Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: Defining T Cell Responses As Potential Correlates of Immunity
Identifieur interne : 000019 ( Istex/Corpus ); précédent : 000018; suivant : 000020Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: Defining T Cell Responses As Potential Correlates of Immunity
Auteurs : Nina D. Russell ; Michael G. Hudgens ; Richard Ha ; Colin Havenar-Daughton ; M. Juliana McelrathSource :
- The Journal of Infectious Diseases [ 0022-1899 ] ; 2003.
Abstract
There is evidence in both simian immunodeficiency virus and human immunodeficiency virus (HIV) type 1 infection that class I major histocompatibility complex–restricted CD8+ cytotoxic T lymphocytes play a pivotal role in controlling infection and, potentially, in protecting by immunization. Progress has been made in designing strategies to elicit these responses with HIV-1 vaccines, but methods to reproducibly quantify them have posed difficulties. An interferon-γ enzyme-linked immunospot assay, using peptide pools spanning the HIV-1 genes, was developed and standardized. This method is rapid (2 days), sensitive (threshold of detection, ⩾0.005%), quantitative, feasible using cryopreserved cells, and able to define epitope specificities. When this assay was applied to 36 HIV-1–seropositive and 10 HIV-1–seronegative subjects, it proved to be robust (specificity, 100%). When responses in natural infection were compared with vaccine-induced responses, vaccine recipient responses were ⩾1 log lower, which confirms the importance of using this sensitive assay as an initial screen in vaccine protocols
Url:
DOI: 10.1086/367702
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<journal-title>The Journal of Infectious Diseases</journal-title>
<abbrev-journal-title>The Journal of Infectious Diseases</abbrev-journal-title>
<issn pub-type="ppub">0022-1899</issn>
<issn pub-type="epub">1537-6613</issn>
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<subject>Major Articles</subject>
<subj-group subj-group-type="heading">
<subject>HIV/AIDS</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: Defining T Cell Responses As Potential Correlates of Immunity<xref ref-type="fn" rid="fn1"></xref>
</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Russell</surname>
<given-names>Nina D.</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="aff" rid="aff3">3</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Hudgens</surname>
<given-names>Michael G.</given-names>
</name>
<xref ref-type="aff" rid="aff2">2</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ha</surname>
<given-names>Richard</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Havenar-Daughton</surname>
<given-names>Colin</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>McElrath</surname>
<given-names>M. Juliana</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="aff" rid="aff3">3</xref>
</contrib>
<aff id="aff1">
<label>1</label>Program in Infectious Diseases, Clinical Research Division, and</aff>
<aff id="aff2">
<label>2</label>Program in Biostatistics, Public Health Science Division, Fred Hutchinson Cancer Research Center, and</aff>
<aff id="aff3">
<label>3</label>Department of Medicine, University of Washington, Seattle</aff>
</contrib-group>
<author-notes>
<corresp id="cor1">Reprints or correspondence: Dr. M. Juliana McElrath, Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, PO Box 19024, Seattle, WA 98109 (<email>kd@u.washington.edu</email>)</corresp>
</author-notes>
<pub-date pub-type="ppub">
<day>15</day>
<month>1</month>
<year>2003</year>
</pub-date>
<volume>187</volume>
<issue>2</issue>
<fpage>226</fpage>
<lpage>242</lpage>
<history>
<date date-type="received">
<day>23</day>
<month>7</month>
<year>2002</year>
</date>
<date date-type="rev-recd">
<day>10</day>
<month>10</month>
<year>2002</year>
</date>
</history>
<copyright-statement>© 2003 by the Infectious Diseases Society of America</copyright-statement>
<copyright-year>2003</copyright-year>
<abstract>
<p>There is evidence in both simian immunodeficiency virus and human immunodeficiency virus (HIV) type 1 infection that class I major histocompatibility complex–restricted CD8<sup>+</sup> cytotoxic T lymphocytes play a pivotal role in controlling infection and, potentially, in protecting by immunization. Progress has been made in designing strategies to elicit these responses with HIV-1 vaccines, but methods to reproducibly quantify them have posed difficulties. An interferon-γ enzyme-linked immunospot assay, using peptide pools spanning the HIV-1 genes, was developed and standardized. This method is rapid (2 days), sensitive (threshold of detection, ⩾0.005%), quantitative, feasible using cryopreserved cells, and able to define epitope specificities. When this assay was applied to 36 HIV-1–seropositive and 10 HIV-1–seronegative subjects, it proved to be robust (specificity, 100%). When responses in natural infection were compared with vaccine-induced responses, vaccine recipient responses were ⩾1 log lower, which confirms the importance of using this sensitive assay as an initial screen in vaccine protocols</p>
</abstract>
</article-meta>
</front>
<body>
<p>There is an urgent global need to develop an effective human immunodeficiency virus (HIV) type 1 vaccine. A major challenge in this undertaking has been to better define the components of immunity that are elicited by immunization and that may confer protection. It is presumed that HIV-1 prevention by immunization will be mediated substantially, although probably not exclusively, by class I major histocompatibility complex (MHC)–restricted HIV-1–specific CD8<sup>+</sup> T cells. Evidence to support this is based on the significant contribution of HIV-1–specific CD8<sup>+</sup> T cells in controlling infection and, possibly, in protecting against transmission in seronegative persons with repeated high-risk HIV-1 exposures [<xref ref-type="bibr" rid="ref1">1</xref>
<xref ref-type="bibr" rid="ref2"></xref>
<xref ref-type="bibr" rid="ref3"></xref>
<xref ref-type="bibr" rid="ref4"></xref>
<xref ref-type="bibr" rid="ref5"></xref>
<xref ref-type="bibr" rid="ref6"></xref>–<xref ref-type="bibr" rid="ref7">7</xref>]. HIV-1–specific CD8<sup>+</sup> T cells exert antiviral activities through cytolysis of infected cells and by release of cytokines, particularly interferon (IFN)–γ, and chemokines, such as RANTES, macrophage inflammatory protein (MIP)–1α, and MIP-1β. Thus, an important goal of HIV-1 vaccine clinical trials is to elicit high-frequency, cytolytic, and IFN-γ–secreting memory CD8<sup>+</sup> T cells that are capable of recognizing multiple epitopes within diverse HIV-1 strains</p>
<p>Recent studies in nonhuman primate models and in early phase 1 and 2 clinical trials have indicated that the administration of live vectors, particularly recombinant pox viruses, can induce class I MHC–restricted cytotoxic T lymphocytes that recognize epitopes expressed by the HIV-1 gene inserts. However, the magnitude, breadth, and persistence of these responses have been variable [<xref ref-type="bibr" rid="ref8">8</xref>
<xref ref-type="bibr" rid="ref9"></xref>
<xref ref-type="bibr" rid="ref10"></xref>–<xref ref-type="bibr" rid="ref11">11</xref>]. Clinical trials with other promising viral vectors and DNA constructs are also under way, and a method to systematically compare these regimens in phase 1 and 2 trials is needed. More important, the decision to proceed with a large-scale efficacy trial that uses a vaccine strategy designed to protect primarily through the induction of CD8<sup>+</sup> T cell effectors will be based on the precise identification of the specificities and frequencies of responses sufficient to render this immune defense. Thus, it is imperative to establish a standardized, sensitive approach to measure HIV-1–specific CD8<sup>+</sup> T lymphocyte activities that can be employed in field trial settings and used to compare the relative immunogenicities of various vaccine regimens in phase 1 and 2 trials</p>
<p>Detection of CD8<sup>+</sup> cytotoxic T lymphocyte (CTL) activity has traditionally relied on the lysis of autologous target cells expressing HIV-1 antigens in a chromium release assay, which indicates target cell membrane disruption resulting from release of perforin and granzymes by CTLs. This assay has been used in numerous vaccine studies [<xref ref-type="bibr" rid="ref12">12</xref>
<xref ref-type="bibr" rid="ref13"></xref>–<xref ref-type="bibr" rid="ref14">14</xref>] but has several disadvantages. Because the level of responses are lower in HIV-1–uninfected vaccine recipients than in HIV-1–infected persons, optimal detection of antigen-specific CD8<sup>+</sup> T cells by these methods requires fresh, rather than cryopreserved, peripheral blood mononuclear cells (PBMC). Real-time assays become problematic in the conduct of multicenter international trials. Moreover, quantitation of cytolytic activities currently relies on the use of a limiting dilution assay, which requires laborious in vitro stimulation and expansion steps that may be less reflective of the in vivo state</p>
