Patients accessing antiretroviral treatment (ART) programmes in sub-Saharan Africa frequently have very advanced immunodeficiency. Previous data suggest that such patients may have diminished capacity for CD4 cell count recovery.Rates of CD4 cell increase were determined over 48 weeks among ART-naïve individuals (n = 596) commencing ART in a South African community-based ART programme.The CD4 cell count increased from a median of 97 cells/μl at baseline to 261 cells/μl at 48 weeks and the proportion of patients with a CD4 cell count <100 cells/μl decreased from 51% at baseline to just 4% at 48 weeks. A rapid first phase of recovery  was followed by a slower second phase . Compared to patients with higher baseline counts, multivariate analysis showed that those with baseline CD4 counts <50 cells/μl had similar rates of phase 1 CD4 cell recovery (P = 0.42), greater rates of phase 2 recovery (P = 0.007) and a lower risk of immunological non-response (P = 0.016). Among those that achieved a CD4 cell count >500 cells/μl at 48 weeks, 19% had baseline CD4 cell counts <50 cells/μl. However, the proportion of these patients that attained a CD4 count 200 cells/μl at 48 weeks was lower than those with higher baseline CD4 cell counts.Patients in this cohort with baseline CD4 cell counts <50 cells/μl have equivalent or greater capacity for immunological recovery during 48 weeks of ART compared to those with higher baseline CD4 cell counts. However, their CD4 counts remain <200 cells/μl for a longer period, potentially increasing their risk of morbidity and mortality in the first year of ART. The World Health Organisation (WHO) estimated that in June 2005 4.7 million people living in sub-Saharan Africa were in urgent need of antiretroviral treatment (ART) . DespitePatients enrolling into ART programmes with very low CD4 cell counts have heightened risk of morbidity and mortality both before and during the initial months of ART -8. MoreoIn this study we have examined determinants of CD4 cell count recovery among patients accessing a community-based antiretroviral programme in South Africa. We focus on the hypothesis that advanced pre-treatment immunodeficiency diminishes the capacity for CD4 cell count recovery during ART as determined by rates of CD4 cell count increase and risk of immunological non-response.We studied patients accessing ART at the Gugulethu Community Health Centre, Cape Town, South Africa . The vasThe first-line ART regimen was comprised of stavudine, lamivudine plus a non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine). The second-line regimen for those failing first-line treatment was comprised of lopinavir/ritonavir, zidovudine and didanosine. Treatment adherence exceeds 90% at one year . All trePlasma HIV-1 load was measured at baseline and 4 monthly during treatment using Versant™ HIV-1 RNA 3.0 branched chain DNA assay  with a lower limit of detection of 50 RNA copies/ml. Blood CD4 cell counts were measured at the same time-points by flow cytometry using FACSCount™ . These assays were all performed in a single nationally accredited laboratory which has rigorous quality assurance procedures.Structured clinical and laboratory records were maintained on all patients screened on entry to the ART programme and this information was transferred on a weekly basis to a computer database. Data were analysed from the start of the programme in September 2002 until data censorship in August 2005. All treatment-naive patients aged over 15 years and with at least a 16-week follow-up time point were included in the analysis. Study of these patients was in compliance with the Declaration of Helsinki and was approved by the Research Ethics Committee of the University of Cape Town; all patients enrolled gave written informed consent.Data were analysed using Stata version 9.0 . We calculated absolute responses in CD4 cell counts during three intervals , as well as rates of CD4 cell increase (cells/μl/month) during each interval. The first interval was used as an estimate of the initial (phase 1) response to ART. The CD4 cell count responses observed during the latter two intervals (16 to 32 and 32 to 48 weeks after treatment initiation) were very similar, and were combined into a single measure of the second phase of CD4 cell responses (phase 2).We further evaluated CD4 cell responses in the following ways: i) whether patients failed to attain an absolute CD4 cell count increase from baseline of at least 50 cells/μl at 48 weeks ; ii) whether patients achieved an absolute CD4 count of 200 cells/μl at the 48 week visit; iii) and whether patients had achieved an absolute CD4 cell count of 500 cells/μl at 48 weeks (super-responders).In bivariate analyses, medians were compared using Wilcoxon rank-sum and sign-rank tests; proportions were compared using chi-square tests. All statistical tests were two-sided at alpha = 0.05. Separate multiple linear regression models were used to examine factors associated with rates of CD4 cell count increase per month during the first and second phases. Baseline CD4 cell counts were categorised as follows: <50, 50–99, 100–149 and >150 cells/μl. Multiple logistic regression was used to assess factors associated with the binary outcomes of a CD4 cell increase of ≥ 50 cells/μl and ≥ 100 cells/μl during the 48 weeks of ART, as well as achieving an absolute CD4 cell count of ≥ 200 cells/μl during follow-up. Variables were included in multivariate models if they demonstrated a persistent association with the outcome of interest, or if their removal affected appreciably associations involving other variables .Of 698 individuals who commenced ART between September 2002 and April 2005, 596 (85%) had completed a 16-week follow-up visit at the time of data censorship, 34 (5%) were awaiting this appointment, 48 (7%) had died and 20 (3%) were either transferred out or were lost to follow-up. Of the 596 individuals who met the inclusion criteria for the study, 580 (97%) remained within the programme at study censorship, 11 (2%) died and 7 (1%) were lost from the programme.10 RNA copies/ml  and 58% of patients had a viral load >100,000 copies/ml. The median blood CD4 cell count was 97 cells/μl  and the proportions of patients with CD4 cell counts within the ranges <50, 50–99, 100–149 and ≥ 150 cells/μl were 25%, 26%, 23% and 26%, respectively. Eighty per cent of patients had symptomatic disease, with 53% and 27% of patients having WHO stages 3 and 4 disease, respectively. During follow-up, only 7 (1%) patients switched to the second-line drug regimen.At baseline the median age was 32 years  and 75% were female. The median plasma viral load was 4.88 logVirological responses to treatment were excellent with viral load suppressed <400 copies/ml in ≥ 94% of patients at each of the follow-up time-points Table . As a reThe rate of CD4 cell count increase in the first 16 week period greatly exceeded