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A thermostable therapeutic vaccine is able to break immune tolerance in a mouse model of chronic hepatitis B

Open AccessPublished:October 12, 2022DOI:https://doi.org/10.1016/j.jhepr.2022.100603

      Highlights

      • -
        Heterologous protein-prime and MVA-vector boost can break immune tolerance to HBV
      • -
        Exposure to higher temperature can damage protein- and vector-vaccine components
      • -
        An amino acid-based formulation can preserve integrity of vaccine components
      • -
        The formulated vaccine proves stable for three months at 40°C and one year at 25°C
      • -
        Despite heat-exposure, stabilized TherVacB triggered strong HBV-specific immunity

      Abstract

      Background and Aims

      Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. Therapeutic hepatitis B vaccine TherVacB combines a protein-prime with a Modified-Vaccinia-virus-Ankara (MVA) vector-boost boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport posing logistic and financial challenges to vaccine applications. In a systemic approach, we aimed at identifying an optimal formulation to maintain stability and immunogenicity of protein- and vector-vaccine components.

      Methods

      We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg) used as protein-antigens and the MVA-vector and investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV infected mice.

      Results

      In vitro analysis proved stability against mechanical stress during lyophilization and long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40°C for three and at 25°C for 12 months. Vaccination of HBV-naïve and AAV-HBV infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4°C/-80°C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high-titer HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice.

      Conclusion

      SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide.

      Lay summary

      Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may allow to cure chronic hepatitis B. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and allow inducing immune responses and control the virus in preclinical mouse models even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many hepatitis B patients worldwide.

      Graphical abstract

      Keywords

      Abbreviations:

      AAV (adeno-associated virus), ALT (alanine aminotransferase), anti-HBc (hepatitis B core antibodies), anti-HBs (hepatitis B surface antibodies), BHK-21 (baby hamster kidney cells), cccDNA (covalently closed circular DNA), c-di-AMP (bis-(3’,5’)-cyclic dimeric adenosine monophosphate), CHB (chronic hepatitis B), CTC (controlled temperature chain), Ctrl (control), DF-1 (chicken embryo fibroblast cells), DLS (dynamic light scattering), ECTC (extended controlled temperature conditions), EMA (European medicines agency), GzmB (granzyme B), HBcAg (hepatitis B core antigen), HBeAg (hepatitis B e antigen), HBsAg (hepatitis B surface antigen), ICS (intracellular cytokine staining), IFNα (interferon alpha), i.m. (intramuscular), MVA (Modified Vaccinia virus Ankara), NAGE (native agarose gel electrophoresis), NK (natural killer), RH (relative humidity), RT (room temperature), SAA (stabilizing amino acids), SEC-HPLC (size exclusion-high performance liquid chromatography), SMCtrl (starting material control), TCID50 (median tissue culture infection dose), TEM (transmission electron microscopy), TherVacBCtrl (non-lyophilized, non-stressed, non-stabilized TherVacB), WHO (world health organization)

      Conflict of interest statement

      UP is a co-founder and shareholder of SCG Cell Therapy, and an ad hoc scientific advisor to Abbvie, Arbutus, Biontech, Gilead, GSK, J&J, Roche, Sanofi, Sobi, Vaccitech and VIR Biotechnology. UP and AK are named as inventors on a patent application describing the therapeutic vaccination scheme of TherVacB (PCT/EP2017/050553). KK, MS, JA are or were employees of LEUKOCARE AG. UP is member of the advisory board of LEUKOCARE AG.

      Financial support statement

      The study was supported by the German Center for Infection Research via TTU 05.715 and 05.813, the German Research Foundation via TRR179, project TP18, and by the Federal Ministry of Education and Research (BMBF) via the program Research KMU Innovativ16 – StabVacHepB AZ 031B0094C. JSa received a stipend from the “Stiftung der deutschen Wirtschaft“ (sdw, German industrial foundation), JSu from the Chinese Scholarship Council, and HK was supported by the TRR179 with an MD student scholarship.

      Author’s contribution

      JSa, AK, KK, KS, JA, MS and UP designed the study; JSa, AK, KK and SL performed the experiments; MK, LW, ML and GS produced critical components and reagents; HK, EAO and JSu supported mouse experiments; SE provided essential instruments and support; JSa, AK, KK and UP wrote the paper. All authors reviewed and confirmed the final version of the manuscript.

      Introduction

      With more than 257 million chronic carriers and more than 880,000 HBV-related deaths worldwide, chronic hepatitis B (CHB) represents a major health problem

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      Design of therapeutic vaccines: hepatitis B as an example.
      . Recently, we have developed a heterologous protein-prime/MVA boost vaccination strategy, termed TherVacB, that aims at inducing HBV-specific B-cell as well as helper and effector T-cell responses

      Backes S, Jäger C, Dembek CJ, Kosinska AD, Bauer T, Stephan A-S, et al. Protein-prime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice. 2016;34:923-932.

      ,
      • Kosinska A.D.
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      Therapeutic vaccination for chronic hepatitis B.
      . We have previously shown that TherVacB is able to break HBV-specific immune tolerance in HBV transgenic and adeno associated virus (AAV)-HBV mouse models

      Backes S, Jäger C, Dembek CJ, Kosinska AD, Bauer T, Stephan A-S, et al. Protein-prime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice. 2016;34:923-932.

      ,
      • Kosinska A.D.
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      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      . In TherVacB, priming with particulate HBsAg and HBcAg leads to the induction of HBV-specific B- and helper CD4+ T-cell responses, but also primes effector CD8+ T cells. The following boost with an MVA-vector virus aims at amplifying the HBV-specific effector CD8+ T cells. Neutralizing antibodies prevent viral spread and multifunctional and multispecific T-cell responses finally control HBV by eliminating virus-infected hepatocytes
      • Kutscher S.
      • Bauer T.
      • Dembek C.
      • Sprinzl M.
      • Protzer U.
      Design of therapeutic vaccines: hepatitis B as an example.
      .
      A major challenge in vaccine application worldwide is a lack of functional cooling chains, especially in low- and middle-income countries or hard-to-reach areas, due to infrastructure gaps, high costs and logistical problems

      Levin A, Wang SA, Levin C, Tsu V, Hutubessy R. Costs of Introducing and Delivering HPV Vaccines in Low and Lower Middle Income Countries: Inputs for GAVI Policy on Introduction Grant Support to Countries. 2014;9:e101114.

      • Chen D.
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      Opportunities and challenges of developing thermostable vaccines.

      WHO. Meeting report. WHO/Paul-Ehrlich-Institut Informal Consultation on Scientific and Regulatory Considerations on the Stability Evaluation of Vaccines under Controlled Temperature Chain (CTC). Langen 2013.

      . Consequently, vaccines may lose their potency due to thermal instability of the vaccine components
      • Chen D.
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      Opportunities and challenges of developing thermostable vaccines.
      ,

      WHO. Temperature sensitivity of vaccines. Geneva 2006. Ordering code: WHO/IVB/06.10.