<p>Enumeration of virus-specific CD8<sup>+</sup> T cells by techniques such as IFN-γ ELISPOT, MHC-peptide tetramer staining, and flow cytometric analysis of intracellular cytokine production offers the advantage of improved sensitivity and more-rapid delineation of class I MHC–restricted epitopes, compared with that of chromium-release CTL assays. In addition, these approaches are simpler to perform, require fewer cells, and obviate the need to expand and clone antigen-specific T lymphocytes and establish autologous B lymphoblastoid cell lines [<xref ref-type="bibr" rid="ref15">15</xref>
<xref ref-type="bibr" rid="ref16"></xref>
<xref ref-type="bibr" rid="ref17"></xref>
<xref ref-type="bibr" rid="ref18"></xref>–<xref ref-type="bibr" rid="ref19">19</xref>]. The IFN-γ ELISPOT assay offers an advantage over tetramer staining in its ability to provide a functional analysis of the HIV-1–specific response in populations spanning multiple class I HLA types without prior knowledge of epitope recognition. In addition, advanced operator training and expensive equipment are not necessary for measuring the frequency of responses by ELISPOT, which makes this technique potentially more feasible in a field setting than flow-based detection of intracellular cytokine secretion</p>
<p>For these reasons, we chose to focus on the development and validation of an IFN-γ ELISPOT assay for the screening and quantitation of HIV-1–specific CD8<sup>+</sup> T cells induced by vaccination. Lymphocytes from 6 chronically HIV-1–infected individuals were used to optimize the assay and to ensure its sensitivity as a screening test in vaccine studies. Assay performance was then assessed in persons at various stages of HIV-1 infection, as well as in vaccinated and unvaccinated HIV-1–uninfected subjects. Our results indicate that the IFN-γ ELISPOT assay can provide a quantitative measurement of the breadth of the HIV-specific CD8<sup>+</sup> T cell response and can define HIV-1 epitopes. This highly sensitive and specific approach appears to be feasible for application in large-scale HIV-1 vaccine clinical trials, to determine the role of vaccine-induced CD8<sup>+</sup> T cells in conferring protection against infection</p>
<sec id="S1">
<title>Population, Materials, and Methods</title>
<p>
<bold>
<italic>Study population</italic>
</bold>To establish optimal conditions for the performance of the IFN-γ ELISPOT assay in HIV-1 vaccine recipients, fresh and cryopreserved PBMC purified from a single leukapheresis were analyzed from 6 HIV-1–infected subjects. One patient (MP0471) had chronic HIV-1 infection, and 5 others (NP1, NP2, NP13, NP14, and NP15) were categorized as long-term nonprogressors (LTNPs), as defined elsewhere [<xref ref-type="bibr" rid="ref20">20</xref>]. HIV-1–specific responses were also evaluated in PBMC from HIV-1–uninfected persons participating in a double-blind, placebo-controlled vaccine trial sponsored by the AIDS Vaccine Evaluation Group (AVEG protocol 022), subsequently known as the HIV Vaccine Trials Network. The immunogen was a recombinant canarypox vector containing HIV-1 genes encoding Env, Gag, and protease (vCP205; Aventis Pasteur) with or without a recombinant gp120 boost (rgp120/SF-2, Chiron; rgp120/MN, VaxGen)</p>
<p>To establish criteria to define a positive response, assays were performed using PBMC from 36 HIV-1–seropositive and 10 HIV-1–seronegative individuals. There were 6 untreated, primary HIV-1–infected patients whose cells were obtained at a mean of 71 days after infection, 12 untreated chronically HIV-1–infected patients, 8 treated chronically HIV-1–infected subjects, and 10 LTNPs. The 10 seronegative individuals had been previously enrolled in AVEG protocol 201; prevaccination cryopreserved PBMC from 5 high-risk and 5 low-risk study participants were used for these experiments after ∼8.2 years of storage in liquid nitrogen [<xref ref-type="bibr" rid="ref21">21</xref>]</p>
<p>
<bold>
<italic>Peptides</italic>
</bold>The National Institutes of Health (NIH) AIDS Research and Reference Reagent Program provided synthetic HIV-1 20-mer peptides with 10-aa overlaps. These included 49 Gag 20-mers based on the HIV-1 subtype B consensus sequence (HXB2), 80 Env (MN), 100 Pol (HXB2), and 20 Nef (BRU/LAI). In addition, 122 HIV-1 Gag 15-mers overlapping by 11 aa (SF2) were generously provided by Dr. Louis Picker (Oregon Health Sciences University, Portland), as well as the NIH AIDS Reagent Program (HXB2). Two hundred twelve HIV-1 Env (MN), 248 Pol (HXB2), and 49 Nef (BRU) 15-mers overlapping by 11 aa were obtained from SynPep</p>
<p>Negative control wells contained either media alone (no peptide) or a pool of 5 irrelevant peptides (purchased from Mimotopes) derived from highly conserved regions of actin and HLA class I α chain precursor. Each peptide was reconstituted in 100% dimethyl sulfoxide (DMSO) and used at a final concentration of 2 μg/mL (<1% DMSO), unless otherwise indicated</p>
<p>
<bold>
<italic>IFN-γ ELISPOT assays</italic>
</bold>An ELISPOT assay was used to detect HIV-1–specific IFN-γ–producing T cells from either fresh or cryopreserved PBMC. Ninety-six–well hydrophobic polyvinylidene difluoride membrane-bottomed plates (Millipore) were coated with 50 μL of 10 μg/mL anti–IFN-γ monoclonal antibody (MAb; 1-D1K, mouse IgG1; Mabtech) overnight at 4°C. After washing 3 times with PBS (pH 7.2), plates were blocked with RPMI/HEPES with 10% fetal bovine serum (FBS) (R10) at room temperature for 1 h</p>
<p>PBMC were added in 100 μL of R10 to precoated plates at concentrations of 1×10<sup>5</sup> or 2×10<sup>5</sup> cells/well. Cryopreserved PBMC were thawed and incubated overnight in RPMI 1640 medium with 10% FBS before being added to plate. To determine the cell subset producing IFN-γ, T cell depletions were performed using anti-CD8<sup>+</sup> and anti-CD4<sup>+</sup> antibody-coated immunomagnetic beads (Microbeads, Miltenyi Biotec; Dynabeads, Dynal), according to the manufacturers’ instructions. HIV-1–specific peptides and control peptides were added to desired wells to give a final concentration of 2 or 5 μg/mL. Cells stimulated with phytohemagglutinin (1 μg/mL) served as a positive control. Responses to individual or pooled peptides were tested in duplicate. For some experiments, a matrix of Gag peptide pools was used to map the response to the single peptide level, as described elsewhere [<xref ref-type="bibr" rid="ref22">22</xref>, <xref ref-type="bibr" rid="ref23">23</xref>]</p>
<p>Plates were incubated overnight (16–20 h) at 37°C in 5% CO<sub>2</sub>, washed with PBS containing 0.05% Tween-20, and incubated at room temperature (RT) for 2 h with a secondary biotinylated anti–IFN-γ MAb at 1 μg/mL (7-B6-1, mouse IgG1; Mabtech). Avidin biotinylated enzyme complex (Vectastain ABC Elite Kit, PK-6100; Vector Laboratories) was added at RT for 1 h, followed by AEC peroxidase substrate (Vectastain). After developing plates for ∼7 min, reaction was stopped by washing with water, and plates were air-dried. Colored spot-forming cells (SFCs) were counted manually under a stereomicroscope (Stemi 2000; Carl Zeiss) or using an automated ELISPOT reader (Immunospot; Cellular Technology). Responses were considered to be positive if (1) ⩾10 SFCs were detected/2×10<sup>5</sup> cells after subtraction of the negative control and (2) SFCs were ⩾2-fold than those in the negative control wells</p>
<p>
<bold>
<italic>Statistical analysis</italic>
</bold>Simple summary statistics, tests, and plots were used for initial data exploration. To determine the qualitative agreement between 2 methods (e.g., overnight vs. 2-day incubation time), standard methods for 2×2 contingency tables were used [<xref ref-type="bibr" rid="ref24">24</xref>]. Specifically, McNemar’s test was used to assess marginal homogeneity, and the tetrachoric correlation coefficient (TCC) was used to assess the degree of positive association. Quantitative agreement was assessed using net responses calculated by subtracting the mean control response from the mean experimental response and converting to 200,000 cells/well, if necessary. Statistical measures of quantitative agreement focused on the concordance correlation coefficient (CCC) and the total deviation index (TDI) [<xref ref-type="bibr" rid="ref25">25</xref>]. The CCC measures agreement along the identity line, in which a value of 1 represents perfect agreement (Y=X), a value of −1 represents perfect disagreement (Y=-X), and a value of 0 represents no agreement. The CCC factors in a product of accuracy and precision coefficients where the accuracy coefficient is 1 if and only if the marginal distribution of <italic>Y</italic> and <italic>X</italic> are equal and the precision coefficient is simply the Pearson’s correlation coefficient (ρ). The accuracy coefficient measures how far the least-squares line is from the line of equality, whereas ρ measures how well the least-squares line fits the data. The TDI is defined such that the absolute value of the differences between the 2 methods is <sub>1−α</sub>, with probability 1-α. To assess agreement, outcomes for the same PBMC across different peptide pools were assumed to be independent, and sensitivity analysis was performed by stratifying the data according to nonoverlapping peptide pools (pools of peptides that did not contain the same individual peptide). Mixed-effects models were also used to account for possible dependency among repeated observations on the same PBMC [<xref ref-type="bibr" rid="ref26">26</xref>]. All tests were computed for a significance level of α=.05. All statistical computations were performed using S-PLUS (version 6.0; Insightful) or SAS (version 8.1; SAS Institute) software</p>
