Discovery of a monoclonal, high-affinity CD8+ T-cell clone following natural hepatitis C virus infection.

CD8+ T cells recognizing their cognate antigen are typically recruited as a polyclonal population consisting of multiple clonotypes with varying T-cell receptor (TCR) affinity to the target peptide-major histocompatibility complex (pMHC) complex. Advances in single-cell sequencing have increased accessibility toward identifying TCRs with matched antigens. Here we present the discovery of a monoclonal CD8+ T-cell population with specificity for a hepatitis C virus (HCV)-derived human leukocyte antigen (HLA) class I epitope (HLA-B*07:02 GPRLGVRAT) which was isolated directly ex vivo from an individual with an episode of acutely resolved HCV infection. This population was absent before infection and underwent expansion and stable maintenance for at least 2 years after infection as measured by HLA-multimer staining. Furthermore, the monoclonal clonotype was characterized by an unusually long dissociation time (half-life = 794 s and koff = 5.73 × 10-4) for its target antigen when compared with previously published results. A comparison with related populations of HCV-specific populations derived from the same individual and a second individual suggested that high-affinity TCR-pMHC interactions may be inherent to epitope identity and shape the phenotype of responses which has implications for rational TCR selection and design in the age of personalized immunotherapies.

B-cell characteristics define HCV reinfection outcome.

BACKGROUND & AIMS: In individuals highly exposed to HCV, reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. Strong evidence of antigen imprinting was found, and the B-cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single-cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Identification of human progenitors of exhausted CD8+ T cells associated with elevated IFN-γ response in early phase of viral infection.

T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of individuals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections.

Longitudinal Characterization of Phagocytic and Neutralization Functions of Anti-Spike Antibodies in Plasma of Patients after Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Phagocytic responses by effector cells to opsonized viruses have been recognized to play a key role in antiviral immunity. Limited data on coronavirus disease 2019 suggest that the role of Ab-dependent and -independent phagocytosis may contribute to the observed immunological and inflammatory responses; however, their development, duration, and role remain to be fully elucidated. In this study of 62 acute and convalescent patients, we found that patients with acute coronavirus disease 2019 can mount a phagocytic response to autologous plasma-opsonized Spike protein-coated microbeads as early as 10 d after symptom onset, while heat inactivation of this plasma caused 77-95% abrogation of the phagocytic response and preblocking of Fc receptors showed variable 18-60% inhibition. In convalescent patients, phagocytic response significantly correlated with anti-Spike IgG titers and older patients, while patients with severe disease had significantly higher phagocytosis and neutralization functions compared with patients with asymptomatic, mild, or moderate disease. A longitudinal subset of the convalescent patients over 12 mo showed an increase in plasma Ab affinity toward Spike Ag and preservation of phagocytic and neutralization functions, despite a decline in the anti-Spike IgG titers by >90%. Our data suggest that early phagocytosis is primarily driven by heat-liable components of the plasma, such as activated complements, while anti-Spike IgG titers account for the majority of observed phagocytosis at convalescence. Longitudinally, a significant increase in the affinity of the anti-Spike Abs was observed that correlated with the maintenance of both the phagocytic and neutralization functions, suggesting an improvement in the quality of the Abs.

Maintenance of broad neutralizing antibodies and memory B cells 1 year post-infection is predicted by SARS-CoV-2-specific CD4+ T cell responses.

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity is critical for predicting protection against reinfection. In an age- and gender-matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralizing antibody breadth against the majority of the variants of concern, including the Delta variant. Modeling these immune responses against vaccine efficacy data indicate a 45%-76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralization breadth and RBD-specific MBC levels at 12 months post-infection.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Long-term persistence of RBD+ memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection.

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.

A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers.

