Biomarkers predictive of response to pembrolizumab in head and neck cancer.

BACKGROUND: We performed an integrated biomarker evaluation in pembrolizumab-treated patients with R/M HNSCC enrolled in KEYNOTE-012 or KEYNOTE-055. The relationship between biomarkers and HPV status was explored. METHODS: We evaluated PD-L1 (combined positive score [CPS]), TMB, T-cell-inflamed gene expression profile (Tcellinf GEP), and HPV status. Associations between biomarkers were evaluated by logistic regression (ORR) and Cox regression (PFS, OS). RESULTS: Two hundred and fifty-seven patients (KEYNOTE-012, n = 106; KEYNOTE-055, n = 151) had TMB data available; of these, 254 had PD-L1 and 236 had Tcellinf GEP. TMB, PD-L1, and Tcellinf GEP were each significantly associated with ORR (p < 0.01). Kaplan-Meier curves at prespecified cutoffs generally showed PFS and OS separation in the anticipated direction for these biomarkers, except for OS and TMB. TMB did not correlate with PD-L1 or Tcellinf GEP (Spearman ρ = -0.03 and ρ = -0.13, respectively); PD-L1 and Tcellinf GEP were moderately correlated (Spearman ρ = 0.47). In multivariate models, TMB, PD-L1, and Tcellinf GEP were each independently predictive for ORR (p < 0.001). ORR was higher in patients with high versus low levels of biomarkers when dichotomized using prespecified cutoffs; patients with higher versus lower levels of TMB and PD-L1 or TMB and Tcellinf GEP had the highest ORRs. Within HPV subgroups, higher versus lower distributions of biomarkers (PD-L1, TMB, and Tcellinf GEP) were associated with response. HPV detection by p16-immunohistochemistry and WES showed good concordance (81%); results were generally similar by HPV status, regardless of the detection method. CONCLUSIONS: TMB and the inflammatory biomarkers PD-L1 and Tcellinf GEP, assessed alone or together, may be useful for characterizing clinical response to pembrolizumab in R/M HNSCC.

Influence of tumor mutational burden, inflammatory gene expression profile, and PD-L1 expression on response to pembrolizumab in head and neck squamous cell carcinoma.

BACKGROUND: To characterize genomic determinants of response to pembrolizumab in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) in the KEYNOTE-012 study. METHODS: Associations between biomarkers (tumor mutational burden (TMB), neoantigen load (NL), 18-gene T-cell-inflamed gene expression profile (TcellinfGEP), and PD-L1 combined positive score (CPS)) and clinical outcomes with pembrolizumab were assessed in patients with R/M HNSCC (n=192). Tumor human papillomavirus (HPV) status was also evaluated with the use of p16 immunohistochemistry and whole exome sequencing (WES; HPV+, mapping >20 HPV reads) in pretreatment tumor samples (n=106). RESULTS: TMB, clonality-weighted TMB, and TcellinfGEP were significantly associated with objective response (p=0.0276, p=0.0201, and p=0.006, respectively), and a positive trend was observed between NL and PD-L1 CPS and clinical response (p=0.0550 and p=0.0682, respectively). No correlation was observed between TMB and TcellinfGEP (Spearman ρ=-0.026) or TMB and PD-L1 (Spearman ρ=0.009); a correlation was observed between TcellinfGEP and PD-L1 (Spearman ρ=0.511). HPV status by WES and p16 immunohistochemistry showed concordance (84% Ò¡=0.573) among patients whose HPV results were available using both methods. CONCLUSIONS: TMB and inflammatory biomarkers (TcellinfGEP and PD-L1) may represent distinct and complementary biomarkers predicting response to anti-programmed death 1 therapies in HNSCC; further study of these relationships in randomized clinical trials is needed. TRIAL REGISTRATION NUMBER: NCT01848834.