</sec>
<sec id="S2">
<title>Results</title>
<p>To standardize and optimize the identification and quantitation of HIV-1–specific CD8<sup>+</sup> T cells, 6 HIV-1–infected patients (NP1, NP2, NP13, NP14, NP15, and MP0471) were chosen for detailed study with the IFN-γ ELISPOT assay. These individuals had previously demonstrated consistent HIV-1–specific class I MHC–restricted CTLs recognizing ⩾1 epitopes. Clinical and virological profiles of the patients included the following: median age, 39 years (range, 33–55 years); median duration of infection, 13 years (range, 6–16 years); median CD4<sup>+</sup> cell count, 505 cells/mm<sup>3</sup> (range, 366–1225 cells/mm<sup>3</sup>); and median plasma HIV-1 RNA load at the time of leukapheresis, 462 copies/mL (range, <50–39,565 copies/mL). Only 1 (MP0471) of 6 patients had received antiretroviral therapy at the time of study. Several technical parameters of the assay were varied to establish maximal sensitivity. Conditions such as cell concentration, peptide concentration, incubation time, peptide pool size, and peptide length were tested to improve sensitivity while maintaining a simple, rapid, and relatively low-cost methodology. Assays were then optimized to identify epitope specificities and the MHC restricting allele. Subsequent analyses were performed on HIV-1–infected patients with acute and chronic infection, as well as HIV-1–uninfected vaccine recipients, to determine the frequency and range of responses</p>
<p>
<bold>
<italic>Cell number per well</italic>
</bold>To determine the optimal cell number per well to detect HIV-1–specific CD8<sup>+</sup> responses in the low frequency range, PBMC from 4 donors were diluted serially (8.0×10<sup>5</sup>–0 cells/well) and stimulated with single Gag 20-mer (<xref ref-type="fig" rid="Fig1">figure 1<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig1">1<italic>B</italic>
</xref>) or 15-mer peptides (<xref ref-type="fig" rid="Fig1">figure 1<italic>C</italic>
</xref> and <xref ref-type="fig" rid="Fig1">1<italic>D</italic>
</xref>) containing class I MHC–restricted HIV-1 epitopes identified in previous experiments. As shown in 4 representative examples in <xref ref-type="fig" rid="Fig1">figure 1</xref>, positive responses were commonly detected with >50,000 PBMC/well, and increases, often linear, were observed up to 400,000–600,000 PBMC/well. At greater cell numbers, the frequency of HIV-1–specific IFN-γ SFCs reached a plateau and, in some cases, declined in conjunction with increasing IFN-γ SFC frequencies in the control wells (<xref ref-type="fig" rid="Fig1">figures 1<italic>C</italic>
</xref> and <xref ref-type="fig" rid="Fig1">1<italic>D</italic>
</xref>). These findings were confirmed with repeated testing that used both 20- and 15-mers</p>
<p>Cell numbers from clinical trial participants will be limiting if analyses include the identification of specific T cell subsets recognizing multiple gene products and definition of optimal epitopes within these regions. We anticipate that ⩽200,000 cells/well will be available from cryopreserved specimens without exceeding the blood volume restrictions for vaccine trials. Thus, on the basis of the above results, a comparison between 100,000 and 200,000 cryopreserved PBMC/well from 5 HIV-1–infected donors was performed after overnight stimulation with pools containing 50, 25, or 10 Gag 20-mer peptides. Positive responses were easily detected with both cell concentrations when the frequency of IFN-γ SFCs was higher (>50 SFCs/well; data not shown). However, responses clearly positive with 200,000 cells/well were frequently below or at the threshold for positivity with 100,000 PBMC/well. In repeated experiments in which responses were at low frequency, 200,000 cells/well were necessary to consistently detect IFN-γ SFCs distinct from background responses in the control wells. Therefore, the use of 200,000 PBMC/well may be more advantageous in demonstrating vaccine-induced HIV-1–specific T cells present in lower frequencies than those recognizing immunodominant epitopes typically found in infected persons</p>
<p>
<bold>
<italic>Duration of stimulation</italic>
</bold>To establish the time sufficient to stimulate antigen-specific T cells to secrete IFN-γ, we analyzed SFC frequencies in 4 patients, using a total of 65 peptide pools, and compared overnight (16–20 h) incubation with 2-day (36–40 h) incubation (<xref ref-type="fig" rid="Fig2">figure 2</xref>). Of note, there was no substantial increase in response to the control peptides with the longer incubation in any of the experiments performed. When cryopreserved PBMC were tested from MP0471, the response to the pool of all 122 Gag 15-mers decreased by 49 SFCs (20.4%) with the longer stimulation time, whereas the response to a pool of 25 Gag 15-mers increased by 30 SFCs (16.5%) (<xref ref-type="fig" rid="Fig2">figure 2<italic>A</italic>
</xref>). Only 1 of 4 peptide pools recognized by PBMC from NP15 generated a greater number of spots with the longer stimulation time (<xref ref-type="fig" rid="Fig2">figure 2<italic>B</italic>
</xref> pool 1–25). PBMC from NP13 and NP1, known low-level responders, were similarly tested in repeat experiments with Gag 20-mers in pools of 50, 25, and 10 peptides (data not shown)</p>
<p>When data from all 65 peptide pool comparisons were stratified according to positive and negative responses, there was excellent qualitative agreement between the 2 incubation times. Only 4 of 65 comparisons yielded discordant results, with higher frequency responses (>25 SFCs/well) detected regardless of incubation time. For the 30 comparisons that used pools of 10 Gag 20-mer peptides, there was near-perfect agreement, with only 2 discordant outcomes (TCC, 0.97; 95% confidence interval [CI], 0.90–1.00; P=1.00, McNemar’s test). Similar results held for other pools. Analysis of quantitative agreement between the 2 incubation times focused on the 31 comparisons in which both incubation times yielded positive responses. The sign and Wilcoxon signed-rank tests on the differences in net responses gave mixed results (P=.15 and P=.04, respectively). <xref ref-type="fig" rid="Fig2">Figure 2<italic>C</italic>
</xref> shows a scatterplot of the 31 comparisons with the solid least-squares line (intercept β<sub>0</sub>=16.1 [95% CI, 2.7–29.5]; slope β<sub>1</sub>=0.7 [95% CI, 0.6–0.8]) and the dotted 45° line of perfect agreement. The CCC equaled 0.85 with 1-sided lower 95% confidence limit (CL) of 0.79, indicating good agreement between the 2 methods. The accuracy and precision (Pearson’s correlation) coefficients were 0.94 and 0.91, respectively, and the TDI<sub>0.9</sub> estimate was 53.7 (1-sided upper 95% CL, 66.5), which suggests that, on average, 90% of the paired experiments differed by <54 SFCs. Quantitative agreement analysis considering only nonoverlapping peptide pools (pools of peptides that did not contain the same individual peptide) gave similar results. Taken together, little difference both qualitatively and quantitatively was observed between the 2 stimulation periods. Thus, for convenience, we chose the shorter overnight period of stimulation</p>
<p>
<bold>
<italic>Peptide concentration</italic>
</bold>To assess the optimal concentration of each peptide within a pool, we sought to determine the lowest concentration that preserves sensitivity but conserves reagents. Of note, lower IFN-γ SFC frequencies were commonly observed when peptide pools contained >1% DMSO (data not shown); thus, final DMSO concentrations were subsequently maintained at <1%, to avoid cellular toxicity. Initial experiments examined responses to 15- and 20-mers titrated from 0 to 20 μg/mL using both individual peptides and pools of peptides containing the individual peptide (<xref ref-type="fig" rid="Fig3">figure 3<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig3">3<italic>B</italic>
</xref> representative examples in 2 donors). In general, IFN-γ SFCs were detected on stimulation with ⩾0.1 μg/mL of peptide. The IFN-γ SFC frequency rose with increasing concentrations of the individual peptide, up to 5–10 μg/mL, after which a plateau was reached (<xref ref-type="fig" rid="Fig3">figure 3<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig3">3<italic>B</italic>
</xref>). When responses to the peptide pools were tested, similar or decreased frequencies were observed with peptide concentrations ⩾5 μg/mL, and a striking fall in SFC frequencies was noted when 20 μg/mL of each peptide was used (<xref ref-type="fig" rid="Fig3">figure 3<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig3">3<italic>B</italic>
</xref>)</p>
