From 669e44b967c2bb97113a29ed056a115b7d817d44 Mon Sep 17 00:00:00 2001 From: Gabrielle Lemire <43865993+lemireg@users.noreply.github.com> Date: Wed, 21 Aug 2024 11:15:46 -0700 Subject: [PATCH] incorporate Bhavesh's comments on PR. some comments still outstanding to review with Alicia before adding. --- .../methods-adcc/adcc-biological-endpoints.Rmd | 6 +++--- inst/templates/methods-adcc/adcc-lab-methods.Rmd | 12 ++++++++---- .../methods-adcc/adcc-statistical-methods.Rmd | 6 +++--- 3 files changed, 14 insertions(+), 10 deletions(-) diff --git a/inst/templates/methods-adcc/adcc-biological-endpoints.Rmd b/inst/templates/methods-adcc/adcc-biological-endpoints.Rmd index 6ab066f..11c979b 100644 --- a/inst/templates/methods-adcc/adcc-biological-endpoints.Rmd +++ b/inst/templates/methods-adcc/adcc-biological-endpoints.Rmd @@ -5,10 +5,10 @@ title: "biological-endpoints" -# Biological Endpoints GTL +## Biological Endpoints GTL -ADCC-mediated antibody responses were measured using Luciferase GranToxiLux (GTL) assays from specimens obtained at *[describe visits; include visit number, timepoint in weeks or months, and relation to SPA. E.g. week 26 (2 weeks post-4th vaccination, visit 10)]*. The GTL ADCC assay measures percent Granzyme B activity, defined as the percentage of antigen-coated target cells positive for proteolytically active Granzyme B out of the total viable target cell population. Endpoints are the response rate and magnitude of ADCC-mediated antibody responses against a panel of *[number of antigens]* HIV-1 antigens representing *[include description of viruses: those included in the vaccine product (vaccine-matched), Env matched in clade to vaccine products, and other Env to identify the breadth of the responses against HIV-1 subtypes]*. +ADCC-mediated antibody responses were measured using GranToxiLux (GTL) assays from specimens obtained at *[describe visits; include visit number, timepoint in weeks or months, and relation to SPA. E.g. week 26 (2 weeks post-4th vaccination, visit 10)]*. The GTL ADCC assay measures percent Granzyme B activity, defined as the percentage of antigen-coated target cells positive for proteolytically active Granzyme B out of the total viable target cell population. Endpoints are the response rate and magnitude of ADCC-mediated antibody responses against a panel of *[number of antigens]* HIV-1 antigens representing *[include description of viruses: those included in the vaccine product (vaccine-matched), Env matched in clade to vaccine products, and other Env to identify the breadth of the responses against HIV-1 subtypes]*. -# Biological endpoints Luciferase +## Biological endpoints Luciferase ADCC-mediated antibody responses were measured using Luciferase ADCC assays from specimens obtained at *[describe visits; include visit number, timepoint in weeks or months, and relation to SPA. E.g. week 26 (2 weeks post-4th vaccination, visit 10)]*. The Luciferase ADCC assay tests reactivity against Infectious Molecular Clone (IMC)-infected target cells by measuring percent reduction in Relative Luminescence Units (RLUs), reported as percentage specific killing. Endpoints are the response rate and magnitude of ADCC-mediated antibody responses against a panel of *[number of IMCs]* HIV-1 IMC expressing Env representing *[include description of IMCs: those included in the vaccine product (vaccine-matched), Env matched in clade to vaccine products, and other Env to identify the breadth of the responses against HIV-1 subtypes]*. diff --git a/inst/templates/methods-adcc/adcc-lab-methods.Rmd b/inst/templates/methods-adcc/adcc-lab-methods.Rmd index e7c8ee5..80c1349 100644 --- a/inst/templates/methods-adcc/adcc-lab-methods.Rmd +++ b/inst/templates/methods-adcc/adcc-lab-methods.Rmd @@ -5,13 +5,17 @@ title: "lab-methods" -# Lab Methods GTL +## Lab Methods GTL -The qualified GranToxiLux Antibody-Dependent Cell-Mediated Cytotoxicity (GTL-ADCC) assay was performed as previously described [@Pollara2014]. Target cells were a clonal isolate of the