      . Addressing this problem, the world health organization (WHO) has presented guidelines on “Controlled temperature conditions” (CTC) and “Extended controlled temperature condition” (ECTC)

      WHO. Meeting report. WHO/Paul-Ehrlich-Institut Informal Consultation on Scientific and Regulatory Considerations on the Stability Evaluation of Vaccines under Controlled Temperature Chain (CTC). Langen 2013.

      ,

      WHO. Annex 5. Guidelines on the stability evaluation of vaccines for use under extended controlled temperature conditions. 2006. Technical report series No 999.

      . The WHO CTC guideline requires vaccines to resist 40°C for a minimum of 3 days without losing efficacy. This facilitates vaccine application worldwide, reduces the costs of vaccination campaigns and prevents administration of inactive vaccines
      • Chen D.
      • Kristensen D.
      Opportunities and challenges of developing thermostable vaccines.
      ,

      WHO. Meeting report. WHO/Paul-Ehrlich-Institut Informal Consultation on Scientific and Regulatory Considerations on the Stability Evaluation of Vaccines under Controlled Temperature Chain (CTC). Langen 2013.

      .
      In this study we aimed to develop a lyophilized, thermostable version of the different vaccine components of TherVacB. For this purpose, a series of stabilizing amino acid (SAA)-based formulations were designed using the excipient database from LEUKOCARE. SAA-formulated vaccine components were heat-challenged to demonstrate compliance with WHO criteria. In addition, a storage period of up to one year at 25°C was evaluated. Comprehensive in vitro analyses of antigen integrity and MVA-vector infectivity proved suitable SAA-formulations. In addition, immunogenicity and efficacy of SAA-formulated vaccine despite heat-stress was demonstrated in a preclinical model of HBV infection.

      Materials and methods

      For additional, detailed descriptions please refer to Supporting Information.

      Vaccine components

      HBcAg (HBV genotype D, subtype ayw) was expressed in E. coli (APP Latvijas Biomedicinas, Riga, Latvia), HBsAg (HBV genotype A, subtype adw) in yeast (Biovac, South Africa). MVA-S/C vector contains the coding sequences for the small envelop protein (S) of HBV genotype A2, subtype adw, and core protein of HBV genotype D, subtype ayw, connected by a P2A site and was produced as reported
      • Kremer M.
      • Volz A.
      • Kreijtz J.H.
      • Fux R.
      • Lehmann M.H.
      • Sutter G.
      Easy and efficient protocols for working with recombinant vaccinia virus MVA.
      .

      Sample preparation

      Vaccine components were diluted with SAA-formulations or PBS. After lyophilisation, the samples were stored under specific defined temperature conditions according to the International Council for Harmonisation (ICH) guidelines at 40°C/75% relative humidity (RH), 25°C/60% RH or at 5°C. Samples were reconstituted with the required volume of H2O (in vitro) or PBS (in vivo).

      Sandwich ELISA

      HBcAg-specific ELISA: coating with 1 μg/mL anti IFA HepBCore (CIGB, Havanna, Cuba), detection with HRP-labelled anti-HepBcore (1:7000; CIGB). HBsAg-specific ELISA: coating with 1 μg/mL HBV-specific single-chain antibody c8 (scFv C8)
      • Bohne F.
      • Chmielewski M.
      • Ebert G.
      • Wiegmann K.
      • Kurschner T.
      • Schulze A.
      • et al.
      T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.
      , detection with 5 μg/well HBs-specific murine antibody 5F9
      • Golsaz-Shirazi F.
      • Amiri M.M.
      • Farid S.
      • Bahadori M.
      • Bohne F.
      • Altstetter S.
      • et al.
      Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).
      and goat anti-human IgG Fc (HRP-labelled; Abcam, Cambridge, UK, 1:1000). 15 ng of HBcAg and 30 ng of HBsAg were analysed.

      Native Agarose Gel Electrophoresis (NAGE)

      2.5 μg native protein sample per lane was run for 90 min through a 1% agarose gel (Peqlab, Erlangen, Germany) at 150V. Nucleic acid content was analysed by UV-light in the Fusion FX7 (Peqlab, Erlangen, Germany) after Roti®-Safe GelStain staining. Protein content was visualized by Coomassie staining.

      Dynamic light scattering (DLS)

      5 μg protein sample diluted in 10 mM NaCl was analysed using a Malvern Zetasizer Nano ZS (Malvern Panalytical GmbH, Herrenberg, Germany) or DynaPro Nanostar DLS instrument (Wyatt Technology Europe GmbH, Dernbach, Germany). Per measurement, 50 individual scans, each 10 s long, were analysed.

      Size exclusion-high performance liquid chromatography (SE-HPLC)

      Antigens were analysed by SE-HPLC (UV-280 nm; UHPLC system UltiMate3000 Thermo Scientific, Germany). A size exclusion column TSK-gel® G5000 SWXL 300 mm x 7.8 I.D. column (100 nm pore size; Tosoh Bioscience, Tokyo, Japan) with a flow rate of 0.6 mL/min at 25°C and an injection volume of 100 μL was used. The mobile phase was a 50 mM phosphate buffer pH 7.0. Chromatograms were progressed using Chromeleon 7 Chromatography Data Software (Thermo Scientific, Germany).

      Transmission electron microscopy (TEM)

      MVA-S/C inactivated with 2% paraformaldehyde was absorbed for 15 min, antigens for 5 min at RT onto a copper grid with a formvar/carbon film (400 mesh), negatively stained with 1% phosphotungstic acid and imaged using a Libra 120 transmission electron microscope (Zeiss, Oberkochen, Germany).

      Determination of MVA-S/C titer

      MVA-S/C was titrated in serial dilutions on BHK-21 cells. The MVA-S/C titer was determined by half-maximal median tissue culture infection dose (TCID50) of two repetitions
      • Bohne F.
      • Chmielewski M.
      • Ebert G.
      • Wiegmann K.
      • Kurschner T.
      • Schulze A.
      • et al.
      T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.
      .

      Animal experiments - ethical statement

      Mouse experiments were performed according to the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA), the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the 3R rules. Experiments were approved by the local Animal Care and Use Committee of Upper Bavaria (permission number: ROB-55.2-2532.Vet_02-18-24) according to the institution's guidelines. All animals received human care, studies conform to Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and comply with institution's guidelines. Animals were maintained under pathogen-free conditions and experiments were performed during the light phase of the day. Male C57BL/6J mice were purchased from Janvier (Le Genest-Saint-Isle, France). To establish persistent HBV replication, mice were intravenously infected four weeks before vaccination with 4x109 genome equivalents of the adeno-associated virus (AAV)-HBV1.2 vector encoding 1.2-fold overlength HBV genome of genotype D
      • Dion S.
      • Bourgine M.
      • Godon O.
      • Levillayer F.
      • Michel M.L.
      Adeno-Associated Virus-Mediated Gene Transfer Leads to Persistent Hepatitis B Virus Replication in Mice Expressing HLA-A2 and HLA-DR1 Molecules.
      .

