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a) Schematic drawings depicting the chemical structure of the AMP-DNA adjuvants. The ssDNA variant, dT 50 consists of 50 phosphorothioate (PS)-linked deoxythymidines. When annealed to a 50-mer deoxyadenosine DNA the resulting dsDNA forms dA:dT 50 . The diacyl lipid (AMP-tail) is conjugated to the 5’-end of dT 50 in both ssDNA and dsDNA forms. b) Vaccine formulation consists of the protein antigen (OVA or <t>RBD)</t> admixed with the candidate DNA-adjuvants depicted in a) and ), and ISD or HSV 60 . c) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg OVA protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. d) ELISpot analysis of splenocytes restimulated with <t>OVA</t> <t>OLPs</t> overnight and assayed for IFNγ production. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. e) FluoroSpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα and IL2 production. Shown is the frequency of SFCs per 10 6 splenocytes that produce at least one cytokine. Donut charts represent percentages of polyfunctional versus single cytokine secreting cells. f) Multiplexed proteomics analysis (Luminex) of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα, IL2, GM-CSF and GzmB production. Depicted are quantities of secreted cytokines in the culture supernatant. g) Peripheral blood CD8 + T cells were assayed ex vivo for OVA reactive TCRs using a SIINFEKL-specific tetramer. h-i) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + (h) T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry scatter plots of IFNγ and TNFα positive CD8 + T cells. j) Serum IgG titers were determined against OVA protein. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected

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1) Product Images from "Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates"

Article Title: Amphiphile-engineered DNA adjuvants stimulate strong type I IFN production in lymph nodes via cytosolic danger-sensing to induce potent cellular and humoral immunity in mice and non-human primates

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Legend Snippet: a) Schematic drawings depicting the chemical structure of the AMP-DNA adjuvants. The ssDNA variant, dT 50 consists of 50 phosphorothioate (PS)-linked deoxythymidines. When annealed to a 50-mer deoxyadenosine DNA the resulting dsDNA forms dA:dT 50 . The diacyl lipid (AMP-tail) is conjugated to the 5’-end of dT 50 in both ssDNA and dsDNA forms. b) Vaccine formulation consists of the protein antigen (OVA or RBD) admixed with the candidate DNA-adjuvants depicted in a) and ), and ISD or HSV 60 . c) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg OVA protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. d) ELISpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ production. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. e) FluoroSpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα and IL2 production. Shown is the frequency of SFCs per 10 6 splenocytes that produce at least one cytokine. Donut charts represent percentages of polyfunctional versus single cytokine secreting cells. f) Multiplexed proteomics analysis (Luminex) of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα, IL2, GM-CSF and GzmB production. Depicted are quantities of secreted cytokines in the culture supernatant. g) Peripheral blood CD8 + T cells were assayed ex vivo for OVA reactive TCRs using a SIINFEKL-specific tetramer. h-i) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + (h) T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry scatter plots of IFNγ and TNFα positive CD8 + T cells. j) Serum IgG titers were determined against OVA protein. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected

Techniques Used: Variant Assay, Formulation, Enzyme-linked Immunospot, Luminex, Ex Vivo, Flow Cytometry, Vaccines

a) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. b) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. c) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. d-g) Flow cytometric analysis of cytokine production by CD8 + (d, f) and CD4 + T cells (e, g) in peripheral blood and perfused lung tissue. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry dot plots of TNFα and IFNγ positive T cells. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Figure Legend Snippet: a) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. b) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. c) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. d-g) Flow cytometric analysis of cytokine production by CD8 + (d, f) and CD4 + T cells (e, g) in peripheral blood and perfused lung tissue. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry dot plots of TNFα and IFNγ positive T cells. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Techniques Used: Enzyme-linked Immunospot, Flow Cytometry, Vaccines

