PD-L1 and PD-1 Are Associated with Clinical Outcomes and Alveolar Immune Cell Activation in ARDS.

The relationship between the Programmed Death-Ligand 1 (PD-L1)/Programmed Death-1 (PD-1) pathway, lung inflammation, and clinical outcomes in acute respiratory distress syndrome (ARDS) is poorly understood. We sought to determine whether PD-L1/PD-1 in the lung or blood is associated with ARDS and associated severity. We measured soluble PD-L1 (sPD-L1) in plasma and lower respiratory tract samples (ARDS1 (n = 59) and ARDS2 (n = 78)) or plasma samples alone (ARDS3 (n = 149)) collected from subjects with ARDS and tested for associations with mortality using multiple regression. We used mass cytometry to measure PD-L1/PD-1 expression and intracellular cytokine staining in cells isolated from bronchoalveolar lavage fluid (BALF) (n = 18) and blood (n = 16) from critically-ill subjects with or without ARDS enrolled from a fourth cohort. Higher plasma levels of sPD-L1 were associated with mortality in ARDS1, ARDS2, and ARDS3. In contrast, higher levels of sPD-L1 in the lung were either not associated with mortality (ARDS2) or were associated with survival (ARDS1). Alveolar PD-1POS T cells had more intracellular cytokine staining compared with PD-1NEG T cells. Subjects without ARDS had a higher ratio of PD-L1POS alveolar macrophages to PD-1POS T cells compared with subjects with ARDS. We conclude that sPD-L1 may have divergent cellular sources and/or functions in the alveolar vs. blood compartments given distinct associations with mortality. Alveolar leukocyte subsets defined by PD-L1/PD-1 cell-surface expression have distinct cytokine secretion profiles, and the relative proportions of these subsets are associated with ARDS.

Cross-reactive and mono-reactive SARS-CoV-2 CD4+ T cells in prepandemic and COVID-19 convalescent individuals.

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.

Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins.

Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

CD4+ T cells recognize unique and conserved 2009 H1N1 influenza hemagglutinin epitopes after natural infection and vaccination.

Influenza A/California/4/2009 (H1N1/09) is a recently emerged influenza virus capable of causing serious illness or death in otherwise healthy individuals. Serious outcomes were most common in young adults and children, suggesting that pre-existing heterologous immunity may influence the severity of infection. Using tetramers, we identified CD4(+) T-cell epitopes within H1N1/09 hemagglutinin (HA) that share extensive homology with seasonal influenza and epitopes that are unique to H1N1/09 HA. Ex vivo tetramer staining revealed that T cells specific for conserved epitopes were detectable within the memory compartment, whereas T cells specific for unique epitopes were naive and infrequent prior to infection or vaccination. Following infection, the frequencies of T cells specific for unique epitopes were 11-fold higher, reaching levels comparable to those of T cells specific for immunodominant epitopes. In contrast, the frequencies of T cells specific for conserved epitopes were only 2- to 3-fold higher following infection. In general, H1HA-reactive T cells exhibited a memory phenotype, expressed CXCR3 and secreted IFN-γ, indicating a predominantly Th1-polarized response. A similar Th1 response was seen in vaccinated subjects, but the expansion of T cells specific for HA epitopes was comparatively modest after vaccination. Our findings indicate that CD4(+) T cells recognize both strain-specific and conserved epitopes within the influenza HA protein and suggest that naive T cells specific for HA epitopes undergo significant expansion, whereas memory T cells specific for the conserved epitopes undergo more restrained expansion.

Mediators of monocyte chemotaxis and matrix remodeling are associated with the development of fibrosis in patients with COVID-19.

