1. Introduction

2. Materials and Methods

2.2. Serological Assays

Anti-HBs antibody levels were determined using the Architect anti-HBs quantitative assay (Abbott Laboratories, Germany). Recombinant preS (preS1 + preS2) and preS-derived synthetic peptides spanning the whole preS sequence of HBV genotype A2 (GenBank: AAT28735), as well as peptides covering the NTCP attachment site of genotypes A-H (Figure S1) were produced as described [7]. The preS- and peptide-specific IgG, IgG_1-4, IgM, IgA determination as well as IgG₁ and IgG₄ quantification were performed by enzyme-linked immunosorbent assay (ELISA) as described in [7]. If not stated otherwise, plots and charts were created using Prism 6.0 software (GraphPad, CA, USA).

Table S1. Lists of synthetic preS-derived peptides. Shown are the amino acid sequences of the peptides used in the assessment of preS-specific immune response.

Table S1. Lists of synthetic preS-derived peptides. Shown are the amino acid sequences of the peptides used in the assessment of preS-specific immune response.

preS-derived peptides	Accession№.	Sequence
Peptides mapping the N-terminal epitopes of preS
Peptide A (aa 1-29) Peptide B (aa 30-61) Peptide C (aa 17-51)	AAT28735	CMGGWSSKPRKGMGTNLSVPNPLGFFPDHQ
		CLDPAFGANSNNPDWDFNPIKDHWPAANQVGVG
		CSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDH
Overlapping peptides spanning the whole preS sequence
P1 (aa 2-31) P2 (aa 22-51) P3 (aa 42-71) P4 (aa 62-91) P5 (aa 82-111) P6 (aa 102-131) P7 (aa 122-151) P8 (aa 142-174)	AAT28735	GGWSSKPRKGMGTNLSVPNPLGFFPDHQLD
		LGFFPDHQLDPAFGANSNNPDWDFNPIKDH DWDFNPIKDHWPAANQVGVGAFGPGLTPPH AFGPGLTPPHGGILGWSPQAQGILTTVSTI QGILTTVSTIPPPASTNRQSGRQPTPISPP GRQPTPISPPLRDSHPQAMQWNSTAFHQAL WNSTAFHQALQDPRVRGLYFPAGGSSSGTV PAGGSSSGTVNPAPNIASHISSISARTGDPVTN
Peptides covering the NTCP attachment site of genotypes A-H
Genotype A Genotype B Genotype C Genotype D Genotype E Genotype F Genotype G Genotype H	APD28359	GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDH
	BAA88276	GTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDN
	BAA32833	GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDH
	BAD02320 BAC65105 AAG49720 BAB64320 BAB69786	GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDT GKNHSTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDH GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNKNKDN GKNLSTSNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDP GQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDN

2.3. preS Micro-Array Production and Sample Analysis

Glass slides containing 6 micro-arrays formed by oval epoxy frames (Paul Marienfeld GmbH & Co. KG, Germany) were coated with amino-reacting polymer MCP-2 (Lucidant Polymers, CA, USA). The recombinant preS and preS-derived synthetic peptides were diluted and re-buffered for the following spotting conditions: concentration 0.5-1 mg/mL, buffer 75 mM Na₂HPO₄, pH 8.4. Afterwards they were spotted in triplicates on the pre-activated glass slides with a SciFlexArrayer S12 (Scienion AG, Germany) as described in [9]. The micro-array layout is depicted in Figure S1.

Figure S1. Layout and exemplary measurements of the preS micro-array. (a) Spotting scheme of a preS micro-array showing the order of dots (triplicates). (b, c) Scanned images depicting fluorescence of the (b) pre-immune and (c) and a post-vaccination serum sample.

Figure S1. Layout and exemplary measurements of the preS micro-array. (a) Spotting scheme of a preS micro-array showing the order of dots (triplicates). (b, c) Scanned images depicting fluorescence of the (b) pre-immune and (c) and a post-vaccination serum sample.