<p>Obvious advantages in using lower peptide concentrations include the ability to test more peptides in each pool without increasing DMSO content and to economize on reagents. Thus, additional studies were performed to determine whether 2 μg/mL, rather than 5 μg/mL, was sufficient for detection of HIV-1–specific T cells. Cryopreserved PBMC from 5 donors were stimulated with 20-mers and from those 2 donors with 15-mers. No benefit in the ability to detect a positive response was observed with use of the higher peptide concentration (<xref ref-type="fig" rid="Fig3">figure 3<italic>C</italic>
</xref> and <xref ref-type="fig" rid="Fig3">3<italic>D</italic>
</xref> representative experiments). For the 2 donors tested with 15-mers, 9 experiments were done for each donor at both 2 and 5 μg/mL. Of these 18 pairs, there was only one discordant response (positive at 5 μg/mL and negative at 2 μg/mL), which indicates excellent qualitative agreement between the 2 methods (TCC=1.00; P=1.00, McNemar’s test). For the 13 paired samples with concordant positive responses, the CCC was 0.98 (1-sided lower 95% CL, 0.97), indicating excellent agreement between the 2 methods. The accuracy and precision (Pearson’s correlation) coefficients equaled 0.99 and 1.00, respectively, and the TDI<sub>0.9</sub> estimate was 77.8 (1-sided upper 95% CL, 108.4). The especially high correlation coefficients are partially attributable to the broad range of the response data (10–851 net SFC/10<sup>5</sup> PBMC), for which the intersample variability is much greater than the intrapair variability. Sign and Wilcoxon signed-rank tests were both significant (P<.01), indicating a stronger response with the higher peptide concentration. Using a mixed effect model on positive responses, stimulation with the higher peptide concentration resulted in a small percentage increase in SFCs/2×10<sup>5</sup> PBMC recognizing pools of 25 15-mers (16%; P=.03) and individual 15-mers (12%, P=.06). This benefit was not appreciated, however, after stimulation with the larger pool of 122 peptides (P=.63). On the basis of these findings, peptide concentrations of 2 μg/mL are sufficient for the detection of HIV-1–specific IFN-γ SFCs, particularly when screening large peptide pools, although slightly lower SFC frequencies may result when testing individual or smaller peptides pools</p>
<p>
<bold>
<italic>Peptide pool size</italic>
</bold>Next, we determined the optimal number of peptides in a pool that could afford detection of responses to multiple epitopes while conserving cell numbers. The SFC frequencies from 6 donors were compared using 20-mer peptide pools spanning Gag: 1 large pool of 49 20-mers (1–49), 2 pools of 25-mers (1–25 and 26–49), 5 pools of 10-mers (1–10, 11–20, 21–30, 31–40, and 41–49), and 25 pools of 2 20-mers. Positive responses to individual 20-mers were generally maintained by stimulation with pools of all sizes (<xref ref-type="fig" rid="Fig4">figure 4</xref>, representative examples). However, individual peptide responses were frequently greater (31%–220%) when the 20-mer was contained in a smaller pool (10 or 2 peptides), compared with the larger pools (25 or 50 peptides) (<xref ref-type="fig" rid="Fig4">figure 4<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig4">4<italic>B</italic>
</xref>). Thus, stimulation with 10 20-mers within a pool may be optimal to detect low-frequency responses, although responses were clearly detectable when larger pools of 25–50 20-mers were used. Similar comparisons were performed with Gag 15-mers in pools of 122, 25 (spanning the same amino acid sequence as pools containing 10 20-mers), or individually and revealed fewer differences. However, there tended to be an advantage in the detection of higher SFC frequencies when using pools of 25, rather than 122, 15-mers (data not shown)</p>
<p>To determine whether there is an inhibitory effect when using peptides pools containing >1 recognized epitope, we compared SFC frequencies with pools of 10 20-mers with those detected using the individual peptides comprising the pool. Experiments in 2 donors using Env and Pol 20-mer peptides are depicted in <xref ref-type="fig" rid="Fig4">figure 4<italic>C</italic>
</xref> and <xref ref-type="fig" rid="Fig4">4<italic>D</italic>
</xref>. The SFC frequency measured after stimulation with the peptides mixed in the pool was not necessarily the sum of the responses seen with the individual peptides, but it was generally greater than those recognized by the individual peptides. Thus, there was no evidence of a significant inhibitory effect by combining >1 responding peptide in a pool</p>
<p>
<bold>
<italic>Amino acid length of peptide</italic>
</bold>To determine the optimal peptide length to stimulate and detect both CD8<sup>+</sup> and CD4<sup>+</sup> HIV-1–specific T cells, we compared 15-mers overlapping by 11 aa (n=122) and 20-mers overlapping by 10 aa (n=49) in repeated experiments in 5 seropositive donors. We observed up to ⩾2-fold SFC frequencies after stimulation with the pool of Gag 15-mers, compared with the pool of 20-mers (<xref ref-type="fig" rid="Fig5">figure 5<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig5">5<italic>B</italic>
</xref> representative examples). Even greater differences were observed with the individual 15-mers, compared with those of the 20-mers containing the same epitope (<xref ref-type="fig" rid="Fig5">figure 5<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig5">5<italic>B</italic>
</xref>). Of note, a response considered to be negative after stimulation with a 20-mer peptide (6 SFC for 20-mer 15-QMVHQAISPRTLNAWVKVVE) was easily detectable after stimulation with the corresponding, embedded 15-mer peptide (62 SFC for 15-mer 37-QAISPRTLNAWVKVV) (<xref ref-type="fig" rid="Fig5">figure 5<italic>B</italic>
</xref>)</p>
<p>To quantify differences in SFC frequencies after stimulation with peptides of different lengths, data from all experiments in 5 donors were analyzed. As seen in the scatterplot in <xref ref-type="fig" rid="Fig5">figure 5<italic>C</italic>
</xref> every net response was greater when 15-mers were used. All responses are included in <xref ref-type="fig" rid="Fig5">figure 5<italic>C</italic>
</xref> except one experiment (NP14), which generated >1000 SFCs/2×10<sup>5</sup> PBMC using 15-mer peptides. Over the range of data in <xref ref-type="fig" rid="Fig5">figure 5<italic>C</italic>
</xref> the CCC was 0.60 (1-sided lower 95% CL, 0.40), with precision and accuracy coefficients of 0.92 and 0.66, respectively, indicating a good fit regarding the least-squares line but a change in scale between the 2 assays. Both the sign and Wilcoxon signed-rank tests were highly significant (P<.001), indicating higher response rates by stimulation with 15-mers. Similarly, the solid least-squares regression line in <xref ref-type="fig" rid="Fig5">figure 5<italic>C</italic>
</xref> had a slope significantly <1 (β<sub>1</sub>=0.64 [95% CI, 0.48–0.80]), which implies that 15-mers tend to give ∼1.6 times the number of spots as 20-mers. In summary, these data suggest that stimulation with pools of 15-mer peptides increases the ability to detect low-level responses and leads to the detection of higher SFC frequencies in this assay</p>
<p>
<bold>
<italic>Fresh versus frozen PBMC</italic>
</bold>To discern the sensitivity of the IFN-γ ELISPOT assay using frozen cells, we compared results using fresh and cryopreserved PBMC obtained from a single leukapheresis in our 6 HIV-1–seropositive patients. For these and subsequent experiments using cells thawed between 9 and 62 days after cryopreservation, cell recovery averaged 60% and cell viability averaged 90%. Of note, improved spot quality and higher spot counts were observed by incubating thawed cells overnight at 37°C (data not shown), which led to a mean increase of 5% viability and mean loss of 13% of cells. Although the SFC frequencies from some frozen populations were diminished, compared with the SFC frequencies from fresh populations, positive responses were consistently maintained (<xref ref-type="fig" rid="Fig6">figure 6<italic>A</italic>
</xref> and <xref ref-type="fig" rid="Fig6">6<italic>B</italic>
</xref>). In addition, the overall proportion of positive responses observed with the fresh PBMC was not significantly different than that observed with the frozen cells. For example, of the 144 pools of 2 Gag 20-mer peptides tested across all 6 donors, 13.9% were positive when using frozen PBMC, compared with 11.8% positive when using fresh cells (P=.61, McNemar’s test). Similar results were observed with the pools of 10 and 25 Gag 20-mer peptides. In addition, there was a strong positive association between fresh and cryopreserved assays. For example, the TCC was 0.86 (95% CI, 0.63–1.00) for the 30 pools of 10 Gag 20-mer peptides</p>
<p>To determine the impact of the duration of cryopreservation on SFC frequency, Gag-specific responses from the same leukapheresis were tested in 5 donors using fresh PBMC and PBMC thawed after several time intervals spanning 442 days. As seen in the representative data shown in <xref ref-type="fig" rid="Fig6">figure 6<italic>C</italic>