the rates in both the 16–32 week and 32–48 week intervals, but the rates did not differ comparing the latter two intervals. Thus, the pattern of CD4 cell count increase was divided into 2 phases: a ra–pid phase 1  and a slower phase 2 .Baseline characteristics and rates of CD4 cell count change in phase 1 and phase 2 did not differ when comparing the results of analyses of all eligible patients with those restricted to subjects who had data for every time-point (n = 292); this was also the case for all subsequent stratified analyses. Use of data from the larger cohort (n = 596) was therefore validated.10 copies/ml the phase 1 CD4 cell slope was 21.3 cells/μl/month compared to 31.0 cells/μl/month among those with baseline viral load of >5 log10 copies/ml (P < 0.001).Rates of phase 1 CD4 cell increase were similar across all CD4 cell count strata Figure . SubsequContrary to our initial hypothesis, the rate of phase 2 CD4 cell increase was greater among those with a baseline CD4 cell count <50 cells/μl compared to those with higher baseline counts Figure . MultivaIn the multivariate model predicting rates of phase 2 CD4 cell increase, factors associated with the response to ART during the first 16 weeks were also included. A lower rate of phase 1 CD4 cell increase and full suppression of viral load at 16 weeks were both strongly associated with greater rate of phase 2 CD4 cell increase. Viral load suppression <400 copies/ml at 16 weeks was associated with a subsequent rate of CD4 cell increase of 6.8 cells/μl/month compared to 0.7 cells/μl/month among those whose viral load remained >400 copies/ml (P < 0.001).We next examined whether baseline CD4 cell count was a risk factor associated with immunological non-response (defined as a CD4 cell increase of <50 cells/μl at 48 weeks). Among those followed to 48 weeks n = 311), the blood CD4 cell count increased by <50 cells/μl among 30 (9.7%) patients; of these, the viral load was suppressed <50 copies/ml among 22 patients, representing a treatment discordance rate of 7%. Contrary to our initial hypothesis, low baseline CD4 cell counts were not associated with increased risk of immunological non-response. Among patients with baseline CD4 cell counts of <50, 50–99, 100–149 and 150 cells/μl, the proportions of patients who were immunological non-responders were 5%, 4%, 11% and 19%, respectively. Furthermore, in multivariate analyses, an increment of <50 cells/μl was independently associated with higher baseline CD4 cell count as well as older age, lower baseline viral load, and a viral load >400 copies/ml at any follow-up time-point  achieved an absolute CD4 cell count of >500 cells/μl. These super-responders were principally characterised by age <40 years and by all having follow-up viral loads persistently suppressed <50 copies/ml and a CD4 cell count of 150 cells/μl after 16 weeks of ART. This group of patients had a wide distribution of baseline CD4 cell counts, and included among them were 4 (19%) who had baseline CD4 cell counts of <50 cells/μl. Thus, a low baseline CD4 cell count did not preclude patients from having an excellent immunological response.To our knowledge this is the first analysis to examine the determinants of CD4 cell count recovery among patients receiving ART in resource limited settings. These data indicate that those with baseline CD4 cell counts <50 cells/μl had similar rates of phase 1 CD4 recovery (0–16 weeks) and greater rates of phase 2 recovery 16–48 weeks) compared to rates among those with higher baseline CD4 cell counts. Moreover, those with the lowest baseline counts were least likely to be immunological non-responders to ART. Despite these observations, those with baseline CD4 cell counts <50 cells/μl were nevertheless least likely to attain a CD4 cell count 200 cells/μl at 48 weeks. Taken together, these results suggest that although patients with very low baseline CD4 cell counts retain capacity for similar or slightly greater rates of CD4 cell count recovery compared to those with higher counts, they are nevertheless likely to require a longer period of time to attain a CD4 cell count threshold of 200 cells/μl. Thus, a prolonged period below a 'safe' CD4 cell count threshold rather than a diminished rate of immunological recovery is likely to underlie the high rates of morbidity and mortality observed among those with advanced disease during the early months of ART [ weeks coThe findings of this analysis are strengthened by the relatively homogeneous study population receiving treatment at a single facility using standardised clinical protocols. Patients were all ART-naïve and received a standard triple-drug regimen with uniform follow-up time points. Quality-assured laboratory assays were all performed in a single nationally accredited laboratory. In contrast, previous studies of the determinants of CD4 cell count recovery have examined heterogeneous study populations in multiple centres and used diverse treatment regimens. Moreover, these studies included many patients with prior ART exposure -22 and set al.[et al. showed a similar but non-significant trend when comparing patients with baseline CD4 cell counts <200 cells/μl with those with higher counts [et al. found that CD4 cell increases in the first year of ART were similar comparing those with baseline counts <100 cells/μl with those with counts 100–199 cells/μl [The immunological response to ART among those with low CD4 cell counts was excellent with the proportion of patients with a CD4 cell count <100 cells/μl decreasing from 51% at baseline to just 4% at 48 weeks. However, our most important finding was that in multivariate analysis baseline CD4 cell counts <50 cells/μl were independently associated with similar rates of phase 1 CD4 cell recovery and greater rates of phase 2 CD4 cell recovery compared to individuals with higher baseline CD4 cell counts. This has not been clearly highlighted in previous publications from Europe and North America although comparison of our data with previous studies is difficult in view of differing cohort compositions. However, this overall observation is consistent with the findings of Bennett et al., and Le cells/μl .Survival bias could potentially have affected our findings. We have previously shown in this cohort that patients with the lowest baseline CD4 cell counts had a higher risk of death  and immuWe have previously shown that low baseline CD4 count at entry to an ART programme was associated with increased risks of tuberculosis and of mortality during the first year of ART ,8. Resul5 log10 copies/ml had 11-fold greater CD4 count increases than those with lower viral loads. Immune dysregulation and immune cell activation promote sequestration of CD4+CD45Ro+ memory T cells in lymphoid tissue; suppression of viral replication then triggers rapid redistribution of this cell pool and a reduction in apoptotic cell death during