Association of BRAF V600E/K Mutation Status and Prior BRAF/MEK Inhibition With Pembrolizumab Outcomes in Advanced Melanoma: Pooled Analysis of 3 Clinical Trials.

Importance: The optimal sequencing of immune checkpoint inhibitors and targeted therapy for BRAF V600E/K-mutant melanoma is not well established. Objective: To assess the association of BRAF wild-type (WT) or BRAF V600E/K-mutant status and BRAF inhibitor (BRAFi) with or without MEK inhibitor (MEKi) therapy with response to pembrolizumab. Design, Setting, and Participants: This study is a post hoc subgroup analysis of pooled data from 3 multinational, multisite studies: KEYNOTE-001 (data cutoff September 1, 2017), KEYNOTE-002 (data cutoff May 30, 2018), and KEYNOTE-006 (data cutoff December 4, 2017). Patients included in this analysis were adults with advanced melanoma and known BRAF V600E/K tumor status who had received pembrolizumab. Interventions: Patients received pembrolizumab in dosages of 2 mg/kg every 3 weeks, 10 mg/kg every 2 weeks, or 10 mg/kg every 3 weeks. Main Outcomes and Measures: End points were objective response rate (ORR) and progression-free survival (PFS) assessed by Response Evaluation Criteria in Solid Tumors, version 1.1, and overall survival (OS). Objective response rates, 4-year PFS, and OS rates were compared in the following patient subgroups: BRAF WT vs BRAF V600E/K-mutant melanoma and BRAF V600E/K-mutant melanoma with vs without previous treatment with BRAFi with or without MEKi therapy. Results: The overall study population (N = 1558) included 944 men (60.6%) and 614 women (39.4%). The mean (SD) age was 60.0 years (14.0). The ORR was 38.3% (596/1558), 4-year PFS rate was 22.0%, and 4-year OS rate was 36.9%. For patients with BRAF WT (n = 1124) and BRAF V600E/K-mutant melanoma (n = 434), ORR was 39.8% (n = 447) and 34.3% (n = 149), 4-year PFS rate was 22.9% and 19.8%, and 4-year OS rate was 37.5% and 35.1%, respectively. Patients with BRAF V600E/K-mutant melanoma who had (n = 271) vs had not (n = 163) previously received BRAFi with or without MEKi therapy had baseline characteristics with worse prognosis; ORR was 28.4% (n = 77) and 44.2% (n = 72), 4-year PFS rate was 15.2% and 27.8%, and 4-year OS rate was 26.9% and 49.3%, respectively. Conclusions and Relevance: Results of this subgroup analysis support the use of pembrolizumab for treatment of advanced melanoma regardless of BRAF V600E/K mutation status or receipt of prior BRAFi with or without MEKi therapy.

The pharmacogenetics of OATP1B1 variants and their impact on the pharmacokinetics and efficacy of elbasvir/grazoprevir.

Aim: To evaluate the effect of SLCO1B1 genetic variants on grazoprevir pharmacokinetics and efficacy. Methods: A retrospective analysis of 1578 hepatitis C virus-infected participants from ten Phase II/III clinical trials. Results: Relative to noncarriers of the risk allele, geometric mean ratios (95% CI) of grazoprevir area under curve (AUC)0-24 were: rs4149056 (risk allele C), one copy, 1.13 (1.06-1.21), two copies, 1.43 (1.16-1.77); and rs11045819 (risk allele A), one copy, 0.93 (0.87-1.00); two copies, 0.78 (0.61-1.00). The rs2306283 variant was not associated with grazoprevir exposure. None of the SLCO1B1 variants were associated with sustained virologic response. Conclusion: Genetic variants in SLCO1B1 were associated with modest changes in grazoprevir pharmacokinetics, but not with meaningful differences in efficacy.

A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks. It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment. We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma. We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and that this response would correlate with disease-free survival. We identified a rapid and potent anti-tumor response, with 8 of 27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease free. These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks, with reinvigoration in the blood observed as early as 1 week. Transcriptional analysis demonstrated a pretreatment immune signature (neoadjuvant response signature) that was associated with clinical benefit. In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution. Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma. Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.

Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) checkpoint blockade immunotherapy elicits durable antitumor effects in multiple cancers, yet not all patients respond. We report the evaluation of >300 patient samples across 22 tumor types from four KEYNOTE clinical trials. Tumor mutational burden (TMB) and a T cell-inflamed gene expression profile (GEP) exhibited joint predictive utility in identifying responders and nonresponders to the PD-1 antibody pembrolizumab. TMB and GEP were independently predictive of response and demonstrated low correlation, suggesting that they capture distinct features of neoantigenicity and T cell activation. Analysis of The Cancer Genome Atlas database showed TMB and GEP to have a low correlation, and analysis by joint stratification revealed biomarker-defined patterns of targetable-resistance biology. These biomarkers may have utility in clinical trial design by guiding rational selection of anti-PD-1 monotherapy and combination immunotherapy regimens.

Pembrolizumab in patients with thymic carcinoma: a single-arm, single-centre, phase 2 study.

BACKGROUND: Treatment options are limited for patients with thymic carcinoma. These aggressive tumours are not typically associated with paraneoplastic autoimmune disorders, and strong PD-L1 expression has been reported in thymic epithelial tumours. We aimed to assess the activity of pembrolizumab, a monoclonal antibody that targets PD-1, in patients with advanced thymic carcinoma. METHODS: We completed a single-arm phase 2 study of pembrolizumab in patients with recurrent thymic carcinoma who had progressed after at least one line of chemotherapy. This was a single-centre study performed at Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA. Key inclusion criteria were an Eastern Cooperative Oncology Group performance status of 0-2, no history of autoimmune disease or other malignancy requiring treatment or laboratory abnormality, and adequate organ function. Patients received 200 mg of pembrolizumab every 3 weeks for up to 2 years. The primary objective of the study was the proportion of patients who had achieved a response assessed with Response Evaluation Criteria in Solid Tumors version 1.1. Analysis was per protocol, in all eligible patients. The study is registered with ClinicalTrials.gov, number NCT02364076, and is closed to accrual; we report the final analysis. FINDINGS: 41 patients were enrolled from March 12, 2015, to Dec 16, 2016, of whom 40 were eligible and evaluable and one was excluded because of elevated liver enzymes at screening. The median follow-up was 20 months (IQR 14-26). The proportion of patients who achieved a response was 22·5% (95% CI 10·8-38·5); one (3%) patient achieved a complete response, eight (20%) patients achieved partial responses, and 21 (53%) patients achieved stable disease. The most common grade 3 or 4 adverse events were increased aspartate aminotransferase and alanine aminotransferase (five [13%] patients each). Six (15%) patients developed severe autoimmune toxicity, including two (5%) patients with myocarditis. There were 17 deaths at the time of analysis, but no deaths due to toxicity. INTERPRETATION: Pembrolizumab is a promising treatment option in patients with thymic carcinoma. Because severe autoimmune disorders are more frequent in thymic carcinoma than in other tumour types, careful monitoring is essential. FUNDING: Merck & Co.

Tolerability of two sequential electroporation treatments using MedPulser DNA delivery system (DDS) in healthy adults.

