A whole recombinant yeast-based therapeutic vaccine elicits HBV X, S and Core specific T cells in mice and activates human T cells recognizing epitopes linked to viral clearance

Abstract

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18-27 and HBs183-91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

Figure 1. Structure and expression of the X-S-Core fusion protein expressed in the GS-4774 Tarmogen.

(A) Schematic representation of the ∼73 kDa HBV X-S-Core fusion protein. MADEAP: sequence imparting metabolic stability in yeast; XAg: 60 (non-contiguous) amino acids of the HBxAg; SAg: 399 contiguous amino acids of HBsAg (entirety of large SAg except for N terminal methionine); Core Ag: 182 contiguous amino acids of HBcAg, lacking the N-terminal methionine; H₆ hexahistidine epitope tag. (B) Western blot probed with anti-his tag mAb, showing expression of the S-Core and X-S-Core fusion proteins in GS-4774 yeast. NS3-his std: purified recombinant, his tagged HCV NS3 protein for quantification of X-S-Core, H and L: high (6 µg) and low (3 µg) levels of total protein loaded per lane.

Figure 2. Ex vivo lymphocyte proliferation assays of T cells from GS-4774-immunized mice.

(A) Proliferation of inguinal LN cells harvested from GS-4774- or Yvec-immunized BALB/c mice. LN cells from 5 immunized mice were pooled and placed into in vitro stimulation with the indicated antigens for 4 days, followed by a 20 h ³H-thymidine uptake assay. In vitro stimulants are: Pichia pastoris yeast expressed, purified HBsAg and HBcAg, 3 µg/mL each; IPQSLDSWWTSL (HBsAg L^d restricted peptide) and AYRPPNAPI (HBcAg L^d restricted peptide), 10 µg/mL each; recombinant E. coli- expressed full length HBxAg, 3 µg/mL. P values, GS-4774 vs. Yvec: yeast expressed HBsAg & HBcAg, 0.0001; IPQSLDSWWTSL & AYRPPNAPI peptides, 0.089; recombinant HBxAg, 0.002. (B) Proliferation of CD4⁺ T cells isolated from splenocytes of GS-4774- or Yvec-immunized BALB/c mice. MACS-isolated splenic CD4⁺ T cells were stimulated with the indicated HBV antigens and assayed as in (A). Yeast expressed HBsAg & HBcAg stimulants: same as for panel A; GYHGSSLY, a MHC class II HBsAg mimetic peptide plus WGPSLYSIL, a 2-D^d restricted HBsAg peptide (10 µg/mL each). P values, GS-4774 vs. Yvec: yeast expressed HBsAg & HBcAg, 0.034; GYHGSSLY and WGPSLYSIL, 0.023. ³H-thymidine uptake is reported with medium background subtracted counts per minute (cpm) for each antigen stimulation condition. Error bars: standard error (s.e.) for replicate stimulations of the pooled immune cells.

Figure 3. IFNγ and IL2 ELISpot responses in GS-4774-immunized mice.

(A) LN cells from 5 pooled GS-4774- or 5 pooled Yvec-immunized BALB/c mice were stimulated with a 1∶1 mixture of E. coli or Pichia pastoris yeast-expressed HBsAg and HBcAg (cS&C, and yS&C, 3 µg/mL each). Control “no stim” wells contained growth medium plus 10% FBS only. Left, IFNγ; right, IL2. P values, GS-4774 vs. Yvec: cS&C-IFNγ, 0.0001; yS&C-IFNγ, 0.001; cS&C-IL2, 0.004; yS&C-IL2, 0.027. (B) IFNγ ELISpot response in GS-4774-or Yvec-immunized C57BL/6 mice. LN cells of 5 immunized mice per group were pooled and stimulated as in (A). P values, GS-4774 vs. Yvec: cS&C, 0.020; yS&C, 0.0081. (C) IFNγ ELISpot responses specific for acute-resolved, MHC class I restricted epitopes in GS-4774- or Yvec-immunized HLA-A*02∶01 tg mice (n = 5 mice per group). HBcAg 11–27: ATVELLSFLPSDFFPSV; HBsAg 14–22: VLQAGFFLL; yS&C and no stim, see panel A. (D) IL2 ELISpot responses to novel HBxAg epitopes in BALB/c mice. Splenocytes from 10 immunized mice were pooled and stimulated with 7 µM of 44 different 9-mer and 32 different 15-mer peptides for 4 days followed by a 24 h IL2 ELISpot assay. Only positive responding peptides are shown here (see also Fig. S2 for full results) and are defined as those with > 40 spots per million splenocytes in GS-4774 immunized mice and for which the GS-4774/Yvec response ratio was >2.5. Sequences of positive responding peptides: VLHKRTLGL, AHQFLPKVLHKRTLG or HKRTLGLSAMSTTDL. P values, GS-4774 vs. Yvec: VLHKRTLGL, 0.005; AHQFLPKVLHKRTLG, 0.061; HKRTLGLSAMSTTDL, 0.034. Error bars: s.e. for replicate stimulations of the pooled immune cells.

