected into both tibialis anterior muscles). Four weeks post vaccination serum samples were analyzed for HBsAg-specific antibodies using a micro-particle enzyme immunoassay (IMX AUSAB kit; Abbott Diagnostics) following the manufacturer’s instructions. Values are presented as mIU/ml. The statistical significance of differences between the two groups was determined by the unpaired Student’s t-test. ns, not significant.

larly, the pCI/S vaccine efficiently primed Ld /S28–39 - (but only barely detectable Dd /S201–209 -, Kb /S190–197 - and Kb /S208–215 -) specific CD8 T cell responses in F1dxb (BALB/c × B6) mice (Fig. 4, group 1) while high numbers of Dd /S201–209 -, Kb /S190–197 - and Kb /S208–215 -specific CD8 T cells were induced by the pCI/SL39V DNA vaccine (Fig. 4, group 2). A similar pattern of specific CD8 T cell numbers was found in spleens and livers of immunized mice at days 12–21 post vaccination (data not shown). Notably, the pCI/S and the pCI/SL39V

vaccine induced comparable Dd /S201–209 - and Kb /S190–197 -specific CD8 T cell responses in Ld -negative BALB/cdm2 and B6 mice (Fig. 3B and C). Hence, the dominant Ld -restricted CD8 T cell response to HBsAg downregulates subdominant CD8 T cell responses to other epitopes of this antigen. Inactivation of this dominant, Ld /S28–39 specific CD8 T cell response can thus reveal alternative responses to subdominant epitopes of HBsAg extending our previous findings [22].

Fig. 2. Immunogenicity of recombinant vaccines. (A) BALB/c mice (four mice per group) were immunized with 100 ␮g pCI, pCI/S, or pCI/SL39V DNA. 12 days after injection epitope-specific CD8 T cell responses were determined by Ld /S28–39 -specific pentamer (Proimmune, Oxford, UK) staining. The mean % of Ld /S28–39 pentamer+ CD8 T cells in the splenic CD8 T cell population (±SD) of a representative experiment (out of 3 independent experiments performed) are shown. The statistical significance of differences between groups was determined by the unpaired Student’s t-test. *P < 0.05. (B) Kinetics of CD8 T cell responses primed in BALB/c mice by pCI/S DNA. Mice were immunized once i.m. with 100 ␮g pCI/S. At the indicated time points post vaccination, splenic Ld /S28–39 -specific CD8 T cell numbers were determined by specific ex vivo stimulation with the antigenic Ld /S28–39 peptide (and an irrelevant Ld -binding control peptide) followed by determination of IFN␥+ CD8 T cell frequencies. The mean numbers of IFN␥+ CD8 T cells per 105 splenic CD8 T cells (±SD) are shown. (C) BALB/c mice (four mice per group) were immunized once i.m. with 100 ␮g pCI/S or pCI/SL39V DNA. 12 days post vaccination, splenic T cells were stimulated ex vivo with the Ld /S28–39 , Ld /S28–39/L39V and a control peptide, followed by determination of IFN␥+ CD8 T cell frequencies as described above.

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Fig. 3. Characterization of HBsAg-specific CD8 T cell responses in different mouse strains. BALB/c (A), BALB/cdm2 (B) and B6 (C) mice (four mice per group) were immunized with 100 ␮g of the indicated DNAs. Spleen cells obtained 12 days post vaccination were either stimulated ex vivo for 5 h with the indicated peptides (A and B) or directly stained with Kb /S190–197 tetramers (C) followed by determination of IFN␥+ CD8 T cell frequencies or tetramer+ CD8 T cells. The statistical significance of differences between groups was determined by the unpaired Student’s t-test. *P < 0.05; ns, not significant.

Fig. 4. Characterization of immunodominance hierarchies. F1dxb (BALB/c × B6) mice (four mice per group) were immunized with 100 ␮g of the indicated DNAs. DNA was injected into either the right or left tibialis anterior muscle. Spleen cells obtained 12 days post vaccination were either stimulated ex vivo for 5 h with the indicated peptides followed by determination of IFN␥+ CD8 T cell frequencies. The statistical significance of differences between groups was determined by the unpaired Student’s t-test. *P < 0.05; ns, not significant.