the initial weeks of ART [Mycobacterium tuberculosis[Cryptococcus neoformans[Rates of phase 1 and phase 2 CD4 cell increase were similar in magnitude to those previously reported from high-income countries ,20. The s of ART ,27. A poerculosis, Cryptoceoformans that is Greater phase 2 CD4 cell recovery was strongly associated with age as reported elsewhere ,24. SustPatients with the lowest CD4 counts in this setting do not have diminished capacity for immune recovery. Although patients with low baseline CD4 counts have increased risk of acute morbidity and mortality, if such patients survive the initial months of ART and fully suppress the viral load, their chances of immunological recovery are good during the first year. Future studies are required to determine the long-term prospects for immune recovery among patients treated in ART programmes in sub-Saharan Africa.The author(s) declare that they have no competing interests.SDL and RW initiated and designed the study. LM did the statistical analyses. LGB and RW established the study cohort and data collection systems. SDL wrote the manuscript, which RW, LM and LGB all helped to revise.The pre-publication history for this paper can be accessed here:


Veazey and Lackner discuss a new study which found that most patients who start antiretroviral drugs as early as possible after HIV infection still do not experience complete restoration of intestinal CD4+ T cells to baseline levels. These studies confirmed earlier reports in macaques infected with simian immunodeficiency virus (SIV) [+ T cells in the body. However, profound losses can only be detected when examining mucosal tissues . Recognition of the mucosal immune system as a principal target of early HIV infection constitutes a fundamental change in our understanding of HIV pathophysiology, with potential implications for therapeutic monitoring and vaccine development [Recent studies have confirmed that acute HIV infection results in a rapid and profound loss of memory CD4nfection ,2. Sinceus (SIV)  and demo+ T cells imparts profound disturbances in mucosal immunity. However, since mucosal tissues are at best inconvenient to sample, little is known regarding the impact of the loss of this important T cell subset on host immunity or disease progression. In addition, the potential for antiretroviral therapy (ART) to restore mucosal CD4+ T cells has been difficult to assess, particularly in acute infection. In a new paper by Mehandru et al. [n = 54) of HIV-infected patients soon after infection. These samples were examined and compared with the same samples obtained from HIV-uninfected volunteers to determine whether early therapeutic intervention could restore intestinal CD4+ T cells to baseline levels. Prior to this study, few had examined the effects of ART on HIV-infected humans or SIV-infected macaques, particularly inearly infection. A study by Guadalupe et al. had suggested near complete restoration of mucosal CD4+ T cells in HIV-infected patients when therapy was initiated early, but only three acutely infected patients were examined [+ T cells, but again, few animals were examined [+ T cell repopulation in patients on ART, but this was a cross-sectional study and only eight patients were examined [+ T cells in the peripheral blood have been reported to fully reconstitute in patients on ART, there is considerable controversy regarding the capacity for restoration of intestinal CD4+ T cells, particularly for patients treated in the early stages of infection. This is more than an academic issue, since acutely infected patients are seldom started on ART. Treatment guidelines generally recommend waiting to initiate therapy until blood CD4 cell counts have fallen, in order to reduce the risk of medication side effects as well as the potential for selecting drug-resistant HIV.Clearly, the massive loss of intestinal memory CD4u et al. , lymphocexamined . Studiesexamined . In contexamined . In summThe mucosal immune system is a principal target of early HIV infection.+ T cells was observed on ART, the results clearly demonstrate that restoration is only partial in the majority of patients compared to uninfected controls, even after several years of “fully suppressive” ART. In addition, these studies demonstrate increased levels of activation in the intestinal immune system of patients on ART, even though immune activation in the peripheral blood often returns to baseline levels. The combined results of this new study suggest that ongoing viral replication and CD4+ T cell destruction occur in the intestine of patients on ART, despite what appears to be complete suppression of viral replication in the blood.In the current paper, 54 patients in the acute stage of HIV infection were examined and compared with 18 uninfected volunteers. Of the acutely infected patients, 14 were examined only prior to ART, 18 were examined prior to ART and then sequentially through one to three years of uninterrupted therapy, and 22 were examined cross-sectionally at time points ranging from less than one year to seven years after starting ART. Although partial restoration of mucosal CD4+ T cells, whereas macaque studies are usually performed using samples from the jejunum. The rectum/colon is a mix of immune inductive (organized lymphoid tissues) and effector sites (diffuse lamina propria) whereas the jejunum contains almost no immune inductive sites. This is reflected in the lymphoid composition of each tissue: the jejunum contains mostly memory CD4+ T cells, while the colon contains a larger proportion of naïve CD4+ T cells. This makes it difficult to extrapolate comparisons of T cell subsets from these disparate sites. Third, the percentages of “activated” T cell subsets remaining after treatment need to be interpreted with caution, as these are “snapshot” data observed during what is now believed to be a very dynamic process. For example, detection of increased percentages of “activated” CD4+ T cells in a tissue that has undergone massive CD4+ T cell depletion could be interpreted either as an immune response to the infection or as a selective loss of “resting” CD4+ T cells. However, given that the study also found that higher activation levels were associated with poorer mucosal reconstitution, the more logical interpretation is that HIV infection results in continual activation, turnover, and destruction of intestinal CD4+ T cells. Finally, the low level of infected cells detected in the intestine of patients on ART should be interpreted with caution, as the conclusion was based on small biopsy samples taken from the largest immune organ in the body. Detecting even a small number of infected cells in these pinch biopsies could reflect a very large number of infected cells throughout the intestine.Given the enormous practical challenges involved in obtaining mucosal biopsies from acutely infected patients, the work presented