For specific antibody detection, micro-arrays were washed for 1 min with phosphate-buffered saline containing 1% Tween 20 (PBST) and dried with a Sigma centrifuge and MTP-11113 rotor (both Sigma Laborzentrifugen GmbH, Germany). For IgG, IgG₁, IgG₄, IgM detection serum was diluted 1:100 and for IgA detection 1:20 with a sample diluent (ImmunoCAP Specific IgA/IgG Sample Diluent, Phadia Ab, Sweden). Aliquots of 30 µL were added per array and incubated for 2 h at 22°C. After another washing step, corresponding detection antibodies labelled with DyLight 550 (Cat. 62269, Pierce, IL, USA) with concentration 1.8 µg/mL targeting various types of immunoglobulins were applied: IgG (Cat. 009-000-003, Jackson ImmunoResearch, PA, USA), IgG₁ (Cat.555868, BD Biosciences, NJ, USA), IgG₄ (Cat. 555878, BD), IgA₁/A₂ (Cat. 555886, BD), IgM (Cat. 555856, BD), and incubated for 30 min at 22 ◦C. After incubation, the micro-arrays were washed with PBST and with ddH₂O to remove unbound antibodies and salts and then scanned with a Tecan Powerscanner (Tecan Trading AG, Switzerland). Detection was performed as described in [10]. MAPIX version 8.5.0 (Innopsys, France) software was used to analyze the scanned images. The specific antibody levels are expressed in fluorescence intensity (FI) units, the FI of sample diluent alone was subtracted for each antigen. Depicted are means of triplicate measurements.

2.5. Virus Neutralization Assay

A brief summary of the in vitro virus neutralization assay is depicted in Figure S2. In order to generate the infectious HBV preparation in vitro, a human hepatoma cell line (HepG2) was stably transfected with a plasmid encoding a 1.1 over-length HBV genome of genotype D3 (GenBank: NC_003977). Cells were incubated at 37°C, 5% CO₂ in William’s E medium (Thermo Fisher Scientific, Germany) containing 2% DMSO (Carl Roth, Germany), 2% fetal calf serum (FCS; Thermo Fisher Scientific), 1 µg/mL doxycycline (Carl Roth), 1 µg/mL G418 (Carl Roth) and 1µg/mL puromycin (Carl Roth) for several weeks with continuous media exchanges. Supernatant was collected and ultra-filtrated to enrich viral particles. Viral load was determined to be 3.7x10¹¹ Ge/mL by a highly-sensitive qPCR as previously described [14]. HBsAg in the viral stock was determined to be 4080 IU/mL by the quantitative Architect HBsAg assay (Abbott Laboratories, Germany).

Figure S2. Scheme of the in vitro virus neutralization assay. A human hepatoma cell line (HepG2), stably expressing the HBV high affinity entry receptor NTCP, was infected with in vitro generated HBV (genotype D) after pre-incubation with immune sera at specified concentrations. HBV-infected cells secrete the soluble HBeAg, which is an accepted quantitative marker for productive HBV infection in cell culture experiments. To authenticate the generated HBV infectivity parameter HBeAg, the conventionally used qualitative immunofluorescence of HBV-infected cells was used. Here, newly produced intracellular HBV core protein (HBcAg) after HBV infection was stained and analyzed fully-automated to gain additional semi-quantitative data on the number of HBV-infected cells.

Figure S2. Scheme of the in vitro virus neutralization assay. A human hepatoma cell line (HepG2), stably expressing the HBV high affinity entry receptor NTCP, was infected with in vitro generated HBV (genotype D) after pre-incubation with immune sera at specified concentrations. HBV-infected cells secrete the soluble HBeAg, which is an accepted quantitative marker for productive HBV infection in cell culture experiments. To authenticate the generated HBV infectivity parameter HBeAg, the conventionally used qualitative immunofluorescence of HBV-infected cells was used. Here, newly produced intracellular HBV core protein (HBcAg) after HBV infection was stained and analyzed fully-automated to gain additional semi-quantitative data on the number of HBV-infected cells.

Next, HepG2-NTCP cells [15] were pre-treated with 6 µg/mL doxycycline to induce NTCP expression at least 3 days prior to infection and subsequently seeded into 24-well plates the day before infection. Cells were infected with virus inoculum (5x10⁹ Ge; 55.9 IU HBsAg) in hepatocyte growth medium (HGM) consisting of William’s E medium supplemented with 1× insulin-transferrin-selenium (Thermo Fisher Scientific, Germany), 2 mM l-glutamine (Thermo Fisher Scientific), 100 µg/mL gentamicin (Thermo Fisher Scientific), 10 nM dexamethasone (Sigma-Aldrich, Germany), 1 mM sodium pyruvate (Thermo Fisher Scientific), 0.2% bovine serum albumin (BSA; Carl Roth, Germany) in the presence of 4% PEG-8000 (Sigma-Aldrich), 2% DMSO (Carl Roth) and 100 ng/mL EGF (PeproTech, Germany) as final concentration (f.c.) each. NTCP expression was routinely controlled by uptake of the green-fluorescent bile salt NBD-TC (4-nitrobenzo-2-oxa-1,3-diazole taurocholic acid) as previously described [16].

