A new approach for therapeutic vaccination against chronic HBV infections

Vaccine xxx (xxxx) xxx

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A new approach for therapeutic vaccination against chronic HBV infections Tobias Zahn a,b, Sami Akhras a, Catrina Spengler a, Robin Oliver Murra a, Thomas Holzhauser c, Eberhard Hildt a,b,⇑ a b c

Paul-Ehrlich-Institut, Division of Virology, D-63325 Langen, Germany German Center for Infection Research (DZIF), Gießen-Marburg, Langen, Germany Paul-Ehrlich-Institut, Division of Allergology, D-63325 Langen, Germany

a r t i c l e

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Article history: Received 8 November 2019 Received in revised form 14 February 2020 Accepted 20 February 2020 Available online xxxx Keywords: Virus-like particles Vaccine platform Transdermal vaccination chronic HBV infection Therapeutic vaccination Neutralizing antibodies CTL response TLM-peptide HBcAg capsid Oral vaccination

a b s t r a c t There are currently about 257 million people suffering from chronic HBV infection worldwide. In many cases, an insufficient T cell response is causative for establishment of a chronic infection. To ensure a robust cellular immune response and induction of neutralizing antibodies a novel vaccine platform based on modified cell–permeable HBV capsids was utilized. Cell permeability was achieved by fusion of the membrane–permeable TLM-peptide to HBV core monomers, assembling the capsids. Insertion of a Strep-tagIII into the spike tip domain that protrudes from the capsid surface enables flexible loading with antigens that are fused to streptavidin. In this study, HBV surface antigen-derived PreS1PreS2 domain, fused to monomeric streptavidin, served as cargo antigen. Binding between antigen and capsids was characterized by surface plasmon resonance spectroscopy, electron microscopy and density gradient centrifugation. Confocal immunofluorescence microscopy and in vivo imaging of immunized mice demonstrated membrane permeability of cargo-loaded carriers and spread of antigen over the whole organism. Immunization experiments of mice revealed a robust induction of a specific cellular immune response, leading to destruction of HBV-positive cells and induction of HBV-specific neutralizing antibodies. Membrane permeability of these carriers allows needle-free application of antigen-loaded capsids as evidenced by induction of an HBV-specific CTL response and HBV-specific B cell response after oral or transdermal vaccination. These data indicate that cell–permeable antigen carriers, based on HBV capsids and loaded with HBV antigen, have the capacity to induce a cellular and a neutralizing humoral immune response. In addition, cell permeability of the vaccine platform enables antigen transfer across several cell layers, that could allow oral or transdermal immunization. Ó 2020 Published by Elsevier Ltd.

1. Introduction Approximately 257 million people worldwide are suffering from chronic hepatitis B virus (HBV) infection. Chronic HBV infection represents one of the major causes of liver fibrosis/cirrhosis

Abbreviations: HCC, hepatocellular carcinoma; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; EC, effector cell; TC, target cell; I.P., intraperitoneal; LC, Langerhans cell; TLM, translocation motif; CTL, cytotoxic T lymphocyte; PD-1, programmed cell death 1; DC, dendritic cell; APC, antigen presenting cell; LPS, lipopolysaccharide; mSA, monomeric streptavidin. ⇑ Corresponding author at: Paul-Ehrlich-Institut, Division of Virology, D-63325 Langen, Germany. E-mail address: [email protected] (E. Hildt).

and hepatocellular carcinoma (HCC) [1]. After acute HBV infection, depending on the age of the infected person, up to 20% develop a persistent infection, frequently characterized by chronic inflammation of the liver (hepatitis) [2]. For treatment of chronic active HBV, (PEGylated) interferon-a or nucleoside and nucleotide analogues are instrumental to suppress viral replication. Major problems, however, are represented by episomal covalently closed circular DNA (cccDNA), persisting as a minichromosome with very high stability, and by integrated viral DNA [3]. cccDNA represents a replicative intermediate and stays inside nuclei of infected cells [4]. While the virus itself has almost no direct cytopathic effects, destruction of liver tissue during HBV infection is mainly caused by the immune response. In case of an effective and robust immune response infected hepatocytes are eliminated and the

https://doi.org/10.1016/j.vaccine.2020.02.063 0264-410X/Ó 2020 Published by Elsevier Ltd.

Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063

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infection is cleared. Chronic hepatitis B is frequently associated with permanent inflammation of the liver, due to an insufficient immune response leading to a partial destruction of infected hepatocytes. The remaining, infected cells re-infect hepatocytes generated by liver regeneration – leading to a circle of destruction and regeneration that finally ends up in an imbalance and in fibrosis and cirrhosis. [5,6]. This ineffectiveness in immune response and therefore insufficient clearance of virus in chronic infections is frequently caused by T cell depletion and exhaustion. In some patients, constitutive exposition and excessive existence of viral proteins and peptides, presented to T cells, in combination with various viral immune escape strategies, leads to a lack of maturation of HBV-specific T cells and an inefficient antiviral response [7–9]. In addition, exposure to viral antigens over years of chronicity causes expression of inhibitory receptors, like programmed cell death 1 (PD-1) by T cells, resulting in eventual apoptosis of these cells [8,10,11]. An important additional aspect of hepatitis B chronicity is an accumulative amount of dysfunctional or defective dendritic cells (DCs). Moreover, HBV is apparently inhibiting maturation of DCs by down-regulation of TLR-9 and therefore impaired production of IL-6 and IFNa [12–14]. This decreased DC function affects in turn activation of CD8 + CTLs and CD4 + Th cells. Therefore, improving antigen uptake, processing and presentation on MHC class I/II by antigen presenting cells (APCs) is one of the major strategies for increasing number and functionality of HBV-specific T cells. Virus-like particles (VLP) are highly ordered viral protein structures that can be used as vaccine platforms for delivering antigens. VLPs based on HBV capsids are well-known and structurally characterized vaccine templates [15–20]. Antigens loaded on the surface of VLPs are therefore presented in a highly ordered structure, too. This favors the induction of a robust B cell response [21]. In former studies, HBV capsids as VLPs with a Strep-tagIII inserted into the spike-tip region (aa 79–82) of hepatitis B core proteins (HBcAg) were established [20]. Assembled capsids can subsequently be loaded onto their spike-tip region with antigens fused to streptavidin by streptavidin-Strep-tagIII interaction. For an efficient uptake of capsids and antigens by APCs, the cell permeability-mediating properties of the translocation motif (TLM) were used [20]. TLM is a 12 amino acid amphipathic ahelix that was identified as a part of preS2 domain of hepatitis B surface antigen (HBsAg) [22]. Fusion of TLM to the N-terminus of HBcAg leads to cell-permeable VLPs (TLMcapsids) that can be used for an efficient transfer of nucleic acids, after DNA packaging into capsids, as well as transport of streptavidin-fused proteins into the cytoplasm of cells. Therefore, TLMcapsids represent cellpermeable, universal antigen carriers for an efficient antigen delivery into APCs [20,22–24]. In this study, capacity of cell-permeable TLMcapsids loaded with the PreS1PreS2 domain of HBV surface antigen (HBsAg), to induce a humoral and cellular immune response against HBV, was analyzed. Moreover, the potential of antigen loaded TLMcapsids to enable alternative, non-invasive immunization routes by oral and transdermal antigen delivery was studied.