here is certainly valuable. However, a few limitations are inherent in interpreting the results and especially in comparing these data to those from SIV-infected macaques. First, neither pre-infection CD4 levels nor the exact timing of the initial infection were known, as patients were deemed to be in acute stage of infection based on viremia in the face of negative serology. In contrast, studies of SIV-infected macaques were based on restoration to pre-inoculation CD4 levels. Furthermore, therapy in the macaques was initiated at six weeks of infection , which i+ T cells to baseline levels, regardless of the duration of therapy. Instead, HIV infection results in a continuous state of activation in the intestinal immune system that is not reflected in peripheral lymphoid tissues. Combined, the data provide evidence that intestinal inflammation and continual infection, destruction, and turnover of CD4+ T cells occur in patients on ART, suggesting that the major battle against HIV occurs in sequestered mucosal tissue sites. This important observation suggests a number of potential therapeutic strategies for further research; for example, the investigation of drugs with better intestinal tissue distribution and perhaps the exploration of mechanisms to reduce immune activation in mucosal tissues to more effectively combat HIV infection.In conclusion, the findings reported by Mehandru et al. indicate that most patients who initiate therapy as early as possible after HIV infection still do not experience complete restoration of intestinal CD4


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.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.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. We took advantage of an unusually immunodominant, HLA-DR1 restricted CD4+ T cell epitope which we had characterized in detail by specific CD4+ T cell clones from three different patients and which was complexed into an HLA-DR1 tetramer. This novel tetramer was used to explore the frequency, phenotype and function of hepatitis C virus (HCV)-specific CD4+ T cells in seven patients with acute hepatitis C during the most critical phase of viral elimination or persistence. We identified a novel pattern of dynamics of helper cell populations associated with viral persistence, which is likely to be critical in the evolution of HCV towards persistent infection.Seven HLA-DR1 positive patients with acute hepatitis C were included in this study. All patients were available for study within the first 3 months after onset of symptoms. Acute hepatitis C was diagnosed by documented seroconversion to anti-HCV antibodies or all of the following: acute onset of hepatitis in previously healthy individual, aminotransferases at least 10× the upper limit of normal, exclusion of other infectious, metabolic, or toxic causes of hepatitis, recent exposure or source of infection identified. The clinical characteristics of the patients are summarized in Five HLA-DR1 positive patients with established chronic hepatitis C  and two long-term recovered patients  were studied for CD4+ T cell responses in peripheral blood . In six Four healthy and four HIV-infected HLA-DR1-positive individuals, as well as two healthy and five acutely HCV-infected HLA-DR1-negative individuals (n = 15) served as controls.www.shcs.ch) that have all given informed consent for use of clinical data, viral sequencing and genetic analyses. The HIV positive controls were recruited from an ethically approved study of St Mary's Hospital, London, UK (protocol 99/IA/161E).All patients gave informed consent to participate in the study and the protocol and the procedures of the study were conducted in conformity with the ethical guidelines of the Declaration of Helsinki. The patients with acute and chronic hepatitis C were recruited at the University Hospital Munich or at the University of Freiburg  comprising the non-structural protein 4 (NS4) or/and the non-structural protein 3 (NS3) region of the HCV polyprotein . Proteins were expressed as COOH-terminal fusion proteins with human superoxide dismutase in yeast (Saccharomyces cerevisiae). Protein was >90% pure.Eighty-three overlapping peptides (20mers) covering the region amino acid (aa) 1207–2014 were synthesized by Chiron Mimotopes  and 301 overlapping peptides (15mers) covering the entire HCV polyprotein were synthesized by EMC .4 cells/well) were incubated in 96-well U-bottom plates  for 5 days in the presence of HCV proteins  in 150 µl of tissue culture medium  containing 2 mM L-glutamine, 1 mM sodium pyruvate , 100 U of penicillin  per ml, 100 µg of streptomycin  per ml and 5% human AB serum . Cultures were labelled by incubation for 16 h with 2 µCi of 3H-thymidine . Triplicate cultures were assayed routinely, and the results expressed as mean counts per minute. The stimulation index (SI) was calculated as the ratio of counts per minute obtained in the presence of antigen to that obtained without antigen. A SI of >3 was considered significant.Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Hypaque gradients  and washed four times in phosphate buffered saline (PBS). PBMCs . After 48 h incubation, cells were washed off and the biotin-conjugated anti-IFN gamma antibody  was added. After an incubation period  unbound antibody was washed off and 100 µl of the streptavidin-alkaline phosphatase  was added for 1 h. Unbound conjugate was washed off and, finally, 100µl of 5-bromo-4-chloro-indolylphosphate/nitroblue tetrazolium substrate solution  was added for 2 h. The colour reaction was stopped by extensive washing. After drying the number of spots was scored by use of a dissection microscope.Nitrocellulose-bottom 96-well millititer HA plates  were coated with 100 µl of IFN-gamma monoclonal antibody , at a concentration of 15 µg/ml in PBS. Plates were incubated at 4°C overnight; before use unbound antibody was removed by excessive washing with PBS. The coated wells were filled with 100 µl tissue culture medium containing 2×10a Protein-conjugated anti-CD14, Peridinin Chlorophyll-a Protein-conjugated anti-CD19, Viaprobe (Becton Dickinson), and FITC-conjugated anti-CD38 monoclonal antibody (Becton Dickinson) were added after at least 20 minutes of incubation. Cells were washed twice and then incubated with anti-PE microbeads for 20 minutes at 4°C. Cells were then washed once and 90% of cells were applied to MS columns  according to the manufacturer̀s instructions. The other 10% were reserved for FACS analysis. The PE positive cells were eluted from the column (post-enrichment sample) and analyzed by FACS. Cells were gated on the CD4+, CD14-, CD19- and Viaprobe-population.The DRB1*0101 tetramer complexed with HCV 1806-1818 (TLLFNILGGWVAA) was custom synthesized by Beckman Coulter Immunomics . PBMC were stained in 100 µl medium  with 3 µl of Phycoerythrin (PE)-conjugated MHC class II tetramer for 2 hours at room