Figure 4. GS-4774 immunization inhibits growth of syngeneic, HBV-Ag expressing tumors in mice.

C57BL/6 mice were thrice immunized and then challenged s.c. with syngeneic EL4 tumors expressing HBV antigens. Kaplan-Meier analyses correspond to mice challenged with tumors expressing: (A) S-Core fusion (n = 10 recipient mice per group); (B) HBcAg (n = 14/group), or; (C) HBxAg (n = 10/group). hr, hazard ratio (hazard rate GS-4774/hazard rate Ovax; see methods). P values : see Figure. Ovax: Control Tarmogen expressing chicken ovalbumin.

Figure 5. GS-4774 induces maturation of human monocyte-derived dendritic cells (moDCs).

CD14⁺ monocytes were isolated from healthy donors and cultured with GM-CSF + IL-4 for 6 days to generate immature moDCs which were then incubated for 24 h with 10 Tarmogens per 1 moDC. The moDCs were stained with dye-coupled antibodies recognizing A, CD80; B, CD83; C, CD86; D, HLA-DR or; E, HLA-A, B, & C and evaluated by flow cytometry.

Figure 6. Cross-presentation of HBV antigens to T cells by GS-4774-pulsed human DCs.

Tarmogen (GS-4774 or Yvec)-pulsed DCs (TPDCs) were incubated in an IFNγ ELISpot plate with HBs183–91 or HBc18–27 TCR re-directed CD8⁺ T cells at a 2∶1 effector:target ratio (10,000 T cells:5000 moDC). P- values, GS-4774 vs. Yvec: HBc18–27, 0.048; HBs183–91: 0.08. The experiment was conducted twice and similar results were obtained each time.

Figure 7. T cell responses to Tarmogen-pulsed DCs in ENGERIX-B vaccinated donor samples.

TPDCs were used to stimulate autologous PBMCs over 3 rounds to expand HBV-specific T cells. (A) IFNγ ELISpot response of a donor immunized with Engerix-B six months prior to TPDC expansion. S-Core Tarmogen: identical to GS-4774 except for the absence of HBxAg. P values (TPDC plus rHBsAg only): GS-4774 vs. Yvec, 0.0001; S-Core vs. Yvec, 0.0001. (B) ICS staining of donor cells collected 20 years post Engerix-B immunization to evaluate the LAMP1 phenotype of IFNγ-secreting CD4⁺ and CD8⁺ T cells. HBV peptides for CD4⁺ T cell response: PICPGYRWMCLRRFIIFL, FFLLTRILTIPQSLD, SGFLGPLLVLQAGFFLLTR, TRILTIPQSLDSWWTSLNF 10 µg/mL each. HBV peptides for CD8⁺ T cell responses: VLQAGFFLL, FLLTRILTI, LLDYQGMLPV, WLSLLVPFV, SIVSPFIPLL 10 µg/mL each. P values (TPDC plus HBV peptides), S-Core vs. Yvec: CD8, 0.0001; CD4, 0.0002.

Figure 8. GS-4774-emergent IFNγ responses in healthy subjects or CHB patients on adefovir therapy.

(A) TPDCs were used to stimulate autologous PBMCs (two rounds), followed by IFNγ ELISpot assay in the presence of HBV peptide pools. n = 5 subjects/group. P values (healthy vs. CHB): GS-4774, <0.0001; S-Core, 0.44; Yvec, 0.95 (B) IFNγ ELISpot response of TPDC-expanded T cells in a chronic HBV donor. P values, S-Core vs. Ovax: TPDC, 0.00051; TPDC plus rS&C, 0.0051. (C) Both CD4⁺ and CD8⁺ T cells from the CHB donor produce IFNγ in response to TPDC stimulation. P values, S-Core vs. Ovax: CD4, 0.0001; CD8, 0.0001. X-axis label abbreviations: TPDC, expansion with TPDCs only; TPDC plus rHBsAg or HBV peptides, expansion with TPDC followed by 24 hour incubation with rHBsAg or HBV peptides during the ELISpot or ICS assay. X-S-Core, fusion protein expressed in GS-4774.