For neutralization, virus inoculum was pre-incubated with sera at defined concentrations in a dilution with HGM for 1 h, 37°C and under regular agitation. Pre-incubation with mouse-derived monoclonal anti-preS1 antibody MA18/7 detecting an epitope DPXF in the preS1 amino acids 20 to 23 (31 to 34 in genotype A) [17] was performed in the same manner and served as neutralization control. Twenty hours after virus inoculation, cells were washed three times with HGM and further incubated for 9 days in HGM supplemented with 2% DMSO with medium changed every 2 days. As a first outcome parameter of the HBV virus neutralization assay, HBeAg secreted from infected cells into the cell culture supernatant was determined in supernatants collected from day 7 to 10 post infection (p.i)., using the Architect HBeAg assay (Abbott Laboratories, Germany).

As a second outcome parameter, immunofluorescence of the infected cells was measured as follows: At day 10 p.i., cells were fixed by incubation with 3.7% formaldehyde in PBS for one hour at 4°C, washed with PBS and permeabilized using 0.2% Triton X-100 in PBS for 30 min at room temperature (RT). Cells were washed again, and blocked with 10% FCS in PBS for 60 min at RT. As primary antibody, a polyclonal α-HBc antiserum from an immunized guinea pig (Eurogentec, Belgium) was used in a 1:500 dilution for 1 h at 37°C and cells were then washed five times. For detection, cells were incubated with Alexa-488 coupled α-guinea-pig-IgG-antibodies (Thermo Fisher Scientific, Germany) in a 1:400 dilution for 30 min at 37°C in the dark and subsequently washed five times. Nuclei were stained by incubation of cells with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL f.c.) for one hour at 37°C in the dark. Analysis of immunofluorescence was performed with the ImageXpress Pico automated cell-imaging system (Molecular Devices, CA, USA). The total area analyzed was 12% of the total well size.

IC₅₀ values were determined using the integrated “Absolute IC₅₀, X is concentration” analysis integrated into Graphpad Prism version 9.4.1. Results are displayed as IC₅₀ (± 95% CI). In the neutralization experiments serum from a subject immunized with HBsAg-based vaccines containing 2600 IU/L anti-HBs was included for control purposes.

3. Results

3.2. VVX001 Induces Robust HBV-Specific Antibody Responses Directed Mainly to the N-Terminus of the preS-Containing NTCP Binding Site

Subcutaneous immunization with VVX001 was started in November 2018. No relevant immediate or late-phase side effects were observed after each of the five injections containing 20 µg VVX001 adsorbed to Aluminum hydroxide when applied in monthly intervals. The vaccination with VVX001 induced a strong and sustained preS-specific IgG response peaking already after the third immunization (Figure 2b, Figure S3) as well as distinct increases of Phl p 5-specific IgG antibodies (Figure S4). During the ongoing VVX001 immunization we also observed a sharp increase of anti-HBs antibodies which vanished almost completely after one month, although VVX001 did not contain SHBs (Figure 2a).

Figure S3. The preS-specific antibody response was dominated by IgG₁. Shown are the OD values of preS-specific IgG, IgG₁, IgG₂, IgG₃, IgG₄, IgM, IgA measured by ELISA (y-axis) over the time.

Figure S3. The preS-specific antibody response was dominated by IgG₁. Shown are the OD values of preS-specific IgG, IgG₁, IgG₂, IgG₃, IgG₄, IgM, IgA measured by ELISA (y-axis) over the time.

Figure S4. Phl p 5-specific IgG were lower than preS-specific IgG levels. Shown are the OD values of preS- and Phl p 5-specific IgG levels measured by ELISA over the time.

Figure S4. Phl p 5-specific IgG were lower than preS-specific IgG levels. Shown are the OD values of preS- and Phl p 5-specific IgG levels measured by ELISA over the time.

The analysis of preS-specific antibody isotypes and subclasses showed that the preS-specific antibody response was composed mainly of IgG₁ (Figure S3). The epitope specificity of the induced antibodies assessed with overlapping synthetic peptides P1-P8 (Figure 3a) showed that IgG antibodies were mainly directed to the N-terminal part of preS which contains the liver cell attachment site of HBV (Figure 3b). Antibodies were measured by ELISA and by micro-array technology.

