Invasive versus non-invasive management of older patients with non-ST elevation myocardial infarction (SENIOR-NSTEMI): a cohort study based on routine clinical data.

BACKGROUND: Previous trials suggest lower long-term risk of mortality after invasive rather than non-invasive management of patients with non-ST elevation myocardial infarction (NSTEMI), but the trials excluded very elderly patients. We aimed to estimate the effect of invasive versus non-invasive management within 3 days of peak troponin concentration on the survival of patients aged 80 years or older with NSTEMI. METHODS: Routine clinical data for this study were obtained from five collaborating hospitals hosting NIHR Biomedical Research Centres in the UK (all tertiary centres with emergency departments). Eligible patients were 80 years old or older when they underwent troponin measurements and were diagnosed with NSTEMI between 2010 (2008 for University College Hospital) and 2017. Propensity scores (patients' estimated probability of receiving invasive management) based on pretreatment variables were derived using logistic regression; patients with high probabilities of non-invasive or invasive management were excluded. Patients who died within 3 days of peak troponin concentration without receiving invasive management were assigned to the invasive or non-invasive management groups based on their propensity scores, to mitigate immortal time bias. We estimated mortality hazard ratios comparing invasive with non-invasive management, and compared the rate of hospital admissions for heart failure. FINDINGS: Of the 1976 patients with NSTEMI, 101 died within 3 days of their peak troponin concentration and 375 were excluded because of extreme propensity scores. The remaining 1500 patients had a median age of 86 (IQR 82-89) years of whom (845 [56%] received non-invasive management. During median follow-up of 3·0 (IQR 1·2-4·8) years, 613 (41%) patients died. The adjusted cumulative 5-year mortality was 36% in the invasive management group and 55% in the non-invasive management group (adjusted hazard ratio 0·68, 95% CI 0·55-0·84). Invasive management was associated with lower incidence of hospital admissions for heart failure (adjusted rate ratio compared with non-invasive management 0·67, 95% CI 0·48-0·93). INTERPRETATION: The survival advantage of invasive compared with non-invasive management appears to extend to patients with NSTEMI who are aged 80 years or older. FUNDING: NIHR Imperial Biomedical Research Centre, as part of the NIHR Health Informatics Collaborative.

Restriction of V3 region sequence divergence in the HIV-1 envelope gene during antiretroviral treatment in a cohort of recent seroconverters.

BACKGROUND: Dynamic changes in Human Immunodeficiency Virus 1 (HIV-1) sequence diversity and divergence are associated with immune control during primary infection and progression to AIDS. Consensus sequencing or single genome amplification sequencing of the HIV-1 envelope (env) gene, in particular the variable (V) regions, is used as a marker for HIV-1 genome diversity, but population diversity is only minimally, or semi-quantitatively sampled using these methods. RESULTS: Here we use second generation deep sequencing to determine inter-and intra-patient sequence heterogeneity and to quantify minor variants in a cohort of individuals either receiving or not receiving antiretroviral treatment following seroconversion; the SPARTAC trial. We show, through a cross-sectional study of sequence diversity of the env V3 in 30 antiretroviral-naive patients during primary infection that considerable population structure diversity exists, with some individuals exhibiting highly constrained plasma virus diversity. Diversity was independent of clinical markers (viral load, time from seroconversion, CD4 cell count) of infection. Serial sampling over 60 weeks of non-treated individuals that define three initially different diversity profiles showed that complex patterns of continuing HIV-1 sequence diversification and divergence could be readily detected. Evidence for minor sequence turnover, emergence of new variants and re-emergence of archived variants could be inferred from this analysis. Analysis of viral divergence over the same time period in patients who received short (12 weeks, ART12) or long course antiretroviral therapy (48 weeks, ART48) and a non-treated control group revealed that ART48 successfully suppressed viral divergence while ART12 did not have a significant effect. CONCLUSIONS: Deep sequencing is a sensitive and reliable method for investigating the diversity of the env V3 as an important component of HIV-1 genome diversity. Detailed insights into the complex early intra-patient dynamics of env V3 diversity and divergence were explored in antiretroviral-naïve recent seroconverters. Long course antiretroviral therapy, initiated soon after seroconversion and administered for 48 weeks, restricts HIV-1 divergence significantly. The effect of ART12 and ART48 on clinical markers of HIV infection and progression is currently investigated in the SPARTAC trial.