monomeric streptavidin and the PreS1PreS2 domain (HBV genotype D). 2.2. Protein purification, in vitro assembly of carrier capsids and loading of capsids with mSA_preS1/2 Expression, purification and assembly of TLMcapsids and DTLMcapsids was performed as described in [20]. Cargo antigen mSA_preS1/2 was produced in BL21 DE3 E. coli and found in inclusion bodies. After isolation of inclusion bodies by differential centrifugation, recombinant hexa-his-tagged proteins were enriched by affinity chromatography on a Ni-NTA affinity column using an AEKTA chromatography system (GE Healthcare, Amersham, UK). Proteins were refolded by a linear gradient against a native buffer on the column and finally eluted by imidazole. For details, see supplementary section. After affinity chromatographic purification of TLMcore, DTLMcore and mSA_preS1/2, samples were cleared from bacterial lipopolysaccharides (LPS) by using Pierce High Capacity Endotoxin Removal Resin (Thermo Fisher Scientific, Karlsruhe, Germany) according to manufacturer’s instructions. To load mSA_preS1/2 onto the surface of carrier capsids by streptavidinStrep-tagIII interaction, both components were incubated in a mass ratio of 1:4 (cargo: capsid), unless stated otherwise, for 1 h at room temperature or overnight for 16 h at 4 °C in PBS, with gentle shaking. 2.3. Sucrose density gradient centrifugation To analyze binding between cargo (mSA_preS1/2) and carrier capsids (TLMcapsids or DTLMcapsids) and to investigate particle formation of in vitro assembled TLMcapsids and self-assembling DTLMcapsids, sucrose density gradient centrifugation was performed. Therefore, 1 ml of mSA_preS1/2 alone, TLMcapsids alone, DTLMcapsids alone or mSA_preS1/2 bound to carrier capsids was loaded on top of a sucrose gradient. The gradient was layered from bottom to top with 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% w/v sucrose in PBS, with a volume of 1 ml each. Tubes were centrifuged for a minimum of 6 h at 41,000 rpm, 4 °C, in a SW41 Ti rotor (Beckman Coulter, Krefeld, Germany). Afterwards, 9 fractions of 1 ml were collected from top to bottom and analyzed via SDS-PAGE (reducing/denaturing conditions) and western blot using anti-HBcAg (MAB16990, Merck Millipore, Darmstadt, Germany) for capsids and anti-LHBs (MA18/7, [26]) for mSA_preS1/2. 2.4. Surface plasmon resonance (SPR) spectroscopy To analyze interactions between TLMcapsids and mSA_preS1/2 or DTLMcapsids and mSA_preS1/2, kinetic binding parameters were determined by using surface plasmon resonance (SPR) spectroscopy with Biacore T200 (GE Healthcare, Freiburg, Germany). We investigated the binding properties between Strep-tagIII on the surface of TLMcapsids/DTLMcapsids and monomeric streptavidin of mSA_preS1/2. For details see supplementary section. 2.5. Microscopy

2. Methods 2.1. Construction of bacterial expression vector pRSET_mSA_preS1_preS2 Construction of bacterial expression vector pRSET_mSA_preS1_preS2 was performed using standard procedures [25]. A detailed description is provided in the supplementary section. The plasmid encodes a fusion protein of an N-terminal hexa-his-tag,

For microscopic, negative stain visualization of TLMcapsids and DTLMcapsids, transmission electron microscopy (TEM) was used. 15 ml of samples were pipetted on a carbon coated, glam discharged formvar grid and immobilized for 5 min at room temperature. Grids were washed twice with 25 ml ddH2O and subsequently incubated with 10 ml of 2% uranylacetate in ddH2O or 2% phosphotungstic acid in ddH2O for 10 s at room temperature. Grids were dried and analyzed by TEM (EM 109, Zeiss, Jena, Germany).

Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063

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Indirect immunofluorescence staining was visualized by confocal laser scanning microscopy (CLSM, CLSM 510 Meta, Carl Zeiss, Jena, Germany) and analyzed by software ZEN 2009. For details see supplementary section. 2.6. Mice For all in vivo and ex vivo experiments C57BL/6N wild-type mice were used. For in vivo imaging BALB/c wild-type mice were used to facilitate visualization. C57/BL6N and BALB/c mice were obtained from Charles River, Freiburg, Germany. For invasive procedures, animals were sedated with isoflurane. Housing of animals was conducted under pathogen-free conditions in the facilities of PaulEhrlich-Institut, Langen, Germany. All animal experiments were performed in accordance with institutional policies and had been approved by the local veterinary authorities of Darmstadt, Germany. The animal testing license number is F107-K5970. 2.7. Immunization of C57BL/6N mice Female C57BL/6N mice (8–10 weeks old) were vaccinated in a two-boost schedule with ten days between prime and first boost and first boost and second boost (all experiments with n = 3 mice). Blood was collected by retro-orbital bleeding every 10 days. 21 days after second boost, mice were sacrificed for organ dissection and final blood collection. Animals were immunized with 10 mg TLMcapsids or DTLMcapsids or 2,5 mg mSA_preS1/2 or the same amounts of proteins in a coupled approach. Protein amounts were determined via spectral photometry using NanoDrop One (Thermo Scientific, Karlsruhe, Germany). Three different routes were tested: Intraperitoneal route with 100 ml injection volume, oral-mucosal route with 15 ml sample volume and transdermal route with 20 ml sample volume. All routes differ only in volume, but not in indicated amounts of proteins. Volumes for oral and transdermal applications where adjusted according to practical feasibility. 2.8. In vitro infection and neutralization experiments HBV was enriched from supernatant of HepAD38 (ATCC CRL12077) by PEG precipitation as described in [27]. Differentiated HepaRG cells [27] were infected with HBV gtD MOI 1 for 16 h at 37 °C in the presence of 4% PEG8000. Afterwards, cells were washed four times with PBS and cultivated for 14 days. For blocking of HBV infections in vitro, sera of TLMcapsids + mSA_preS1/2, DTLMcapsids + mSA_preS1/2, mSA_preS1/2 and PBS intraperitoneal, transdermal and oral immunized mice were preincubated with HBV in HepaRG medium in dilutions of 1:20, 1:50, 1:100 and 1:500 proportional to media for 2 h at 37 °C prior to in vitro infection as described above (all experiments with n = 3, except for transdermal TLMcapsids + mSA_preS1/2n = 1). During 2 weeks of cultivation, supernatants were collected on day 1, 2, 4, 6, 8, 10, 12 and 14 and HBsAg levels were analyzed by HBsAgELISA (Enzygnost, Siemens, Schwalbach, Germany) according to manufacturer’s instructions. 2.9. Elisas and epitope mapping For detection of antibodies against mSA_preS1/2 in sera of TLMcapsids + mSA_preS1/2, DTLMcapsids + mSA_preS1/2, mSA_preS1/2 and PBS I.P., oral and transdermal immunized mice, mSA_preS1/2 was coated onto wells of a 96-well plate and incubated with sera (n = 3 mice). After additional incubation with HRP-conjugated anti-mouse IgG antibodies, tetramethylbenzidine (TMB; Sigma, Seelze, Germany) was added and reaction was stopped with 1 N H2SO4. For details see supplementary section.