</xref> with the exception of responses to 1 pool of 10 Gag peptides (Gag 11–20), the SFC frequency was greater when testing fresh PBMC than previously frozen PBMC. A moderate decline in SFC was noted after 4 months of freezing. However, consistently positive responses were maintained over the 14 months when examining all responding peptide pools. Using a repeated-measures model on positive responses from all 5 donors, approximately one-third fewer SFCs resulted with the use of previously cryopreserved PBMC than fresh PBMC (P=.10). However, the length of cryopreservation did not have a significant effect on the frequency of SFCs (P=.22)</p>
<p>
<bold>
<italic>Interoperator reproducibility and intraoperator variability</italic>
</bold>Next, we looked at the reproducibility and variability of responses measured both within the same and across different operators. To examine the variability in SFC frequency across multiple assays performed by a single operator, we had one technician perform 5 identical tests on cells from each of 2 donors. A single PBMC thaw was used for all 5 tests in which responses to 5 different pools of 25 Gag 15-mers were measured (data not shown). For 9 of 10 combinations of donor and peptide pools, the 5 tests were in perfect qualitative agreement; the 1 discordant result had 4 negative responses and 1 positive response. Quantitative analysis of positive responses using a random effects model revealed that only ∼1% of the variability of the responses was attributable to the different tests—that is, the ELISPOT procedure itself</p>
<p>To assess the amount of variability introduced when multiple operators perform the assay, we compared SFC frequencies obtained when 5 different technicians performed identical assays in cells from each of 3 donors. Again, a single PBMC thaw was used for each donor assay, for which responses to pools of 25 Gag 15-mers were assessed (data not shown). The 5 operators were in perfect qualitative agreement for all 15 combinations of donor and peptide pool (7 negative and 8 positive). Quantitative agreement between the operators was excellent, with an analysis of positive responses using a random effects model revealing that <0.5% of the variability of the responses was attributable to the different operators. Thus, there was excellent reproducibility both within and across operators</p>
<p>
<bold>
<italic>IFN-γ ELISPOT for fine mapping of HIV-1–specific T cell responses</italic>
</bold> IFN-γ ELISPOT assays that use peptide pools spanning multiple HIV-1 genes and a pool of optimal peptides as stimulating antigens can be readily applied as a rapid screening tool to assess vaccine-induced T cell responses. Once a positive response is detected, the assay can be repeated to characterize this response down to the individual 15- or 20-mer or ideal immunodominant 8–10-mer peptide, as shown in <xref ref-type="fig" rid="Fig7">figure 7</xref> in patient MP0471, whose class I HLA type was A24, B*1501, B27. In this case, responses to 1 Gag peptide (27-IYKRWIILGLNKIVRMYSPT), 1 Env peptide (74-IVELLGRRGWEVLKYWWNLL), 5 Pol peptides (33-KILEPFRKQNPDIVIYQYMD, 36-GQHRTKIEELRQHLLRWGLT, 49-IAEIQKQGQGQWTYQIYQEP, 50-QWTYQIYQEPFKNLKTGKYA, and 99-KIIRDYGKQMAGDDCVASRQ), and the immunodominant, HLA B-15–restricted, Nef 9-mer were characterized. Of note, the Gag 20-mer peptide 27 includes an amino acid sequence matching a HLA-B15 CTL epitope described elsewhere (GLNKIVRMY) and an HLA-B27–restricted epitope (KRWIILGLNK) [<xref ref-type="bibr" rid="ref27">27</xref>, <xref ref-type="bibr" rid="ref28">28</xref>], and the Env 20-mer peptide 74 includes an HLA-B27–restricted epitope (GRRGWEALKY) [<xref ref-type="bibr" rid="ref29">29</xref>]</p>
<p>
<bold>
<italic>Cross-sectional analysis of HIV-1–specific IFN-γ responses in HIV-1–infected subjects</italic>
</bold>After optimizing the IFN-γ ELISPOT assay conditions for performance using cryopreserved PBMC, frozen cells from 36 HIV-1–seropositive subjects and 10 HIV-1–seronegative subjects were tested for both CD8<sup>+</sup> and CD4<sup>+</sup> T cell responses to HIV-1 Gag, Env, Pol, and Nef using pools of 15-mer peptides (<xref ref-type="table" rid="tb1">table 1</xref>). For these experiments, anti-CD4<sup>+</sup> antibody–coated immunomagnetic beads were used to define T cell subsets. CD4<sup>+</sup> responses were based on the positively selected CD4<sup>+</sup> T cell fraction, and CD8<sup>+</sup> responses were based on the CD4<sup>+</sup>-depleted PBMC population. The seropositive individuals included 6 untreated primary HIV-1–infected patients whose PBMC were obtained at a mean of 71 days after infection, 10 LTNPs, 12 untreated chronically HIV-1–infected patients, and 8 treated chronically HIV-1–infected subjects. The 10 HIV-1–seronegative individuals were study participants in AVEG protocol 201, whose PBMC were drawn at a baseline prevaccination visit. As expected, median plasma HIV-1 RNA levels in the LTNPs and treated patients were lower (<1500 copies/mL) than those with untreated primary and chronic infection (>10,000 copies/mL)</p>
<p>All but one individual tested had a CD8<sup>+</sup> IFN-γ response to at least one of the HIV-1 gene products. By contrast, CD4<sup>+</sup> T cell responses were found in fewer patients (42%). With the exception of the LTNPs, the levels of CD4<sup>+</sup> T cell responses were 0.5–1.5 log lower than the CD8<sup>+</sup> T cell responses in the same patient groups (data not shown). Overall, the response in the chronically HIV-1–infected subjects undergoing treatment was of a greater magnitude than the responses observed in the other groups, with a median total number of SFCs of 6006 cells/10<sup>6</sup> CD8<sup>+</sup>-enriched PBMC. However, the chronically HIV-1–infected subjects who were untreated had a broader response, with a median of 50% of the 15-mer pools recognized. The group of primary infected subjects had a narrower and less robust response, with only 18% of the 15-mer pools recognized and a median total number of SFCs of 866 cells/10<sup>6</sup> CD8<sup>+</sup>-enriched PBMC. Of the 10 HIV-1–seronegative subjects tested, not one demonstrated a positive response to any of the HIV-1 gene products tested. The median number of SFCs observed was 0 cells/10<sup>6</sup> CD8<sup>+</sup>-enriched PBMC, and the maximum number of SFCs observed was 4.7 cells/10<sup>6</sup> CD8<sup>+</sup>-enriched PBMC. On the basis of these results, the IFN-γ assay can easily distinguish positive CD4<sup>+</sup> and CD8<sup>+</sup> T cell responses to multiple HIV-1 epitopes over a broad range of SFC frequencies</p>
<p>Observations from these 46 experiments were analyzed to establish a statistical basis for a criterion for positivity in the IFN-γ ELISPOT assay. A formal statistical criterion (<xref ref-type="fig" rid="Fig8">figure 8</xref>) based on an exact binomial test was used to determine whether the number of SFCs in the experimental wells of these assays was significantly greater than in the control wells. A Bonferroni correction was used to control for the statistical false-positive rate observed with multiple peptide pool comparisons. Two lines are depicted in <xref ref-type="fig" rid="Fig8">figure 8</xref> to represent the different numbers of peptide pools used for the experiments in HIV-1–seronegative subjects (26 pools) versus HIV-1–seropositive subjects (14 pools). The positive criterion curve was generated by comparing the total number of SFCs in the experimental and control wells for a particular peptide pool with the proportion of the total number of SFCs attributable to the experimental wells. By this analysis, a volunteer is considered to be a positive responder if the proportion of SFCs attributable to at least one peptide pool is above the curve. For the seronegative subjects tested, all were negative responders by this criterion (<xref ref-type="fig" rid="Fig8">figure 8</xref>). Thus, this statistical criterion can easily distinguish true positive responses in HIV-1–infected persons from true negative responses in HIV-1–uninfected persons</p>
<p>A comparison was made at both the subject and peptide pool level between the results obtained using this statistical criterion and the standard criterion of twice or greater background and ⩾10 SFCs/2×10<sup>5</sup> cells after subtraction of the negative control that is described in <xref ref-type="sec" rid="S1">Materials and Methods</xref>. For 93% of the 764 peptide pools tested, the statistical criterion gave the same result as the standard criterion. Of the 51 discordant peptide pool responses, all were positive by the statistical criterion and negative by the standard criterion, which suggests a possible increase in the sensitivity of the assay when using the new statistical method. Subjects were categorized as positive responders when at least one peptide pool was positive, and, at the subject level, there was perfect agreement between the results obtained using the 2 criteria. Thus, this statistical criterion provides a sensitive alternative to the standard approach for the categorization of positive and negative responses by IFN-γ ELISPOT</p>
<p>
<bold>
<italic>IFN-γ ELISPOT response in HIV-1 vaccine recipients</italic>
