Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials

Moodie, Zoe; Huang, Yunda; Gu, Lin; Hural, John; Self, Steven G.

doi:10.1016/j.jim.2006.07.015

Cited by 60 publications

(55 citation statements)

References 29 publications

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“…Following recent CIP suggestions we also used a nonparametric statistical distribution-free resampling (DFReq) test method together with the recommended R based web tool (http://www.scharp.org/zoe/runDFR/) suitable to detect moderate immune response magnitudes (Moodie et al . 54–56 ) for assessing immune responses in the 42 samples meeting the CIMT criteria. Response rates obtained with the DFR(eq) method are shown in Supplementary Figure 2A .…”

Section: Resultsmentioning

confidence: 99%

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

et al. 2018

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Mutated proteins arising from somatic mutations in tumors are promising targets for cancer immunotherapy. They represent true tumor-specific antigens (TSAs) as they are exclusively expressed in tumors, reduce the risk of autoimmunity and are more likely to overcome tolerance compared to wild-type (wt) sequences. Hence, we designed a panel of long peptides (LPs, 28–35 aa) comprising driver gene mutations in TP35 and KRAS frequently found in gastrointestinal tumors to test their combined immunotherapeutic potential. We found increased numbers of T cells responsive against respective mutated and wt peptides in colorectal cancer patients that carry the tested mutations in their tumors than patients with other mutations. Further, active immunization of HLA(-A2/DR1)-humanized mice with mixes of the same mutated LPs yielded simultaneous, polyvalent CD8+/CD4+ T cell responses against the majority of peptides. Peptide-specific T cells possessed a multifunctional cytokine profile with CD4+ T cells showing a TH1-like phenotype. Two mutated peptides (Kras[G12V], p53[R248W]) induced significantly higher T cell responses than corresponding wt sequences and comprised HLA-A2/DR1-restricted mutated epitopes. However, vaccination with the same highly immunogenic LPs strongly increased systemic regulatory T cells (Treg) numbers in a syngeneic sarcoma model over-expressing these mutated protein variants and resulted in accelerated tumor outgrowth. In contrast, tumor outgrowth was delayed when vaccination was directed against tumor-intrinsic Kras/Tp53 mutations of lower immunogenicity. Conclusively, we show that LP vaccination targeting multiple mutated TSAs elicits polyvalent, multifunctional, and mutation-specific effector T cells capable of targeting tumors. However, the success of this therapeutic approach can be hampered by vaccination-induced, TSA-specific Tregs.

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Section: Resultsmentioning

confidence: 99%

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

et al. 2018

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“…Custom peptide manufacturers and suppliers have responded to this demand by increasing their supply capacity to meet the growing demand in both research and therapeutics markets (15). Since peptide libraries used for screening cellular immune responses may contain hundreds or even thousands of individual peptides, and each peptide is synthesized and purified independently, QA/QC of peptide libraries used in both basic research and clinical research protocols has become a significant and daunting task (4,16,24,27,28). Stringent QA/QC is an absolute requirement for clinical trials where biological assay outcome may be the end-point determinant for advancement or nonadvancement of a vaccine or therapeutic product (10,25).…”

Section: Discussionmentioning

confidence: 99%

Peptide Impurities in Commercial Synthetic Peptides and Their Implications for Vaccine Trial Assessment

