Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: PBMC from pre-vaccination (Day 0) and post-final vaccination (FV) time points were analysed for B cell responses by flow cytometry; gating strategies are as described in Methods and and . Frequencies of DBPRII-specific B cells – identified by probe staining – were compared between vaccine platforms ( A-B ) or protein/adjuvant dosing regimens ( C-D ) within both plasma cell ( A, C ) or memory IgG+ B cell ( B, D ) populations. Similarly, frequencies of RH5-specific B cells were compared between protein/adjuvant dosing regimens within plasma cell ( E ) and memory IgG+ B cells ( F ). IgM+, IgA+, activated and resting memory B cell responses are shown in and . VV = ChAd63-MVA viral vectors; PA = PvDBPII protein/adjuvant [PA-M and PA-D]; PA-M = PvDBPII protein/adjuvant monthly dosing; PA-D = PvDBPII protein/adjuvant delayed booster dosing; PA-DB = PvDBPII protein/adjuvant delayed booster dosing with extra booster; M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. Post-vaccination comparisons were performed between DBPRII platforms ( A-B ) or RH5 dosing regimens ( E-F ) with Mann-Whitney U tests, or between PvDBPII protein/adjuvant dosing regimens by Kruskal Wallis test with Dunn’s correction for multiple comparisons ( C-D ). Sample sizes for all assays were based on sample availability; each circle represents a single sample. ( A-B ) VV/PA: Day 0 = 8/5-6, FV+7 = 8/12, FV+14 = 8/12, FV+28 = 8/10. ( C-D ) PA-M/PA-D/PA-DB: Day 0 = 3-4/2/na, FV+7 = 4/8/5, FV+14 = 4/8/4, FV+28 = 4/6/5. ( E-F ) M/D: Day 0 = 5/1-4, FV+7 = 4-5/3-4, FV+14 = 4/6, FV+28 = 4/4. PA-D vaccinees returning in the PA-DB group are connected by lines. Bars represent medians. * p < 0.05, ** p < 0.01.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Staining, Adjuvant, Clinical Proteomics, MANN-WHITNEY

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: PBMC from pre-vaccination (Day 0) and post-final vaccination (FV) time points were analysed for B cell responses by flow cytometry. ( A ) Gating strategy shows identification of CD19+ B cells within live single (B cell-enriched) lymphocytes, and definition of CD38+CD27+ plasma cells within this population. Total memory cells are defined as CD27+ non-plasma cells (using a NOT gate to exclude plasma cells; indicated with thick black box); activated and resting memory B cells are more specifically categorised as CD21-CD27+ or CD21+CD27+, respectively. IgG+, IgM+ or IgA+ populations are subsequently gated within total memory cells. Proliferating (Ki67+) or vaccine-specific (those co-staining with RH5-PE and RH5-APC probes as indicated by thick black box) cells are defined within the plasma cell or isotype-specific memory B cell populations. Example shows Ki67 expression of plasma cells and RH5-specific gating on memory IgG+ B cells. A FV+14 sample (blue) is overlaid on a matched Day 0 sample (red) for all plots. Frequencies of Ki67+ cells shown within plasma cells ( B ), activated IgG+ memory B cells ( C ), and resting IgG+ memory B cells ( D ). M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. Post-vaccination comparisons were performed between dosing regimens with Mann-Whitney U tests. Sample sizes for all assays were based on sample availability; each circle represents a single sample. ( B-D ) VV/PA: Day 0 = 5/4-5, FV+7 = 5/3-4, FV+14 = 4/6, FV+28 = 4/4. Bars represent medians.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Clinical Proteomics, Staining, Expressing, Adjuvant, MANN-WHITNEY

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: PBMC from pre-vaccination (Day 0) and post-final vaccination (FV) time points were analysed for B cell responses by flow cytometry; gating strategies are as described in Methods and . Frequencies of RH5-specific B cells – identified by probe staining – were compared within activated memory IgG+ B cells ( A ), resting memory IgG+ B cells ( B ), total memory IgA+ B cells ( C ), and total memory IgM+ B cells ( D ) between dosing regimens. M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. Post-vaccination comparisons were performed between dosing regimens with Mann-Whitney U tests. Sample sizes for all assays were based on sample availability; each circle represents a single sample. ( B-D ) VV/PA: Day 0 = 5/4-5, FV+7 = 5/4 FV+14 = 4/6, FV+28 = 4/4. Bars represent medians. * p < 0.05.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Staining, Adjuvant, MANN-WHITNEY