temperature. Allophycocyanin-conjugated anti-CD4, Peridinin Chlorophyll-Frequencies of tetramer-positive CD4+ T cells were determined as described previously 6 irradiated autologous PBMCs as feeder cells.Biopsies were obtained in patients with chronic hepatitis as clinically indicated. From material not needed for histological analysis, lymphocytes were isolated and expanded as explained previously Viral RNA was extracted from plasma samples using either the QIAamp Viral RNA Mini Kit (QIAGEN) or the COBAS AMPLICOR HCV Specimen Preparation Kit v2.0 (Roche) according to manufacturer's instructions. An initial RT-PCR using the primers 2412F2 (CACCTCCACCARAACATYGT) and 9192R (GGAGTGAGTTTGAGCTTGGT) in combination with the SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase PCR Kit (Invitrogen) was performed. The first-round product was then used in nested 2nd round PCRs to amplify the relevant NS4 region with the following primer pairs, HCV-5040F/HCV-HCV-6121R (CACATAGATGCCCACTTCCT/GCCCCGGGAGGCRAASGC) and HCV-6076F/HCV-7636R (CTGTGCARTGGATGAACMG/TGATGAGGGCSCCKGTCCA) or alternatively the genotype 1-specific primers 5517M13F/6531M13R (GAGCARTTCAAGCAGAAGGCSCTC/AGGGGCCCGTGGTGTABGCGTT) together with the Platinum Taq DNA Polymerase High Fidelity Kit (Invitrogen). PCR products were purified using the Exosap method (Amersham) and BigDyeTerminator (v3.1) sequencing reactions were set up with PCR or M13 primers. The samples were run on an ABI 3730 Genetic Analyser and electropherograms analysed using Seqscape v2.0 (ABI).DNA was obtained from whole blood using the QIAamp DNA Blood Mini Kit following the manufacturer's guidelines. HLA Class II typing was performed by PCR and/or direct DNA sequencing using previously described methods 6 CD4+ T cells For tetramer synthesis we selected the epitope HCV-1806-1818 which has previously been shown to be a highly targeted epitope in HLA-DR1 positive patients with acute or resolved hepatitis C 6 CD4+ T cells that were analysed, corresponding to a mean frequency of 0.00016±0.00017% of CD4+ T cells (In the controls a mean of 0.73±0.75 tetramer+ CD4+ T cells (range 0–2) were found among a mean of 0.5×10 T cells .Representative dotblots of the HLA class II tetramer staining in patients AR1 and AC1 before and after enrichment are shown in A cohort of seven HLA-DR1 positive persons with acute hepatitis C was available for study . Five ofThe two other HLA-DR1 positive individuals with acute hepatitis C were available for study at a single time point one and four weeks after onset of disease, respectively. One was still viremic after 4 months and was treated successfully with antiviral therapy (AC4), and the other was lost to follow-up (AX).In four of the five longitudinally studied patients  proliferation assays with overlapping peptide libraries were performed to define the hierarchy of immunodominant epitopes . One pepTetramer+ CD4+ T cells were detected in all seven patients with acute hepatitis C, irrespective of clinical outcome, with frequencies ranging from 0.007% to 0.65% (mean 0.16%+/−0.2). Representative stainings from patients AR1 and AC1 showing both pre-enrichment and post-enrichment samples are displayed in In patient AR1, the highest frequency of tetramer+ CD4+ T cells  was measured at the earliest time point which was 2 weeks after spontaneous clearance of HCV-RNA from the serum. Tetramer+ CD4+ T cells subsequently declined to a level of about 0.1% and remained stable for more than 3.5 years of follow-up. Very strong peptide specific T cell proliferation and NS4-specific IFN-gamma secretion were present during the first 6 months after disease onset, and -despite some fluctuations-robust responses were maintained during long-term follow-up.In patient AR2, relatively high levels of tetramer+ CD4+ T cells (0.12%) and significant T cell proliferation were present during the phase of viral clearance. At week 12 the frequency of tetramer+ CD4+ T cells declined by one log and proliferation was lost. Despite the continuing absence of HCV-RNA from the serum at that time, the constantly elevated ALT levels suggested a high risk of viral relapse and interferon-alpha treatment was initiated. The frequency of tetramer+ CD4+ T cells recovered to the initial levels and finally remained around 0.05% during the follow-up after antiviral therapy. T cell proliferation was detected only intermittently during and after interferon-alpha treatment.All three patients with chronic evolution went through a phase of undetectable serum HCV-RNA in the first six months of disease. In all three, tetramer+ CD4+ T cells and significant antigen-specific proliferation were present during viral clearance and the first phase of viral control; antigen-specific proliferation, however, was completely lost in all three patients when viral relapse occurred. In patients AC2 and AC3 simultaneous IFN-γ Elispot assays revealed that IFN-γ secretion was absent even earlier. In patient AC3 this occurred three weeks before the loss of proliferation; in patient AC2 IFN-γ secretion could never be detected. Importantly, in AC2 and AC3 the loss of function occurred while the frequency of circulating tetramer+ CD4+ T cells was relatively stable at levels between 0.04% and 0.14%. While in patient AC2 the frequency of tetramer+ CD4+ T cells subsequently declined and eventually specific cells disappeared, successful interferon therapy in AC3 was associated with stable levels of tetramer+ CD4+ T cells although only a modest recovery of function was measurable. A different pattern was seen in patient AC1: here, the tetramer+ CD4+ T cells had already disappeared from the peripheral blood before viral recrudescence and only very low frequencies (0.003%) could be detected, intermittently, during the subsequent months. Importantly, in no case was the loss of HCV-specific CD4+ T cell function accompanied by a relevant decrease of recall antigen (tetanus toxoid) or mitogen (PHA) responses.Longitudinal phenotyping for CD38 was routinely performed with all tetramer stainings; in the first samples that could be studied, between 5.8 and 32% of tetramer positive cells expressed CD38. In contrast, at all time points beyond 6 months, CD38 expression was negative in both recovered and chronically infected patients. Representative experiments are shown in In HLA-DR1 positive patients with chronic hepatitis C a mean of 0.0032%+/− 0.0025 of CD4+ T cells stained tetramer+ , which is more than 1.5 logs below the mean frequency of tetramer+ CD4+ T cells in patients in the early phase of acute hepatitis C, although still in some cases higher than the control stainings (DR1+HCV- and DR1-HCV+) which were all <0.001%  whereas in parallel, using similar antigen-independent stimulation techniques, in the same cohort of patients substantial class I tetramer+ CD8+ T cell populations were visualised http://www.bioafrica.net/GDElinux/GDEmicrobial.html).Another explanation