a-f) C57BL/6J mice (n = 10) were immunized twice with 5 μg WH-01 RBD protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (a,c,e), or dT 50 (b,d,f). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with RBD OLPs overnight at the indicated time points. a,b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. c,d) Peripheral blood CD8 + T cells were assayed for RBD reactive TCRs using a VNFNFNGL-specific tetramer. Shown are Tetramer + cells among CD8 T cells. e,f) Tetramer + CD8 + T cells from peripheral blood were stained for CD44 and CD62L to determine memory phenotype. g-h) C57BL/6J mice (n = 10) were immunized twice with 5 μg OVA protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (g), or dT 50 (h). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with OVA OLPs overnight at the indicated time points. Statistics in a-d) and g-h) compare AMP- to SOL DNA treatment groups. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Figure Legend Snippet: a-f) C57BL/6J mice (n = 10) were immunized twice with 5 μg WH-01 RBD protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (a,c,e), or dT 50 (b,d,f). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with RBD OLPs overnight at the indicated time points. a,b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. c,d) Peripheral blood CD8 + T cells were assayed for RBD reactive TCRs using a VNFNFNGL-specific tetramer. Shown are Tetramer + cells among CD8 T cells. e,f) Tetramer + CD8 + T cells from peripheral blood were stained for CD44 and CD62L to determine memory phenotype. g-h) C57BL/6J mice (n = 10) were immunized twice with 5 μg OVA protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (g), or dT 50 (h). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with OVA OLPs overnight at the indicated time points. Statistics in a-d) and g-h) compare AMP- to SOL DNA treatment groups. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques Used: Vaccines, Staining

C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvant. Cells were assayed 7 days post booster dose. a) Schematic describing the modifications of the DNA adjuvants. b-h) Evaluation of the effect of DNA modifications on immune activation. Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes; The assessed DNA modifications included: b) single stranded versus double stranded DNA, (bold letters indicate the AMP-conjugated strand); c-d) HSV 60 and ISD sequences in single stranded (c) and double stranded (d) form, e-f) DNA length variants for dT (e) and dAdT (f); g-h) phosphodiester (PO)- vs phosphorothioate (PS)-linked bases for dT (g) and dAdT (h). Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. Statistical analyses in panels e and f compare each AMP-modified length variant to its soluble counterpart.

Figure Legend Snippet: C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvant. Cells were assayed 7 days post booster dose. a) Schematic describing the modifications of the DNA adjuvants. b-h) Evaluation of the effect of DNA modifications on immune activation. Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes; The assessed DNA modifications included: b) single stranded versus double stranded DNA, (bold letters indicate the AMP-conjugated strand); c-d) HSV 60 and ISD sequences in single stranded (c) and double stranded (d) form, e-f) DNA length variants for dT (e) and dAdT (f); g-h) phosphodiester (PO)- vs phosphorothioate (PS)-linked bases for dT (g) and dAdT (h). Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. Statistical analyses in panels e and f compare each AMP-modified length variant to its soluble counterpart.

Techniques Used: Adjuvant, Activation Assay, Enzyme-linked Immunospot, Vaccines, Modification, Variant Assay

C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-dAdT:dAdT adjuvant. Cells were assayed 7 days post booster dose. a) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. Mock vaccines contained antigen only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Figure Legend Snippet: C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-dAdT:dAdT adjuvant. Cells were assayed 7 days post booster dose. a) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. Mock vaccines contained antigen only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques Used: Adjuvant, Enzyme-linked Immunospot, Vaccines

Cellular and humoral responses to WH-01 RBD and AMP-DNA adjuvant were assessed in Rhesus macaques. a) 3 animals were immunized at week 0 (Baseline, BL) and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . b-d) PBMCs were collected at BL and week 6 and stimulated with WH-01 RBD OLPs overnight. b) IFNγ ELISpot analysis. Shown are IFNγ SFCs per 1 x 10 6 PBMCs. c, d) Flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells. Shown are frequencies of combined IFNγ, TNFα and IL2 cytokine production. e) For antibody response assessment serum was collected at BL and weeks 2, 4, 6, and 8. RBD-specific serum IgG binding antibody units (BAU) were assessed for WH-01 at each time point. f) For neutralizing antibody (nAb) response assessment through pseudovirus inhibition serum was collected at BL and week 6. Shown are ID 50 values for WH-01 in comparison to convalescent human plasma (CHP). The dashed lines in e-f) represent the lower limit of detection discriminating between samples positive or negative for seroconversion. The dotted line in f) represents the mean value observed for the human plasma comparators. g) For GC B cell assessment LN fine needle aspirates (FNA) were collected at BL and weeks 2 and 6. Shown are frequencies of RBD-specific GC B cells (CD20 + BcL6 + Ki-67 + ) in individual animals over time with corresponding representative flow cytometry scatter plots of RBD-specific B cells. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Figure Legend Snippet: Cellular and humoral responses to WH-01 RBD and AMP-DNA adjuvant were assessed in Rhesus macaques. a) 3 animals were immunized at week 0 (Baseline, BL) and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . b-d) PBMCs were collected at BL and week 6 and stimulated with WH-01 RBD OLPs overnight. b) IFNγ ELISpot analysis. Shown are IFNγ SFCs per 1 x 10 6 PBMCs. c, d) Flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells. Shown are frequencies of combined IFNγ, TNFα and IL2 cytokine production. e) For antibody response assessment serum was collected at BL and weeks 2, 4, 6, and 8. RBD-specific serum IgG binding antibody units (BAU) were assessed for WH-01 at each time point. f) For neutralizing antibody (nAb) response assessment through pseudovirus inhibition serum was collected at BL and week 6. Shown are ID 50 values for WH-01 in comparison to convalescent human plasma (CHP). The dashed lines in e-f) represent the lower limit of detection discriminating between samples positive or negative for seroconversion. The dotted line in f) represents the mean value observed for the human plasma comparators. g) For GC B cell assessment LN fine needle aspirates (FNA) were collected at BL and weeks 2 and 6. Shown are frequencies of RBD-specific GC B cells (CD20 + BcL6 + Ki-67 + ) in individual animals over time with corresponding representative flow cytometry scatter plots of RBD-specific B cells. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Techniques Used: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison, Flow Cytometry