      Heterologous protein-prime/MVA-boost vaccination

      Mice were immunized i.m. using a heterologous protein-prime/MVA-boost vaccine as described

      Backes S, Jäger C, Dembek CJ, Kosinska AD, Bauer T, Stephan A-S, et al. Protein-prime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice. 2016;34:923-932.

      . Two protein immunizations consisted of a mixture of 10 μg particulate HBcAg, 10 μg particulate HBsAg and 10 μg bis-(3’,5’)-cyclic dimeric adenosine monophosphate (c-di-AMP; InvivoGen, San Diego, CA) two weeks apart. Booster immunization was performed with 5×107 TCID50 MVA-S/C two weeks after.

      Intracellular cytokine staining (ICS)

      Single-cell suspensions of splenocytes and liver associated lymphocytes (LALs) were prepared as described
      • Kosinska A.D.
      • Moeed A.
      • Kallin N.
      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      ,
      • Stross L.
      • Günther J.
      • Gasteiger G.
      • Asen T.
      • Graf S.
      • Aichler M.
      • et al.
      Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice.
      . For ICS, lymphocytes were stimulated ex vivo with 1 μg/mL MVAB8R, OVAS8L, HBV S280 and C93 or peptide pools HBV Spool or Cpool
      • Kosinska A.D.
      • Moeed A.
      • Kallin N.
      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      (Tables S1 and 2). After 1-h stimulation, brefeldin A was added for 14 h before staining with Fixable-Viability-Dye eFluorTM 780 (eBioscience, Frankfurt, Germany), anti-CD4-APC (clone GK1.5, eBioscienceTM) and anti-CD8a-Pb (clone 53-6.7, BD PharmingenTM). For ICS, we used anti-IFNγ (clone XMG1.2; BD PharmingenTM). Data were acquired on a CytoflexS flow cytometer (Beckmann Coulter, Brea, CA, USA) and analysed with FlowJo software (Tree Star, Ashland, OR, USA).

      Multimer staining

      HBV-specific CD8+ T cells were detected through staining with MHC class I multimers conjugated with the H-2Kb-restricted HBV-derived peptides C93–100 (C93, MGLKFRQL) and S190-197 (S190, VWLSAIWM), as described previously
      • Kosinska A.D.
      • Moeed A.
      • Kallin N.
      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      ,
      • Michler T.
      • Kosinska A.D.
      • Festag J.
      • Bunse T.
      • Su J.
      • Ringelhan M.
      • et al.
      Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice.
      . As a control, staining with multimer conjugated with the ovalbumin-derived peptide S8L257 (OVAS8L, SIINFEKL) was performed. Prior to use, C93 and OVAS8L multimers (kindly provided by Dirk Busch, Technical University of Munich, Germany) were labeled with Streptactin-PE (IBA Lifesciences, Göttingen, Germany), as previously described
      • Kosinska A.D.
      • Moeed A.
      • Kallin N.
      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      ,
      • Michler T.
      • Kosinska A.D.
      • Festag J.
      • Bunse T.
      • Su J.
      • Ringelhan M.
      • et al.
      Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice.
      .

      Serological and virological analyses

      HBeAg, HBsAg and anti-HBs were quantified using the ArchitectTM platform (Abbott Laboratories, Wiesbaden, Germany), anti-HBc using Enzygnost® Anti-HBc monoclonal test (Siemens Healthcare Diagnostics, Erlangen, Germany). Alanine aminotransferase (ALT) activity was determined after 1:4 dilution in PBS by Reflotron® GPT/ALT tests (Roche Diagnostics, Mannheim, Germany).
      DNA was extracted from 50 μL mouse serum using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany) or 20 mg of liver tissue using a NucleoSpin Tissue DNA Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer’s instructions. The quantification of HBV-DNA was performed through real-time PCR with SYBR green as previously described
      • Kosinska A.D.
      • Festag J.
      • Muck-Hausl M.
      • Festag M.M.
      • Asen T.
      • Protzer U.
      Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.
      .

      Immunohistochemistry

      Liver tissue samples were fixed in 4% buffered formalin for 48 h and embedded in paraffin. Immunohistochemistry was performed with anti-HBcAg antibody (Diagnostic Biosystems, Pleasanton, CA; 1:50 dilution) according to the protocol described previously
      • Kosinska A.D.
      • Festag J.
      • Muck-Hausl M.
      • Festag M.M.
      • Asen T.
      • Protzer U.
      Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.
      . HBcAg-positive hepatocytes were determined in 10 random view fields (40x magnification) and quantified per mm2.

      Statistical analysis

      Data were analysed using GraphPad Prism version 5.01 (GraphPad Software Inc., San Diego, CA) using Mann-Whitney test or Students t-test. P-values <0.05 were considered significant.

      Results

      Loss of functional integrity of protein and vector vaccine components after exposure to thermal stress

      To analyse thermal stability of TherVacB components, antigens and MVA-S/C in solution were exposed to a temperature gradient ranging from 25°C (representing room temperature (RT)) up to 40°C (WHO CTC guideline

      WHO. Meeting report. WHO/Paul-Ehrlich-Institut Informal Consultation on Scientific and Regulatory Considerations on the Stability Evaluation of Vaccines under Controlled Temperature Chain (CTC). Langen 2013.

      ) for three, 14 and 28 days. As control, the antigens were stored at 4°C, the MVA at -80°C.
      Antigen integrity was assessed by ELISA. HBsAg was stable for three days at 25°C, but had lost 33% of the ELISA signal at day 14 and 80% at day 28 (Fig. 1A). Storage at 36 to 40°C resulted in rapid loss of HBsAg detection by ELISA already after three days. (Fig. 1A). HBcAg did not show any obvious loss of antigen integrity for up to 14 days at 25°C, but after 28 days 25% of the ELISA signal was lost (Fig. 1B). Storage at 36, 38 and 40°C resulted in increasing, up to 60% loss of antigen integrity in ELISA at day three, 87% at day 14 and an almost complete loss over time (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1Effect of heat-exposure on TherVacB vaccine components. Particulate HBsAg, HBcAg and MVA-S/C kept in solution were stored at 25°C, 36°C, 38°C and 40°C and as control at 5°C or -80°C, respectively, as indicated. (A) HBsAg- and (B) HBcAg-specific ELISA after three (left), 14 (middle) and 28 days of storage (right). (C) MVA-S/C titres were determined by TCID50. (D-G) HBcAg, HBsAg and MVA-S/C were formulated with SAA-based formulation F1.1 or PBS, lyophilized and analysed directly after lyophilization. Cake structure of antigens (D) MVA-S/C (E) after lyophilization. (F-G) For control (Ctrl), antigens were stored at 5°C, MVA-S/C at -80°C without stabilization, lyophilization and temperature exposure. (F) Quantification of HBcAg- and HBsAg by ELISA relative to Ctrl (set to 100%). (G) Hydrodynamic radius of antigens determined by DLS. (H) TCID50 of MVA-S/C. Data are given as mean±SD. Statistical analysis applied unpaired t-test. *p<0.05; **p≤0.01; ***p≤0.001; ns - not significant.
      Integrity of MVA-S/C expressing HBV core and S proteins was determined by infectivity in cell culture. TCID50 did not significantly drop until day 14 at 25°C. Over 28 days TCID50 dropped by approx. 1-log10 (Fig. 1C). However, increasing the temperature to 38 or 40°C resulted in a >1-log10 reduction of MVA-S/C titers already after three days, and storage at 36, 38 and 40°C resulted in a complete loss of MVA-S/C infectivity after 14 days (Fig. 1C). This indicated that stabilization of the vaccine components is crucial to store the TherVacB vaccine components at RT or higher temperatures.