3.4. Delivery of different HBsAg-specific vaccines to different sites induced a multispecific CD8 T cell response to dominant and subdominant epitopes Differences in antigen expression or processing are unlikely to explain the immunodominance hierarchy within the HBsAg. Priming a CD8 T cell response to the dominant Ld /S28–39 epitope by injecting the pCI/S DNA vaccine into the right leg of F1dxb (BALB/c × B6) mice did not suppress priming of CD8 T cell responses to the subdominant Dd /S201–209 , Kb /S190–197 and Kb /S208–215 epitopes by injecting the pCI/SL39V DNA vaccine into the left leg (Fig. 4). We have thus evidence for a local, Ld /S28–39 -specific immune suppression mechanism. Notably, immunization of mice with pCI/S and pCI/SL39V DNA into different sites efficiently primed CD8 T cells specific for dominant (Ld /S28–39 ) and subdominant (Dd /S201–209 , Kb /S190–197 and Kb /S208–215 ) epitopes (Fig. 4, group 3). Thus, this simple immunization strategy induced the broadest repertoire of CD8 T cells in F1dxb (BALB/c × B6) mice. 4. Discussion As the establishment of robust, multispecific CD8 T cell responses is a key priority in novel vaccine designs for the control of viruses, evaluating immunodominance phenomena will always be a major task. We used a DNA-based vaccination approach to demonstrate under well-controlled experimental conditions (i.e., inactivation/deletion of an immunodominant Ld -restricted epitope from the HBsAg; mouse strains that express or do not express the

Ld -molecule) that Ld /S28–39 -mediated CD8 T cells suppressed the multispecific CD8 T cell responses to Dd - and Kb -restricted epitopes. A single amino acid exchange L to V in the immunodominant Ld /S28–39 epitope (i.e., mutant SL39V ) did not alter HBsAg-specific expression and its immunogenicity for B cells, but had a strong impact on priming multispecific CD8 T cell responses. Single residue exchanges in the sequence of antigenic, MHC class I-binding epitopes can drastically change their immunogenicity for CD8 T cells and play a role in the outcome of CTL escape variants of certain viruses [30,31]. We here showed that a single point mutation within the HBsAg can completely silence dominant, Ld /S28–39 -specific CD8 T cells thereby facilitating priming of a multispecific CD8 T cell response to subdominant epitopes. Similarly, single amino acid exchanges within different, HBsAg-specific epitopes (or in the epitope-flanking regions) either abrogated or enhanced their immunogenicity for CD8 T cells. As a consequence, three natural variants of the HBV surface antigen induced distinct patterns of CD8 T cell responses [28,32]. The cellular and molecular mechanisms mediating this immune suppression are unknown. We previously showed that Ld restricted epitopes from two unrelated viruses, i.e., murine CMV and lymphocytic choriomeningitis virus, induced immunodominance hierarchies in multiepitope vaccines [33]. This implied that immunodominance is a prominent property of Ld -restricted CD8 T cell responses. Ld -mediated immunodominance could be overcome at least partially by expressing multiepitope vaccines as chimeric antigens that endogenously capture constitutively expressed stress proteins (Hsp73) [33]. This may be related to specific effects of Hsp73. Hsp73-binding efficiently enhance and stablize antigen expression, facilitate cross-priming, and introduce antigens into alternative processing pathways [27,34]. These data suggested that Hsp73-bound protein antigens produced in situ by DNA vaccines display exceptional immunogenicity and can (at least partially) escape suppressive Ld -mediated immunoregulation. However, suppression of T cell responses to subdominant HBsAg epitopes is not limited to the dominant Ld /S28–39 epitope. We have shown that dominant H-2b -restricted, adenovirus-specific CD8 T cell responses suppress Dd /S201–209 -specific CD8 T cells because a recombinant, HBsAg-expressing Ad5 vector efficiently elicited Dd /S201–209 specific CD8 T cell responses in homozygous Balb/cdm2 mice but not in heterozygous Ld- F1dxb (Balb/cdm2 × B6) mice [26]. Immunization of mice with HBsAg-expressing pCI/S plasmid DNA into the left leg and an ‘empty’ Ad5 into the right leg did not impair priming of Dd /S201–209 -specific CD8 T cell responses by pCI/S DNA vaccines [26]. There is thus strong evidence for a local, Ad5-specific immune suppression mechanism. Similarly, delivering the same antigen (HBsAg) by Ad- or DNA-based vectors to HLA-A*0201 transgenic mice (that express only one transgene-encoded HLA-A*0201 MHC