</bold>Finally, vaccine-induced responses defined by the IFN-γ ELISPOT assay were examined in cryopreserved PBMC (>2 years) from HIV-1 vaccine recipients identified previously to have had significant CD8<sup>+</sup> Gag-specific lysis by chromium release assays. Responses were evaluated in AVEG protocol 022 recipients of a recombinant canarypox vector vaccine (vCP205) and rgp120/SF-2 boost [<xref ref-type="bibr" rid="ref30">30</xref>, <xref ref-type="bibr" rid="ref31">31</xref>]. One vaccine recipient was identified to have had CD8<sup>+</sup> Gag–specific lysis at an effector:target ratio of 50:1 of 20% and 50% from the 546- and 728-day time points (6 and 12 months after the final vaccine boost). Using PBMC from day 728, borderline-positive IFN-γ responses were detected in the pool containing 20-mer peptides 1–10 and the pool containing peptides 1, 11, 21, 31, and 41 (pools 1–41), both of which contained Gag peptide 1 as their only peptide in common (<xref ref-type="fig" rid="Fig9">figure 9<italic>A</italic>
</xref>). Therefore, by this assay, the response was mapped to a single HIV-1 Gag 20-mer peptide (1-MGARASVLSGGELDRWEKIR). When the experiment was repeated using CD4<sup>+</sup>- and CD8<sup>+</sup>-depleted PBMC from the earlier day 546 time point, the PBMC SFC frequency was reduced by >50% with the depletion of CD8<sup>+</sup> T cells and thus was confirmed to be a CD8<sup>+</sup> T cell response (<xref ref-type="fig" rid="Fig9">figure 9<italic>B</italic>
</xref>)</p>
<p>Cryopreserved PBMC from a different AVEG protocol 022 vaccine recipient obtained 3 months (day 455) after the final vaccination were enriched for CD8<sup>+</sup> T cells and tested for a response to pools of 10 Gag 20-mer peptides. A low-level positive response of 16 SFCs/2×10<sup>5</sup> CD8<sup>+</sup>-enriched PBMC was detected to Gag pool 21–30 (<xref ref-type="fig" rid="Fig9">figure 9<italic>C</italic>
</xref>). This response was mapped to 15-mer peptide 66 (IYKRWIILGLNKIVR) by using an overlapping matrix of pools (A–J) of 5 Gag 15-mer peptides that corresponded to the amino acid sequences contained in the positive 20-mer pool (<xref ref-type="fig" rid="Fig9">figure 9<italic>D</italic>
</xref>). All 15 amino acids contained in 15-mer peptide 66 are also contained in 20-mer peptide 27, a peptide found in the Gag 20-mer pool 21–30 shown in <xref ref-type="fig" rid="Fig9">figure 9<italic>C</italic>
</xref> and a peptide to which positive responses were confirmed in subsequent experiments (data not shown). Thus, these experiments indicated that the IFN-γ ELISPOT assay can detect, quantify, and determine the specificity of low-frequency vaccine-induced CD8<sup>+</sup> T cells in cryopreserved PBMC in HIV-1–uninfected clinical trial participants</p>
</sec>
<sec id="S3">
<title>Discussion</title>
<p>The challenge to develop an efficacious HIV-1 preventative vaccine is enormous and unprecedented. Heretofore, protective immunity imparted by vaccines that curtail infections of public health importance largely has been attributed to the induction of antibodies that are easily measured by standardized serological testing. The compelling evidence that CD8<sup>+</sup> T cells in primates may play a crucial role in vaccine efficacy against simian immunodeficiency virus infection [<xref ref-type="bibr" rid="ref32">32</xref>
<xref ref-type="bibr" rid="ref33"></xref>–<xref ref-type="bibr" rid="ref34">34</xref>] in conjunction with strong support that T cells are vital in controlling HIV-1 infection calls for vaccine strategies that can induce HIV-1–specific CD8<sup>+</sup> T cells. Yet, determining the precise T cell responses that correlate with vaccine effect and relying on these measurements in the conduct of large-scale trials that will determine vaccine efficacy and licensure is unparalleled in vaccine development. In addition, there is increasing recognition that other intracellular pathogens and tumors may require antigen-specific T cells to prevent or control disease. To this end, our investigation provides a framework for examining antigen-induced T cell responses in human trials, particularly with respect to HIV-1 vaccine development, but it is also relevant to clinical immunotherapeutic and vaccine studies designed to protect or control other infectious agents and neoplasms</p>
<p>Our findings provide evidence that the IFN-γ ELISPOT assay is a simple and sensitive approach to measure CD8<sup>+</sup> T cell responses induced by either vaccination or natural HIV-1 infection. We demonstrate that the variability inherent in the methodology is acceptably low and that the reproducibility among several operators is high. Therefore, if such an approach is instituted and standardized among various networks conducting vaccine trials, the data should be comparable across protocols. This is important in accelerating the evaluation of multiple vaccine strategies in phase 1–3 trials. Perhaps one of the weakest links in assay reproducibility lies in the interpretation of the spots formed on the plates as true IFN-γ–secreting cells. In this regard, the use of a commercial image analyzer offers an attractive alternative to visual enumeration of spots by light microscopy. However, these units still require a considerable amount of subjective operator input to accurately distinguish “true“ spots from background. This becomes particularly relevant in the analysis of vaccine-induced T cells from HIV-1–uninfected persons, which may result in smaller and less intense spots than those more easily recognized in testing HIV-1–infected patients. Thus, consistency in the detection and interpretation of SFCs will be necessary. In addition, to ensure the ongoing quality control of assay reagents and the standardization of spot-counting parameters and to aid in comparability of assay results across laboratories using samples from international sites, it will be critical to identify reagents that can be used as positive controls. A recently described pool of peptides whose sequences span 23 optimal CTL epitopes within influenza virus, cytomegalovirus, and Epstein-Barr virus will be useful to stimulate memory T cells whose IFN-γ secretion patterns can be contrasted to those induced by the vaccines. These are recognized by ∼85% of the general US population [<xref ref-type="bibr" rid="ref35">35</xref>], and studies are in progress to develop similar panels of epitopic peptides that are recognized globally. Finally, the criterion for positivity formulated here and described in more detail in forthcoming work can be successfully applied using data from multiple laboratories, and considerations are built in to account for the infrequent responder with unusually high background cytokine secretion, as well as plate-to-plate and well-to-well variability</p>
<p>Practical considerations are paramount as well in large-scale vaccine trials, and the IFN-γ ELISPOT assay satisfies many criteria for feasibility. We demonstrate excellent concordance in the ability to detect positive responses in cryopreserved versus freshly isolated CD8<sup>+</sup> T cells. Although the IFN-γ SFC frequencies observed in cryopreserved CD8<sup>+</sup> T cells are, on average, one-third lower than in fresh CD8<sup>+</sup> T cells obtained at the same venipuncture, fortunately the duration of cryopreservation does not diminish the SFC frequencies. The importance of timely, meticulous processing of blood and PBMC cryopreservation cannot be underestimated. The use of PBMC with viabilities of at least 85% on thawing will be necessary to consistently detect IFN-γ–secreting CD8<sup>+</sup> T cells, particularly if the frequencies are low. Another advantage to emphasize with the IFN-γ ELISPOT procedure is the ability to detect responses using pools of peptides at low concentrations (1–2 μg/mL). In addition, with acceptable blood volumes (∼50 mL) from one venipuncture, it is feasible over 3–4 days to screen responses by stimulating with peptide pools and to define the CD4<sup>+</sup> or CD8<sup>+</sup> epitopic responses to all HIV-1 gene products within a 8–15 aa range. Taken together, these attributes offer a tremendous advantage over the previous use of chromium release assays in the detection of MHC-restricted CD8<sup>+</sup> CTL responses</p>