Prognostic significance of troponin level in 3121 patients presenting with atrial fibrillation (The NIHR Health Informatics Collaborative TROP-AF study).

Background Patients presenting with atrial fibrillation (AF) often undergo a blood test to measure troponin, but interpretation of the result is impeded by uncertainty about its clinical importance. We investigated the relationship between troponin level, coronary angiography, and all-cause mortality in real-world patients presenting with AF. Methods and Results We used National Institute of Health Research Health Informatics Collaborative data to identify patients admitted between 2010 and 2017 at 5 tertiary centers in the United Kingdom with a primary diagnosis of AF. Peak troponin results were scaled as multiples of the upper limit of normal. A total of 3121 patients were included in the analysis. Over a median follow-up of 1462 (interquartile range, 929-1975) days, there were 586 deaths (18.8%). The adjusted hazard ratio for mortality associated with a positive troponin (value above upper limit of normal) was 1.20 (95% CI, 1.01-1.43; P<0.05). Higher troponin levels were associated with higher risk of mortality, reaching a maximum hazard ratio of 2.6 (95% CI, 1.9-3.4) at ≈250 multiples of the upper limit of normal. There was an exponential relationship between higher troponin levels and increased odds of coronary angiography. The mortality risk was 36% lower in patients undergoing coronary angiography than in those who did not (adjusted hazard ratio, 0.61; 95% CI, 0.42-0.89; P=0.01). Conclusions Increased troponin was associated with increased risk of mortality in patients presenting with AF. The lower hazard ratio in patients undergoing invasive management raises the possibility that the clinical importance of troponin release in AF may be mediated by coronary artery disease, which may be responsive to revascularization.

Association of troponin level and age with mortality in 250 000 patients: cohort study across five UK acute care centres.

OBJECTIVE: To determine the relation between age and troponin level and its prognostic implication. DESIGN: Retrospective cohort study. SETTING: Five cardiovascular centres in the UK National Institute for Health Research Health Informatics Collaborative (UK-NIHR HIC). PARTICIPANTS: 257 948 consecutive patients undergoing troponin testing for any clinical reason between 2010 and 2017. MAIN OUTCOME MEASURE: All cause mortality. RESULTS: 257 948 patients had troponin measured during the study period. Analyses on troponin were performed using the peak troponin level, which was the highest troponin level measured during the patient's hospital stay. Troponin levels were standardised as a multiple of each laboratory's 99th centile of the upper limit of normal (ULN). During a median follow-up of 1198 days (interquartile range 514-1866 days), 55 850 (21.7%) deaths occurred. A positive troponin result (that is, higher than the upper limit of normal) signified a 3.2 higher mortality hazard (95% confidence interval 3.1 to 3.2) over three years. Mortality varied noticeably with age, with a hazard ratio of 10.6 (8.5 to 13.3) in 18-29 year olds and 1.5 (1.4 to 1.6) in those older than 90. A positive troponin result was associated with an approximately 15 percentage points higher absolute three year mortality across all age groups. The excess mortality with a positive troponin result was heavily concentrated in the first few weeks. Results were analysed using multivariable adjusted restricted cubic spline Cox regression. A direct relation was seen between troponin level and mortality in patients without acute coronary syndrome (ACS, n=120 049), whereas an inverted U shaped relation was found in patients with ACS (n=14 468), with a paradoxical decline in mortality at peak troponin levels >70×ULN. In the group with ACS, the inverted U shaped relation persisted after multivariable adjustment in those who were managed invasively; however, a direct positive relation was found between troponin level and mortality in patients managed non-invasively. CONCLUSIONS: A positive troponin result was associated with a clinically important increased mortality, regardless of age, even if the level was only slightly above normal. The excess mortality with a raised troponin was heavily concentrated in the first few weeks. STUDY REGISTRATION: ClinicalTrials.gov NCT03507309.