Figure 3. VVX001-induced IgG antibodies react mainly with the N-terminus of preS1. Shown is (a) the localization scheme of preS-derived peptide within the preS sequence (amino acid numbering indicated for genotype A) and levels of preS/peptide-specific IgG measured by (b) ELISA (OD) and (c) by micro-array technology (fluorescence intensity, FI).

Figure 3. VVX001-induced IgG antibodies react mainly with the N-terminus of preS1. Shown is (a) the localization scheme of preS-derived peptide within the preS sequence (amino acid numbering indicated for genotype A) and levels of preS/peptide-specific IgG measured by (b) ELISA (OD) and (c) by micro-array technology (fluorescence intensity, FI).

The design of the preS microarray containing preS and preS-derived peptides (six micro-arrays on each glass slide) and images of scanned images are depicted in Figure S1. The results from the micro-array measurements were in good agreement with results obtained by ELISA and showed a similar pattern of IgG reactivity focusing mainly to the N-terminus of preS. However, stronger signals were obtained with the micro-array for the peptides as compared to the complete preS protein (Figure 3b, 3c). The levels of preS-specific IgM and IgA (Figure S5) were low and also directed mainly to N-terminal peptides of preS.

Figure S5. IgM and IgA levels specific for preS and preS-derived peptides. Shown are levels of preS/peptide-specific (a) IgM and (b) IgA (fluorescence intensity, FI) measured with the preS micro-array.

Figure S5. IgM and IgA levels specific for preS and preS-derived peptides. Shown are levels of preS/peptide-specific (a) IgM and (b) IgA (fluorescence intensity, FI) measured with the preS micro-array.

The micro-array measurements of preS- and peptide-specific IgG₁ and IgG₄ confirmed that IgG₁ was the dominating IgG subclass with maximal levels present 1 month after the last injection of VVX001 (Figure S6a). The induction of IgG₄ became also visible, but followed a different kinetic. The build-up phase of IgG₄ was longer and maximal antibody levels were reached 3 months later than for IgG₁ (i.e., 4 months after the last VVX001 injection) (Figure S6b). The quantification of IgG₁ and IgG₄ specific to preS showed that the preS-specific IgG₁ serum concentration reached 1.9 mg/mL one month after the last injection and preS-specific IgG₁ at a concentration of 0.68 mg/mL was still present one year after the course of immunizations (Table 1). The concentrations of preS-specific IgG₄ were approximately 100-fold lower and, accordingly, could not be detected after the single immunization (Table 1). Nonetheless, preS-specific IgG₄ were still detectable by micro-array measurements one year later (Figure S6b). The quantification of N-terminal peptide-specific antibody levels suggested that the induced antibodies reacted to both N-terminal epitopes of preS which are important for HBV infectivity with most of the reactivity directed towards the region defined by peptide A (Table 1).

Figure S6. IgG₁ and IgG₄ reactivity to preS and preS-derived peptides. Shown are levels of preS/peptide-specific (a) IgG₁ and (b) IgG₄ (fluorescence intensity, FI) measured with the preS micro-array.

Figure S6. IgG₁ and IgG₄ reactivity to preS and preS-derived peptides. Shown are levels of preS/peptide-specific (a) IgG₁ and (b) IgG₄ (fluorescence intensity, FI) measured with the preS micro-array.

3.3. VVX001-Induced Antibodies Cross-React with HBV Genotypes A-H

To address the question of genotype cross-reactivity, we have synthesized eight peptides representing the liver cell attachment site of HBV genotypes A-H (Table S1) as described [7]. According to both ELISA and micro-array measurements, IgG cross-reactivity to the eight peptides representing the NTCP attachment site was at all time points detectable for genotypes A-H with somewhat lower reactivity to genotypes D, E and G (Figure 4). The genotype cross-reactivity was also assessed using recombinant SVPs composed of large, middle and small HBV surface proteins (LHBs, MHBs, SHBs) of HBV genotypes A2, B2, C2, D1, E, F4, H as well as a control SVPs composed of genotype D3 SHBs only (Figure 5). The pre-immune serum of the subject of the current study did not show any SVP reactivity before VVX001 immunization. The sample obtained one month after the last immunization reacted to all SVPs tested with the exception of the control SVPs of genotype D3 which contained no preS. Both of the two conventionally vaccinated subjects who had received the standard HBsAg-based immunization and had high anti-HBs levels within two years prior to the blood donation (subject 1: anti-HBs > 4000 IU/L, subject 2: anti-HBs > 6500 IU/L) reacted to all SVPs including control genotype D3 comparably. Serum obtained from the study subject obtained one month (05.04.2019) after the last immunization with VVX001 showed higher reactivity to all LHBs-containing SVPs than the sera from the two control subjects (Figure 5).