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After co-cultivation of lymphocytes and splenocytes of TLMcapsids + mSA_preS1/2, DTLMcapsids + mSA_preS1/2, mSA_preS1/2 and PBS I.P., oral and transdermal immunized mice with HBV–transfected AML12, supernatant was collected and analyzed via IFNc-ELISA (Invitrogen, Thermo Fisher Scientific, Karlsruhe, Germany) according to manufacturer’s instructions. For mapping of sequential stretches of mSA_preS1/2 recognized by antibodies of TLMcapsids + mSA_preS1/2, DTLMcapsids + mSA_ preS1/2, mSA_preS1/2 and PBS I.P immunized mice, in approximation of epitopes, 72 synthetic overlapping peptides of 15 aa in length with an offset of 4 aa between each peptide, representing the sequence of mSA_preS1/2, were spotted as quadruples onto adhesive microscope slides and incubated with sera of mice. For details see supplementary section.

2.10. In vivo imaging To analyze biodistribution of mSA_preS1/2 loaded onto TLMcapsids or on DTLMcapsids, as well as free mSA_preS1/2, TLMcapsids and DTLMcapsids, proteins were labeled with sulfoNHS-Cy7 (Lumiprobe, Hannover, Germany). For details of labeling procedure see supplementary section. Labeled proteins were applied systemically via intraperitoneal route into BALB/c mice. To study the distribution of the labeled proteins fluorescence was measured at 745/800 nm for a period of 6 h with 9 different time points and monitored by in vivo imaging using the IVIS Imaging System 200 (Caliper Life Sciences, Waltham, MA, USA).

2.11. Biodistribution analysis in the liver after I.P. Injection Female, 8–10 weeks old C57BL/6N mice were injected intraperitoneally with TLMcapsids + mSA_preS1/2 (10 mg + 2.5 mg), DTLMcapsids + mSA_preS1/2 (10 mg + 2.5 mg) or PBS in a volume of 100 ml. After 2.5 h mice were sacrificed and a hepatectomy was performed. Liver was frozen in optimal cutting temperature compound (OCT compound) and cutted into 8 mm thick slices. Slices were stained with preS1/2-specific antisera, DAPI and phalloidin-633 and visualized via confocal laser scanning microscopy (Leica TCS SP8, Wetzlar, Germany).

2.12. In vitro killing assay of HBV-transfected AML12 Murine hepatocyte cell line AML12 was transfected with an 1.2xHBV gtD construct for 72 h and co-incubated with lymphocytes (of I.P. and transdermal vaccinated mice) or splenocytes (of oral vaccinated mice) for 24 h in a TC:EC ratio of 1:10 (I.P. and oral groups with n = 3 mice, transdermal n = 1). Isolation of the lymphocytes and splenocytes is described in detail in the supplementary section. Supernatant was collected and analyzed by IFNcELISA. Target cells were visualized using indirect immunofluorescence staining to determine amount of remaining HBV-positive cells after potential cytotoxic effects of HBV-specific CD8+ CTLs. For details see supplementary section.

2.13. Statistical analysis Results were analyzed using one-way ANOVA (GraphPad Prism software) and described as means ± standard deviation (SD). Statistical significances are indicated as stars representing p-values as stated in figure legends.

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3. Results 3.1. Generation of membrane permeable carrier capsids loaded with HBV-specific antigen PreS1PreS2 domain of HBV surface protein was used as an antigen. To couple this antigen onto the surface of either TLMharboring (TLMcapsids) or TLM-lacking (DTLMcapsids) HBV capsids by streptavidin-Strep-tagIII interaction, antigen was produced as a fusion protein with monomeric streptavidin referred to as mSA_preS1/2. Purification of mSA_preS1/2 was performed by NiNTA affinity chromatography as the recombinant protein harbours an N-terminal hexa-his-tag (Fig. S1). Coomassie-stained SDS-PAGE and western blot analysis showed the identity and purity of isolated mSA_preS1/2 (Fig. 1a). TLMcapsids and DTLMcapsids harboring a Strep-tagIII in the spike-tip region (aa 79–82) were produced, purified and assembled as described in [20]. Proper assembly of the antigen carriers was shown by electron microscopy (Fig. 1b). LPS removal from cargo antigen and carrier capsids was performed as described in the methods section. For characterization of binding between the mSA_preS1/2 cargo antigen and carrier capsids sucrose density gradient centrifugation was performed. For coupling, cargo antigen and carrier capsids were mixed in a mass ratio of 1:4. The coupling product was sub-

jected to sucrose density gradient centrifugation. For control experiments uncoupled mSA_preS1/2 and unloaded capsids were used. Fractions of the gradient were analyzed by western blot analysis using the PreS1-specific monoclonal (MA18/7) or a corespecific antiserum (Fig. 1c). The blots show that after coupling of mSA_preS1/2 to carriers, cargo antigen co-migrates with carriers into the gradient, while free mSA_preS1/2 floats on top of the gradient. Co-migration of cargo and carrier indicates that antigen indeed binds to carriers. In addition, differences in size of loaded and unloaded TLMcapsids could be visualized via electron microscopy (Fig. 1d). For a more detailed analysis of this interaction, the dissociation constant (KD) for binding of mSA_preS1/2 to carrier capsids was determined by surface plasmon resonance spectroscopy (SPR). TLMcapsids or DTLMcapsids were immobilized onto a CM5-chip and different concentrations of mSA_preS1/2 were added (7500 mM – 12 mM), to measure KD in a multi cycle kinetic. mSA_preS1/2 binds to both immobilized carrier capsids (TLMcapsids and DTLMcapsids) with a KD of 6 * 10–7 for mSA_preS1/2 binding to TLMcapsids and with a KD of 3.5 * 10 7 for binding of mSA_preS1/2 to DTLMcapsids (Fig. 1e). Taken together, these data indicate that Strep-tag-III insertion into the spike-tip of carrier capsids allows loading of carriers with cargo antigen by a high-affinity binding.