Hepatitis C (HCV) is a rapidly mutating RNA virus, with a strong propensity to cause chronic infection and progressive liver disease. Recent evidence has shown that early appearance of neutralizing antibodies in primary infection is associated with clearance. Little is known about the characteristics of HCV-specific B cells and their correlation with outcomes in primary infection, as there is a lack of sensitive tools for HCV-specific B cells which are present at very low frequency. We describe the development and optimisation of tetramer staining for flow cytometric detection of HCV-specific B cells using a cocktail of two recombinant HCV Envelope-2 (rE2) glycoproteins (from genotype 1a and 3a; Gt1a and Gt3a) and streptavidin dyes. The optimal weight to weight (w/w) ratio of streptavidin-phycoerythrin (PE) and rE2 proteins were determined for sensitive detection using HCV E2-specific hybridoma cell lines and peripheral blood mononuclear cells (PBMC) from HCV-infected individuals. In a cross-sectional set of PBMC samples collected from 33 subjects with either chronic infection or previous clearance, HCV E2-specific B cells (CD19+CD20+CD10-IgD-tetramer+) were detected in 29 subjects (87.8%), with a mean frequency of 0.45% (0.012-2.20%). To validate the specificity of tetramer staining, 367 HCV E2-specific B cells were single cell sorted from 9 PBMC samples before monoclonal antibodies (mAbs) were synthesised, with 87.5% being reactive to E2 via ELISA. Of these mAbs, 284 and 246 clones were reactive to either Gt1a or Gt3a E2 proteins, respectively. This is a sensitive and robust method for future studies investigating B cell responses against the HCV Envelope protein.

Immune dysregulation in patients with carpal tunnel syndrome.

Peripheral immunity plays a key role in maintaining homeostasis and conferring crucial neuroprotective effects on the injured nervous system, while at the same time may contribute to increased vulnerability to neuropathic pain. Little is known about the reciprocal relationship between entrapment neuropathy and peripheral immunity. This study investigated immune profile in patients with carpal tunnel syndrome (CTS), the most prevalent entrapment neuropathy. All patients exhibited neurophysiological abnormalities in the median nerve, with the majority reporting neuropathic pain symptoms. We found a significant increase in serum CCL5, CXCL8, CXCL10 and VEGF, and in CD4+ central and effector memory T cells in CTS patients, as compared to healthy controls. CCL5 and VEGF were identified as having the highest power to discriminate between patients and controls. Interestingly, and contrary to the prevailing view of CCL5 as a pro-nociceptive factor, the level of circulating CCL5 was inversely correlated with neuropathic pain intensity and median nerve motor latency. In contrast, the level of central memory T cells was positively associated with abnormal neurophysiological findings. These results suggest that entrapment neuropathy is associated with adaptive changes in the homeostasis of memory T cells and an increase in systemic inflammatory modulating cytokines/chemokines, which potentially regulate neuropathic symptoms.

HIV infection is associated with higher levels of monocyte chemoattractant protein-1 and eotaxin among people with recent hepatitis C virus infection.

BACKGROUND: Human immunodeficiency virus (HIV) infection leads to more rapid progression of hepatitis C virus (HCV)-related liver fibrosis, which could be linked to differences in the severity of liver inflammation among HIV/HCV co-infected individuals compared to HCV mono-infected individuals. This study assessed the association of HIV co-infection with pro-inflammatory and pro-fibrogenic cytokines and chemokines during recent HCV infection. METHODS: Participants from the ATAHC study, a prospective cohort of recent HCV infection, with detectable HCV RNA at the time of acute HCV detection were included. Concentrations of 27 plasma cytokines and chemokines were measured by multiplex immunoassays and compared between those with, and without, HIV co-infection. RESULTS: Out of 117 individuals with recent HCV infection included in analysis, 73 had HCV mono-infection and 44 had HIV/HCV co-infection. Individuals with HIV/HCV co-infection had significantly higher mean levels of eotaxin (1.79 vs. 1.62 log pg/mL; P < 0.001), monocyte chemotactic protein 1 (MCP-1; 2.10 vs. 1.98 log pg/mL; P < 0.001), and interferon-gamma inducible protein-10 (IP-10; 3.11 vs. 2.98 log pg/mL; P = 0.013). Linear regression analyses adjusting for age, alanine transaminase (ALT), HCV RNA levels, and assay run, higher eotaxin levels were independently associated with HIV/HCV co-infection (adjusted ß: 0.12; 95%CI: 0.01, 0.24; P = 0.039). Higher MCP-1 levels were also independently associated with HIV/HCV co-infection in adjusted analysis (adjusted ß: 0.11; 95%CI: 0.03, 0.18; P = 0.009). CONCLUSIONS: During recent HCV, those with HIV/HCV co-infection had a stronger pro-fibrogenic mediator profile compared to those with HCV mono-infection. These findings may provide a potential explanation for accelerated liver fibrosis in HIV/HCV co-infection. TRIAL REGISTRATION: Australian Trial in Acute Hepatitis C (ATAHC) study was registered with ClinicalTrials.gov registry on September 11, 2005. NCT00192569 .

Alanine aminotransferase, HCV RNA levels and pro-inflammatory and pro-fibrogenic cytokines/chemokines during acute hepatitis C virus infection.