Figure 4. VVX001-induced antibodies cross-react to the NTCP binding site of preS of HBV genotypes A-H. Shown are the IgG levels to the synthetic peptides representing the NTCP attachment site of HBV genotypes A-H measured by (a) ELISA (OD) and (b) micro-array technology (fluorescence intensity, FI).

Figure 4. VVX001-induced antibodies cross-react to the NTCP binding site of preS of HBV genotypes A-H. Shown are the IgG levels to the synthetic peptides representing the NTCP attachment site of HBV genotypes A-H measured by (a) ELISA (OD) and (b) micro-array technology (fluorescence intensity, FI).

Figure 5. VVX001-induced antibodies react to SVPs of different HBV genotypes. Shown are IgG levels (OD) to SVPs of HBV genotypes A2, B2, C2, D1, E, F4, H (LHBs, MHBs, SHBs) and D3 (SHBs only) in serum samples of the study subject before and after immunization as well as in two subjects successfully vaccinated with conventional HBsAg-based vaccines.

Figure 5. VVX001-induced antibodies react to SVPs of different HBV genotypes. Shown are IgG levels (OD) to SVPs of HBV genotypes A2, B2, C2, D1, E, F4, H (LHBs, MHBs, SHBs) and D3 (SHBs only) in serum samples of the study subject before and after immunization as well as in two subjects successfully vaccinated with conventional HBsAg-based vaccines.

3.4. VVX001-Induced Antibodies Strongly Neutralize HBV Infection In Vitro

A neutralizing ability of the antibodies induced by the vaccination was assessed for the samples obtained from the subject after vaccination with Engerix (February 2018) as well as before, during and after immunization with VVX001 (Figure 2c). A well-established diagnostic HBeAg assay was used to determine in vitro virus neutralization with a high range between positive controls (i.e., HBV infection without addition of serum “PC” HBeAg: 158.82-166.09 S/CO (signal/cutoff) and negative control (i.e., uninfected cells HBeAg <1 S/CO). There was no relevant increase of virus neutralization after the last HBsAg booster injection with Engerix-B from February 2018 to November 2018 (Figure 2c). Immunization with VVX001 resulted in a strong increase in HBV neutralization with a strong neutralization observed between the time points February 2019 (1 month after the 3^rd VVX001 injection) and May 2019 (2 months after the 5^th VVX001 injection). The subsequent samples showed a decrease of neutralization potential but neutralization effects were observed until October 2019. After October 2019 no virus neutralization was observed in the in vitro virus neutralization test although anti-preS antibodies were still present (Figure 2b,c).

Next, titration experiments were performed to determine IC₅₀ values (Figure S7). For titration the serum obtained 1 month after the last VVX001 injection on 05.04.2019 was used. It showed an IC₅₀ corresponding to 1.44% (± 0.48) of serum in the virus pre-incubation mix (Figure 6a). As a control, an immune serum containing 2600 IU/L of anti-HBs antibodies after conventional vaccination was used which yielded an IC₅₀ corresponding to 0.92% (± 0.64) of serum in the virus pre-incubation mix (Figure 6b). Based on this comparison we estimated that the subject´s serum taken in April 2019 contained “functionally” neutralizing antibodies corresponding to approximately 1661 IU/L of anti-HBs antibodies. The monoclonal anti-preS1 antibody MA18/7 was already 97% neutralizing at 1 µg/mL and fully neutralizing at all higher concentrations (Figure S7a,b). Therefore, the calculation of an IC₅₀ of the MA18/7 could not be performed.

Figure S7. Additional outcome parameters of in vitro HBV neutralization assays. (a) HBeAg secretion and (b) de novo expressed HBcAg (baseline correction of immunofluorescence was performed by subtracting the value of the uninfected control) of infected cells after pre-incubation of HBV inoculum with titrated sera after vaccination. Data are presented as % compared to positive control (PC, infection w/o serum pre-incubation). Neutralization: ≥90% (strong neutralization; green), ≥50% (partial neutralization; blue), ≥10% (weak neutralization; magenta); HBeAg below cut-off (grey). Ø = 0% HBcAg-positive cells after baseline-correction.

