sj-docx-1-tag-10.1177_17562848221123980 – Supplemental material for Association of oral microbiome and pancreatic cancer: a systematic review and meta-analysis

Supplemental material, sj-docx-1-tag-10.1177_17562848221123980 for Association of oral microbiome and pancreatic cancer: a systematic review and meta-analysis by Mengyao Yuan, Ying Xu and Zhimin Guo in Therapeutic Advances in Gastroenterology</p

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

<div><p>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.</p></div

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

<p>(A) LN cells from 5 pooled GS-4774- or 5 pooled Yvec-immunized BALB/c mice were stimulated with a 1∶1 mixture of <i>E. coli</i> or <i>Pichia pastoris</i> 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. <b><u>P values</u></b>, 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). <b><u>P values</u></b>, 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101904#pone.0101904.s002" target="_blank">Fig. S2</a> 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. <b><u>P values</u></b>, 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.</p

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

<p>(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: <i>Pichia pastoris</i> 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 <i>E. coli-</i> expressed full length HBxAg, 3 µg/mL. <b><u>P values</u></b>, 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). <b><u>P values</u></b>, 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.</p

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

<p>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). <b><u>P- values</u></b>, GS-4774 vs. Yvec: HBc18–27, 0.048; HBs183–91: 0.08. The experiment was conducted twice and similar results were obtained each time.</p

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

<p>(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.</p

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

<p>C57BL/6 mice (n = 8 per group) were thrice immunized and then challenged s.c. with syngeneic EL4 tumors expressing HBV antigens. Tumor diameters correspond to size at days 7, 8, 9, or 10 post-challenge. EL4/S-Core-Adoptive (day 7): data are from adoptive transfer of splenocytes from GS-4774- or Ovax-immunized mice to <i>scid</i> recipients prior to tumor challenge. “Mice with tumors”: fraction of mice with measureable tumors at the indicated day.</p><p>*<b><u>P-values</u></b>: applicable to comparison of tumor diameters for GS-4774 vs. Ovax for each experiment (ANOVA).</p

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

<p>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. <b><u>P values</u></b> (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. <b><u>P values</u></b> (TPDC plus HBV peptides), S-Core vs. Yvec: CD8, 0.0001; CD4, 0.0002.</p

Th1 cytokine responses in CD4⁺ and CD8⁺ T cells from GS-4774 vs. Ovax-immunized C57BL/6 mice.

<p>Intracellular cytokine staining was used to assess the production of IFNγ, IL2, and TNFα by CD8⁺ T cells in the presence of peptide HBs190–197 (VWLSVIWM; top), and the production of IFNα in CD4⁺ T cells in the presence of peptide HBcAg 120–140 VSFGVWIRTPPAYRPPNAPIL (bottom). <b><u>P values</u></b>: in parentheses. n.t., not tested. Ovax: control Tarmogen expressing chicken ovalbumin. Splenocytes from 7 vaccinated mice per group were pooled for the analysis.</p