HIV-1-specific CD4+ T lymphocyte turnover and activation increase upon viral rebound.

HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1-specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned.

Synergistic inhibition of HIV-1 infection by combinations of soluble polyanions with other potential microbicides.

Several polyanionic compounds with potential for use as topically applied microbicides to prevent HIV-1 sexual transmission, such as PRO 2000, are currently in phase III clinical efficacy trials. Microbicidal formulations may well comprise combinations of inhibitors to increase potency, reduce dose and minimize problems of HIV-1 resistance. We have therefore evaluated in vitro, the anti-HIV-1 activity of two leading polyanionic microbicides combined with other antiretroviral agents with microbicidal potential. Dextran sulfate (DS) and PRO 2000 were combined with the neutralizing antibody IgG1b12, the peptide-based fusion inhibitor T20, the CCR5 antagonist TAK779 and the cyanobacterial protein cyanovirin-N. Anti-HIV-1 activity was assessed in a single cycle replication assay using pseudoviruses carrying a luciferase reporter gene and the envelope glycoproteins from HIV-1 isolates JR-FL (R5) and HxB2 (X4), against both immortalized and primary CD4+ cell targets. The data were analyzed for synergy using Calcusyn software. Results indicate that PRO 2000 and DS can act synergistically with most inhibitors tested, although the degree of synergy depends on inhibitor concentration and combination. These data provide a rational basis for testing of microbicide combinations in vivo.

Direct measurement of in-vivo vaginal microbicide levels of PRO 2000 achieved in a human safety study.

OBJECTIVES: To directly measure the cervico-vaginal lavage (CVL) and plasma PRO 2000 concentrations achieved by vaginal dosing. DESIGN: A sub-study of a prospective randomized, double-blind phase 1 trial of a candidate vaginal microbicide, PRO 2000 gel. METHODS: Thirty-six sexually abstinent women self-administered 4% PRO 2000 gel, 0.5% PRO 2000 gel or placebo gel twice on day 0 and then once daily for a further 12 days. RESULTS: There was no evidence of systemic absorption of PRO 2000. PRO 2000 concentrations in CVL exceeded 25 microg/ml in all women in both the 4 and 0.5% groups at 2 h post-first dose, and in 10 of 12 of the women in the 4% gel group compared with five of 12 of women in the 0.5% group at 12 h post-seventh dose. Single use of both 4 and 0.5% PRO 2000 gels was therefore associated with levels of PRO 2000 in CVL that would be capable of preventing HIV infection in vitro, although the 4% gel gave a greater margin of excess. Levels substantially in excess of the target concentration were present 12 h after repeated dosing in twice as many 4% gel recipients compared with 0.5% gel recipients. CONCLUSIONS: Both PRO 2000 gel strengths provided satisfactory in-vivo HIV inhibitory concentrations. However, our observations show that higher concentrations of PRO 2000 are likely to provide a greater margin of potential efficacy in the context of sexual intercourse provided safety issues are equivalent for differing concentrations of the agent.

Zidovudine plus lamivudine in Human T-Lymphotropic Virus type-I-associated myelopathy: a randomised trial.