for the clear loss of tetramer+ populations, and loss of responsiveness associated with chronic infection would potentially be immune escape within the target epitope. To address this question, we sequenced early and late viral samples from patients AC1 and AC2. In patient AC3 no HCV sequence could be obtained probably due to the low viral load before antiviral therapy was initiated. In both early and late samples the sequence within epitope 1805-1824 corresponded to the prototype sequence . To furtIn this study we used a novel MHC Class II peptide tetramer for the detection of HCV-specific CD4+ T cells in HLA DR1+ patients with different stages and courses of HCV infection. This is the first HCV specific HLA class II tetramer to be tested and validated using epitope specific CD4+ T cell clones and we could demonstrate that this tetramer possesses similar sensitivity and specificity to HIV or influenza specific HLA class II tetramers characterized in a similar manner The major novel aspect of this study was the analysis of tetramer+ populations at early time-points after HCV infection, and most importantly, longitudinal analysis of the fate of these virus-specific populations in patients with a different clinical outcome of acute hepatitis C. The data clearly show that regardless of the long term clinical outcome, CD4+ T cells specific for this immunodominant epitope are present in all patients with acute disease. Interestingly, the frequency of tetramer+ populations in our cohort with acute hepatitis C was among the highest ever reported with HLA class II tetramers in viral infection studied ex vivo The subsequent decline of tetramer+ CD4+ T cells mirrors the kinetics of HCV specific CD8+ T cells in acute hepatitis C that have been studied with HLA class I tetramers Possibly the most informative facet of acute hepatitis C is the phase of transient viral control and subsequent viral recrudescence in the majority of patients who eventually develop chronic hepatitis C. It has previously been described that viral relapse is associated with a loss of T cell proliferative responses to HCV antigens In conclusion, using a novel HLA-DR1 tetramer complexed with an immunodominant CD4+ T cell epitope, we could show that HCV specific CD4+ T cells are induced in each patient with acute hepatitis C irrespective of outcome. Whereas spontaneous viral clearance is associated with long term maintenance of tetramer+ CD4+ T cells, early loss of function followed by depletion of tetramer+ CD4+ T cells is observed in patients with evolving chronic hepatitis C. The impact of such T cell loss may be very significant, undermining both CD8+ T cell-mediated and B cell mediated immune responses in the long term which can be associated with accentuated escape from CD8+ T cells as has been observed in model and natural infection


Patients with HIV infection are at risk of co-infection with HBV, as the routes of transmission are shared and thus immunization with HBV vaccine could be protective in them. The aim of the present study was to assess the efficacy of recombinant vaccine in treatment-naive HIV positive patients and healthy controls, and to dissect out differences if any, in different limbs of immune response.Forty HIV positive patients and 20 HIV negative controls, negative for HBsAg, HBsAbs and HBcAbs were vaccinated with three doses of 40μg and 20μg of vaccine respectively. Patients were divided into high CD4 and low CD4 group based on CD4+ lymphocytes of 200 and < 200/mm3 respectively. Group II consisted of healthy controls. Detection of phenotypic markers was done by flowcytometry. Cytokine estimation was done by sandwich ELISA. HBsAbs were estimated in serum by ELISA.4+, CD8+ and CD3+ cells increased significantly in all the groups. There was no increase in NK cell activity in patients with high CD4+ lymphocytes and only a marginal increase in patients with low CD4+ lymphocytes (170 to 293/mm3) whereas a marked increase was observed in controls (252 to 490/mm3). After vaccination, although an increase in memory cells was observed in HIV positive patients, yet HBsAb levels were significantly lower than controls (P < 0.05) indicating a functional defect of memory cells in HIV/AIDS patients. Basal IFN-γ levels were also significantly lower in HIV/AIDS patients (P < 0.01). Although the levels increased after vaccination, the peak level remained lower than in controls. HBsAb titers were much lower in HIV positive patients compared to controls. . IL-4 and IL-10 were low in patients.After vaccination, CD4+ counts and lack of T help, accounted for low HBsAb levels. Vaccination in patients with CD4+ lymphocytes < 50/mm3 was ineffective. Thus early immunization is advocated in all HIV positive patients at a stage when they are still capable of mounting an adequate immune responseDespite a double dose in patients, IL-4 and IL-10, which regulate antibody response, were also lower in patients, and this together with low CD By the end of year 2004 nearly 5.1 million people were living with HIV/AIDS in India .Acute hepatitis caused by HBV is milder in HIV infected patients but chronic disease is more frequent with a poorer prognosis and increased infectivity . PatientAlthough substantial literature is available regarding antibody response to different HBV vaccines in Caucasians, yet precise events pertaining to cellular immune responses, which are crucial for an optimal immune response are either sparse or their correlation with antibody response is imperfect. Further, since immune responses are linked to environmental, genetic and nutritional factors, these are likely to vary in different geographical areas in different clades and different population groups. Thus, data obtained from the western countries cannot be reliably extrapolated to Indian population. The present study was aimed to assess the efficacy of an indigenous HBV vaccine in HIV positive patients harboring mainly clade C  and to sDiagnosis of HIV was established as per National AIDS Control Organization (NACO) guidelines (WHO criteria adopted by NACO) . HIV inf4+ lymphocytes, group IA, high CD4 group with an absolute CD4+ lymphocyte count of ≥ 200/mm3 and group 1B, low CD4 group with an absolute CD4+ lymphocyte count of < 200/mm3. Group II consisted of 20 normal healthy control subjects.HIV infected patients and controls were screened for HBsAg and HBsAb by ELISA using Monalisa HBsAg and Monalisa antiHBs 3.0 kits . Subjects positive for HBsAg, HBsAb and HBcAb were excluded from the study. A total of 40 asymptomatic HIV positive patients and 20 control subjects were included in the vaccination and immune assessment study under 2 groups. HIV positive patients were subdivided into two groups on the basis of their CDAll the patients and controls received three doses of recombinant Senvac™ HBV vaccine  in the deltoid region at 0, 1 and 6 months. All HIV positive patients