<p>Our results indicate that the threshold for detection of a positive response by ELISPOT is ∼50 IFN-γ SFCs/10<sup>6</sup> PBMC (0.005%). A significant feature in improving the detection of HIV-1–specific CD8<sup>+</sup> T cells was the use of 15-mer rather than 20-mer peptides for cell activation. Our results indicate that there was an average increase in IFN-γ SFCs/10<sup>6</sup> PBMC of >50% with the use of the 15-mers, compared with that of the 20-mers. The sensitivity of the assay is within the range to detect vaccine-induced responses, as based on the representative findings in 2 vaccine recipients with concordant CTL responses (<xref ref-type="fig" rid="Fig9">figure 9</xref>) and on evidence from previous studies that memory cells that remain after antigen clearing of infection comprise <0.1% of PBMC [<xref ref-type="bibr" rid="ref36">36</xref>]. The ability to identify such low-level responses by ELISPOT provides an advantage over intracellular cytokine staining or tetramer binding by flow cytometry, because it is unlikely that the current flow-based methods can routinely distinguish 0.005% antigen-specific staining cells from negative or irrelevant control staining cells. Nevertheless, there remains the possibility that such low-frequency responses may not be relevant for vaccine protection, because the interval of time necessary for activation and proliferation of sufficient numbers of effector cells may exceed the window of time in which containment of viral replication is required for a vaccine effect. Certainly, we demonstrate that the total HIV-specific IFN-γ–secreting cells in HIV-1–infected persons, regardless of stage of infection, are present in frequencies ⩾1 log greater than those induced by the canarypox vector vaccines. This difference reflects, in part, the persistence of HIV-1 replication, which maintains the memory population, but it remains to be clarified whether this frequency, which provides only partial and certainly not durable control, is relevant to that needed for protection</p>
<p>Understanding the effectiveness of CD8<sup>+</sup> T cells in vaccine protection may require information beyond the mere measurement of the frequencies of antigen-specific cells induced at peak time intervals. The state of activation and differentiation of the memory T cells may be key to protection invoked by the secondary response, and the effector cells must be capable of homing to the site of active viral replication. To this end, the flow-based cytometric methods distinguishing cytokine secretion, as well as phenotypic characteristics, will be important to use after the ELISPOT screening. Thus, we propose an algorithm to screen PBMC for the recognition of HIV-1 epitopes using 15-mer peptides spanning the gene products contained in the vaccine regimen. After this, the epitopes recognized by vaccine-induced T cells can be identified using individual 15-mers within the peptide pool, as well as with smaller optimal 8–10-mers. Additional studies to identify the CD4<sup>+</sup> or CD8<sup>+</sup> T cell subset mediating the response, the MHC restricting molecule and the phenotypic properties (memory, differentiation, activation, and homing molecular expression) can be performed by flow cytometric studies that incorporate intracellular cytokine staining after 6 h of stimulation with the individual 15-mer or optimal peptide. In this sequence, there is no necessity to deplete T cell subsets to distinguish the particular one mediating the response, obviating larger cell requirements and the theoretical possibility that cytokine secretion by CD8<sup>+</sup> T cells will be suboptimal if the CD4<sup>+</sup> helper cells and antigen-presenting cells are removed prior to the ELISPOT assay</p>
<p>Finally, until there is a vaccine efficacy trial that substantially induces IFN-γ–secreting cells, it remains unclear whether these are the immune cells that will afford the greatest protection. Lytic function or secretion of other cytokines (interleukin-2 or tumor necrosis factor–α) or chemokines may be just as pertinent. It is envisioned that many of these functions can be routinely measured by flow cytometric analysis once the optimal epitope is identified. Moreover, no matter how high the frequency and avidity of the T cell response, if only a narrow response is elicited, this may increase the likelihood for viral escape soon after infection, which may forfeit any of the early protective effect afforded by the vaccine. In addition, induction of T cell responses that are not cross-reactive with the epitopes in the infecting strain may render the vaccine-induced responses ineffective. The ability to examine diverse epitopic responses throughout the HIV-1 genome, as well as across viral subtypes, is easily accomplished with the IFN-γ assay described here, and such studies are currently in progress</p>
<p>In conclusion, the IFN-γ ELISPOT assay that we describe provides a practical, sensitive, and validated instrument for the assessment of cellular immune responses to HIV-1 immunogens in clinical vaccine trials. It is a simple assay that can be performed using PBMC that have been cryopreserved for varying lengths of time, so that multiple time points can be examined side by side in a single experiment. The feasibility of this approach was recently demonstrated when IFN-γ ELISPOT assays were used in a large-scale phase 2 vaccine trial. The data from this protocol are the subject of a pending publication, but preliminary results indicate that the ELISPOT was able to detect low-level CD8<sup>+</sup> T cell responses, in addition to providing excellent discrimination between vaccine and placebo recipients. As we move forward to phase 3 clinical trials with more effective immunogens, the IFN-γ ELISPOT assay provides us with an opportunity to analyze vaccine responses to establish an immune correlate of protection from HIV-1 infection</p>
</sec>
</body>
<back>
<ack>
<title>Acknowledgments</title>
<p>We thank the volunteers for their participation in our study, Jean Lee for arranging for the leukapheresis of our study subjects, Ya-Lin Chiu for help with data management and statistical programming, and Alicia Cerna for assistance with manuscript preparation</p>
</ack>
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<p>Titration of cell no./well. Serial dilutions (8.0×10<sup>5</sup>–0 cells/well) of cryopreserved peripheral blood mononuclear cells (PBMC) obtained from 4 human immunodeficiency virus (HIV) type 1–infected donors—MP0471 <italic>(A)</italic> NP13 <italic>(B)</italic> NP14 <italic>(C)</italic> and NP15 <italic>(D)</italic>—were added to the wells. Cells were stimulated in triplicate with a single Gag peptide, either a 20-mer <italic>(A</italic> and <italic>B)</italic> or a 15-mer <italic>(C</italic> and <italic>D)</italic> peptide containing a class I major histocompatibility complex–restricted HIV-1 epitope identified in previous experiments or with a pool of negative control peptides. Mean total of interferon (IFN)–γ spot-forming cells (SFCs) is depicted for each cell concentration</p>
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<p>Comparison of peptide stimulation times. Cryopreserved peripheral blood mononuclear cells (PBMC) from 2 human immunodeficiency virus (HIV) type 1–infected donors, MP0471 <italic>(A)</italic> and NP15 <italic>(B)</italic>, were stimulated with Gag 15-mer peptide pools for 1 (16–20 h) or 2 days (36–40 h), and the interferon (IFN)–γ spot-forming cell (SFC) frequency is shown. Cells were stimulated with either a pool of all 122 Gag 15-mer peptides, pools of 25 Gag 15-mers, or the negative control peptide pool. Each bar represents the mean (±SD) no. of IFN-γ SFCs in duplicate wells. <italic>C</italic> Scatterplot of stimulation times for all pools tested in 4 donors. PBMC from MP0471 and NP15 were stimulated with Gag 15-mers, and PBMC from NP1 and NP13 were stimulated with Gag 20-mers. Thirty-one positive concordant experiments are shown by patient, with least-squares line <italic>(solid line)</italic> and line of equality <italic>(dashed line)</italic>
</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig002.tif"></graphic>
</fig>
<fig id="Fig3" position="float">
<label>Figure 3</label>
<caption>
<p>Stimulation with varying peptide concentrations/well. Cryopreserved peripheral blood mononuclear cells (PBMC) from 2 donors, NP15 <italic>(A</italic> and <italic>C)</italic> and NP14 <italic>(B</italic> and <italic>D)</italic> were thawed and stimulated with Gag 15-mers at varying concentrations. <italic>A</italic> and <italic>B</italic> interferon (IFN)–γ spot-forming cell (SFC) frequencies after stimulation with Gag peptides alone or in combination, with each peptide concentration titrated from 0 to 20 μg/mL. <italic>C</italic> and <italic>D</italic> Comparison of IFN-γ SFC frequencies when PBMC were stimulated with either 2 or 5 μg/mL of peptide. Both individual Gag 15-mers and pools of 15-mers were tested. Bars represent the mean (±SD) nos. of IFN-γ SFC in duplicate wells</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig003.tif"></graphic>
</fig>
<fig id="Fig4" position="float">
<label>Figure 4</label>
<caption>
<p>Peptide pool size comparisons. <italic>A</italic> and <italic>B</italic> Pools of 50, 25, 10, and 2 Gag 20-mer peptides were used to stimulate cryopreserved peripheral blood mononuclear cells (PBMC) from 2 human immunodeficiency virus (HIV) type 1–infected donors, <italic>(A)</italic> NP13 and <italic>(B)</italic> MP0471. In these experiments, only 1 recognized epitope was contained within each of the larger pools. <italic>C</italic> and <italic>D</italic> Interferon (IFN)–γ responses to pools of 10 Gag 20-mer peptides were compared with the responses observed with multiple individual peptides contained within those pools that stimulated a positive response. Bars represent the mean (±SD) no. of spot-forming cells (SFCs) in duplicate wells</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig004.tif"></graphic>
</fig>