BACKGROUND: This study assessed the association of alanine-aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels with pro-inflammatory and pro-fibrogenic cytokines and chemokines during acute HCV infection to provide further insight into the potential HCV immunopathogenesis. METHODS: Participants in the ATAHC study, a prospective study of recent HCV infection, with detectable HCV RNA at the time of HCV detection were included. Plasma levels of 27 cytokines and chemokines were measured and their correlation with ALT and HCV RNA levels were assessed. Log10 transformed cytokines and ALT values were used in the analysis. RESULTS: Among 117 individuals, the plasma levels of interferon-gamma inducible protein-10 (IP-10) and macrophage inflammatory protein-1beta (MIP-1ß) were positively correlated with ALT levels (IP-10: r = 0.42, P < 0.001; MIP-1ß: r = 0.29, P = 0.001) and HCV RNA levels (IP-10: rs = 0.44, P < 0.001; MIP-1ß: rs = 0.43, P < 0.001). Using linear regression, after adjusting for sex, age, infection duration, symptomatic infection, HIV co-infection, interferon-lambda rs12979860 genotype, HCV genotype, and assay run, higher ALT levels (ß = 0.20; 95 % CI: 0.07, 0.32; P = 0.002) and HCV RNA levels >400,000 IU/mL (vs. <8,500 IU/mL; ß = 0.16; 95 % CI: 0.03, 0.28; P = 0.014) were independently associated with higher IP-10 levels. HCV RNA levels >400,000 IU/mL (vs. <8,500 IU/mL; ß = 0.16; 95 % CI: 0.01, 0.31; P = 0.036) were associated with higher MIP-1ß levels. CONCLUSIONS: During acute HCV infection, high ALT and HCV RNA levels were associated with increased IP-10 levels, while high HCV RNA levels were also associated with increased MIP-1ß levels. These data suggest that IP-10 and MIP-1ß may have a role in HCV immuno-pathogenesis starting early in acute HCV infection.

Herpes simplex virus type 2-infected dendritic cells produce TNF-α, which enhances CCR5 expression and stimulates HIV production from adjacent infected cells.

Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because <1% of cells were dually infected, this was a field effect. Virus-stripped supernatants from HSV-2-infected MDDCs were shown to enhance HIV-1 infection, as measured by HIV-1-DNA and p24 Ag in MDDCs. Furthermore these supernatants markedly stimulated CCR5 expression on both MDDCs and LCs. TNF-α was by far the most prominent cytokine in the supernatant and also within HSV-2-infected MDDCs. HSV-2 infection of isolated immature epidermal LCs, but not keratinocytes, also produced TNF-α (and low levels of IFN-ß). Neutralizing Ab to TNF-α and its receptor, TNF-R1, on MDDCs markedly inhibited the CCR5-stimulating effect of the supernatant. Therefore, these results suggest that HSV-2 infection of DCs in the skin during primary or recurrent genital herpes may enhance HIV-1 infection of adjacent DCs, thus contributing to acquisition of HIV-1 through herpetic lesions.

A novel assay for detection of hepatitis C virus-specific effector CD4(+) T cells via co-expression of CD25 and CD134.

Hepatitis C virus (HCV)-specific CD4(+) effector T cell responses are likely to play a key role in the immunopathogenesis of HCV infection by promoting viral clearance and maintaining control of viraemia. As the precursor frequency of HCV-specific CD4(+) T cells in peripheral blood is low, favoured assay systems such as intracellular cytokine (ICC) or tetramer staining have limited utility for ex vivo analyses. Accordingly, the traditional lymphocyte proliferation assay (LPA) remains the gold standard, despite detecting responses in only a minority of infected subjects. Recently, we reported development and validation of a novel whole blood CD4(+) effector T cell assay based on ex vivo antigen stimulation followed by co-expression of CD25 and CD134 on CD4(+) T cells. Here we report adaptation of this assay to assessment of HCV-specific responses in cryopreserved peripheral blood mononuclear cells using standardised antigens, including peptide pools, viral supernatants and recombinant viral proteins. The assay allowed detection of HCV-specific CD4 responses in donors with both resolved and chronic infection. Responses were highly correlated with those revealed by LPA. Application of this assay will further define the role of CD4(+) T cells in the immunopathogenesis of HCV infection.

Pulmonary interleukin-23 gene delivery increases local T-cell immunity and controls growth of Mycobacterium tuberculosis in the lungs.