Cellular and humoral responses to RBD and AMP-DNA adjuvant were assessed in rhesus macaques as in . Three animals were immunized at week 0 and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . a-c) T cell cross-reactive responses were compared upon overnight restimulation with RBD OLPs to variants of concern (VOC) at week 6 by a) IFNγ ELISpot analysis [shown are IFNγ SFCs per 1 x 10 6 PBMCs], and b-c) flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells [shown are frequencies of IFNγ, TNFα and IL2 cytokine production]. d) RBD-specific serum IgG binding antibody units (BAU) were assessed for VOCs at each sample collection time point and e) compared among VOCs at week 6. f, g) Neutralizing antibody responses were assessed through pseudovirus inhibition at week 6. Shown are ID 50 values for Delta and Omicron variants in comparison to convalescent human plasma (CHP). The dashed lines represent the lower limit of detection discriminating between samples positive or negative for seroconversion and dotted lines represent the mean value observed for the human plasma comparators where appropriate. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Figure Legend Snippet: Cellular and humoral responses to RBD and AMP-DNA adjuvant were assessed in rhesus macaques as in . Three animals were immunized at week 0 and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . a-c) T cell cross-reactive responses were compared upon overnight restimulation with RBD OLPs to variants of concern (VOC) at week 6 by a) IFNγ ELISpot analysis [shown are IFNγ SFCs per 1 x 10 6 PBMCs], and b-c) flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells [shown are frequencies of IFNγ, TNFα and IL2 cytokine production]. d) RBD-specific serum IgG binding antibody units (BAU) were assessed for VOCs at each sample collection time point and e) compared among VOCs at week 6. f, g) Neutralizing antibody responses were assessed through pseudovirus inhibition at week 6. Shown are ID 50 values for Delta and Omicron variants in comparison to convalescent human plasma (CHP). The dashed lines represent the lower limit of detection discriminating between samples positive or negative for seroconversion and dotted lines represent the mean value observed for the human plasma comparators where appropriate. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Techniques Used: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison

a) C57BL/6J mice (n = 5) were immunized once with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-conjugated dT 50 . Lymph nodes were collected and processed for protein extraction and analysis by Luminex at 6 and 24 hours post immunization. Analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentrations (pg/ml). Analytes are annotated by functional category: (1) growth factors, (2) inflammatory cytokines, (3) chemokines, (4) inflammasome, and (5) IFN-I. Statistical analysis compares AMP-DNA treated groups with time point-matched SOL DNA groups. b) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control 24 hours prior to immunization as in a). Protein from lymph nodes was extracted 24 hours post immunization. Heatmap representation and analyte annotation as in a). Statistical analysis compares IFNAR1-Ab treated groups with isotype-Ab treated groups. c-d) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. c) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. d) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with RBD OLPs. Mock immunized animals received PBS vehicle only. e-f) TBK1 fl/fl x Vav-iCre +/- mice (TBK1 -/- , n = 8) and TBK1 fl/fl x Vav-iCre -/- mice (TBK1 + , n = 5) were immunized twice with 5 μg OVA protein and 5 nmol AMP-dT 50 . Cells were assayed 7 days post booster dose. e) Splenocytes were restimulated with SIINFEKL peptide overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. f) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with SIINFEKL peptide overnight. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Figure Legend Snippet: a) C57BL/6J mice (n = 5) were immunized once with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-conjugated dT 50 . Lymph nodes were collected and processed for protein extraction and analysis by Luminex at 6 and 24 hours post immunization. Analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentrations (pg/ml). Analytes are annotated by functional category: (1) growth factors, (2) inflammatory cytokines, (3) chemokines, (4) inflammasome, and (5) IFN-I. Statistical analysis compares AMP-DNA treated groups with time point-matched SOL DNA groups. b) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control 24 hours prior to immunization as in a). Protein from lymph nodes was extracted 24 hours post immunization. Heatmap representation and analyte annotation as in a). Statistical analysis compares IFNAR1-Ab treated groups with isotype-Ab treated groups. c-d) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. c) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. d) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with RBD OLPs. Mock immunized animals received PBS vehicle only. e-f) TBK1 fl/fl x Vav-iCre +/- mice (TBK1 -/- , n = 8) and TBK1 fl/fl x Vav-iCre -/- mice (TBK1 + , n = 5) were immunized twice with 5 μg OVA protein and 5 nmol AMP-dT 50 . Cells were assayed 7 days post booster dose. e) Splenocytes were restimulated with SIINFEKL peptide overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. f) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with SIINFEKL peptide overnight. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques Used: Protein Extraction, Luminex, Functional Assay, Blocking Assay, Control, Enzyme-linked Immunospot

a-e) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control one day prior to immunization with one dose of 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Protein from lymph nodes was extracted 24 hours post immunization. a) Luminex assessed analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentration (pg/ml). Functional group annotations as in . b-e) Concentrations of measured analytes from panel a) for functional groups 1 to 5 as indicated. f-k) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. f) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. g, h) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + T cells (h) in peripheral blood. k) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. Mock immunized animals received vehicle only. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Figure Legend Snippet: a-e) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control one day prior to immunization with one dose of 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Protein from lymph nodes was extracted 24 hours post immunization. a) Luminex assessed analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentration (pg/ml). Functional group annotations as in . b-e) Concentrations of measured analytes from panel a) for functional groups 1 to 5 as indicated. f-k) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. f) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. g, h) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + T cells (h) in peripheral blood. k) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. Mock immunized animals received vehicle only. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Techniques Used: Blocking Assay, Control, Luminex, Concentration Assay, Functional Assay, Enzyme-linked Immunospot

Image Search Results

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) Schematic drawings depicting the chemical structure of the AMP-DNA adjuvants. The ssDNA variant, dT 50 consists of 50 phosphorothioate (PS)-linked deoxythymidines. When annealed to a 50-mer deoxyadenosine DNA the resulting dsDNA forms dA:dT 50 . The diacyl lipid (AMP-tail) is conjugated to the 5’-end of dT 50 in both ssDNA and dsDNA forms. b) Vaccine formulation consists of the protein antigen (OVA or RBD) admixed with the candidate DNA-adjuvants depicted in a) and ), and ISD or HSV 60 . c) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg OVA protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. d) ELISpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ production. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. e) FluoroSpot analysis of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα and IL2 production. Shown is the frequency of SFCs per 10 6 splenocytes that produce at least one cytokine. Donut charts represent percentages of polyfunctional versus single cytokine secreting cells. f) Multiplexed proteomics analysis (Luminex) of splenocytes restimulated with OVA OLPs overnight and assayed for IFNγ, TNFα, IL2, GM-CSF and GzmB production. Depicted are quantities of secreted cytokines in the culture supernatant. g) Peripheral blood CD8 + T cells were assayed ex vivo for OVA reactive TCRs using a SIINFEKL-specific tetramer. h-i) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + (h) T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry scatter plots of IFNγ and TNFα positive CD8 + T cells. j) Serum IgG titers were determined against OVA protein. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected

Article Snippet: For ex vivo re-stimulation of cells from animals immunized with RBD antigen, custom 15mer OLPs with 11 amino acid overlap were generated spanning the SARS-CoV-2 Spike RBD WH-01 protein (amino acids R319-S591, GenScript).