      Selection of stabilizing excipients for the formulation of protein and vector vaccine components

      Excipients for stabilizing the vaccine components were selected from a database established by LEUKOCARE AG, Martinsried, Germany, based on experience with thermal stabilization of lyophilized HBcAg and an MVA-vector expressing HBV core protein using a mixture of seven amino acids (alanine, arginine, glutamic acid, glycine, lysine, histidine and tryptophan) in combination with trehalose.
      A first round of optimizing the SAA-formulation aimed at maintaining a pharmaceutically desired cake structure during lyophilization by addition of dextran and elimination of amino acids glycine and lysine. Antioxidative excipients, e.g. methionine, osmotic amino acids, e.g. proline, glutamine, metal chelating excipients, e.g. citric acid, and a mixture of sugars and sugar alcohols, as well as small amounts of surfactant were assessed. Based on this, four SAA-based formulations (F1.1-F1.4, Table S1) were selected to stabilize TherVacB components.

      Impact of lyophilization on particulate antigen and vector integrity

      To facilitate distribution of TherVacB worldwide, without the necessity of a cooling chain, we chose to lyophilize the vaccine components. We therefore first determined the impact of thermal stress during lyophilization.
      For stabilization, we used SAA-formulation F1.1 (Table S1) as an exemplary formulation for either individual HBsAg and HBcAg or a combination thereof and the MVA-vector vaccine component. After lyophilization, pharmaceutical cake structure remained intact for F1.1-formulated in contrast to non-formulated proteins (Fig. 1D) and the MVA-vector (Fig. 1E). Antigen integrity was studied using an ELISA developed as potency assay (Fig. 1F).
      HBsAg integrity after lyophilization was reduced when formulated with PBS, but fully preserved by F1.1-formulation (Fig. 1F, left panel). HBcAg lyophilized in PBS completely lost its integrity, which could to a large part be prevented by F1.1.-formulation (Fig. 1F, right panel). Interestingly, combining HBcAg with HBsAg also preserved HBcAg integrity to a large extend.
      DLS and SE-HPLC were used to evaluate the particulate structure of the antigens. High-speed centrifugation before SE-HPLC reduced larger particles as shown by a reduced peak absorbance if antigens were not SAA-formulated before lyophilization, and a shoulder before the elution of HBsAg, suggesting the presence of larger particles (Fig. S1). In line with this observation, DLS showed an increase in hydrodynamic radius (Rh) of HBsAg particles. For HBcAg particles, the correlation function could not be defined anymore indicating the formation of larger aggregates if HBcAg was not formulated with F1.1 or combined with HBsAg (Fig. 1G). MVA-S/C infectivity remained high for all samples after lyophilization indicating intrinsic stability of the vector (Fig. 1H).
      Together, this indicated that homogeneity of HBcAg and HBsAg particles was lost during lyophilization if antigens were not formulated with SAA. From these data we concluded that the integrity of particulate protein-antigens, HBcAg and HBsAg, as well as the MVA-vector can be maintained during lyophilization by an SAA-based formulation, and that combining HBcAg and HBsAg is of advantage.

      Stabilization of HBcAg and HBsAg during short-term thermal stress

      To test if our lyophilized TherVacB vaccine components fulfill the requirements of the WHO CTC guideline, we thermally stressed them at 40°C/75% RH for three days. By ELISA, NAGE, DLS and SE-HPLC we verified that formulation with SAA was able to maintain full antigen integrity for three days at 40°C (Fig. 2A-D). PBS-formulated antigen samples lost their integrity, but SAA-formulation F1.1 prevented this and allowed fulfilling the WHO CTC criteria for vaccine development. In contrast to the protein-antigens, F1.1 formulation only showed a moderate effect to prevent temperature damage to MVA-S/C (Fig. 2E).
      Figure thumbnail gr2
      Fig. 2Effect of short-term thermal stress exposure of TherVacB vaccine components. Vaccine components were formulated with SAA-based formulation F1.1 or PBS, lyophilized and stressed for three days at 40°C/75% RH. Control (Ctrl) vaccine components were without stabilization, lyophilization and temperature exposure (set to 100% in A). (A) Quantification of HBcAg- and HBsAg by ELISA relative to Ctrl. (B) NAGE of HBcAg. (C) DLS and (D) SE-HPLC of both protein-antigens. (E) TCID50 of MVA-S/C after 3 day-storage at 5 and 40°C. Data are given as mean±SD. Statistical analysis applied unpaired t-test. **p≤0.01; ***p≤0.001; ns – not significant.

      Selection of the SAA-based vaccine formulation for storage of lyophilized vaccine components

      To challenge the SAA-formulation, we extended the recommended storage time of lyophilized vaccine antigens at 40 °C from three to 28 days and compared the modified formulations F1.1, F1.2, F1.3 and F1.4 (Table S1) in vitro and in vivo. Non-lyophilized, non-stressed and non-stabilized vaccine components constantly stored at either 4°C (HBsAg, HBcAg) or -80°C (MVA-S/C) served as control (TherVacBCtrl).
      All four SAA-based formulations, in contrast to PBS, preserved HBcAg and HBsAg integrity at 40°C/75% RH for 28 days in ELISA-based analyses and proved superior to storage in PBS at 5°C in ELISA-based analyses (Fig. 3A). NAGE revealed the typical migration pattern of intact, particulate HBcAg in all SAA-formulated samples, in contrast to non-stabilized samples stored at 40°C or 5°C where protein aggregates prevented that HBcAg run into the gel (Fig. 3B). In addition, the loss of infectivity of MVA-S/C was prevented in the presence of all four selected SAA-based formulations (Fig. 3C).
      Figure thumbnail gr3
      Fig. 3Selection of the most suitable SAA-formulation. TherVacB was formulated with four different SAA-based formulations (F1.1-F1.4, see Suppl. Tab.1) or PBS only, lyophilized and stored at 5°C or 40°C/75% RH for 28 days as indicated. Control (Ctrl) vaccine components were without stabilization, lyophilization and temperature exposure (set to 100% in A). (A) HBsAg- and HBcAg-specific ELISA, (B) NAGE of HBcAg and (C) TCID50 assay of MVA-S/C. Data are given as mean±SD. (D-G) C57BL/6J mice (n=5) were immunized with SAA-formulated or PBS-formulated TherVacB components. Non-vaccinated mice (-) or mice immunized with control (Ctrl) vaccine components not stabilized, lyophilized and constantly stored at 5°C (protein) or -80°C (MVA) served as controls. (D) Vaccination scheme. One week after boost, (E) serum anti-HBs (left panel) and anti-HBc (right panel) levels were quantified using the ArchitectTM platform. Frequencies of S-specific (left panel) and core-specific (right panel) IFNγ+ splenic CD4+ (F) and CD8+ T-cells (G) determined by flow cytometry after ICS. Data are given as mean±SEM. Statistical analysis applied unpaired t-test (A, B) and Mann-Whitney test (E, F). *p<0.05; **p≤0.01; ***p≤0.001; ns - not significant.
      To complete the analysis, we vaccinated naïve, male C57BL/6J mice with the SAA-formulated, heat-exposed vaccine components using a heterologous protein-prime/MVA-vector boost vaccination (Fig. 3D)