CEM.NKRCCR5 CD4+ T-cell line (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from Dr. Alexandra Trkola [@Trkola1999]. These cells were coated with recombinant gp120s representing the HIV-1 envelopes of the subtype [specify subtype and antigens, e.g. C (TV1 and 1086c)]. Effector cells were PBMCs obtained from a HIV-seronegative donor with heterozygous for Fc$\gamma$R3A at position 158 (158F/V). PBMCs were obtained by leukapheresis to collect enough cells for completion of the study with a single donation, minimizing potential effector cell population variability effects on the study outcome. PBMCs were used at an effector cell to target cell ratio of 30:1. Serum samples were tested after five-fold serial dilutions starting at 1:50 (1:50, 1:250, 1:1250, 1:6250, 1:31250, and 1:156250). Each plate has one standardized positive control in duplicate and one standardized negative control in duplicate. +The qualified GranToxiLux Antibody-Dependent Cell-Mediated Cytotoxicity (GTL-ADCC) assay was performed as previously described [@Pollara2014]. Target cells were a clonal isolate of the CEM.NKRCCR5 CD4+ T-cell line (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from Dr. Alexandra Trkola [@Trkola1999]. These cells were coated with recombinant gp120s representing the HIV-1 envelopes of the subtype [specify subtype and antigens, e.g. C (TV1 and 1086c)]. + +Effector cells were peripheral blood mononuclear cells (PBMCs) obtained from a HIV-seronegative donor heterozygous for Fc$\gamma$R3A at position 158 (158F/V). PBMCs were obtained by leukapheresis to collect enough cells for completion of the study with a single donation, minimizing potential effector cell population variability effects on the study outcome. + +PBMCs were used at an effector cell to target cell ratio of 30:1. Serum samples were tested after five-fold serial dilutions starting at 1:50 (1:50, 1:250, 1:1250, 1:6250, 1:31250, and 1:156250). Each plate has one standardized positive control in duplicate and one standardized negative control in duplicate. ADCC is quantified as net percent granzyme B activity, which is the percent of target cells positive for GTL (an indicator of granzyme B uptake) minus the percent of target cells positive for GTL when incubated with effector cells in the absence of a source of antibodies. Flow cytometry is used to quantify the frequency of granzyme B positive cells. -# Lab Methods Luciferase +## Lab Methods Luciferase We utilized a modified version of a previously published ADCC luciferase procedure [@Pollara2014; @Fisher2019] . Briefly, CEM.NKRCCR5 cells [@Trkola1999] were used as targets for ADCC luciferase assays after infection by one of the following HIV-1 [vaccine-matched, if all IMCs are vaccine-matched; if not, indicate match in table below] IMCs: Complete IMC name Accession Number Abbreviated name Vaccine Match @@ -19,6 +23,6 @@ Complete IMC name Accession Number Abbreviated name Vaccine Match Peripheral blood mononuclear cells (PBMCs) were obtained from a HIV-seronegative donor by leukapheresis and cryopreserved until the day of the assay. After thawing and overnight resting in RPMI 1640 supplemented with antibiotics, $10\%$ fetal bovine serum (R10), and 10 ng/mL of IL-15, the PBMCs were used as effector cells at an effector-to-target ratio of 30:1. -Target and effector cells were plated in white 96-well half-area plates and co-cultured with 4-fold serial dilutions of trial participant serum starting at the 1:50 dilution. For each sample, percent specific killing was measured in duplicate at dilutions of 1:50, 1:200, and 1:800, 1:3200, 1:12800, and 1:51200. Co-cultures were incubated for 6 hours at $37^{\circ}$C in $5\%$ CO2. The final readout was the reduction of luminescence intensity generated by the presence of residual intact target cells that had not been lysed by the effector population in the presence of ADCC-mediating serum antibodies. The percentage of killing was calculated using the formula: percent specific killing = $100 * \frac{\text{RLU