Immunotherapy against infectious agents and malignant tumors requires efficient priming of effector cells through direct expression and/or efficient cross-presentation of antigens by antigen-presenting cells. Electroporation is a new procedure aimed at transiently increasing cell membrane permeability and direct delivery of antigen or antigen-encoding nucleic acids inside targeted cells. We evaluated the tolerability including compliance with repeated electroporation treatments using MedPulser DDS in 24 healthy adults. Pain severity was evaluated at time of electroporation treatment, and at 1, 5, 10, and 20 minutes, and 24 hours thereafter, using two clinically validated questionnaires: McGill Pain Questionnaire (MPQ) (Present Pain Intensity) and Brief Pain Inventory (BPI). Electroporation treatments were generally well tolerated. Twenty-two out of 24 subjects returned for the second electroporation treatment 14 days after first treatment. Only two subjects reported a treatment-related systemic adverse experience following either electroporation application. For both pain assessment tools, maximum pain and/or discomfort were mostly reported immediately (within 5 minutes) after electroporation; Furthermore, no difference was observed when comparing peak-pain scores after first and second electroporation treatments. This study supports the clinical application of MedPulser DDS for the improvement of antigen-induced immune responses for prophylactic or therapeutic vaccines, especially in gene-based therapies for cancer.

Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial.

BACKGROUND: Observational data and non-human primate challenge studies suggest that cell-mediated immune responses might provide control of HIV replication. The Step Study directly assessed the efficacy of a cell-mediated immunity vaccine to protect against HIV-1 infection or change in early plasma HIV-1 levels. METHODS: We undertook a double-blind, phase II, test-of-concept study at 34 sites in North America, the Caribbean, South America, and Australia. We randomly assigned 3000 HIV-1-seronegative participants by computer-generated assignments to receive three injections of MRKAd5 HIV-1 gag/pol/nef vaccine (n=1494) or placebo (n=1506). Randomisation was prestratified by sex, adenovirus type 5 (Ad5) antibody titre at baseline, and study site. Primary objective was a reduction in HIV-1 acquisition rates (tested every 6 months) or a decrease in HIV-1 viral-load setpoint (early plasma HIV-1 RNA measured 3 months after HIV-1 diagnosis). Analyses were per protocol and modified intention to treat. The study was stopped early because it unexpectedly met the prespecified futility boundaries at the first interim analysis. This study is registered with ClinicalTrials.gov, number NCT00095576. FINDINGS: In a prespecified interim analysis in participants with baseline Ad5 antibody titre 200 or less, 24 (3%) of 741 vaccine recipients became HIV-1 infected versus 21 (3%) of 762 placebo recipients (hazard ratio [HR] 1.2 [95% CI 0.6-2.2]). All but one infection occurred in men. The corresponding geometric mean plasma HIV-1 RNA was comparable in infected male vaccine and placebo recipients (4.61 vs 4.41 log(10) copies per mL, one tailed p value for potential benefit 0.66). The vaccine elicited interferon-gamma ELISPOT responses in 75% (267) of the 25% random sample of all vaccine recipients (including both low and high Ad5 antibody titres) on whose specimens this testing was done (n=354). In exploratory analyses of all study volunteers, irrespective of baseline Ad5 antibody titre, the HR of HIV-1 infection between vaccine and placebo recipients was higher in Ad5 seropositive men (HR 2.3 [95% CI 1.2-4.3]) and uncircumcised men (3.8 [1.5-9.3]), but was not increased in Ad5 seronegative (1.0 [0.5-1.9]) or circumcised (1.0 [0.6-1.7]) men. INTERPRETATION: This cell-mediated immunity vaccine did not prevent HIV-1 infection or reduce early viral level. Mechanisms for insufficient efficacy of the vaccine and the increased HIV-1 infection rates in subgroups of vaccine recipients are being explored.

HIV seroconversion without infection after receipt of adenovirus-vectored HIV type 1 vaccine.