BACKGROUND: No therapies have been proven to persistently improve the outcome of HTLV-I-associated myelopathy. Clinical benefit has been reported with zidovudine and with lamivudine in observational studies. We therefore conducted a randomised, double blind, placebo controlled study of six months combination therapy with these nucleoside analogues in sixteen patients. RESULTS: Primary outcomes were change in HTLV-I proviral load in PBMCs and clinical measures. Secondary endpoints were changes in T-cell subsets and markers of activation and proliferation. Six patients discontinued zidovudine. No significant changes in pain, bladder function, disability score, gait, proviral load or markers of T-cell activation or proliferation were seen between the two arms. Active therapy was associated with an unexplained decrease in CD8 and non-T lymphocyte counts. CONCLUSION: Failure to detect clinical improvement may have been due irreversible nerve damage in these patients with a long clinical history and future studies should target patients presenting earlier. The lack of virological effect but may reflect a lack of activity of these nucleoside analogues against HTLV-I RT in vivo, inadequate intracellular concentrations of the active moiety or the contribution of new cell infection to maintaining proviral load at this stage of infection may be relatively small masking the effects of RT inhibition.

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

BACKGROUND: The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. METHODS AND FINDINGS: To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. CONCLUSION: Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Comparative response of African HIV-1-infected individuals to highly active antiretroviral therapy.

OBJECTIVE: Few data exist on the virological response to antiretroviral therapy of individuals infected with African HIV-1 subtypes. Our objective was to compare the response, in our clinic, of African HIV-1-infected patients with their British and European contemporaries treated with the same regimes. DESIGN: The St Mary's Hospital HIV database was used to identify drug-naive African and European patients starting a highly active antiretroviral therapy (HAART) regimen. METHODS: HIV-1 subtype was determined by phylogenetic analysis of pol sequences. Kaplan-Meier survival analysis was used to estimate the proportion of patients achieving undetectable viral loads (< 500 copies/ml). The longer-term response to therapy was assessed by changes in CD4 cell counts and viral loads from baseline. RESULTS: A total of 265 patients were classified as 'European' and 97 as 'African', confirmed by sequence. The time to first undetectable viral load was similar for the two groups (P = 0.9). Although there were no statistically significant differences in the CD4 cell count responses (P = 0.11), there was evidence of an increase in viral load after 9 months for the African group, resulting in a widening viral load gap between the two cohorts; the effect of ethnic group was statistically significant (P < 0.001). CONCLUSION: The initial virological and immunological responses of the African and European cohorts to HAART were similar; although the longer-term virological response was poorer in the African cohort, which may be related to adherence. On the basis of these findings, there is no justification for withholding HAART from Africa on virological grounds.

Residual HIV-specific CD4 and CD8 T cell frequencies after prolonged antiretroviral therapy reflect pretreatment plasma virus load.

BACKGROUND: Virus-specific cellular immune responses mediated by CD4 and CD8 T lymphocytes are thought to be central to the effective control of HIV-1 replication in vivo. However, quantitative correlations between HIV-specific T lymphocyte frequencies and plasma virus load (pVL) have proved difficult to establish in infected human individuals. This most likely reflects the complex interactions between the virus and these immune effector cells in the absence of treatment. OBJECTIVE: To assess frequencies of HIV-specific T lymphocytes after prolonged suppression of viral replication, i.e., under conditions where the effects of virus on the immune response are standardized and minimized, thereby fixing an important variable in a dynamic multivariate system. METHODS: HIV-specific CD4 and CD8 T lymphocyte frequencies were measured in 122 individuals after prolonged periods of successful combination antiretroviral therapy (ART) administered during chronic HIV-1 infection. RESULTS: The residual frequency of both CD4 and CD8 T lymphocytes specific for HIV-1 was inversely related to the pretreatment pVL. This relationship appeared to be non-linear, indicating the presence of a threshold pretreatment pVL level above which HIV-specific CD4 and CD8 T lymphocyte responses could not be maintained when antigenic drive was suppressed. Substantial populations of functional HIV-specific CD4 and CD8 T lymphocytes were generally detectable after prolonged ART only in those individuals with a pretreatment plasma HIV-1 RNA < 100,000 copies/ml. CONCLUSION: These findings identify a quantitative immune associate of host-virus interactions in established HIV-1 infection.