Acute respiratory distress syndrome (ARDS) has a fibroproliferative phase that may be followed by pulmonary fibrosis. This has been described in patients with COVID-19 pneumonia, but the underlying mechanisms have not been completely defined. We hypothesized that protein mediators of tissue remodeling and monocyte chemotaxis are elevated in the plasma and endotracheal aspirates of critically ill patients with COVID-19 who subsequently develop radiographic fibrosis. We enrolled COVID-19 patients admitted to the ICU who had hypoxemic respiratory failure, were hospitalized and alive for at least 10 days, and had chest imaging done during hospitalization ( n = 119). Plasma was collected within 24h of ICU admission and at 7d. In mechanically ventilated patients, endotracheal aspirates (ETA) were collected at 24h and 48-96h. Protein concentrations were measured by immunoassay. We tested for associations between protein concentrations and radiographic evidence of fibrosis using logistic regression adjusting for age, sex, and APACHE score. We identified 39 patients (33%) with features of fibrosis. Within 24h of ICU admission, plasma proteins related to tissue remodeling (MMP-9, Amphiregulin) and monocyte chemotaxis (CCL-2/MCP-1, CCL-13/MCP-4) were associated with the subsequent development of fibrosis whereas markers of inflammation (IL-6, TNF-α) were not. After 1 week, plasma MMP-9 increased in patients without fibrosis. In ETAs, only CCL-2/MCP-1 was associated with fibrosis at the later timepoint. This cohort study identifies proteins of tissue remodeling and monocyte recruitment that may identify early fibrotic remodeling following COVID-19. Measuring changes in these proteins over time may allow for early detection of fibrosis in patients with COVID-19.

Phase 2, randomized, double-blind, placebo-controlled multi-center trial of the clinical and biological effects of anti-CD14 treatment in hospitalized patients with COVID-19 pneumonia.

BACKGROUND: Severe COVID-19 is associated with innate immunopathology, and CD14, a proximal activator of innate immunity, has been suggested as a potential therapeutic target. METHODS: We conducted the COVID-19 anti-CD14 Treatment Trial (CaTT), a Phase II randomized, double-blind, placebo-controlled trial at 5 US-sites between April 12, 2021 and November 30, 2021 (NCT04391309). Hospitalized adults with COVID-19 requiring supplemental oxygen (<30 LPM) were randomized 1:1 to receive 4 daily doses of intravenous IC14, an anti-CD14 monoclonal antibody, or placebo. All participants received remdesivir. The primary outcome was time-to-resolution of illness, defined as improvement on the 8-point NIH-Ordinal COVID-19 Scale to category ≤3. Secondary endpoints were safety and exploratory endpoints were pro-inflammatory and antiviral mediators in serum on days 0-5 & 7. The trial was stopped after 40 patients were randomized and treated due to slow enrollment. FINDINGS: 40 participants were randomized and treated with IC14 (n = 20) or placebo (n = 20). The median time-to-recovery was 6 days (95% CI, 5-11) in the IC14 group vs. 5 days (95% CI, 4-10) in the Placebo group (recovery rate ratio: 0.77 (95% CI, 0.40, 1.48) (log-rank p = 0.435). The number of adverse events was similar in each group, and no IC14-attributable secondary infections occurred. In repeated-measures mixed-effects analyses, IC14 treatment increased serum sCD14 concentrations, an expected pharmacodynamic effect. Pre-planned, exploratory analyses suggested that IC14 treatment decreased the trajectories of circulating MIP-1ß and TNF-α. INTERPRETATION: IC14 treatment did not improve time-to-resolution of illness in hypoxemic patients with COVID-19 in this small trial. Results of exploratory analyses suggested IC14 had biologic effects that warrant future clinical investigation. FUNDING: National Institute of Allergy and Infectious Diseases.

Early Prognostic Indicators of Subsequent Hospitalization in Patients with Mild COVID-19.