Currier

Galley

Wenschuh

et al. 2008

Clin Vaccine Immunol

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The advent of T-cell assay methodologies that are amenable to high throughput coupled with the availability of large libraries of overlapping peptides have revolutionized the fields of vaccine efficacy testing and cellular immune response assessment. Since T-cell assay performance is critically dependent upon the quality and specificity of the stimulating peptides, assurance of high-quality and reliable input peptides is an important aspect of assay validation. Herein, we demonstrate that individual peptides from large human immunodeficiency virus (HIV)-based peptide library sets obtained directly from two independent custom peptide suppliers contained contaminating peptides capable of giving false-positive results, which were consistent with nominal antigen-specific CD8 ؉ T-cell responses. In-depth investigation of the cellular response in terms of responding CD8 ؉ T-cell frequency and human leukocyte antigen (HLA) restriction led to the conclusion that one set of HIV type 1 (HIV-1)-derived peptides was contaminated with a peptide from human cytomegalovirus (HCMV), which is commonly used in cellular immunology research applications. Analytical characterization of the original stock of the suspect HIV-1 peptide confirmed the presence of ϳ1% by weight of the HCMV peptide. These observations have critical implications for quality assurance (QA) and quality control (QC) of peptides used in clinical trials where cellular immune-based assays are important end-point determinants. We propose a simple schema of biological QA/QC protocols to augment the standard biochemical QA/QC analyses as a means to circumvent this and other problems that can affect cellular immune-based assay outcome and interpretation.The identification and characterization of immunogenic Tcell epitope-containing regions of the proteomes of infectious agents and candidate cancer-and tumor-specific proteins are an essential phase in the rational development of prophylactic vaccines and immunotherapeutic strategies. Recent methodological advances, particularly enzyme-linked immunospot (ELISPOT) assays and cytokine-based flow cytometry (CFC) assays coupled with overlapping pooled peptide technology, give the opportunity for detailed and precise analyses of specific cellular immune responses (1,3,11,19,22,29). As cellular immune response assays proceed from being used primarily as research tools to be being used as tools for clinical evaluation and assessment of end points in different phases of vaccine development, the required standards of quality assurance (QA)/quality control (QC) for these assays rise dramatically (4,10,16,17,21,27,28). While assay techniques and equipment can be standardized by employing standard operating procedures and assay reagents and can be standardized by use of manufacturer-certified assay kits, a critical component of the assay that is not typically subject to standardized QA/QC is the synthetic peptides used for stimulating the T cells. Synthetic peptides are usually specific to the particular application of the assay, d...

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“…Frozen STCL were thawed at 37°C and immediately washed, permeabilized with Perm Solution 2 (BD) for 10 min at room temperature, washed again, and stained for 30 Statistical analysis. The ex vivo ELISPOT readouts were analyzed as described previously (25). Cultured ELISPOT response rates were analyzed using classical two-group proportion tests (z-statistics) and the Fisher exact test.…”

Section: Methodsmentioning

confidence: 99%

Quantitative and Qualitative Differences in the T Cell Response to HIV in Uninfected Ugandans Exposed or Unexposed to HIV-Infected Partners

Pala

Serwanga

Watera

et al. 2013

J Virol

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d HIV-exposed and yet persistently uninfected individuals have been an intriguing, repeated observation in multiple studies, but uncertainty persists on the significance and implications of this in devising protective strategies against HIV. We carried out a cross-sectional analysis of exposed uninfected partners in a Ugandan cohort of heterosexual serodiscordant couples (37.5% antiretroviral therapy naive) comparing their T cell responses to HIV peptides with those of unexposed uninfected individuals. We used an objective definition of exposure and inclusion criteria, blinded ex vivo and cultured gamma interferon (IFN-␥) enzymelinked immunospot assays, and multiparameter flow cytometry and intracellular cytokine staining to investigate the features of the HIV-specific response in exposed versus unexposed uninfected individuals. A response rate to HIV was detectable in unexposed uninfected (5.7%, 95% confidence interval [CI] ‫؍‬ 3.3 to 8.1%) and, at a significantly higher level (12.5%, 95% CI ‫؍‬ 9.7 to 15.4%, P ‫؍‬ 0.0004), in exposed uninfected individuals. The response rate to Gag was significantly higher in exposed uninfected (10/50 [20.%]) compared to unexposed uninfected (1/35 [2.9%]) individuals (P ‫؍‬ 0.0004). The magnitude of responses was also greater in exposed uninfected individuals but not statistically significant. The average number of peptide pools recognized was significantly higher in exposed uninfected subjects than in unexposed uninfected subjects (1.21 versus 0.47; P ‫؍‬ 0.0106). The proportion of multifunctional responses was different in the two groups, with a higher proportion of single cytokine responses, mostly IFN-␥, in unexposed uninfected individuals compared to exposed uninfected individuals. Our findings demonstrate both quantitative and qualitative differences in T cell reactivity to HIV between HESN (HIV exposed seronegative) and HUSN (HIV unexposed seronegative) subject groups but do not discriminate as to whether they represent markers of exposure or of protection against HIV infection.

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Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials

Cited by 60 publications

References 29 publications

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses

Peptide Impurities in Commercial Synthetic Peptides and Their Implications for Vaccine Trial Assessment

Quantitative and Qualitative Differences in the T Cell Response to HIV in Uninfected Ugandans Exposed or Unexposed to HIV-Infected Partners

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