Detection of HIV-1 antiretroviral resistance from patients with persistently low but detectable viraemia.

We modified the Abbott diagnostics HIV-1 Viroseq version 2 assay trade mark in order to detect the presence of HIV-1 drug resistance mutations in patients with viraemia below 1000 copies/ml of plasma. One hundred and forty-four patients with a detectable HIV-1 plasma viral load below 1000 copies/ml were selected and HIV-1 genetic analysis carried out using a modification of the Abbott Diagnostics Viroseq 2.0 assay trade mark. The procedure differs from the standard protocol in that a nested PCR amplification step was introduced. The oligonucleotide primers for the first round of PCR were those supplied in the RT-PCR module of the kit. The nested PCR primers were primers A and H taken from the sequencing module. One hundred and twenty-eight out of 144 (89%) plasma samples with an HIV-1 viral load of less than 1000 copies/ml (ranging from 54 to 992 copies) were successfully sequenced. HIV-1 genotypes were obtained from 68 out of 81 (84%) samples with a viral load of greater than 50 but less than 300 copies/ml and 60/63 (95%) of samples with a viral load of greater than 300 but less than 1000 copies/ml. Serial dilution of a sample with a high viral load did not affect the detection of resistance mutations. Multiple sequencing of samples with low viral load did not result in detection of additional mutations, although, in one sample the K103N mutation was detected in 3/6 replicates while wild-type was detected in 2/6 and a mixture of wild-type/mutant in 1/6. Samples from patients infected with both clade B and non-B clades of HIV-1 could be genotyped at low copy number. Modification of the Abbott Viroseq assay allows reproducible sequencing of the HIV-1 genome from patients with low, but detectable, plasma virus burden.

Ciclosporin A proof of concept study in patients with active, progressive HTLV-1 associated myelopathy/tropical spastic paraparesis.

INTRODUCTION: Patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) become progressively impaired, with chronic pain, immobility and bladder, bowel and sexual dysfunction. Tested antiretroviral therapies have not been effective and most patients are offered a short course of corticosteroids or interferon-α, physiotherapy and symptomatic management. Pathogenesis studies implicate activated T-lymphocytes and cytokines in tissue damage. We therefore tested the hypothesis that inhibition of T-cell activation with ciclosporin A would be safe and clinically beneficial in patients with early and/or clinically progressing HAM/TSP. MATERIALS AND METHODS: Open label, proof of concept, pilot study of 48 weeks therapy with the calcineurin antagonist, ciclosporin A (CsA), in seven patients with 'early' (50% deterioration in timed walk during the preceding three months) HAM/TSP. Primary outcomes were incidence of clinical failure at 48 weeks and time to clinical failure. RESULTS: All patients completed 72 weeks study participation and five showed objective evidence of clinical improvement after 3 months treatment with CsA. Two patients exhibited clinical failure over 6.4 person-years of follow-up to week 48. One patient had a >2 point deterioration in IPEC (Insituto de Pesquisa Clinica Evandro Chagas) disability score at weeks 8 and 12, and then stopped treatment. The other stopped treatment at week 4 because of headache and tremor and deterioration in timed walk, which occurred at week 45. Overall pain, mobility, spasticity and bladder function improved by 48 weeks. Two patients recommenced CsA during follow-up due to relapse. CONCLUSIONS: These data provide initial evidence that treatment with CsA is safe and may partially reverse the clinical deterioration seen in patients with early/progressive HAM/TSP. This trial supports further investigation of this agent's safety and effectiveness in larger, randomised controlled studies in carefully selected patients with disease progression.

Epidemiology of non-B clade forms of HIV-1 in men who have sex with men in the UK.