Fig. 1. Purified mSA_preS1/2 can be efficiently loaded on assembled TLMcapsids or DTLMcapsids. (a) Cargo antigen mSA_preS1/2 was enriched via Ni-NTA affinity chromatography under denaturing conditions, with subsequent refolding and elution under native conditions (left). TLMcapsids were purified via streptactin affinity chromatography under native conditions (right). Purity of fractions is shown by Coomassie stained SDS-PAGE and identity by western blot, using anti-HBcAg and anti-LHBs monoclonal antibodies. (b) Transmission electron microscopy of negative stain of TLMcapsids and DTLMcapsids (350 mg/ml) in the presence of 450 mM NaCl (Scale bar: 100 nm). (c) Free mSA_preS1/2 or mSA_preS1/2 bound to TLMcapsids were loaded onto sucrose density gradient for density-dependent separation. Free mSA_preS1/2 floats on top of the gradient; coupling of mSA_preS1/2 to the carrier capsids shifts mSA_preS1/2 from low to high density fractions. (d) mSA_preS1/2 was coupled onto TLMcapsids in a molar ratio of 1:4 and analyzed via EM. Left: TLMcapsids loaded with mSA_preS1/2; right: unloaded TLMcapsids (Scale bar: 100 nm). (e) TLMcapsids or DTLMcapsids were covalently immobilized on CM5-chips and incubated with different concentrations of mSA_preS1/2 (12 nM to 7500 nM) and analyzed by plasmon resonance spectroscopy applying a 1:1 binding model to determine the dissociation constant kD.

Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063

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3.2. Cargo antigen can be transferred across the plasma membrane into cytoplasm by using TLMcapsids as cell-permeable carriers To analyze if coupling of mSA_preS1/2 to cell-permeable carrier capsids enables translocation across cellular membranes, cell permeability assays in HepG2 cells were performed. For this purpose, mSA_preS1/2 was coupled to TLMcapsids. For control experiments, mSA_preS1/2 was coupled to DTLMcapsids or left without carrier capsids. As an additional control, unloaded carrier capsids without cargo were used to address the potential impact of the cargo on membrane permeability. HepG2 cells were incubated with the components described above for 1 h at 37 °C. Internalized proteins were detected by confocal laser scanning microscopy (CLSM) using core- and LHBs-specific antibodies. Confocal immunofluorescence microscopy shows that loading of cargo antigens on TLMcapsids leads to an efficient transfer of antigen into target cells, as reflected by intracellular staining for core and mSA_preS1/2 (Fig. 2). However, if TLM-deficient carrier capsids (DTLMcapsids) were used, no specific intracellular staining was observed – reflecting that no considerable transfer of cargo antigen or carrier into HepG2 cells has occurred. The C-terminal part of PreS2 domain harbors a TLM. In accordance to this, incubation with free mSA_preS1/2 alone showed

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minor signals of internalization. However, coupling of mSA_preS1/2 to DTLMcapsids does not lead to cell permeability of the complex, indicating that single TLM in PreS2 domain is either masked or is less efficient compared to surface exposed tandem TLM, fused via a linker to the N-terminus of the core protein. Taken together, these data indicate that TLM-carrier capsids have the potential to efficiently translocate their antigen cargo across the plasma membrane into cytoplasm of target cells. 3.3. Prolonged systemic distribution of TLMcapsids in mice after intraperitoneal injection To study biodistribution of cell-permeable TLMcapsids in the physiological environment of a living organism, the spread after intraperitoneal injection in BALB/c mice was analyzed. For this purpose, all single components were labeled with Cy7 or only mSA_preS1/2 was labeled when coupled to unlabeled TLM- or DTLMcapsids. As an additional control Cy7 was applied without protein (Fig. S2). By fluorescence based in vivo imaging, biodistribution of applied proteins was monitored for a period of 6 h at 9 different time points in living BALB/c mice. In vivo imaging (Fig. 3a,b) revealed that all separately administered components were remaining inside the surroundings of the peritoneum during

Fig. 2. The membrane permeability of the carrier capsids enables membrane translocation of the cargo antigen. HepG2 cells were incubated with free mSA_preS1/2, TLMcapsids, DTLMcapsids and mSA_preS1/2 coupled onto both carrier capsids. CLSM visualized translocation of TLMcapsids, alone or loaded with mSA_preS1/2, into cytoplasm. Proteins were stained with anti-HBVcore (red) for detection of capsids and anti-LHBs (green) for detection of mSA_preS1/2. Nuclei were stained with DAPI (blue) and F-actin with FITC-phalloidin (turquoise). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Biodistribution of I.P. administered mSA_preS1/2 + TLMcapsids or DTLMcapsids and controls in living C57BL/6N mice. (a) mSA_preS1/2, TLMcapsids and DTLMcapsids were labeled with Cy7 or (b) Cy7-labelled mSA_preS1/2 was loaded onto unlabeled carrier capsids. Labeled proteins were intraperitoneally injected into BALB/c mice. Signals were measured at 745/800 nm and visualized via in vivo imaging. Colored areas show presence of labeled proteins in different intensities (dark red = low intensity, yellow = high intensity). (c) TLMcapsids + mSA_preS1/2, DTLMcapsids + mSA_preS1/2 or PBS were injected I.P. into C57BL/6N mice. After 2.5 h mice were sacrificed, and cryosections of the liver were analyzed via indirect immunofluorescence staining. For better visualization of distribution patterns, brightness was slightly adapted. White arrows prompt to membrane associated preS1/S2. Both pictures of PBS control were adapted concerning brightness in the exact same manner as pictures for TLMcapsids + mSA_preS1/2 or DTLMcapsids + mSA_preS1/2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the first 30 min after injection. After 1 h mSA_preS1/2, if applied without carrier, is already systemically distributed almost in the whole body of the mouse. A stable signal is detected for another 1.5 h before it starts to faint and disappears. Unloaded TLMcapsids and DTLMcapsids without coupled antigen behave similarly within the first hour. Capsids are detected in the environment of the peritoneum, head and extremities. There is no pronounced change in signal intensity on side of DTLMcapsids from this time point on. In contrast to this, TLMcapsids are showing a very distinctive overall-distribution after 2.5 h. This systemic spread is detected without any change, even after 6 h (Fig. 3a). When Cy7-labeled mSA_preS1/2 was coupled onto the surface of TLMcapsids and DTLMcapsids, distribution pattern differed depending on the carrier (Fig. 3b). In case of mSA_preS1/2 loaded on TLMcapsids, we observed a systemic spread almost all over the whole body that slowly disappeared within the last 5 h. In case of mSA_preS1/2 coupled to DTLMcapsids, the spread of mSA_preS1/2 is less pronounced. The signal distributes in the peritoneal cavity within 1 h. After that time point, signal strength slowly fades and detectable protein does not further spread (Fig. 3b). This indicates that loading of cargo antigen on TLMcapsids affects the distribution pattern and promotes systemic spreading of antigen. For a more detailed analysis of distribution, we investigated spreading inside the liver of mice. Therefore C57BL/6N mice were injected intraperitoneally with TLMcapsids + mSA_preS1/2,