<fig id="Fig5" position="float">
<label>Figure 5</label>
<caption>
<p>Comparison of peptide amino-acid length. Individual and pooled 20-mer peptides overlapping by 10 aa and 15-mer peptides overlapping by 11 aa were compared for stimulation of cryopreserved peripheral blood mononuclear cells (PBMC) in 2 donors, NP14 <italic>(A)</italic> and NP1 <italic>(B)</italic>. Also included are individual peptides containing class I major histocompatibility complex–restricted human immunodeficiency virus (HIV) type 1 epitopes recognized by these donors. Each bar represents the mean (±SD) no. of interferon (IFN)–γ spot-forming cells (SFCs) in duplicate wells. <italic>C</italic> Scatterplot of 15-mers vs. 20-mers in 5 donors with line of equality <italic>(dashed line)</italic> and least-squares regression line <italic>(solid line)</italic>
</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig005.tif"></graphic>
</fig>
<fig id="Fig6" position="float">
<label>Figure 6</label>
<caption>
<p>Interferon (IFN)–γ responses in fresh vs. cryopreserved peripheral blood mononuclear cells (PBMC). <italic>A</italic> and <italic>B</italic> Comparison between the no. of IFN-γ spot-forming cells (SFCs) observed after stimulation with pools of Gag 20-mer peptides using fresh vs. cryopreserved PBMC obtained from the same leukapheresis. Bars represent mean (±SD) no. of SFCs in duplicate wells. <italic>C</italic> IFN-γ responses in fresh PBMC after stimulation with pools of 10 Gag 20-mer peptides were compared with responses in cryopreserved cells thawed at multiple timepoints over a 430-day period after leukapheresis</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig006.tif"></graphic>
</fig>
<fig id="Fig7" position="float">
<label>Figure 7</label>
<caption>
<p>Interferon (IFN)–γ ELISPOT assay using peptide pools can be used to delineate epitopes across multiple human immunodeficiency virus (HIV) type 1 genes. Cryopreserved peripheral blood mononuclear cells (PBMC) from patient MP0471 were stimulated with pools of 20-mer peptides and individual 20-mer peptides, in addition to previously described HLA-matched 9-mer peptides from HIV-1 Gag <italic>(A)</italic> Pol <italic>(B)</italic> Env <italic>(C)</italic> and Nef <italic>(D)</italic>. For the individual 9-mers, only positive responses are shown. Bars represent the mean (±SD) no. of spot-forming cells (SFCs) in duplicate wells</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig007.tif"></graphic>
</fig>
<fig id="Fig8" position="float">
<label>Figure 8</label>
<caption>
<p>Positive criterion analysis in human immunodeficiency virus (HIV) type 1–seropositive and –seronegative patients. Cryopreserved peripheral blood mononuclear cells from 36 HIV-1–seropositive and 10 HIV-1–seronegative subjects were CD4<sup>+</sup> depleted and tested for responses to HIV-1 Gag, Env, Pol, and Nef by using pools of 15-mer peptides. The jagged lines are positive criterion curves with a false-positive rate of 5%. Twenty-six pools of 25 15-mer peptides were used to test the HIV-1–seronegative subjects <italic>(thick line)</italic> and 14 pools of 50 15-mer peptides were used to test the seropositive subjects <italic>(thin line)</italic>. The horizontal axis represents the total no. of spot-forming cells (SFCs) from the experimental and the control wells for a particular peptide pool (N=E+C). The vertical axis is the proportion of the total no. of SFCs attributable to the experimental wells (P=E/E+C)</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig008.tif"></graphic>
</fig>
<fig id="Fig9" position="float">
<label>Figure 9</label>
<caption>
<p>Interferon (IFN)–γ ELISPOT responses in CD8<sup>+</sup> T cells from AIDS Vaccine Evaluation Group protocol 022 human immunodeficiency virus type 1 vaccine recipients of vCP205 plus SF-2 rgp120 boost. <italic>A</italic> Cryopreserved peripheral blood mononuclear cells (PBMC) obtained 12 months after final vaccination were stimulated with a matrix of Gag 20-mer peptide pools. <italic>B</italic> Cryopreserved PBMC from 6 months after the final vaccination were CD8<sup>+</sup> depleted and tested for a response to Gag pool 1–10 as well as the individual Gag peptide 1. <italic>C</italic> Cryopreserved PBMC from another vaccine recipient obtained 3 months after final vaccination were enriched for CD8<sup>+</sup> T cells and tested for a response to pools of 10 Gag 20-mer peptides. <italic>D</italic> Positive response to Gag pool 21–30 was mapped to 15-mer peptide 66 using an overlapping 5×5 matrix of pools of 5 Gag 15-mer peptides (depicted above the graph) that corresponded to the amino acid sequences contained in the positive 20-mer pool. Bars represent mean (±SD) no. of spot-forming cells (SFCs) in duplicate wells</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-fig009.tif"></graphic>
</fig>
<fig id="tb1" position="float">
<label>Table 1</label>
<caption>
<p>Summary of cross-sectional analysis of human immunodeficiency virus (HIV) type 1–specific interferon (IFN)–γ–secreting CD8+ T cells in patients with acute and chronic HIV-1 infection</p>
</caption>
<graphic mimetype="image" xlink:href="187-2-226-tab001.tif"></graphic>
</fig>
</sec>
<fn-group>
<fn id="fn1">
<p>Presented in part: 8th Conference on Retroviruses and Opportunistic Infections, Chicago, 4–8 February 2001 (abstract 177)</p>
<p>Written informed consent was obtained from all subjects, and the human experimentation guidelines of the Fred Hutchinson Cancer Research Center institutional review board were followed</p>
<p>Financial support: National Institutes of Health (grants AI-46725, AI-48017, AI-41535, AI-47806, and AI-27757); Burroughs Wellcome Clinical Scientist Award in Translational Research (to M.J.M.)</p>
</fn>
</fn-group>
</back>
</article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: Defining T Cell Responses As Potential Correlates of Immunity</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: Defining T Cell Responses As Potential Correlates of Immunity</title></titleInfo><name type="personal"><namePart type="given">Nina D.</namePart><namePart type="family">Russell</namePart><affiliation>Program in Infectious Diseases, Clinical Research Division, and</affiliation><affiliation>Department of Medicine, University of Washington, Seattle</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Michael G.</namePart><namePart type="family">Hudgens</namePart><affiliation>Program in Biostatistics, Public Health Science Division, Fred Hutchinson Cancer Research Center, and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Richard</namePart><namePart type="family">Ha</namePart><affiliation>Program in Infectious Diseases, Clinical Research Division, and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Colin</namePart><namePart type="family">Havenar-Daughton</namePart><affiliation>Program in Infectious Diseases, Clinical Research Division, and</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="given">M. Juliana</namePart><namePart type="family">McElrath</namePart><affiliation>Program in Infectious Diseases, Clinical Research Division, and</affiliation><affiliation>Department of Medicine, University of Washington, Seattle</affiliation><affiliation>E-mail: kd@u.washington.edu</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>The University of Chicago Press</publisher><dateIssued encoding="w3cdtf">2003-01-15</dateIssued><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><abstract>There is evidence in both simian immunodeficiency virus and human immunodeficiency virus (HIV) type 1 infection that class I major histocompatibility complex–restricted CD8+ cytotoxic T lymphocytes play a pivotal role in controlling infection and, potentially, in protecting by immunization. Progress has been made in designing strategies to elicit these responses with HIV-1 vaccines, but methods to reproducibly quantify them have posed difficulties. An interferon-γ enzyme-linked immunospot assay, using peptide pools spanning the HIV-1 genes, was developed and standardized. This method is rapid (2 days), sensitive (threshold of detection, ⩾0.005%), quantitative, feasible using cryopreserved cells, and able to define epitope specificities. When this assay was applied to 36 HIV-1–seropositive and 10 HIV-1–seronegative subjects, it proved to be robust (specificity, 100%). When responses in natural infection were compared with vaccine-induced responses, vaccine recipient responses were ⩾1 log lower, which confirms the importance of using this sensitive assay as an initial screen in vaccine protocols</abstract><relatedItem type="host"><titleInfo><title>The Journal of Infectious Diseases</title></titleInfo><titleInfo type="abbreviated"><title>The Journal of Infectious Diseases</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">0022-1899</identifier><identifier type="eISSN">1537-6613</identifier><identifier type="PublisherID">jid</identifier><identifier type="PublisherID-hwp">jinfdis</identifier><part><date>2003</date><detail type="volume"><caption>vol.</caption><number>187</number></detail><detail type="issue"><caption>no.</caption><number>2</number></detail><extent 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