      Backes S, Jäger C, Dembek CJ, Kosinska AD, Bauer T, Stephan A-S, et al. Protein-prime/modified vaccinia virus Ankara vector-boost vaccination overcomes tolerance in high-antigenemic HBV-transgenic mice. 2016;34:923-932.

      . One week after boost vaccination, comparably high anti-HBs serum titers (Fig. 3E, left panel) and comparable activation of IFNγ-production CD4+ T-cells (Fig. 3F) were detected in all mice irrespective of the vaccine formulation. In contrast, anti-HBc levels were significantly higher in mice immunized with stabilized vaccine components (Fig. 3E, right panel). TherVacB induced a weak S-specific, but a strong core-specific IFNγ+ CD4+ T-cell response which was comparable between all vaccinated groups (Fig. 3F). Importantly, stabilization of vaccine components with various SAA-formulations resulted in significantly higher HBV core- and S-specific CD8+ T-cell responses (Fig. 3G). Although there was no statistically significant difference between the SAA-formulations evaluated, F1.1 seemed to elicit the most robust T-cell response among the different animals. As F1.1 also contained the least excipients conferring an advantage for clinical vaccine development it was selected for further evaluation.

      Maintenance of antigen and MVA integrity during storage at high temperatures for up to three months

      After successful stabilization of TherVacB components for one month, we wondered whether SAA-formulation would allow exposure to 40°C/75% RH for up to three months. ELISA analysis confirmed intact SAA-formulated HBcAg and HBsAg after 1-month (Fig. S2A) and 3-month storage (Fig. 4A) at 5°C, 25°C and 40°C. In contrast, formulation with PBS led to >65% loss of structural and functional integrity (Fig. 4A, Fig. S2A). DLS and SE-HPLC indicated that SAA-formulated HBsAg and HBcAg did maintain their particulate appearance despite high temperature exposure (Fig. 4B,C; Figs. S2B and C) while large, apparently aggregated particle-structures were detected already after one month in non-stabilized samples (Fig. S2B) that did not run into the NAGE gel (Fig. S2D) and lost integrity of cake structure (Fig. S2F). In contrast, even after three months at 40°C, the particulate structure of SAA-formulated antigens remained intact (Fig. 4A-C; Figs. S2E and F). MVA-S/C lost infectivity over three months and with increasing storage temperature, and SAA-formulation could only partially prevent that loss (Fig. 4D,E, Fig. S2G). TEM demonstrated that F1.1-formulated vaccine components preserved their morphology after heat-exposure while PBS-formulated protein-antigens attached to each other and formed aggregates (Fig. 4F,G).
      Figure thumbnail gr4
      Fig. 4Long-term thermal stress exposure of TherVacB vaccine components. Lyophilized F1.1- or PBS-formulated vaccine components were stored at 5°C, 25°C/60% RH or 40°C/75% RH for one (D,F,G) and three months (A-C,E). Control (Ctrl) vaccine components were without stabilization, lyophilization and temperature exposure. Antigen integrity was analysed after 3 months (A-C). (A) HBsAg- and HBcAg-specific ELISA relative to Ctrl (set to 100%), (B) DLS and (C) SE-HPLC. (D,E) TCID50 of MVA-S/C after storage for (D) one and (E) three months. Data are given as mean±SD. (F) Transmission electron microscopy of negative-stained antigens and (G) MVA-S/C after one-months storage at 40°C/75% RH. Magnification 40,000-fold, scale bars 100 nm. Statistical analysis applied unpaired t-test. *p<0.05; **p≤0.01; ***p≤0.001; ns - not significant.
      Taken together, the results confirm a successful stabilization of all vaccine components by SAA-formulation despite a 25°C or 40°C exposure for up to three months.

      Immunogenicity of SAA-formulated TherVacB after one- and three-months storage

      We next vaccinated naïve, male C57BL/6J mice to verify immunogenicity of thermally stressed TherVacB components. We used vaccine components that had been stored for one or three months at 40°C/75% RH or 25°C/60% RH. Non-lyophilized, non-stabilized and non-stressed vaccine components (TherVacBCtrl) served as positive control. In vitro analysis confirmed preserved integrity of F1.1-formulated HBsAg and HBcAg and infectivity of MVA-S/C (Fig. 5A,B).
      Figure thumbnail gr5
      Fig. 5In vivo immunogenicity of TherVacB after long-term thermal stress exposure. TherVacB vaccine components were formulated with F1.1 or PBS, lyophilized and stored at 25°C/60% RH or 40°C/75% RH for one or three months. Control (Ctrl) vaccine components were without stabilization, lyophilization and temperature exposure. (A) HBsAg- and HBcAg-specific ELISA relative to Ctrl (set to 100%). (B) TCID50 of MVA-S/C. Data are given as mean±SD. Male C57BL/6J mice (n=5) were immunized with F1.1- or PBS-formulated, heat-exposed vaccine components as indicated and analysed one week after MVA-S/C boost. Non-vaccinated mice (-) or mice immunized with control (Ctrl) vaccine components not stabilized, lyophilized and constantly stored at 5°C (protein) or -80°C (MVA) served as controls. (C) Serum anti-HBs/anti-HBc levels. (D) Percentage of core- and S-specific IFNγ+ splenic CD8+ T-cell. Data are given as mean±SEM. Statistical analysis applied unpaired t-test (A, B) and Mann-Whitney test (C,D). *p<0.05; **p≤0.01; ***p≤0.001; ns - not significant.
      One week after MVA-boost vaccination (scheme in Fig. 3D), all vaccinated animals had developed high serum anti-HBc and anti-HBs titers (Fig. 5C). In addition, mice immunized with SAA-formulated TherVacB components or TherVacBCtr, but not with PBS-formulated vaccines elicited strong core-specific CD8+ T-cell responses (Fig. 5D, right panel). S-specific CD8+ T-cell responses were lost when vaccine components were not stabilized, but only slightly reduced in comparison to TherVacBCtr when F1.1-formulated (Fig. 5D, left panel). These results clearly show that SAA-formulation allows to maintain immunogenicity of TherVacB despite exposure to up to 40°C for three months.