DTLMcapsids + mSA_preS1/2 and additionally with PBS as a negative control. After 2.5 h mice were sacrificed, a hepatectomy was performed and cryosections were made. Immunohistochemistry was performed using a PreS1-specific antibody. We observed a slightly weaker signal when TLMcapsids instead of DTLMcapsids were used as carriers. TLMcapsids-bound mSA_preS1/2 is quite evenly intracellularly and extracellularly distributed all over the liver. In contrast to this, the DTLMcapsids-bound mSA_preS1/2, is enriched on the surface of the hepatocytes, but much less is found within the cells (Fig. 3c). This reflects the capacity of the TLMcapsids to deliver their cargo into cells and to distribute over the whole organism. Contrary to this, preS1/2 cargo bound to the non-cell–permeable DTLMcapsids accumulates on the hepatocyte surface. 3.4. Intraperitoneal immunization of C57BL/6N mice with preS1/2loaded TLMcapsids enhances B cell response and results in HBV neutralizing antibodies The experiments described above indicate that loading of mSA_preS1/2 onto cell-permeable capsids results in a different in vivo biodistribution as compared to uncoupled antigen. To investigate the impact on the immune response, wild-type C57BL/6N mice were vaccinated by a prime-boost-protocol via intraperitoneal route. Immunological responses against mSA_preS1/2

Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063

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∆ TLMcapsids + mSA_preS1/2

TLMcapsids + mSA_preS1/2

merge

mSA_preS1/2

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Fig. 3 (continued)

loaded on TLMcapsids were compared to mSA_preS1/2 loaded on DTLMcapsids and to uncoupled mSA_preS1/2. As a negative control, PBS was injected into an additional group. Three weeks after the second boost, levels of mSA_preS1/2-specific antibodies were determined by ELISA (Fig. 4). The significantly highest signals were obtained in case of these mice that were vaccinated with mSA_preS1/2-loaded TLMcapsids compared to all controls. Administration of either free mSA_preS1/2 or mSA_preS1/2 loaded onto DTLMcapsids led to induction of specific antibodies as well, but to significantly lower antibody levels. To analyze antisera specificity in more detail, microarrays were spotted with synthetic overlapping peptides covering the entire amino acid sequence of mSA_preS1/2 and incubated with these sera. The array revealed that comparable sequential stretches, in approximation of epitopes, were recognized by the different sera. However, signal intensity of the array incubated with serum of mSA_preS1/2 is visibly stronger as compared to TLMcapsids + mSA_preS1/2 and DTLMcapsids + mSA_preS1/2 derived sera. But interestingly, antibodies of all three groups bind to peptides within the receptor binding domain – suggesting that these antibodies could have

the capacity to directly block an HBV infection (Fig. 5a). To experimentally examine this, HBV permissive, differentiated HepaRG cells [27] were used. The HBV inoculum was pre-incubated for 2 h with respective mice sera, before infection of differentiated HepaRG cells was performed. Analysis of infection via HBsAgELISA revealed that sera from mice I.P immunized with either free mSA preS1/preS2 or coupled to DTLMcapsids - contain neutralizing antibodies (Fig. 5b, fig. S3). However, these data indicate that loading of mSA_preS1/2 onto cell-permeable TLMcapsids leads to induction of a stronger antibody response compared to free mSA_preS1/2 or mSA_preS1/2 loaded onto DTLMcapsids. 3.5. Robust induction of cellular immune response against HBVexpressing hepatocytes after immunization with mSA_preS1/2-loaded TLMcapsids As described above, it was observed that use of TLMcapsids as antigen carriers leads to a stronger humoral immune response. To study the effect on the cellular immune response, lymphocytes

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Fig. 4. Immunization of C57BL/6N mice with mSA_preS1/2 + TLMcapsids leads to highest amount of anti-mSA_preS1/2 antibodies. C57BL/6N mice (n = 3) were immunized by a two-boost vaccination schedule via intraperitoneal route, with a period of ten days between each administration of proteins, with 10 mg of TLMcapsids and DTLMcapsids and 2,5 mg of mSA_preS1/2 either as free antigen or loaded onto the carrier capsids with a volume of 50 ml. 50 ml of proteins were injected intraperitoneally, n = 3 mice. Statistics were performed via one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

(effector cells, (EC)) from vaccinated mice were isolated as described in the methods section and incubated with HBVexpressing AML12 cells (murine hepatocyte cell line) as target cells (TC). Target cells were incubated with lymphocytes in a TC:EC ratio of 1:10 for 24 h. To analyze the number of remaining HBV-positive cells, attached AML12 target cells were fixed, stained with a corespecific antiserum and analyzed by CLSM. Microscopic analysis revealed a significantly lower number of HBV-positive cells, when these cells were co-incubated with effector cells derived from mice immunized with mSA_preS1/2 + TLMcapsids, compared to mice immunized either with free mSA_preS1/2 or mSA_preS1/2 coupled to DTLMcapsids (Fig. 6a). This was quantified by measurement of HBcAg-specific signal intensity (Fig. 6b). A tenfold lower signal was observed in case of effector cells derived from mice immunized with mSA_preS1/2 coupled to TLMcapsids compared to the other groups. In addition to quantification of HBV-positive cells, levels of IFNc in supernatant from co-cultivation experiments were anlyzed by ELISA (Fig. 6c). Highest amounts of secreted IFNc were significantly found for the mSA_preS1/2 + TLMcapsids group, compared to control groups, reflecting activation of CD8 + T cells. These results provide evidence that TLMcapsids as antigen carriers have the potential to induce a strong cellular immune response against cargo antigen mSA_preS1/2, as reflected by killing of HBV-positive target cells and elevated levels of IFNc. 3.6. TLMcapsids as an opportunity for transdermal immunization Inspired by the ability of TLMcapsids to cross cellular membranes by diffusion and by their pronounced spreading capacity, possibilities of alternative immunization routes were evaluated based on these observations. As a proof of principle experiment, the ability of TLMcapsids to migrate through skin layers of mice ex vivo was analyzed. For this purpose, skin patches of C57BL/6N mice were excised in an area of 1 cm2. Afterwards, coupled or single proteins, in the same set-up as used for in vitro permeability assays, were admin-