BACKGROUND: We analyzed human immunodeficiency virus (HIV) seroresponses from 3 phase I HIV-1 vaccine trials to assess the frequency of vaccine-induced seroconversion. METHODS: HIV-1 and HIV-2 enzyme-linked immunosorbent assay (ELISA) was performed during trials of adenovirus type 5 (Ad5)-vectored clade B HIV-1 monovalent gag and trivalent gag/pol/nef vaccines given to HIV-seronegative adults. Doses were administered at day 1, week 4, and week 26. Results were analyzed by vaccine formulation and dose and were stratified by baseline Ad5 titer. ELISA-positive samples were reflexively tested by Western blotting. RESULTS: Overall, 165 (41%) of 406 evaluable vaccine recipients had positive ELISA results but negative PCR results by week 78. Seroconversion rates were directly related to vaccine dose, were inversely related to baseline Ad5 titer, and were unaffected by vaccine valency. One hundred (89%) of 113 evaluable patients with low baseline Ad5 antibody titers (or=1 dose of vaccine with >or=1 x 10(10) gag-containing Ad5 particles per dose experienced seroconversion. Of 163 vaccine recipients who had positive ELISA results and available Western blot results, 150 (92%) had indeterminate results of Western blot, typically involving bands at p24, p40, and/or p55. Thirteen uninfected patients (8%) had equivocally positive Western blot results, usually because of an additional weak glycoprotein 41 band. Env-specific enzyme immunoassay results were falsely positive for 2 uninfected vaccine recipients. CONCLUSIONS: Positive ELISA results were similarly common for monovalent and trivalent vaccine recipients. Vaccine dose and baseline Ad5 immunity were major determinants of vaccine-induced seroconversion rates. Corresponding Western blots characteristically showed bands directed only at Gag proteins, which helped to distinguish HIV-uninfected vaccine recipients who experienced seroconversion from true HIV-infected patients. If available, an enzyme immunoassay exclusively targeting proteins not expressed by the vaccine should be the screening test of first choice for vaccine recipients.

Safety and immunogenicity of a replication-incompetent adenovirus type 5 HIV-1 clade B gag/pol/nef vaccine in healthy adults.

BACKGROUND: The safety and immunogenicity of the MRK adenovirus type 5 human immunodeficiency virus type 1 clade B gag/pol/nef vaccine, a replication-incompetent adenovirus type 5-vectored vaccine designed to elicit cell-mediated immunity against conserved human immunodeficiency virus proteins, was assessed in a phase 1 trial. METHODS: Healthy adults not infected with human immunodeficiency virus were enrolled in a multicenter, dose-escalating, blind, placebo-controlled study to evaluate a 3-dose homologous prime-boost regimen of the trivalent MRK adenovirus type 5 human immunodeficiency virus type 1 vaccine containing from 3 x 10(6) to 1 x 10(11) viral particles per 1-mL dose administered on day 1, during week 4 and during week 26. Adverse events were recorded for 29 days after each intradeltoid injection. The primary immunogenicity end point was the proportion of study participants with a positive unfractionated Gag-, Pol-, or Nef-specific interferon-gamma enzyme-linked immunosorbent spot response measured 4 weeks after administration of the last dose. RESULTS: Of 259 randomized individuals, 257 (99%) received > or = 1 dose of vaccine or placebo and were included in the safety analyses. Enzyme-linked immunosorbent spot results were available for 217 study participants (84%) at week 30. No serious vaccine-related adverse events occurred. No study participant discontinued participation because of vaccine-related adverse events. The frequency of injection-site reactions was dose dependent. Vaccine doses of > or = 3 x 10(9) viral particles elicited positive enzyme-linked immunosorbent spot responses to > or = 1 vaccine component in > 60% of recipients. High baseline antibody titers against adenovirus type 5 diminished enzyme-linked immunosorbent spot responses at all doses except the 3 x 10(10) viral particle dose. CONCLUSIONS: The vaccine was generally well tolerated and induced cell-mediated immune responses against human immunodeficiency virus type 1 peptides in most healthy adults. Despite these findings, vaccination in a proof-of-concept trial with use of this vaccine was discontinued because of lack of efficacy.

Enhanced rates and magnitude of immune responses detected against an HIV vaccine: effect of using an optimized process for isolating PBMC.