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class I molecule) [35] resulted in a striking difference in the HBsAgspecific CD8 T cell responses. Whereas a multispecific immune response to three well-defined HLA-A*0201-restricted HBsAg epitopes (S20–28 , S185–194 and S208–216 ) was induced by pCI/S the Ad/S vector elicited CD8 T cells only to the S20–28 epitope [26,32]. This epitope has the highest binding affinity to HLA-A*0201 molecules that may favour its presentation to CD8 T cells [7,36]. It is unknown if HLA-A*0201-restricted Ad epitopes directly compete with the presentation of S185–194 and S208–216 epitopes. Notably, a highaffine, HLA-A*0201-restricted Flu58–66 epitope of the influenza A matrix protein coexpressed in a multispecific, HBsAg-encoding vaccine efficiently suppressed HLA-A*0201-restricted CD8 T cell responses to the subdominant (S185–194 , S208–216 ) but not dominant (S20–28 ) epitopes in HLA-A*0201 transgenic mice [37]. Interference between responses to individual epitopes presented by (the same or different) MHC class I molecules hence strikingly limits priming of HBsAg-specific responses with extensive repertoire diversity. The interference between epitopes and CD8 T cell responses may be more complex in the natural HBV infection, because more antigens (core/precore, preS2, preS1, S, polymerase, X-antigen) are expressed and more antigenic epitopes compete for MHC class Ibinding/presentation. We previously primed CD8 T cell responses to multidomain antigens (with CD8 T cell-defined epitopes of the HBsAg, core (C) and polymerase (Pol) proteins). Interestingly, priming of mono- or multispecific, HLA-A*0201- or Kb -restricted CD8 T cell responses by these DNA vaccines differed [38]. Multidomain vaccines encoding five well-defined, HLA-A*0201-restricted epitopes (S20–28 , S185–194 , S208–216 , C18–27 and Pol803–811 ) induced CD8 T cell responses to the immunodominant (S20–28 , C18–27 and Pol803–811 ) but not (or barely) to the subdominant S185–194 and S208–216 epitopes [38]. Moreover, a Kb /S190–197 -specific CD8 T cell response did not allow priming of a subdominant Kb /C93–100 specific CD8 T cell response in B6 mice immunized with the multidomain vaccine. Thus, immunological hierarchies also operate in CD8 T cell responses between different antigens of the same virus. Characterization of T cell responses able to exert anti-viral functions is a complex problem during the different phases of acute and persistent hepatitis B virus (HBV) infection [17,39,40]. A small number of HLA-A*0201-restricted CD8 T cell specificities (e.g., specific for the C18–27 epitope) were found to quantitatively dominate the CD8 T cell response in individual HLA-A*0201+ patients with acute hepatitis [41–43], indicating that HBV infection induces only a limited repertoire of specific CD8 T cell responses (for a recent review see [17]). However, the hierarchies of CD8 T cell responses in individual patients is still incomplete because there is no information available about competition among HBV-specific epitopes restricted by different human HLA-class I molecules [17]. Induction of multispecific CD8 T cell responses is an incompletely understood phenomenon. Little is known about how immunodominant and subdominant determinants are distinguished by the CD8 T cell system and whether the different CD8 T cell specificities (specific for dominant or subdominant epitopes) exerted protective anti-viral or anti-tumour functions in vivo. Immunodominant CD8 T cell responses do or do not correlate with efficient protection from viral infection or tumour growth [22,44–46]. We previously showed that a subdominant (i.e., easily suppressed) Kb /C93–100 -specific CD8 T cell response was efficiently elicited in 1.4HBV-Smut tg B6 mice (that harbour a replicating HBV genome that produces HBV surface, core and precore antigen in the liver). Kb /C93–100 -specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-Smut tg B6 mice and transiently suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific anti-viral effects to HBV-expressing hepatocytes. Induction of a multispecific CD8 T cell repertoire to dominant and subdom-