istered onto the skin by using a transdermal patch system (fig. S4). After 6 h of incubation, skins were sliced into 10 mm thick specimens, stained with HBcAg- and HBsAg-specific antibodies and analyzed by CLSM. Microscopic analysis revealed that TLMcapsids have the ability of crossing membranes and of diffusing through cell layers of the skin (Fig. 7). HBcAg and HBsAg signals were detected exclusively after incubation with mSA_preS1/2 loaded onto cell-permeable TLMcapsids in contrast to the used controls (free mSA_preS1/2 and mSA_preS1/2–loaded DTLMcapsids). This led to the conclusion that TLM-mediated permeability enables a spread through several cell layers of the skin. 3.7. Use of TLMcapsids enables the induction of a robust humoral and cellular immune response against the cargo antigen after oral-mucosal or transdermal vaccination In light of the observation that TLMcapsids loaded with cargo antigen can mediate antigen transport through cell layers, the question arose whether this can be used for oral or transdermal vaccination strategies that finally depend on antigen uptake through cell layers. To experimentally investigate this, C57BL/6N mice were vaccinated with mSA_preS1/2-loaded TLMcapsids, free mSA_preS1/2 or mSA_preS1/2-loaded DTLMcapsids, as well as PBS by oral-mucosal or transdermal route. For oral-mucosal vaccination, samples were applied directly into the mouths of mice, sedated with isoflurane. For transdermal vaccination, patches with respective antigens were used. As described for I.P.-vaccinated mice, sera were analyzed by ELISA for quantification of antipreS1/2 antibodies. Interestingly, induction of preS1/2-specific antibodies exclusively occurred in both non-invasive application routes, if mSA_preS1/2 was loaded onto TLMcapsids (Fig. 8a, b). Neither free mSA_preS1/2 nor mSA_preS1/2 coupled to DTLMcapsids had the capacity to induce mSA_preS1/2-specific antibodies. Additionally, levels of preS1/2-specific antibodies after immunization with TLMcapsids + mSA_preS1/2 were compared between all three used application routes, showing no significant differences between these routes under these conditions (Fig. 8c). Further

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a

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Fig. 5. Induction of neutralizing antibodies after immunization of mice with free mSA_preS1/2 or coupled to carrier capsids. (a) 72 synthetic overlapping peptides (15 aa in length; 4 aa offset) covering the mSA_preS1/2 sequence were spotted as quadruples onto microscope slides. These multi-peptide microarrays were incubated with sera of I.P.immunized mice and stained with fluorophore-labeled anti-mouse IgG antibodies. Green accentuation visualizes the receptor binding domain of HBV. Pre-immune sera of mice were used as a control. (b) The amount of HBsAg in supernatant of HepaRG cells infected either in the presence or absence of the respective mice sera was determined by HBsAg-ELISA. Data are displayed as ratio of signal at OD450 to cut-off (negative control + 0.05; manufacturer’s recommendation). Values under the dotted line are considered as uninfected and above the dotted line as infected. Protein and PBS samples n = 3, infection controls n = 1. Transdermal mSA_preS1/2 + TLMcapsids n = 1. Statistics were performed via one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

analysis of these sera revealed that neutralizing antibodies were generated after oral application and, even though statistically insignificant, after transdermal immunization, as shown by blocking of infection of differentiated HepaRG cells (Fig. 5b). To investigate whether oral-mucosal or transdermal vaccination, based on antigen-loaded TLMcapsids, has the capacity to induce a cellular immune response, the experimental setting for the cytotoxicity assay, as described above, was used. But this time, we used lymphocytes (transdermal route) and splenocytes (oralmucosal route) as effector cells and HBV-expressing AML12 cells

as target cells. Analysis of the number of HBV-positive cells by CLSM revealed a lower amount of HBV-positive cells, if effector cells from mSA_preS1/2 + TLMcapsids immunized mice were used. In both the transdermal and oral-mucosal set-up, this amount was lower compared to all control groups (Fig. 6d, 6 g). For the oralmucosal and transdermal set-up, this was additionally quantified by measurement of HBcAg-specific signal intensity. A significantly lower signal was observed in case of effector cells derived from mice, immunized with mSA_preS1/2 coupled to TLMcapsids as compared to mSA_preS1/2 coupled to DTLMcapsids (Fig. 6e, h).

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a

b I.P.: α-HBVcore signal after co-cultivation ns ns

ns ns

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Fig. 6. Lymphocytes and splenocytes of mSA_preS1/2 + TLMcapsids vaccinated mice show cytotoxic effects against HBV-expressing target cells. (a-c) C57BL/6N mice were vaccinated with mSA_preS1/2 + TLMcapsids via I.P. route and lymphocytes were co-cultivated for 24 h with HBV-transfected AML12 in a EC:TC ratio of 1:10. (a) Number of HBV-positive target cells was determined by immunofluorescence microscopy using an HBcAg-specific antiserum (red fluorescence), nuclei were stained by DAPI (blue). (b) For quantification, HBcAg-specific signal intensities were calculated using Zeiss ZEN microscope software. (c) The amount of IFNc in the supernatant of co-cultivation experiments was determined by ELISA. (d–f) C57BL/6N mice were vaccinated via oral-mucosal route and splenocytes were co-cultivated for 24 h with HBV-positive target cells in a EC:TC ratio of 1:10. (d/e) as described under a/b, the number of HBV-positive cells was determined by immunofluorescence microscopy using a HBcAg-specific serum. HBcAg-specific signals were quantified using Zeiss ZEN microscope software. (f) The amount of IFNc in the supernatant of co-cultivation experiments was determined by ELISA. (g-i) C57BL/6N mice were vaccinated via transdermal route and lymphocytes were co-cultivated with HBV-positive target cells for 24 h in a EC:TC ratio of 1:10. (g/h) as described for a/b and d/e. (i) IFNc amount in the supernatant of co-cultivation was determined by ELISA. (a-f) n = 3, NC of IFNc-ELISA n = 1. (g,h,i) n = 1. Statistics were performed via one-way ANOVA. *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In addition to the quantification of HBV-positive cells by CLSM, levels of IFNc in the supernatant of co-cultivation experiments were analyzed by ELISA. The significantly highest amounts of secreted IFNc was measured for the mSA_preS1/2 + TLMcapsids group as compared to controls in the oral-mucosal set-up (Fig. 6f) – the same group showed highest signals compared to controls in the transdermal set-up (Fig. 6i). Additionally, the mSA_pr eS1/2 + TLMcapsids group showed highest amounts of CD107a, a surrogate marker for T cell activation, in the latter set-up (fig. S5). Taken together, these data indicate that use of TLMcapsids as antigen carriers enables the induction of a robust humoral and

cellular immune response against the cargo antigen. Moreover, membrane permeability of carriers allows antigen transfer across several cell layers and therefore, enables new needle-free, noninvasive vaccination strategies, based on oral-mucosal or transdermal vaccination routes. 4. Discussion Therapeutic vaccination remains one of the most promising strategies against chronic hepatitis B virus infections and therefore against HBV associated pathogenesis. Even though there are sev-