Interleukin-23 (IL-23) is a heterodimeric cytokine that shares IL-12 p40 but contains a unique p19 subunit similar to IL-12 p35. Previous studies indicate a greater importance for intact IL-12/23 p40 expression than IL-12 p35 for immunity against Mycobacterium tuberculosis, suggesting a role for IL-23 in host defense. The effects of IL-23 on the outcome of pulmonary infection with M. tuberculosis have not been described. Here, we show that local delivery of replication-defective adenovirus vectors encoding IL-23 (AdIL-23) greatly stimulated expression of both gamma interferon (IFN-gamma) and IL-17 in lung tissues of otherwise normal mice. When given 72 h prior to infection with M. tuberculosis, AdIL-23 significantly reduced the bacterial burden at 14, 21, and 28 days. Markedly lower levels of lung inflammation were observed at 28 days than in control mice pretreated with control adenovirus (AdNull) or vehicle controls. AdIL-23 pretreatment resulted in increased numbers of CD4(+) CD25(+) activated T cells in lungs and draining lymph nodes compared to control groups and more CD4(+) T cells bearing surface memory markers in lung lymph nodes. IL-23 gene delivery also significantly enhanced host anti-mycobacterial T-cell responses, as shown by elevated levels of IFN-gamma and IL-17 secreted in vitro following restimulation with M. tuberculosis purified protein derivative. Overall, our data show that transient IL-23 gene delivery in the lung is well tolerated, and they provide the initial demonstration that this factor controls mycobacterial growth while augmenting early pulmonary T-cell immunity.

FL-CTL assay: fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells.

Cytotoxic T lymphocytes (CTLs) are crucial effectors against intracellular pathogens and cancer. Accurate and efficient assessment of CTL activity is important for basic and clinical studies. Widely used CTL assays, including the chromium release, JAM test and ELISPOT, involve either radioisotopes or lengthy procedures. Here, we developed a new fluorolysometric CTL assay based on cell-mediated cytolysis of fluorescent protein (GFP or DsRed) expressing cells quantified by one of the fluoro-based methods: flow cytometry, fluorescence microplate reader, or fluorescence microscopy. With flexible detection methods and lentiviral vector transduced stable lines of either GFP+ or DsRed+ cells as targets for antigen presentation and equal number of the other as internal reference for consistency and accuracy, this assay is easy to perform and to scale-up for simultaneous multi-sample analyses. Using two different antigen systems, we demonstrated that this assay is very sensitive to determine primary CTL activity of both in vitro and in vivo primed antigen-specific T cells. Thus, this FL-CTL assay is highly sensitive, reliable, reproducible, economical, convenience and supports broad applications compared to conventional CTL assays.

Effector HIV-specific cytotoxic T-lymphocyte activity in long-term nonprogressors: associations with viral replication and progression.

Ex vivo effector cytotoxic T-lymphocyte (CTL) activity was assessed in 27 members of the Australian Long-Term Nonprogressor cohort and correlated with genetic, virological, and immunological markers. The 27 individuals were antiretroviral naive with CD4(+) T-cell counts of >500 cells/ microl for more than 8 years after human immunodeficiency virus type 1 (HIV-1) infection. Effector CTL activity was determined using a standard ex vivo chromium release assay. Individuals with CTL activity (HIV-1 env(IIIB) or pol or gag) were then compared to those without CTL activity in relation to plasma HIV-1 RNA, ICD p24 antigen, beta(2)-microglobulin, CD4 and CD8 T-cell counts, CCR5 and CCR2b genotypes, and progression to CD4 <500 cells/microl or commencement of antiretroviral treatment. Of the 27 individuals examined, 19 had no detectable effector CTL activity. The eight individuals with detectable CTL activity had significantly higher plasma levels of HIV-1 RNA (P = 0.014), immune complex dissociated p24 antigen (P = 0.006), and beta(2)-microglobulin (P = 0.009). There was increased risk of progression within 4 years of study entry in individuals with detectable effector CTL activity, higher plasma levels of HIV-1 RNA, higher beta(2)-microglobulin levels, and higher immune complex dissociated p24 antigen levels at enrollment (P = 0.017, P = 0.004, P = 0.027, P = 0.008 respectively). Multivariate analysis demonstrated viral load remained the strongest predictor of disease progression within this group (P = 0.017). There were no significant associations between CTL response and chemokine receptor genotype. These findings demonstrate the importance of HIV replication in generating an effector CTL response and show that effector CTL activity may be an early predictor of progression in people with long-term asymptomatic HIV infection.