inant epitopes by vaccination is thus an attractive option for the specific control of chronic viral infections. Here we provided a simple method to induce multispecific CD8 T cell responses. Delivery of different vaccines (with or without a dominant epitope) to different sites (see Fig. 4) or at different times (data not shown) induced the broadest spectrum of antigen-specific CD8 T cells. Considering that CD8 T cell responses vary in individual vaccine-recipients or HBVinfected patients (e.g., depending on their different composition of HLA-class I molecules), our data suggest that at least the known immunodominant HBV epitopes should be delivered on separate vaccines. Acknowledgements We greatly appreciate the expert technical assistance of Katrin Ölberger and Claudia Heilig. This work was supported by a grant from the Deutsche Forschungsgemeinschaft DFG SCHI 505/2-4 and DFG SCHI 505/4-1 to R.S. References [1] Shastri N, Cardinaud S, Schwab SR, Serwold T, Kunisawa J. All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigen-processing pathway. Immunol Rev 2005;207:31–41. [2] Cresswell P, Ackerman AL, Giodini A, Peaper DR, Wearsch PA. Mechanisms of MHC class I-restricted antigen processing and cross-presentation. Immunol Rev 2005;207:145–57. [3] Yewdell JW, Haeryfar SM. Understanding presentation of viral antigens to CD8+ T cells in vivo: the key to rational vaccine design. Annu Rev Immunol 2005;23:651–82. [4] Yewdell JW, Bennink JR. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 1999;17:51–88. [5] Kedl RM, Kappler JW, Marrack P. Epitope dominance, competition and T cell affinity maturation. Curr Opin Immunol 2003;15(1):120–7. [6] Yewdell JW. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity 2006;25(4):533–43. [7] Sette A, Vitiello A, Reherman B, Fowler P, Nayersina R, Kast WM, et al. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J Immunol 1994;153(12):5586–92. [8] Chen W, Anton LC, Bennink JR, Yewdell JW. Dissecting the multifactorial causes of immunodominance in class I-restricted T cell responses to viruses. Immunity 2000;12(1):83–93. [9] Choi EY, Christianson GJ, Yoshimura Y, Sproule J, Jung N, Joyce S, et al. Immunodominance of H60 is caused by an abnormally high precursor T cell pool directed against its unique minor histocompatibility antigen peptide. Immunity 2002;17(5):593–603. [10] Tourdot S, Gould KG. Competition between MHC class I alleles for cell surface expression alters CTL responses to influenza A virus. J Immunol 2002;169(10):5615–21. [11] Basler M, Youhnovski N, Van Den BM, Przybylski M, Groettrup M. Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus. J Immunol 2004;173(6):3925–34. [12] Yewdell JW, Del Val M. Immunodominance in TCD8+ responses to viruses: cell biology, cellular immunology, and mathematical models. Immunity 2004;21(2):149–53. [13] Price DA, Brenchley JM, Ruff LE, Betts R, Hill BJ, Roederer M, et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J Exp Med 2005;202(10):1349–61. [14] La Gruta NL, Kedzierska K, Pang K, Webby R, Davenport M, Chen W, et al. A virusspecific CD8+ T cell immunodominance hierarchy determined by antigen dose and precursor frequencies. Proc Natl Acad Sci USA 2006;103(4):994–9. [15] Rutigliano JA, Ruckwardt TJ, Martin JE, Graham BS. Relative dominance of epitope-specific CD8+ T cell responses in an F1 hybrid mouse model of respiratory syncytial virus infection. Virology 2007;362(2):314–9. [16] Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, et al. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance. Nat Immunol 2009;10(6):636–46. [17] Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. J Gen Virol 2006;87(Pt 6):1439–49. [18] Loirat D, Lemonnier FA, Michel ML. Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization. J Immunol 2000;165(8):4748–55. [19] Depla E, Van der AA, Livingston BD, Crimi C, Allosery K, De B, et al. Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections. J Virol 2008;82(1):435–50. [20] Ishikawa T, Kono D, Chung J, Fowler P, Theofilopoulos A, Kakumu S, et al. Polyclonality and multispecificity of the CTL response to a single viral epitope. J Immunol 1998;161(11):5842–50.

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