      Antiviral effect of stabilized TherVacB vaccine components after exposure to high temperature for one month

      Following successful immunization of naïve mice, we asked whether TherVacB efficacy in breaking immune tolerance in HBV-carrier mice was preserved after exposure to 40°C/75% RH for one month. For this, we took advantage of the AAV-HBV mouse model, in which AAV-HBV infection induces persistent replication of HBV in mouse hepatocytes over months without inducing HBV-specific immunity
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      ,
      • Dion S.
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      Adeno-Associated Virus-Mediated Gene Transfer Leads to Persistent Hepatitis B Virus Replication in Mice Expressing HLA-A2 and HLA-DR1 Molecules.
      ,
      • Kosinska A.D.
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      • Festag M.M.
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      • Protzer U.
      Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.
      .
      9-week-old, male C57BL/6J mice were infected with 4*109 AAV-HBV genome-equivalents known to reproducibly induce persistent HBV replication and immunized with heat-exposed, SAA- or PBS-formulated vaccine components using the TherVacB vaccination scheme (Fig. 6A). In vitro analysis of the stored vaccine components confirmed that the SAA-formulation had maintained antigen integrity and improved MVA-S/C infectivity (Figs. S3A and B).
      Figure thumbnail gr6
      Fig. 6Immunogenicity of SAA-formulated, heat-exposed TherVacB in a mouse model of chronic HBV infection. SAA- and PBS-formulated vaccine components were lyophilized and stored at 40°C/75% RH for one month. (A) Scheme of the experimental set-up. (B) Four weeks after infection, HBV-specific T cells in liver and spleen were identified using sensitive staining with HBV core93-100 (C93) and S190-197 (S190) peptide-loaded MHC-multimers in AAV-HBV infected and HBV-naive control mice. (C–I) Male mice (n=5) infected with AAV-HBV were immunized according to the TherVacB regimen. Non-vaccinated mice (-) or mice immunized with control (Ctrl) vaccine components not stabilized, lyophilized and constantly stored at 5°C (protein) or -80°C (MVA) served as controls. Final analyses were performed four weeks after boost vaccination. (C) Serum anti-HBs/anti-HBc titers. (D) Serum HBsAg kinetics. (E) Serum HBV-DNA levels at week 8. (F) Percentage of IFNγ+ CD4+ T-cell responses in liver. (G) IFNγ+ CD8+ T-cell responses in liver (upper panel) and spleen (lower panel). Left: HBV S-specific, right: HBV core-specific responses. (H) Serum ALT and (I) HBeAg kinetics. Data are given as mean±SEM. Statistical analysis applied Mann-Whitney test. *p<0.05; **p≤0.01; ns - not significant.
      Despite being fully immune competent, AAV-HBV infected mice did not develop a detectable HBV-specific B- or T-cell response (Fig. 6B, Fig. S4). Four weeks after boost vaccination, however, high serum anti-HBc and anti-HBs titers were observed in all vaccinated mice (Fig. 6C). High anti-HBs antibody titers efficiently suppressed serum HBsAg over time in all vaccinated animals (Fig. 6D). At week 8, HBV-DNA in the sera of most of the vaccinated mice became undetectable (Fig. 6E). However, only immunization with SAA-formulated, but not with PBS-formulated vaccine components induced a strong S-specific CD4+ T-cell response in the liver comparable to the “fresh” vaccine (TherVacBCtrl, Fig. 6F). Core-specific CD4+ T-cell responses remained below the level of detection at the examined time point in all groups of mice. SAA-formulated vaccine components induced a strong core- and S-specific CD8+ T-cell responses in liver (Fig. 6G, upper panel) and spleen (Fig. 6G, lower panel) accompanied by a mild but sustained rise in serum ALT activity (Fig. 6H), while without stabilization no CD8+ T-cell response was detected. Consequently, a gradual reduction of HBeAg after immunization was detected (Fig. 6I), which was comparable to that after TherVacBCtrl vaccination.
      Taken together, this demonstrates that even after one month’s storage at 40°C, SAA-formulated TherVacB elicited high-titer HBV-specific antibodies and functional core- and S-specific T-cell responses in AAV-HBV infected mice that efficiently suppressed HBsAg and HBeAg.

      Stability and immunogenicity of vaccine components stored for one year at room temperature

      The International Council for Harmonisation (ICH) protocol requires prolonged storage stability at elevated RT (25°C/60% RH) for vaccines being applied in countries with climate zones 1 and 2

      ICH. ICH Harmonised Tripartite Guidline. Stability testing of new drug substances and pruducts Q1A(R2). step 4 version. 2003.