Techniques: Variant Assay, Formulation, Enzyme-linked Immunospot, Luminex, Ex Vivo, Flow Cytometry, Vaccines

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) Schema showing animal dosing and experimental schedule. C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvants. Cells were assayed 7 days post booster dose. b) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. c) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. d-g) Flow cytometric analysis of cytokine production by CD8 + (d, f) and CD4 + T cells (e, g) in peripheral blood and perfused lung tissue. Shown are percentages of cytokine + cells among CD8 + or CD4 + T cells and representative flow cytometry dot plots of TNFα and IFNγ positive T cells. Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Techniques: Enzyme-linked Immunospot, Flow Cytometry, Vaccines

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a-f) C57BL/6J mice (n = 10) were immunized twice with 5 μg WH-01 RBD protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (a,c,e), or dT 50 (b,d,f). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with RBD OLPs overnight at the indicated time points. a,b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. c,d) Peripheral blood CD8 + T cells were assayed for RBD reactive TCRs using a VNFNFNGL-specific tetramer. Shown are Tetramer + cells among CD8 T cells. e,f) Tetramer + CD8 + T cells from peripheral blood were stained for CD44 and CD62L to determine memory phenotype. g-h) C57BL/6J mice (n = 10) were immunized twice with 5 μg OVA protein adjuvanted with either 100 μg alum or 5 nmol dAdT 50 (g), or dT 50 (h). Mock vaccines contained vehicle only. Peripheral blood samples were collected, processed and stimulated with OVA OLPs overnight at the indicated time points. Statistics in a-d) and g-h) compare AMP- to SOL DNA treatment groups. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques: Vaccines, Staining

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-DNA adjuvant. Cells were assayed 7 days post booster dose. a) Schematic describing the modifications of the DNA adjuvants. b-h) Evaluation of the effect of DNA modifications on immune activation. Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes; The assessed DNA modifications included: b) single stranded versus double stranded DNA, (bold letters indicate the AMP-conjugated strand); c-d) HSV 60 and ISD sequences in single stranded (c) and double stranded (d) form, e-f) DNA length variants for dT (e) and dAdT (f); g-h) phosphodiester (PO)- vs phosphorothioate (PS)-linked bases for dT (g) and dAdT (h). Mock vaccines contained vehicle only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. Statistical analyses in panels e and f compare each AMP-modified length variant to its soluble counterpart.

Techniques: Adjuvant, Activation Assay, Enzyme-linked Immunospot, Vaccines, Modification, Variant Assay

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: C57BL/6J mice (n = 5) were immunized as described in . Immunizations contained 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-dAdT:dAdT adjuvant. Cells were assayed 7 days post booster dose. a) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. b) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood. Shown are percentages of cytokine + cells among CD8 + T cells. Mock vaccines contained antigen only. Values depicted are means±SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques: Adjuvant, Enzyme-linked Immunospot, Vaccines

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: Cellular and humoral responses to WH-01 RBD and AMP-DNA adjuvant were assessed in Rhesus macaques. a) 3 animals were immunized at week 0 (Baseline, BL) and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . b-d) PBMCs were collected at BL and week 6 and stimulated with WH-01 RBD OLPs overnight. b) IFNγ ELISpot analysis. Shown are IFNγ SFCs per 1 x 10 6 PBMCs. c, d) Flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells. Shown are frequencies of combined IFNγ, TNFα and IL2 cytokine production. e) For antibody response assessment serum was collected at BL and weeks 2, 4, 6, and 8. RBD-specific serum IgG binding antibody units (BAU) were assessed for WH-01 at each time point. f) For neutralizing antibody (nAb) response assessment through pseudovirus inhibition serum was collected at BL and week 6. Shown are ID 50 values for WH-01 in comparison to convalescent human plasma (CHP). The dashed lines in e-f) represent the lower limit of detection discriminating between samples positive or negative for seroconversion. The dotted line in f) represents the mean value observed for the human plasma comparators. g) For GC B cell assessment LN fine needle aspirates (FNA) were collected at BL and weeks 2 and 6. Shown are frequencies of RBD-specific GC B cells (CD20 + BcL6 + Ki-67 + ) in individual animals over time with corresponding representative flow cytometry scatter plots of RBD-specific B cells. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Techniques: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison, Flow Cytometry