of target and effector well– RLU of test well)}}{\text{RLU of target and effector well}}$. +Target and effector cells were plated in white 96-well half-area plates and co-cultured with 4-fold serial dilutions of trial participant serum starting at the 1:50 dilution. For each sample, percent specific killing was measured in duplicate at dilutions of 1:50, 1:200, 1:800, 1:3200, 1:12800, and 1:51200. Co-cultures were incubated for 6 hours at $37^{\circ}$C in $5\%$ CO2. The final readout was the reduction of luminescence intensity generated by the presence of residual intact target cells that had not been lysed by the effector population in the presence of ADCC-mediating serum antibodies. The percentage of killing was calculated using the formula: percent specific killing = $100 * \frac{\text{(RLU of target and effector well– RLU of test well)}}{\text{RLU of target and effector well}}$. In this analysis, the Relative Luminescence Units (RLU) of the target plus effector wells represents spontaneous lysis in the absence of any source of antibody and is used to calculate background activity. The monoclonal antibody [insert antibody name from lab study plan, e.g. Synagis] and a cocktail of HIV-1 monoclonal antibodies [insert antibody names from lab study plan, e.g. (A32, 2G12, CH44, and 7B2)] were used as negative and positive controls, respectively. diff --git a/inst/templates/methods-adcc/adcc-statistical-methods.Rmd b/inst/templates/methods-adcc/adcc-statistical-methods.Rmd index 439c97b..cd0130d 100644 --- a/inst/templates/methods-adcc/adcc-statistical-methods.Rmd +++ b/inst/templates/methods-adcc/adcc-statistical-methods.Rmd @@ -12,7 +12,7 @@ Peak net percent granzyme B activity defined as the maximum activity across the ### AUC -Area under the net percent granzyme B activity vs log10 (dilution) curve is ("AUC") calculated using the trapezoidal rule, setting any net percent granzyme B activity below 0\% to 0\%. +Area under the net percent granzyme B activity ("AUC") versus log$_{10}$ (dilution) curve is calculated using the trapezoidal rule, setting any net percent granzyme B activity below 0\% to 0\%. ### Response call @@ -35,13 +35,13 @@ Nonparametric partial Area under the baseline-subtracted curves ("pAUC"), calcul ### Response call -A response is defined as positive if the peak baseline-subtracted \% specific killing activity greater than or equal to 10\% for either the 1:50 or 1:200 dilution. +A response is defined as positive if the baseline-subtracted percent (\%) specific killing activity is greater than or equal to 10\% for either the 1:50 or 1:200 dilutions. # Statistical Methods GTL and Luciferase ## Graphical analysis -Plots of peak activity and AUC show both response rates and the distribution of magnitude. Positive responses are indicated by dots color-coded by treatment group, and negative responses by gray triangles. A boxplot is superimposed on the distribution, including only positive responses. The mid-line of the box denotes the median and the ends of the box denote the $25^{th}$ and $75^{th}$ percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range (i.e., height of the box) or if no value meets this criterion, to the data extremes. +Plots of peak activity and AUC show both response rates and the distribution of magnitude. Positive responses are indicated by dots color-coded by treatment group, and negative responses by gray triangles. A boxplot is superimposed on the distribution of positive responses. The mid-line of the box denotes the median and the ends of the box denote the $25^{th}$ and $75^{th}$ percentiles. The whiskers that extend from the top and bottom of the box extend to the most extreme data points that are no more than 1.5 times the interquartile range (i.e., height of the box) or if no value meets this criterion, to the data extremes. *[If working with durability data and calculating fold change from peak, be sure to specify direction of difference, e.g. $\text{log10(durability visit)} - \text{log10(peak visit)}$.]*