      . Therefore, in further experiments we investigated whether SAA-formulation allows for long-term storage of TherVacB protein-antigens and the MVA-vector at RT for one year.
      ELISA analysis demonstrated that both SAA-formulated HBsAg and HBcAg retained their antigenicity after storage for 6, 9 and 12 months at elevated RT (Fig. 7A,B). In contrast, non-stabilized antigens lost their integrity over time while similar results were obtained when the SAA-formulated samples after storage and the starting material were compared (Fig. 7A-C). DLS (Fig. 7C) revealed an increased particle diameter and NAGE did not allow separation anymore (Fig. S5A) again indicating an aggregation of the particulate antigens that could be prevented by SAA-formulation. SE-HPLC analysis performed after 9 and 12 months of storage confirmed that SAA-formulation was able to maintain HBcAg and HBsAg integrity for up to one year (Fig. 7D). Although a loss of infectivity could not be completely prevented, SAA-formulated MVA-S/C retained significantly higher TCID50 than non-stabilized samples after long-term storage at 25°C (Fig. 7E). It is remarkable that MVA-S/C particles remained infectious at elevated RT for one year even without stabilizing formulation confirming their high intrinsic stability.
      Figure thumbnail gr7
      Fig. 7In vitro analysis of vaccine components after storage at 25°C for 12 months. TherVacB components were formulated using F1.1 or PBS, lyophilized and stored at 5°C and 25°C/60% RH for 6, 9 and 12 months. As control (Ctrl) vaccine components without lyophilization and stored at 5°C (proteins) or -80°C (MVA) were used. Stability of vaccine components were confirmed in vitro by (A) HBsAg- and (B) HBcAg-specific ELISA relative to Ctrl (set to 100%), (C) DLS and (D) SE-HPLC analysis of antigens and (E) TCID50 assay of MVA-S/C. Data given are mean±SD. Statistical analysis applied unpaired t-test. *p<0.05; **p≤0.01; ***p≤0.001; ns - not significant.
      Using the SAA-formulated TherVacB components stored for one year at elevated RT (25°C, Figs. S5B and C), we repeated the immunization (Fig. 6A) in AAV-HBV infected mice as an HBV persistence model to determine the antiviral effect. Four weeks after boost vaccination, all vaccinated mice had high serum anti-HBs titers (Fig. 8A) irrespective of the vaccine formulation. Anti-HBc titers, in contrast, were significantly higher in mice vaccinated with SAA-formulated antigens (Fig. 8B). F1.1-formulated vaccine allowed to improve induction of S-specific and vigorous core-specific IFNγ+ CD8+ T-cell responses in spleen and liver (Fig. 8C,D). In particular, frequencies of HBV-specific granzyme B (GzmB)-positive hepatic CD8+ T cells were higher in F1.1- compared to PBS-immunized mice (Fig. 8E), indicating an improved effector T-cell response elicited by stabilized TherVacB.
      Figure thumbnail gr8
      Fig. 8Immunogenicity and antiviral efficacy of TherVacB in AAV-HBV mice after storage at 25°C for 12 months. TherVacB components were formulated using F1.1 or PBS, lyophilized and stored at 25°C/60% RH for 12 months. AAV-HBV infected, male mice (n=5) were immunized using the TherVacB scheme. Non-vaccinated mice (-) or mice immunized with control (Ctrl) vaccine components not stabilized, lyophilized and constantly stored at 5°C (protein) or -80°C (MVA) served as controls. Final analyses were performed four weeks after boost vaccination. (A) Serum anti-HBs and (B) anti-HBc levels. Percentage of splenic (C) and hepatic (D) IFNγ+ CD8+ T-cells and hepatic GzmB+ CD8+ T-cells (E). Upper panel: HBV S-specific, lower panel: core-specific responses. (F) Serum HBsAg kinetics. (G) Serum HBV-DNA levels at sacrifice. (H) Serum HBeAg kinetics. (I) Quantification of HBcAg-positive hepatocytes per mm2; (J) corresponding representative immunohistochemistry, scale bars 100 μm; (K) intrahepatic HBV-DNA detected in liver tissue at final analysis. Data given are mean±SEM. Statistical analysis applied Mann-Whitney test. *p<0.05; **p≤0.01; ns - not significant.
      All vaccinated groups efficiently suppressed serum HBsAg and HBV-DNA (Fig. 8F,G). Hereby, the drop in HBsAg and in HBV-DNA was significantly stronger in the mice which had received the SAA-formulated vaccine than in those which had received the non-stabilized vaccine (Fig. 8F,G). The strong remaining immunogenicity of SAA-formulated TherVacB stored for one year at elevated RT was also reflected by a decrease in serum HBeAg levels (Fig. 8H) and a significant reduction of HBcAg-positive hepatocytes and intrahepatic HBV-DNA indicating HBV clearance (Fig. 8I-K). This effect was as strong as the effect of TherVacBCtrl that was not lyophilized and continuously stored at optimal conditions.
      Taken together, SAA-formulated TherVacB components remained immunogenic and retained their potential to break immune tolerance in HBV-carrier mice. Even after 1-year storage at elevated RT, SAA-formulated TherVacB protein and MVA-components remained active and elicited a potent, HBV-specific effector T-cell response allowing TherVacB to efficiently control HBV replication and eliminate HBV-infected hepatocytes.

      Discussion

      To address the challenge of transport and storage of a vaccine at elevated temperatures necessary for the application in many regions of the world, we formulated protein and vector vaccine components with SAA-based formulations. SAA-formulation resulted in a long-term thermostable therapeutic hepatitis B vaccine. The SAA-formulated vaccine components of TherVacB, HBsAg, HBcAg and the MVA-vector, fulfilled and exceeded the stability criteria of the WHO CTC guidelines as well as EMA and ICH requirements

      ICH. ICH Harmonised Tripartite Guidline. Stability testing of new drug substances and pruducts Q1A(R2). step 4 version. 2003.

      ,

      WHO. WHO expert committee on specifications for pharmaceutical preparations. Geneva 2009. Technical report series 953.

      . Integrity of the particulate protein-antigens, HBsAg and HBcAg, as well as the MVA-vector was maintained despite a 40°C heat-exposure for up to three months, as well as during long-term storage at 25°C for one year serving as an elevated RT in warmer regions. SAA-formulated vaccine components were well-tolerated and induced a strong HBV-specific antibody and T-cell response in HBV-naïve and AAV-HBV infected mice even after long-term storage. Our results are not only important for the development of a therapeutic hepatitis B vaccine, but also provide a learning example for other vaccination campaigns currently running worldwide.
      Thermal stability is essential for worldwide vaccination campaigns. Thus, development of heat-stable vaccines gained significant interest over the past decade. Ohtake et al. presented a spray-dried measles vaccine which was stable at 37°C for approximately two months
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      . Lyophilized and stabilized herpes simplex virus type 2, Ebola and influenza vaccines proved heat-stable for two to three months at 40°C
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      . Our SAA-formulated hepatitis B vaccine easily withstood these conditions by maintaining stability and immunogenicity in vivo for three months at 40°C. This adds to the evidence that SAA-formulations can stabilize a broad range of various biologics, such as antibodies, adenoviral viral vectors or split-virion influenza vaccine
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      . In addition, we demonstrated that a single SAA-formulation is able to efficiently stabilize very different vaccine components such as the complex, particulate recombinant protein-antigens and the MVA-viral vector used in TherVacB. This will simplify vaccine manufacturing and clinical approval.
      Lyophilization is a widely used drying method to improve the thermal stability of vaccine components during transport and storage. The advantage of HBsAg freeze-drying was already demonstrated in vitro and in vivo
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      . We demonstrate that despite many advantages of lyophilization, the freeze-drying process can also cause significant damage. We show that the negative effects of lyophilization can be prevented by SAA-formulation and by combining TherVacB protein-antigens and thereby confirm earlier reports that a clever selection of cryoprotectants and lyoprotectants helps to prevent vaccine damage
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      . Antigen combination provides an additional benefit for future clinical application as it allows a single “ready to use” formulation.
      After thermal stressing aggregated partially instable vaccine antigens were still sufficient to induce HBV-specific antibodies. Electron microscopy revealed that even after prolonged stressing in high temperature the overall structure of the antigens remained intact. However, our potency ELISA indicated that some of the conformational epitopes had been lost. This confirms recent reports that aggregates with a native-like structure are more immunogenic that those consisting of fully degraded proteins
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      and are capable of inducing antibodies independently of vaccine formulation’s particle diameter
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      . Interestingly, the thermal stress had less effect on antibody and CD4+ T-cell responses than on CD8+ T-cell responses. This may have two reasons. First, infectivity of the MVA vector which is essential to boost CD8+ T-cell responses is affected by lyophilization and thermal stress. In addition, aggregation of the non-protected protein antigens reduces the amount of antigen taken-up and presented by antigen presenting cells, and CD8+ T-cell activation has been reported to critically depend on antigen amounts
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      . Accordingly, we also observed that immunization with lower amounts of antigens or MVA vector significantly diminished vaccine-elicited CD8+ T-cell responses but had a minor effect on the induction of HBV-specific antibodies (unpublished data).
      Recently, MVA has gained considerable interest as a safe vaccine vector facilitating the induction of T-cell responses against encoded antigens

      Volz A, Sutter G. Modified Vaccinia Virus Ankara. Elsevier; 2017. p. 187-243.