Comprehensive data on early prognostic indicators in patients with mild COVID-19 remains sparse. In this single center case series, we characterized the initial clinical presentation in 180 patients with mild COVID-19 and defined the earliest predictors of subsequent deterioration and need for hospitalization. Three broad patient phenotypes and four symptom clusters were characterized, differentiated by varying risk for adverse outcomes. Among 14 symptoms assessed, subjective shortness of breath (SOB) most strongly associated with adverse outcomes (odds ratio (OR) 21.3, 95% confidence interval (CI): 2.7-166.4; p < 0.0001). In combination, SOB and number of comorbidities were highly predictive of subsequent hospitalization (area under the curve (AUC) 92%). Additionally, initial lymphopenia (OR 21.0, 95% CI: 2.1-210.1; p = 0.002) and male sex (OR 3.5, 95% CI: 0.9-13.0; p = 0.05) were associated with increased risk of poor outcomes. Patients with known comorbidities, especially multiple, and those presenting with subjective SOB or lymphopenia should receive close monitoring and consideration for preemptive treatment, even when presenting with mild symptoms.

The COVID-19 immune landscape is dynamically and reversibly correlated with disease severity.

BACKGROUNDDespite a rapidly growing body of literature on coronavirus disease 2019 (COVID-19), our understanding of the immune correlates of disease severity, course, and outcome remains poor.METHODSUsing mass cytometry, we assessed the immune landscape in longitudinal whole-blood specimens from 59 patients presenting with acute COVID-19 and classified based on maximal disease severity. Hospitalized patients negative for SARS-CoV-2 were used as controls.RESULTSWe found that the immune landscape in COVID-19 formed 3 dominant clusters, which correlated with disease severity. Longitudinal analysis identified a pattern of productive innate and adaptive immune responses in individuals who had a moderate disease course, whereas those with severe disease had features suggestive of a protracted and dysregulated immune response. Further, we identified coordinate immune alterations accompanying clinical improvement and decline that were also seen in patients who received IL-6 pathway blockade.CONCLUSIONThe hospitalized COVID-19 negative cohort allowed us to identify immune alterations that were shared between severe COVID-19 and other critically ill patients. Collectively, our findings indicate that selection of immune interventions should be based in part on disease presentation and early disease trajectory due to the profound differences in the immune response in those with mild to moderate disease and those with the most severe disease.FUNDINGBenaroya Family Foundation, the Leonard and Norma Klorfine Foundation, Glenn and Mary Lynn Mounger, and the National Institutes of Health.

Endomembrane targeting of human OAS1 p46 augments antiviral activity.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.

Generation of HIV-1-specific CD8+ cell responses following allogeneic hematopoietic cell transplantation.

This study tested whether donor-derived HIV-specific immune responses could be detected when viral replication was completely suppressed by the continuous administration of highly active antiretroviral therapy (HAART). A regimen of fludarabine and 200 cGy total body irradiation was followed by infusion of allogeneic donor peripheral blood cells and posttransplantation cyclosporine and mycophenolate mofetil. Viral load, lymphocyte counts, and HIV-1-specific CD8(+) cell immune responses were compared before and after hematopoietic cell transplantation (HCT). Uninterrupted administration of HAART was feasible during nonmyeloablative conditioning and after HCT. The HIV RNA remained undetectable and no HIV-associated infections were observed. CD8(+) T-cell responses targeting multiple epitopes were detected before HCT. After HCT a different pattern of donor-derived HIV-specific CTL responses emerged by day +80, presumably primed in vivo. We conclude that allogeneic HCT offers the unique ability to characterize de novo HIV-1-specific immune responses. This clinical trial was registered at ClinicalTrials.gov (identifier: NCT00112593).

Acute limb ischemia as sole initial manifestation of SARS-CoV-2 infection.

We present the case of a patient with acute upper limb ischemia as the sole initial manifestation of severe acute respiratory syndrome associated with coronavirus disease 2 infection, without concomitant respiratory symptoms or pneumonia. Viral infection presumably precipitated the patient's thromboembolic event, causing multifocal vascular occlusions. This case illustrates that coronavirus disease-19 must be considered in the differential diagnosis of patients presenting with signs or symptoms of coagulopathy, even in the absence of respiratory symptoms. We believe that an awareness of the variety of clinical presentations in patients with coronavirus disease-19, particularly extrapulmonary manifestations, is critical for optimal patient management as well as implementation of appropriate infection prevention measures.