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d

e oral: α -HBVcore signal after co-cultivation

*

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Fig. 6 (continued)

eral promising approaches, there is no licensed therapeutic vaccination available that is successfully generating sufficient humoral and cellular immune responses in infected patients to this day [28–32]. So, the crucial point is not just inducing a robust B cell response for neutralizing circulating virions, reducing the viral load and thereby decreasing the number of newly infected hepatocytes, but also to trigger a robust T cell response i.e. activation of HBVspecific CD8+ CTLs for elimination of every infected hepatocyte. VLPs decorated with the antigen of interest on their surface are known to induce a robust B cell response due to the highly ordered presentation of the antigen [33,34]. There were several approaches of using HBc-based VLPs with foreign antigens inserted into the spike-tip region, making them remarkably immunogenic. Blokhina et al. could show that epitopes of different influenza strains can be inserted via fusion of these with the spike-tip-affine peptide GSLLGRMKGA, leading to enhanced immunogenicity [35]. In another approach HBc dimers were produced as polypeptides consisting of two fused monomers (tandem fusion) and assembled to particles. That concept turned out to be beneficial for particle stability and allows the insertion of larger proteins into the spike-tip region [36]. In addition, membrane-permeable VLPs, based on HBV capsids (TLMcapsids), harbouring Strep-tagIII in the spike tip were described to induce a robust cellular immune response in vitro using ovalbumin as a model antigen [20].

Based on this, it was analyzed whether TLMcapsids loaded with an HBV-specific antigen have the potential to induce a humoral and cellular immune response against the cargo antigen. PreS1PreS2 domain of HBV surface protein was chosen as (i) PreS1 domain harbors the receptor binding domain [37,38], (ii) PreS1PreS2 domain contains MHC class I restricted peptides [39,40] and (iii) lack of the S-domain, which contains three transmembrane regions, ensures solubility [41]. The PreS1PreS2 domain was produced as a fusion protein with monomeric streptavidin and a hexa–his–tag. The use of monomeric streptavidin allows a defined binding to Strep-tagIII. Additionally, it avoids steric problems, resulting from the use of tetrameric streptavidin and thus allows a direct analysis of surface plasmon resonance data by established models. KD values of 1.6*10 7 for mSA_preS1/2TLMcapsids and 3.5*10 7 for mSA_preS1/2-DTLMcapsids were determined. This fits well to dissociation constants of streptavidin-ovalbumin to Strep-tagIII and streptavidin to StreptagIII from previous studies (9.57 * 10 7 and 1.06 * 10 7 [20]), as well as to KD of streptavidin to Strep-tagII from literature, with 1.3 * 10 5 [42]. The capacity of TLMcapsids to transfer cargo across cellular membranes has been described for nucleic acids, packaged into the interior of capsids and for ovalbumin bound to the surface of capsids. Moreover the interior of capsids can be loaded with CpG oligonucleotides that function as adjuvants. In this study, it was

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g

h transdermal: α -HBVcore signal after co-cultivation

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Fig. 6 (continued)

observed that TLMcapsids enable the transfer of mSA_preS1/2, if loaded on carrier capsids, across cellular membranes. While free mSA_preS1/2 or mSA_preS1/2 loaded onto DTLMcapsids show no membrane permeability under these conditions. This is surprising, as the PreS2 domain harbors a TLM domain, too (aa41-52) [22]. Either fusion of mSA to PreS1PreS2 impairs dimerization of PreS1Pres2 that is required for membrane permeability or the tandem TLM fused to HBcAg is superior to monomeric TLM present in the PreS2 domain [20,23]. There is evidence that B cell response may be relevant for the control of chronic HBV infection. This can be concluded from treatment of B cell lymphomas with Rituximab that depletes B cells. If patients suffering from controlled HBV infection are treated with Rituximab, reactivation of HBV can occur – indicating the relevance of B cell response for control of chronic infection [43,44]. The use of PreS1PreS2 that leads to induction of antibodies masking the receptor binding domain, as observed here, contributes to an inhibition of the spread of viral infection to so far uninfected hepatocytes [45,46]. It must be mentioned that we observed a slightly weaker signal for the sera of cargo plus carrier immunized mice as compared to the cargo (PreS1PreS2) immunized mice in the peptide array. There has been a variety of attempts to establish a therapeutic vaccine, but none of these efforts has ended so far in a licensed vaccine. A variety of different approaches has been tried, some in clinical trials, using different antigens, including peptide-, protein-, DNA or viral vector-based strategies [47]. A major problem is represented by the fact that in chronic HBV infection HBV-specific T

cells frequently are functionally defective. This includes a large spectrum, encompassing functional inhibition up to deletion. Our study provides proof of principle that a new platform technology, based on membrane-permeable carrier capsids that can be loaded with HBV-specific antigens, could be used as base for a therapeutic vaccine approach. This system combines some advantages of virus-like particles that are known to induce a robust B cell response with vector based strategies that trigger, in addition, cellular immune response. However, no transfer of coding nucleic acids is required nor any replicative forms are transferred: two points that sometimes affect the acceptance of a vaccination strategy by the public [48]. Co-cultivation experiments show that the number of HBV-expressing cells is significantly reduced due to the killing of HBV-positive cells and due to antiviral effects of the interferon response. Besides a transfer of TLMcapsids across cellular membranes, TLMcapsids also enable the spread of cargo antigen across several cellular layers. Both enable transport of cargo antigen into the cytoplasm of APCs, for induction of efficient immunoproteasomal processing and subsequent MHC class I-dependent presentation for robust activation of CD8 + CTLs. This is reflected by a significantly more extended distribution of the applied antigen after intraperitoneal application - resulting in targeting of more APCs. In addition, presented platform based on TLMcapsids could allow needle free vaccination. In our IVIS experiments, visual amounts of injected proteins seem to vary quite strongly inside the peritoneum of individuals, during the first 30 min after injection. This is caused by altering depth of proteins inside the peritoneum. The