      . The induction of potent, antigen-specific T-cell responses is of high importance for the development of vaccines against malaria, tuberculosis or human immunodeficiency virus (HIV) infection
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      , which are also prevalent in countries with high outdoor temperatures. Other MVA-based prophylactic and therapeutic vaccines currently in clinical trials
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      may profit from our findings as we clearly demonstrate that formulation with SAA retained infectivity and immunogenicity of MVA-S/C even after rather tough temperature stressing.
      Very importantly, currently used prophylactic hepatitis B vaccines based on HBsAg must be stored at 2-8°C to maintain vaccine efficacy

      WHO. Temperature sensitivity of vaccines. Geneva 2006. Ordering code: WHO/IVB/06.10.

      . We found a profound gradual loss of antigen integrity over time when non-stabilized HBsAg or HBcAg were exposed to elevated RT. Other studies reported that prophylactic hepatitis B vaccines containing HBsAg were stable even at higher temperatures

      WHO. Temperature sensitivity of vaccines. Geneva 2006. Ordering code: WHO/IVB/06.10.

      . Formulation with aluminium as adjuvants and other excipients may provide an explanation for the differences observed. Despite the potential advantage of alum for HBsAg stability, alum-based vaccine formulations are not suitable to support the induction of efficient CD8+ T-cell responses required to break immune tolerance, eliminate chronic HBV infection
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      or prevent and control infections a number of other viruses. Using c-di-AMP as adjuvant that allows for a balanced Th1/Th2- and subsequent effector T-cell response, our lyophilized HBsAg formulation demonstrated superior temperature and storage stability compared to the alum-based liquid HBsAg

      WHO. Temperature sensitivity of vaccines. Geneva 2006. Ordering code: WHO/IVB/06.10.

      ,
      • Just M.
      • Berger R.
      Immunogenicity of a heat-treated recombinant DNA hepatitis B vaccine.
      ,
      • Van Damme P.
      • Cramm M.
      • Safary A.
      • Vandepapelière P.
      • Meheus A.
      Heat stability of a recombinant DNA hepatitis B vaccine.
      .
      During the course of CHB, HBV-specific T-cells are scarce and progressively become dysfunctional

      Bertoletti A. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. 1994;180:933-943.

      • Chisari F.V.
      • Ferrari C.
      Hepatitis B virus immunopathogenesis.
      • Maini M.K.
      • Boni C.
      • Ogg G.S.
      • King A.S.
      • Reignat S.
      • Lee C.K.
      • et al.
      Direct ex vivo analysis of hepatitis B virus-specific CD8+ T cells associated with the control of infection.
      . We reason that a successful therapeutic vaccine must elicit highly functional virus-specific B- and T-cells de novo. In the AAV-HBV mouse model that develops a strong immune tolerance, therapeutic vaccination is able to induce neutralizing antibodies and newly prime and activate functional effector T cells. In our study we have demonstrated that SAA-formulated TherVacB elicited high anti-HBs titers leading to undetectable circulating HBsAg and serum HBV-DNA levels in most of the mice which represents functional cure of HBV
      • Gehring A.J.
      • Protzer U.
      Targeting Innate and Adaptive Immune Responses to Cure Chronic HBV Infection.
      . Moreover, stabilized vaccine resulted in high numbers of intrahepatic effector HBV-specific CD8+ T cells expressing IFNγ and GzmB which eliminated HBV-positive hepatocytes. However, restoration of effective HBV-specific immunity in chronic HBV-carrier patients might be much harder due to the extremely long-term exposure towards HBV antigens in particular when HBV-infection was acquired around birth.
      In our experiments, clearance of HBV from the liver was accompanied by elevation in ALT levels suggesting an involvement of vaccine-induced, antigen-specific effector cells with cytolytic activity. Besides cytotoxic CD8+ T cells, hepatic natural killer (NK) cells are known to contribute to elimination of HBV-infected cells and HBV-induced liver damage
      • Fisicaro P.
      • Rossi M.
      • Vecchi A.
      • Acerbi G.
      • Barili V.
      • Laccabue D.
      • et al.
      The Good and the Bad of Natural Killer Cells in Virus Control: Perspective for Anti-HBV Therapy.
      . Our recent and previously published data
      • Kosinska A.D.
      • Moeed A.
      • Kallin N.
      • Festag J.
      • Su J.
      • Steiger K.
      • et al.
      Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection.
      ,
      • Kosinska A.D.
      • Festag J.
      • Muck-Hausl M.
      • Festag M.M.
      • Asen T.
      • Protzer U.
      Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.
      implied that TherVacB is capable of inducing hepatic HBV-specific CD8+ T cells with cytotoxic potential. However, a contribution of NK cells to the TherVacB-mediated antiviral effect observed in this study has not been addressed and cannot be excluded.
      Taken together, we here demonstrate that development of a thermostable, highly immunogenic, therapeutic vaccine for the treatment of chronic hepatitis B is feasible. The high thermostability of all vaccine components is of particular importance for future distribution of therapeutic as well as prophylactic vaccines as hepatitis B is predominantly endemic in countries with high outdoor temperatures. Thus, the usage of thermostable vaccine components will be of utmost importance for the worldwide hepatitis B vaccination campaigns but also for other, MVA-vector based vaccines.

      Data Availability

      All data supporting this study is provided in the results section or as supplementary information accompanying this paper.

      Acknowledgments

      We thank Michael Lehmann for support with MVA recombination and purification, Philipp Hagen, Claudia Kahlhofer, Romina Bester, Theresa Asen, Susanne Miko for excellent technical assistance, Marie-Louise Michel (Institute Pasteur, Paris, France) for the AAV-HBV1.2 construct and Carlos Guzman and Thomas Ebensen (Helmholtz Center for Infection Research, Braunschweig, Germany) for advice on c-di-AMP. We appreciate the support and constructive discussion with Sabine Hauck and Frank Thiele. We are grateful to Behnam Naderi Kalali, Ahmed Sadek, Regina Feederle, for their help with antibody production and the Center for Genetic Engineering and Biotechnology (CIGB, Havanna, Cuba), Dieter Glebe (University of Giessen, Germany) and Andris Dislers (Riga, Latvia) for providing antibodies and HBcAg. The work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB-TRR 179/2 2020 –272983813, the European Union's Horizon 2020 research and innovation programme under grant agreement no. 848223, The German Center for Infection Research (DZIF) and the German Ministry for Science as KMU Innovativ project StabVacHepB AZ 031B0094C. JSa received a stipend by the Stiftung der Deutschen Wirtschaft, JSu by the Chinese Scholarship Council.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

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