Ontogeny of different subsets of yellow fever virus-specific circulatory CXCR5+ CD4+ T cells after yellow fever vaccination.

Monitoring the frequency of circulatory CXCR5+ (cCXCR5+) CD4+ T cells in periphery blood provides a potential biomarker to draw inferences about T follicular helper (TFH) activity within germinal center. However, cCXCR5+ T cells are highly heterogeneous in their expression of ICOS, PD1 and CD38 and the relationship between different cCXCR5 subsets as delineated by these markers remains unclear. We applied class II tetramer reagents and mass cytometry to investigate the ontogeny of different subsets of cCXCR5+ T cell following yellow fever immunization. Through unsupervised analyses of mass cytometry data, we show yellow fever virus-specific cCXCR5 T cells elicited by vaccination were initially CD38+ICOS+PD1+, but then transitioned to become CD38+ICOS-PD1+ and CD38-ICOS-PD1+ before coming to rest as a CD38-ICOS-PD1- subset. These results imply that most antigen-specific cCXCR5+ T cells, including the CD38-ICOS-PD1- CXCR5+ T cells are derived from the CXCR5+CD38+ICOS+PD1+ subset, the subset that most resembles preTFH/TFH in the germinal center.

Towards prediction of degenerate CTL epitope recognition.

The cellular immune system is characterized by flexibility with respect to epitope recognition at the level of peptide binding to HLA molecules and HLA-peptide complexes to T-cell receptors (TCRs). For epitopes recognized by cytotoxic T-lymphocytes (CTLs), amino acid substitutions at different positions have varying impact on recognition. By analyzing the frequencies of specific amino acid substitutions at each position in conjunction with HLA-peptide binding and immune-response data, we have developed new methods to predict cross-reactive recognition of epitope variants by CTLs. We derived position-specific substitution matrices (EPSSMs) through the analysis of known HLA ligands and achieved relatively accurate prediction of detrimental and tolerated amino acid substitutions. Initial analysis of amino acid substitutions in CTL epitopes with degenerate recognition showed strong position-specific preferences. This first systematic analysis further suggested that spatial constraint may be the major molecular factor determining the degenerate epitope recognition. As the data cumulates, we anticipate that eventually EPSSMs will be available for prediction of degenerate T-cell epitope recognition.

Abacavir hypersensitivity reaction in primary HIV infection.

OBJECTIVE: To evaluate risk factors associated with the abacavir hypersensitivity reaction during primary HIV infection (PHI). DESIGN: Acute HIV Infection and Early Disease Research Program protocol (AIEDRP) AI-02-001 provided antiretroviral therapy including abacavir. This retrospective analysis evaluated variables potentially associated with hypersensitivity in the cohort enrolled in AI-02-001 at the University of Washington Primary Infection Clinic. METHODS: Cases of suspected hypersensitivity were identified prospectively and reviewed retrospectively using a standardized case definition. Controls were the remaining cohort without hypersensitivity. Univariate analyses were performed by linear logistic regression. RESULTS: Nine (18%) of 50 individuals treated with abacavir developed suspected hypersensitivity. Two of nine cases and no controls were HLA-B5701 positive. When antiretroviral medications were started, cases had lower mean CD8 T-cell percentage and plasma HIV RNA value. After 2 weeks on abacavir, cases had a lower mean HIV RNA value and a trend towards greater decrease in RNA. Cases began abacavir a median of 103 days after HIV acquisition compared to 48 days for controls. There was no significant in vitro abacavir-specific lymphoproliferation or IFN-gamma production in peripheral blood mononuclear cells from individuals following the suspected hypersensitivity reaction. CONCLUSIONS: Abacavir use during PHI may be associated with increased risk of hypersensitivity. As in chronic infection, HLA-B5701 is associated with the abacavir hypersensitivity reaction in PHI. Although levels of CD8 T cells and HIV RNA may be risk factors for hypersensitivity, the observed association may be due to correlation with HLA-B5701. The interesting temporal association of hypersensitivity with initiation of abacavir later in PHI merits future investigation.