Figure S7. Additional outcome parameters of in vitro HBV neutralization assays. (a) HBeAg secretion and (b) de novo expressed HBcAg (baseline correction of immunofluorescence was performed by subtracting the value of the uninfected control) of infected cells after pre-incubation of HBV inoculum with titrated sera after vaccination. Data are presented as % compared to positive control (PC, infection w/o serum pre-incubation). Neutralization: ≥90% (strong neutralization; green), ≥50% (partial neutralization; blue), ≥10% (weak neutralization; magenta); HBeAg below cut-off (grey). Ø = 0% HBcAg-positive cells after baseline-correction.

Figure 6. IC₅₀ determination. IC₅₀ values for serum dilutions determined based on the HBeAg results are presented with 95% confidence interval for (a) the anti-preS-positive human serum obtained in April 2019 and (b) for the anti-S-positive human serum (conventional vaccine, 2600 IU/L anti-HBs).

Figure 6. IC₅₀ determination. IC₅₀ values for serum dilutions determined based on the HBeAg results are presented with 95% confidence interval for (a) the anti-preS-positive human serum obtained in April 2019 and (b) for the anti-S-positive human serum (conventional vaccine, 2600 IU/L anti-HBs).

In fact, an even higher virus neutralization was measured in the subject of this study after another course of preS-based self-vaccination with a preS-RBD SARS-CoV-2 vaccine as described [19]. After the immunization with the preS-RBD vaccine, a 98.6% HBV neutralization (HBeAg 1.185 S/CO in the tested sample vs. HBeAg 83.63 S/CO at baseline) was observed indicating that it is possible to boost the preS-specific virus-neutralizing antibody response by repeated immunizations.

3.5. VVX001 Induces a Sustained preS-Specific CD3⁺CD4⁺ Cellular Response Which Is Accompanied by a Mixed Th2/Th1 Cytokine Response

Vaccination with VVX001 induced a growing and sustained preS-specific CD4⁺ T cell and to a much lower degree of a preS-specific CD8⁺ T cell response in the study subject (Figure 7a). Interestingly, peptide 1 derived from the N-terminus of preS, which was identified as a major B cell epitope, was also identified as a T cell epitope-containing peptide (Figure 7b). All peptides derived from the NTCP attachment site of HBV genotypes A-H induced proliferation of CD4⁺>CD8⁺ T cells from the vaccinated subject although proliferation stimulated with peptides from genotypes D, F, G, H was low (Figure 7c).

Figure 7. PreS-specific T cell responses after vaccination with VVX001. Shown are percentages of proliferated СD3⁺CD4⁺ and CD3⁺CD8⁺ cells upon stimulation with (a) preS over time, (b) preS, P1-P8, equimolar mix of P1-P8 and (c) peptides representing the NTCP attachment site of genotypes A-H at time point 4 months after the last injection (26.06.2019).

Figure 7. PreS-specific T cell responses after vaccination with VVX001. Shown are percentages of proliferated СD3⁺CD4⁺ and CD3⁺CD8⁺ cells upon stimulation with (a) preS over time, (b) preS, P1-P8, equimolar mix of P1-P8 and (c) peptides representing the NTCP attachment site of genotypes A-H at time point 4 months after the last injection (26.06.2019).

Figure S8 shows the induction of preS-specific cytokine responses induced in cultured PBMCs obtained before and after immunization with VVX001 in the study subject. We found that immunization with VVX001 induced a mixed preS-specific Th2/Th1 cytokine secretion profile, which was characterized by the induction of IL-4, IL-5, IL-13 and GM-CSF on the one hand and by the induction of IFN-γ on the other hand (Figure S8). We also noticed the development of preS-specific IL-2 and TNF-α responses and the development of an eventually tolerogenic IL-10 response (Figure S8).

Figure S8. PreS-specific cytokine responses. Shown are the levels of IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-13, GM-CSF and TNF-α measured in supernatants of seven-day PBMC cultures stimulated with preS at baseline (November 2018) and one year after the last injection (April 2020) after subtraction of medium control.

Figure S8. PreS-specific cytokine responses. Shown are the levels of IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-13, GM-CSF and TNF-α measured in supernatants of seven-day PBMC cultures stimulated with preS at baseline (November 2018) and one year after the last injection (April 2020) after subtraction of medium control.

4. Discussion

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