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: Cellular and humoral responses to RBD and AMP-DNA adjuvant were assessed in rhesus macaques as in . Three animals were immunized at week 0 and 4 with 140 µg WH-01 RBD protein admixed with 5 mg of AMP-dT 50 . a-c) T cell cross-reactive responses were compared upon overnight restimulation with RBD OLPs to variants of concern (VOC) at week 6 by a) IFNγ ELISpot analysis [shown are IFNγ SFCs per 1 x 10 6 PBMCs], and b-c) flow cytometric analysis of cytokine production by CD4 + and CD8 + T cells [shown are frequencies of IFNγ, TNFα and IL2 cytokine production]. d) RBD-specific serum IgG binding antibody units (BAU) were assessed for VOCs at each sample collection time point and e) compared among VOCs at week 6. f, g) Neutralizing antibody responses were assessed through pseudovirus inhibition at week 6. Shown are ID 50 values for Delta and Omicron variants in comparison to convalescent human plasma (CHP). The dashed lines represent the lower limit of detection discriminating between samples positive or negative for seroconversion and dotted lines represent the mean value observed for the human plasma comparators where appropriate. Values depicted are mean±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by paired t test.

Techniques: Adjuvant, Enzyme-linked Immunospot, Binding Assay, Inhibition, Comparison

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a) C57BL/6J mice (n = 5) were immunized once with 5 μg WH-01 RBD protein and 5 nmol SOL or AMP-conjugated dT 50 . Lymph nodes were collected and processed for protein extraction and analysis by Luminex at 6 and 24 hours post immunization. Analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentrations (pg/ml). Analytes are annotated by functional category: (1) growth factors, (2) inflammatory cytokines, (3) chemokines, (4) inflammasome, and (5) IFN-I. Statistical analysis compares AMP-DNA treated groups with time point-matched SOL DNA groups. b) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control 24 hours prior to immunization as in a). Protein from lymph nodes was extracted 24 hours post immunization. Heatmap representation and analyte annotation as in a). Statistical analysis compares IFNAR1-Ab treated groups with isotype-Ab treated groups. c-d) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. c) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. d) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with RBD OLPs. Mock immunized animals received PBS vehicle only. e-f) TBK1 fl/fl x Vav-iCre +/- mice (TBK1 -/- , n = 8) and TBK1 fl/fl x Vav-iCre -/- mice (TBK1 + , n = 5) were immunized twice with 5 μg OVA protein and 5 nmol AMP-dT 50 . Cells were assayed 7 days post booster dose. e) Splenocytes were restimulated with SIINFEKL peptide overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. f) Flow cytometric analysis of cytokine production by CD8 + T cells in peripheral blood upon restimulation with SIINFEKL peptide overnight. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis.

Techniques: Protein Extraction, Luminex, Functional Assay, Blocking Assay, Control, Enzyme-linked Immunospot

Journal: bioRxiv

doi: 10.1101/2024.11.01.621501

Figure Lengend Snippet: a-e) C57BL/6J mice (n = 5) were dosed intraperitoneally with IFNAR1 blocking antibody or isotype control one day prior to immunization with one dose of 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Protein from lymph nodes was extracted 24 hours post immunization. a) Luminex assessed analyte concentrations were expressed in a heat map showing mock-subtracted Z-scores of analyte concentration (pg/ml). Functional group annotations as in . b-e) Concentrations of measured analytes from panel a) for functional groups 1 to 5 as indicated. f-k) C57BL/6J mice (n = 5) were immunized twice with 5 μg WH-01 RBD protein and 5 nmol AMP-dAdT 50 . Mice received IFNAR1 blocking antibody or isotype control intraperitoneally one day prior to each dose. Cells were assayed 7 days post booster dose. f) Splenocytes were restimulated with RBD OLPs overnight and assayed for IFNγ production by ELISpot. Shown is the frequency of IFNγ SFCs per 10 6 splenocytes. g, h) Flow cytometric analysis of cytokine production by CD8 + (g) and CD4 + T cells (h) in peripheral blood. k) Serum anti-RBD IgG titers were determined against WH-01 RBD protein. Mock immunized animals received vehicle only. Values depicted are mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA followed by Tukey’s post-hoc analysis. n.d., not detected.

Techniques: Blocking Assay, Control, Luminex, Concentration Assay, Functional Assay, Enzyme-linked Immunospot

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