Cyclophosphamide Treatment for Acquired Factor VIII Inhibitor in a Patient with AIDS-Associated Progressive Multifocal Leukoencephalopathy.

Acquired hemophilia A (AHA) is a severe bleeding disorder with high mortality rates resulting from the development of autoantibodies to factor VIII (FVIII). Patients typically present with hemorrhages in the skin, subcutaneous tissues, and muscles, which are frequently severe. They can also develop life-threatening retroperitoneal hematomas and compartment syndromes. We describe the case of a man with a long history of AIDS complicated by progressive multifocal leukoencephalopathy (PML), who developed AHA while on stable antiretroviral therapy and then presented with new onset bleeding and hypotension. We treated our patient with incrementally increasing doses of cyclophosphamide resulting in resolution of coagulopathy. We review the medical literature for additional cases of HIV-associated AHA and discuss the challenges in the care of our patient, since the immunosuppression needed to eradicate the FVIII inhibitor had the potential to cause recrudescence of his PML.

Effect of adjunct hydroxyurea on helper T cell immunity in HIV type 1-infected patients with virological suppression.

Hydroxyurea (HU) has preferential activity in virus reservoirs not effectively targeted by current antiretroviral drug regimens, but concern for potential toxicity has precluded its routine use. The effect of adjunct HU on T cell proliferative responses and phenotypic markers was examined in a randomized study of 39 chronically HIV-1-infected patients with virological suppression on potent antiretroviral therapy. While patients in the HU arm showed modest declines in the median CD4(+) T cell counts (total, -151 cells/mm(3); naive, -91 cells/mm(3)), no significant differences were noted in the Candida, HIV-1 p24, and HIV-1 gp160 responses between the treatment arms following 24 weeks of therapy. In conclusion, although adjunct HU was associated with modest declines in the CD4(+) T cell counts, there was no significant adverse effect on helper T cell function. Further trials to address the role of HU in HIV-1 treatment may be appropriate after careful selection of HU dose and the adjunct drugs to avoid nonhematological toxicity.

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals.

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

Comprehensive epitope analysis of cross-clade Gag-specific T-cell responses in individuals with early HIV-1 infection in the US epidemic.

To elucidate the mechanisms underlying cross-clade T-cell reactivity, we evaluated responses to Gag peptides based on clades A, B, C, and M-group sequences at the epitope level by IFN-gamma ELISpot assay in 25 subjects following primary clade B infection. T-cell reactivity to CON (consensus), COT (center of tree), and ANC (most recent common ancestor) B peptides was similar and a high level of cross-reactivity was noted to clade A, C, and M-group peptides. T-cell responses to 15 of the 16 epitopes reacted with at least 1 of the 2 heterologous peptides (A or C or both) and 7 epitopes were invariant across all 3 clades. The remaining 9 epitopes were associated with a total of 11 variant sequences, and with the exception of 1, all substitutions were outside the HLA anchor positions. We conclude that Gag-specific cross-clade T-cell responses producing IFN-gamma can be detected in primary HIV-1 infection. Cross-reactivity is attributable to the recognized epitopes being either invariant across clades or differing by single amino acid substitutions outside the HLA anchor sites. Semi-conservative and non-conservative substitutions that presumably involve the T-cell receptor contact sites have significant effects on T-cell recognition. Finally, further studies are needed to determine if the detection of cross-clade IFN-gamma T-cell responses indeed translates to cross-reactive antiviral activity.