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α-HBVcore

α-LHBs

mouse skin + merge

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∆ TLMcapsids +mSA_preS1/2

Fig. 7. Membrane permeability of TLMcapsids enables transfer of the mSA_preS1/2 cargo antigen through skin layers. Isolated skin preparations of C57BL/6N mice were incubated with free mSA_preS1/2, or mSA_preS1/2 coupled to TLMcapsids and DTLMcapsids, in an ex vivo transdermal patch system. Immunostaining and subsequent CLSM visualized migration of the antigen-loaded TLMcapsids through skin layers. Proteins were stained with anti-HBVcore (red) and anti-LHBs (green). Skin was displayed by stain of nuclei by DAPI (gold) and stain of actin by FITC-phalloidin (turquoise). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

higher the distance between Cy7 and most outer skin layers, the weaker are signals that may be detected outside the mice. These differences in depth are equalized after about 30 min. By analysis of biodistribution in the liver, we found that cell permeability of TLMcapsid carrier enables the migration of the mSA_preS1/2 antigen cargo through cell membranes into the cytoplasm of liver cells. In contrast, the non-permeable DTLMcapsids are enriched on the surface of hepatocytes potentially by binding to murine NTCP (Na + -taurocholate cotransporting polypeptide). This might be due to the fact that preS1 harbours the receptor binding domain and tends to bind to outer membrane-located NTCP. Based on the membrane permeability TLMcapsids have also the capacity to transport loaded antigens across cellular layers, accomplishing dermal application or resorption after oral-mucosal application. This is in accordance to previous data that revealed that TLM has no cell- or tissue-specificity. Administration of vaccines via mucosal surfaces is of broad interest nowadays as it is not just leading to mucosal, but also to a systemic response [49,50]. Therefore, applications via intranasal route, for targeting mucosal tissue, have also been reported for HBcAg-VLPs. In one approach, HBcAg particles were co-administered with HBsAg via the intranasal route, leading to a high immunogenicity and an early antibody titer, by stimulating nasal-associated lymphoid tissue (NALT) [51]. In another study, purified plant-derived HBcAg particles, expressed in Nicotiana benthamiana, were administered via i.m. injection followed by a boost of HBcAg derived from transgenic lettuce given by the oral route. A triggering not only a mucosal-intragastric, but also systemic immune response was observed. In this case, HBcAg particles could pass through the acidic environment of the digestive tract and through intestine by use of the protective bioencapsulation into

producer cells, leading to a stimulation of gut-associated lymphoid tissue (GALT) [52]. Transdermal application on the other hand allows an efficient targeting of Langerhans cells (LCs) inside the dermis that are able to express high amounts of MHC class II and are potent APCs that migrate from skin to lymph nodes to present peptides to CD8+ and CD4+ T cells [53–56]. Moreover, Langerhans cells have the ability to transfer targeted antigens to dendritic cells for CD8+ activation by LC-DC clustering [57]. It was found to be immunologically beneficial to administer HBV vaccines intradermally, for lowering the number of non-responders and lowresponders and for reversing nonresponsiveness in HBV vaccination, in contrast to intramuscular injection [58–60]. Although transdermal results in this study are just descriptive due to the low number of animals, this could suggest that transdermal vaccination based on this vaccine carrier may be possible. Interestingly, we could observe no significant differences in signal intensities of antibody levels after TLMcapsids + mSA_preS1/2 administration via intraperitoneal, oral and transdermal routes. This may point to the fact that there might be no disadvantages by use of alternative immunization routes with respect to B cell response concerning the amount of specific antibodies. Significant differences in biodistribution between mSA_preS1/2 bound to TLMcapsids and free or DTLMcapsids-bound mSA_preS1/2 is reflected by strong differences in the immune response. Although induction of PreS1PreS2-specific antibodies with neutralizing potential are observed after all vaccination routes, a significantly higher titer is found in case of mSA_preS1/2 + TLMcapsids immunized mice compared to controls. With regard to cellular immunity, a significantly stronger immune response is observed if mice were immunized with mSA_preS1/2 bound to

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ΔTLMcapsids +preS1/2 mice

PBS mice

Fig. 8. Oral or transdermal immunization of C57BL/6N mice with mSA_preS1/2 + TLMcapsids leads B cell response. C57BL/6N mice were immunized by a two-boost vaccination schedule with a period of ten days between each administration of proteins. Mice were immunized with 10 mg of TLMcapsids and DTLMcapsids and 2.5 mg of mSA_preS1/2 either as free antigen or loaded onto the carrier capsids. (a) 15 ml were pipetted into the mouths of mice for oral-mucosal vaccination and (b) 20 ml were administered onto the shaved skin of mice using a transdermal patch system. (c) Comparison of antibody levels of I.P., oral and transdermal route with mSA_preS1/ 2 + TLMcapsids after second boost. n = 3 mice. Statistics were performed via one-way ANOVA. *p < 0.05.

Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063

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T. Zahn et al. / Vaccine xxx (xxxx) xxx

α -mSA_preS1/2 antibodies: TLMcapsids+mSA_preS1/2 after 2. boost different routes ns

c

ns

1.5

ns

OD450

1.0

0.5

0.0 I.P.

oral

transde rmal

Fig. 8 (continued)

TLMcapsids as compared to free antigen or antigen loaded onto DTLMcapsids.

scopy; Frederic Thalheimer, Patricia Gogesch, Mirco Glitscher, and Fabian Elgner for helpful discussions.

5. Conclusion

Appendix A. Supplementary material

Taken together, these data indicate that this novel vaccine platform has the capacity to induce a robust humoral and cellular immune response that could be the base for a therapeutic vaccine approach to cure chronic HBV infections. Moreover, cell permeability of the vaccine platform could enable needle-free vaccination by antigen transfer across several cell layers allowing oral immunization or may pioneer transdermal immunization.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.02.063.

6. Grant support This work was supported by a grant from DZIF (Deutsches Zentrum für Infektionsforschung) to EH. CRediT authorship contribution statement T. Zahn: Conceptualization, Data curation, Formal analyis, Investigation, Methodology, Visulization, Writing original draft. S. Akhras: Conceptualization, Data curation, Formal analyis, Investigation, Methodology. C. Spengler: Methodology. R. O. Murra: Investigation. T. Holzhauser: Methodology. E. Hildt: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We would like to thank Gert Carra, Regina Eberle, and Stefanie Randow for their excellent technical support. Ute Modlich for many helpful instructions with respect to animal experiments, Klaus Boller for his continuous support concerning electron micro-

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Please cite this article as: T. Zahn, S. Akhras, C. Spengler et al., A new approach for therapeutic vaccination against chronic HBV infections, Vaccine, https:// doi.org/10.1016/j.vaccine.2020.02.063