Quantitative analysis of cell-mediated immune responses induced by candidate HIV vaccines requires robust procedures for collecting and processing human peripheral mononuclear blood cells (PBMCs). We evaluated several parameters in order to optimize a sample handling process that would be suitable for a multicenter clinical trial. Among the findings, systematic increases in the magnitude of IFN-gamma ELISpot responses were observed when the time from blood collection to PBMC freezing was reduced to <12 h. By implementing these improvements within an ongoing clinical trial, the estimated immunologic response rates to an adenovirus- based HIV vaccine increased by more than 20 percentage points to approximately 80% of the vaccine recipients against any of the vaccine antigens and the average levels of T cell response improved more than 3-fold. These studies establish the importance of optimal conditions for PBMC collection and handling to the success of a clinical development program.

Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzyme-linked immunospot assay.

An effective vaccine for HIV is likely to require induction of T-cell-mediated immune responses, and the interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) assay has become the most commonly used assay for measuring these responses in vaccine trials. We optimized and validated the HIV ELISPOT assay using an empirical method to establish positivity criteria that results in a < or =1% false-positive rate. Using this assay, we detected a broad range of HIV-specific ELISPOT responses to peptide pools of overlapping 20mers, 15mers, or 9mers in study volunteers receiving DNA- or adenovirus vector-based HIV vaccines and in HIV-seropositive donors. We found that 15mers generally had higher response magnitudes than 20mers and lower false-positive rates than 9mers. These studies show that our validated ELISPOT assay using 15mer peptide pools and the positivity criteria of > or =55 spots per 10(6) cells and > or =4-fold over mock (negative control) is a sensitive and specific assay for the detection of HIV vaccine-induced cell-mediated immunity.

Analysis of antiretroviral immunotherapy trials with potentially non-normal and incomplete longitudinal data.

For many HIV-infected patients, use of antiretroviral therapy (ART) results in a sustained suppression of plasma viral load to undetectable levels. However, due to lack of antigenic stimulation, this may also result in a gradual loss of cell-mediated immune (CMI) responses that help control HIV infection. In concept, augmenting ART with periodic administrations of an HIV vaccine that boosts CMI responses could enhance control of viral replication. Researchers are designing 'antiretroviral immunotherapy' (ARI) trials to test this hypothesis. In a typical ARI trial, HIV-infected patients with sustained viral suppression will receive inoculations of an experimental HIV vaccine or a placebo, and subsequently stop taking their antiretroviral drugs. The goal is to assess whether plasma viral loads during the ART interruption phase are generally lower in the vaccine group. Assessment of a vaccine effect will be challenging if some subjects resume ART or drop out before the end of the treatment interruption phase. To tackle this 'missing' data problem and potential non-normality of the viral loads in ARI trials, we propose a two-step approach: multiple imputation of the missing values followed by use of the Wei-Lachin method with Wilcoxon scores. We use a numerical example and extensive simulations to illustrate the robustness and power advantages of our proposed method compared with other methods for incomplete longitudinal data, including REML, weighted GEE, last observation carried forward, and 'worst-rank' methods. Our proposed method is general enough for the robust analysis of longitudinal data in other therapeutic areas as well.

A comparison of standard immunogenicity assays for monitoring HIV type 1 gag-specific T cell responses in Ad5 HIV Type 1 gag vaccinated human subjects.

Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.

Cross-clade reactivity of HIV-1-specific T-cell responses in HIV-1-infected individuals from Botswana and Cameroon.

An effective HIV type 1 (HIV-1) vaccine will likely require elicitation of broadly reactive cell-mediated immune (CMI) responses against divergent HIV-1 clades. We compared anti-HIV-1 T-cell immune responses among 363 unvaccinated adults infected with diverse HIV-1 clades. Response rates to clade B Gag and/or clade B Nef in Botswana (95%) and Cameroon (98%) were similar when compared with those in countries previously studied, including Brazil (92%), Thailand (96%), South Africa (96%), Malawi (100%), and the United States (100%). Substantial cross-clade cell-mediated immune responses in Botswana and Cameroon confirm previous findings in a larger, more genetically diverse collection of HIV-1 samples.