OBJECTIVES: To describe the frequency and risk factors of non-B HIV-1 subtypes in men who have sex with men (MSM) in the UK. DESIGN: Observational study. METHODS: MSM diagnosed with HIV-1 infection from 1980-2007, with HIV genotype held in the UK HIV Drug Resistance Database were identified. Protease and reverse transcriptase sequences were collected and viral clade determined using the REGA algorithm. Associations between demographic variables and subtype were analysed using logistic regression. RESULTS: The prevalence of non-B HIV-1 infection amongst MSM in the UK was 5.4% (437/8058). In the UK this increased with year of diagnosis from pre1996 to 2002, and has subsequently remained relatively stable at around 7-9% after 2002, with a recent increase in 2007 to 13%. Multivariate analysis showed that acquisition of non-B HIV-1 infection was independently associated with later year of HIV diagnosis (P < 0.001), black ethnicity (P < 0.001) and non-European country of birth (P = 0.01). Age was also associated with subtype with individuals aged 25-39 years being less likely to have non-B virus than those aged less than 25 years (P = 0.01). Restricting the analysis to white men born in the UK, the association between subtype and year of diagnosis remained statistically significant (P < 0.001), as did the association with age (P < 0.001). DISCUSSION: The number of MSM in the UK infected with non-B clade HIV-1 is increasing, suggesting that the sociodemographic boundaries between HIV-1 viral subtypes globally are diminishing. Should viral subtypes be relevant to clinical disease progression or vaccine design, the changing pattern of distribution will need to be taken into account.

B-cell depletion reveals a role for antibodies in the control of chronic HIV-1 infection.

HIV can be partially contained by host immunity and understanding the basis of this may inform vaccine design. The importance of B-cell function in long-term control is poorly understood. One method of investigating this is in vivo cellular depletion. In this study, we take advantage of a unique opportunity to investigate the role of B cells in an HIV-infected patient. The HIV-1(+) patient studied here was not taking antiretroviral drugs and was treated for pre-existing low-grade lymphoplasmacytoid lymphoma by depletion of CD20+ B cells using rituximab. We demonstrate that B-cell depletion results in a decline in autologous neutralizing antibody (NAb) responses and a 1.7 log(10) rise in HIV-1 plasma viral load (pVL). The recovery of NAbs results in a decline in pVL. The HIV-1 sequences diversify and NAb-resistant mutants are subsequently selected. These data suggest that B-cell function can contribute to the long-term control of pVL, and that NAbs may be more important in controlling chronic HIV-1 infection than previously suspected.

Vaccinia virus strain NYVAC induces substantially lower and qualitatively different human antibody responses compared with strains Lister and Dryvax.

The antibody responses elicited by immunization of humans with vaccinia virus (VACV) strains Lister, Dryvax and NYVAC have been determined and compared. Neutralizing antibodies against intracellular mature virus (IMV) and extracellular enveloped virus (EEV), and binding antibody titres (ELISA) against the EEV protein B5, the IMV proteins A27 and H3, and VACV-infected cell lysate were measured. Lister and Dryvax induced broadly similar antibody titres, consistent with the fact that these vaccines each protected against smallpox. In contrast, antibody titres induced by NYVAC were significantly lower than those induced by both Lister and Dryvax. Moreover, there were qualitative differences with NYVAC-immunized subjects failing to induce A27-specific antibodies. These observations suggest that although NYVAC is a safer VACV strain, it does not induce an optimal VACV-specific antibody response. However, NYVAC strains engineered to express antigens from other pathogens remain promising candidate vaccines for immunization against other diseases.

Precise identification of a human immunodeficiency virus type 1 antigen processing mutant.

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection.

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining.

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.

Human immunodeficiency virus type 1 in illicit-drug solutions used intravenously retains infectivity.

The stability of the human immunodeficiency virus type 1 (HIV-1) strain IIIB in drug solutions was studied. The data demonstrate that HIV-1 infectivity can be retained in drug solutions (e.g. , heroin, "Khanka," and "Vint") for long periods of time. This fact must be taken into account when designing health education programs for the prevention of HIV and AIDS in Eastern Europe.