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: CITRUS was run on single live (B cell-enriched) lymphocyte flow cytometry fcs files to agnostically define the main B cell populations within either DBPRII ( A-C ) or RH5 ( D-E ) trial samples. Clusters identified by CITRUS are visualised in dendrograms ( A , D ), colour-coded for example markers of interest ( A -IgG, CD38; D -IgG). Each node represents a cluster. Median marker expression within each cluster was used to define gating strategies for B cell populations in FlowJo, which were re-analysed for DBPRII-( B-C ) or RH5-specific ( E ) responses through probe staining (gating shown in ). See and – for a full list of populations identified via CITRUS clusters for further analysis. VV-M = ChAd63-MVA viral vector monthly dosing; VV-D ChAd63-MVA delayed booster dosing; PA-M = PvDBPII protein/adjuvant monthly dosing; PA-D = PvDBPII protein/adjuvant delayed booster dosing; PA-DB = PvDBPII protein/adjuvant delayed booster dosing with extra booster; M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. FV = final vaccination. Post-vaccination comparisons were performed between PvDBPII protein/adjuvant dosing regimens by Kruskal Wallis test with Dunn’s correction for multiple comparisons ( B-C ) or RH5 dosing regimens ( E ) with Mann-Whitney U tests. Sample sizes for all assays were based on sample availability; each circle represents a single sample. ( B-C ) VV-M/VV-D/PA-M/PA-D/PA-DB: Day 0 = 6/2/4/2/na, FV+7 = 6/2/4/8/5, FV+14 = 6/2/4/8/4, FV+28 = 6/2/4/6/5. ( E ) M/D: Day 0 = 5/2, FV+7 = 5/4, FV+14 = 4/6, FV+28 = 4/4. PA-D vaccinees returning in the PA-DB group are connected by lines. Bars represent medians. * p < 0.05, ** p < 0.01.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Marker, Expressing, Staining, Plasmid Preparation, Adjuvant, MANN-WHITNEY

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: CITRUS was run on single live (B cell-enriched) lymphocyte flow cytometry fcs files to agnostically define the main B cell populations within either DBPRII or RH5 trial samples. Median marker expression within each cluster was used to define gating strategies for B cell populations in FlowJo, which were re-analysed for DBPRII-or RH5-specific responses through probe staining. ( A ) Gating strategy shows identification of “Population 1” (CD19+CD20+CD21+CD27+CD138-CD38-IgM-IgA-IgG+) and “Population 2” (CD19-CD20-CD21-CD27-CD138-CD38+IgM-IgA-IgG+;) within the DBPRII trial and DBPRII-specific cells within these populations. ( B ) Gating strategy shows identification of “Population 12” (CD19+CD20+CD21+CD27+ CD138-CD38+IgA-IgM-IgG+) within the RH5 trial. A FV+14 sample (blue) is overlaid on a matched Day 0 sample (red) for all plots. See for a full list of populations defined via CITRUS.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Marker, Expressing, Staining

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: CITRUS was run on single live (B cell-enriched) lymphocyte flow cytometry fcs files to agnostically define the main B cell populations within RH5 trial samples. Median marker expression within each cluster was used to define gating strategies for B cell populations in FlowJo, which were re-analysed for RH5-specific responses through probe staining ( A-I ). Population definitions are annotated on individual figures. ( J ) Frequencies of each population within single live B cells (enriched from lymphocytes; see also ) of all samples. M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. FV = final vaccination. Post-vaccination comparisons were performed between dosing regimens with Mann-Whitney U tests ( A-I ). Sample sizes for all assays were based on sample availability; each circle represents a single sample. ( A-G, I ) M/D Day 0 = 4-5/2-6, FV+7 = 4-5/3-4, FV+14 = 4/6, FV+28 = 4/4. ( H ) M/D Day 0 = 3/0, FV+7 = 4/2, FV+14 = 4/4, FV+28 = 2/2. ( J ) n = 38 for all populations. Bars represent medians. * p < 0.05.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Flow Cytometry, Marker, Expressing, Staining, Adjuvant, MANN-WHITNEY

Journal: medRxiv

Article Title: Analyses of vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

doi: 10.1101/2023.03.17.23287040

Figure Lengend Snippet: RH5-specific bone marrow plasma cells were detected in B cells enriched from pre-and post-final vaccination (FV) bone marrow mononuclear cells and assayed by IgG antibody-secreting cell ELISPOT as described in the Methods. The frequency of RH5-specific IgG plasma cell (antibody-secreting cell) per million bone marrow B cells was compared between dosing regimens ( A ). Spearman correlation analyses were performed between RH5-specific bone marrow B cells and matched time point serum IgG ( B ), matched time point frequency of RH5-specific cells within CITRUS-guided “Population 12” [CD19+CD20+CD21+CD27+CD138-CD38+IgM-IgA-IgG+; see , , ] ( C ), and between RH5-specific bone marrow B cells at FV+28 and Population 12 at FV+14 ( D ). M = RH5.1/adjuvant monthly dosing; D = RH5.1/adjuvant delayed booster dosing. Post-vaccination comparisons were performed between dosing regimens by Mann Whitney U test ( A ; not significant). Spearman rho, p values and sample sizes are annotated on individual graphs. Each circle represents a single sample.

Article Snippet: SJD is a named inventor on patent applications relating to RH5 malaria vaccines and adenovirus-based vaccines, and is an inventor on intellectual property licensed by Oxford University Innovation to AstraZeneca.

Techniques: Clinical Proteomics, Enzyme-linked Immunospot, Adjuvant, MANN-WHITNEY