were given 40 μg (double dose) of vaccine in each dose, while controls subjects received 20 μg  of vaccine.Phenotypic characterization of immune cells was done by flowcytometry on EDTA blood using FACSCAN flowcytometer with cell quest software . CD4+ and CD8+ lymphocytes (helper/cytotoxic) were studied using Simultest CD4/CD8 monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC)/phycoerythrin (PE) respectively. CD3+ T cells (pan-T cell) and CD16+56 NK  cells were studied using Simultest CD3 -FITC, CD16+56 -PE monoclonal antibodies. Naïve T lymphocytes were studied using CD45RA-FITC monoclonal antibodies and memory cells were enumerated using CD45RO -PE monoclonal antibodies. The samples for flowcytometry were processed according to the method of Jackson and Warner [Functional characterization of TH1 and TH2 type of T cells was performed on heparinised venous blood according to the method of Luty et al [5/200 μl. Cultures were stimulated with 10% phytohemagglutinin [PHA-P (DIFCO)] in RPMI-1640 medium (Sigma) and without PHA-P (unstimulated). Cultures were incubated for 20 hrs at 37°C in an atmosphere of 5% CO2. The samples were centrifuged at 2000 rpm for 5 minutes and the supernatant stored at -70°C until required for cytokine assay. Cytokines  were estimated in the culture supernatants by sandwich ELISA using reagents from Pharmingen, (USA) as per manufacturer's instructions. Briefly, culture supernatants were distributed in ELISA plates coated with corresponding anti-cytokine antibodies. Corresponding detector antibodies were used to detect cytokine anti-cytokine complexes. The reaction was developed with TMB in 0.1 M sodium acetate solutions and H2O2, OD was recorded at 450 nm. The concentration of the cytokines in culture supernatants were calculated from the standard curve for each cytokine plotted on a log-log paper. Functional characterization of B cells was done by estimation of sequential levels of HBsAb by ELISA.ty et al  with min1 and TH2 subsets of T helper cells and levels of HBsAbs were estimated at baseline (B), before and 11 days after second (C & D) and before and 11 days after the third (E & F) dose of vaccine. The timing was selected to pick up the peak titers  and 11 days after second (D) and the third (F) dose of vaccine. Functional characterization of TH4 count, 17 were males and 10 were females and the age ranged between 22 to 45 years with mean age of 30 ± 6 years. Out of 13 patients with low CD4 counts, 6 were males and 7 were females and the age ranged between 18 – 50 years with mean age of 32 ± 8 years. Of the 20 normal controls, 12 were males and 8 were females and the age ranged between 18 – 45 years with mean age of 29 ± 7 years.Out of 27 patients with high CD4+lymphocyte counts were highest in controls followed by patients with high CD4 counts and those with low CD4 counts in that order  . In controls, the NK cells increased significantly after each dose of vaccination . After vaccination, patients with high CD4 counts showed a significant increase in CD45RA+ naïve cells after each dose of vaccination whereas patients with low CD4 counts and normal control subjects showed increase in CD45RA+ naïve cells after the third dose of vaccination  but 77% of patients had statistically significant levels of HBsAb at stage D i.e. 11 days after second dose . Subsequently 46% of patients had transient lowering of HBsAb levels at stage E (524 ± 1478 mIU/ml p > 0.05). After third dose of vaccination at stage F, 100% of patients with high CD4 counts showed statistically significant levels of HBsAb [8034 ± 14136 mIU/ml, p < 0.01, .In comparison, only 47% of patients who had a CD4 counts where 100% of patients responded, yet the mean peak titers (60000 mIU/ml) were much higher in controls than in patients with high CD4 counts   Table . HBV vac5) Table .Basal IL-10 levels were also significantly higher in normal control subjects as compared to HIV positive patients and even after vaccination, IL-10 levels remained significantly higher in control subjects as compared to HIV positive patients Table . HBV vacBasal IL-12 levels were almost similar in control subjects and HIV positive patients. After vaccination, all patients showed delayed but significant increase in IL-12 levels whereas in controls there was massive though transient increase in IL-12 levels Table .4 counts was insignificant. However after vaccination, levels increased significantly in patients after 6 months. In controls, no further increase of IFN-γ levels could be registered. Yet levels were significantly higher than in patients (Table Baseline IFN-γ levels were much higher in control subjects as compared to HIV positive patients (p < 0.01) but the difference in basal IFN-γ levels in patients with high and low CDts Table .It is known that HIV infection may modify the natural history of HBV infection and HIV positive patients have higher rate of HBV chronicity, higher HBV replication and low rates of sero-conversion to anti-HBe and anti-HBs ,11. Immu4+ lymphocyte number, percentage and decreased CD4: CD8 ratio occurs early during the course of HIV infection and are predictors of disease progression [4+ lymphocytes and changes if any, in the number of CD4+ lymphocytes after HBV vaccination. The basal CD4+ cell count in Indians were found to be lower than Caucasians (743± 214). Response to any vaccine is dependant on adequate function of CD4+ helper cells. In the present study also, response to HBV vaccination was directly correlated with CD4+ lymphocyte number. After the second dose of HBV vaccination, 92% of patients with high CD4 counts, showed a significant increase in the number of CD4+ lymphocytes (peak of 1135/mm3) compared to only 61% of patients with low CD4 counts (peak of 472/mm3). In control subjects, the increase was more impressive after the third dose and peak levels were much higher (1531/mm3). It is important to note that mean CD4+ lymphocytes increased significantly after vaccination at each stage irrespective of the disease status.Decreased absolute CDgression . We anal8+ lymphocytes are responsible for eliminating virus infected cells and act as a second line of defense by becoming activated and eliminating any infected cell, despite the presence of neutralizing antibodies [8+ lymphocytes after HBV immunization. In the present study, average number of total circulating CD8+ lymphocytes in control subjects was 756/mm3, which is higher than the figures (500/mm3) reported in Caucasians [8+ cells was much higher in HIV positive patients. After immunization CD8+ T lymphocytes increased further in all the groups. It has earlier been documented that HIV is associated with a functional defect of CD8+ cells. Wilson et al [8+ CD38+ HLA DR+ T cells. In murine studies it has been observed that repeated exposure to unrelated pathogens, as happens in HIV infected patients, may adversely affect viral specific CD8+ lymphocytes [CDtibodies . They catibodies . Howeverucasians . Mean vaon et al  had repophocytes .4:CD8 ratio in normal control subjects was approximately 1, which is also distinctly different from the values reported in the west where the ratio is close to two [et al [4+ cell number, a higher CD8+ cell count and CD4:CD8 ratio of 1 in healthy North Indians.At baseline, CDe to two . Our dato [et al  who also4+ and CD8+ cells, NK cells are key cells that deal with virally infected cells. Majority of peripheral blood lymphocytes that mediate NK cytotoxic activity in healthy adults express both CD16 and CD56 . Using these two NK cell markers, a decrease in NK cell number has been reported in HIV infected individuals [4 counts and normal control subjects. After HBV vaccination, NK cells in controls increased after each dose unlike in patients with high CD4 counts where no significant difference was observed. Patients with low CD4 counts showed a significant increase only after the last dose of vaccination (170 vs. 293/mm3 p < 0.05). The present study thus illustrates that NK cell response is also compromised in HIV/AIDS.Besides CDividuals ,19. In t4+ lymphocytes with the memory phenotype harbor more HIV provirus and hence are candidates for depletion by HIV cytopathic effects following activation and subsequent stimulation of viral replication [In HIV infection, CDlication . Althouglication , subsequlication ,23.In the present study naïve cells were significantly lower in patients with severe disease while the number of memory cells was increased in HIV/AIDS patients. In spite of this, HBsAb levels were lower than controls thus confirming a functional defect.In our study, HIV infected vaccinated patients failed to elicit adequate IL-4 response. IL4 and IL-10 are the two key cytokines facilitating antibody response. Klein et al  also demIn the present study IFN-γ levels were much higher in controls compared to both the groups of HIV infected patients. Further, the response was delayed in patients and the IFN-γ levels could not match the levels observed in controls indicating a lack of T cell help. IL-12 levels are documented to be low in plasma of HIV infected subjects  but we c4+ lymphocytes and low TH2 type of cytokines i.e., IL-4, IL-10 which promote antibody function and functionally abnormal memory cells possibly accounted for low antibody titers in HIV infected patients.Our study illustrates that HBsAb titers were highest in controls followed by seropositives and minimum in patients with AIDS. Low CD4 counts of > 500/mm3 was 87% while the response rate dropped to 33% in patients with a CD4 count between 200–500/mm3. In our study only 6/40 patients had CD4 counts of > 500/mm3, thus the response rate of 100% in our patients with a CD4 count between 200–500/mm3 is encouraging. These patients also gained weight and depicted a rise of CD4+ lymphocytes. The response rate in controls receiving the conventional dose was 90%. Tedalli et al [4 count of < 200/mm3 should be treated with HAART first and HBV vaccine given preferentially after the cut off of 200/mm3 is achieved. This, however, is not possible in resource poor countries. In an excellent comprehensive study on care of patients with chronic HBV [Diaz et al  stressedonic HBV , the intonic HBV .4 count, in spite of the double dose of vaccine, only 47% were responders and with poor titers against HBV and patients with CD4 counts of < 50/mm3 did not respond at all. Thus early immunization is advocated in all HIV positive patients at a stage when they are still capable of mounting an adequate immune response.In our patients with low CDThe author(s) declare that they have no competing interests.3 and AS recruited the HIV positive patients for the study. SA was the co-guide in the project. RM and BS helped in the documentation and statistical evaluation of the data. SS1 formulated the project and designed the study.NP carried out the phenotype and cytokine assays and the main bench work. UD guided (main guide) and provided the funds. YC did the liver workup of patients and HBV ELISAs. SSThe pre-publication history for this paper can be accessed here:


However, some individuals remain at risk for opportunistic diseases despite prolonged normalization of CD4+ T cell counts. Deficient Epstein-Barr virus (EBV)-specific CD4+ T cell function may explain the occurrence of EBV-associated opportunistic malignancy—such as primary central nervous system (PCNS) lymphoma—despite recovery of absolute CD4+ T cell counts.In chronic HIV infection, antiretroviral therapy–induced normalization of CD4+ T cell counts and EBV-specific CD4+ T cell-dependent interferon-γ production were assessed in six HIV-positive individuals prior to development of PCNS lymphoma (“cases”), and these values were compared with those in 16 HIV-infected matched participants with no sign of EBV-associated pathology (“matched controls”) and 11 nonmatched HIV-negative blood donors. Half of the PCNS lymphoma patients fulfilled IR criteria (defined here as CD4+ T cell counts ≥500/μl blood). EBV-specific CD4+ T cells were assessed 0.5–4.7 y prior to diagnosis of lymphoma. In 0/6 cases versus 13/16 matched controls an EBV-specific CD4+ T cell response was detected . PCNS lymphoma patients also differed with regards to this response significantly from HIV-negative blood donors , but there was no evidence for a difference between HIV-negative participants and the HIV-positive matched controls (p = 0.47).Absolute CD4+ T cell counts, HIV-positive patients who subsequently developed PCNS lymphoma lacked EBV-specific CD4+ T cell function. Larger, ideally prospective studies are needed to confirm these preliminary data, and clarify the impact of pathogen-specific versus surrogate marker-based assessment of IR on clinical outcome.Irrespective of absolute CD4 In a case-control study from the Swiss HIV cohort, Hess and colleagues report that T-helper responses against Epstein-Barr virus are specifically absent in patients developing CNS lymphoma. + T cells that recognize infection and enable other cells of the immune system to respond. Advanced HIV infection, marked by very low numbers of CD4+ cells, is associated with a variety of infections and tumors that are rarely seen in people with intact immune systems. People with advanced HIV who receive highly active antiretroviral treatment (HAART) tend to have increases in their CD4+ cell counts and lose their susceptibility to these so-called opportunistic infections and cancers. For several common opportunistic infections, it is considered safe to discontinue preventive antibiotics after a patient's total CD4+ cell count has returned to normal levels on HAART. Some treated individuals, however, will develop these conditions even after their CD4+ cell counts have returned to normal levels. The reason this happens is unclear.AIDS causes disease by inactivating the body's immune responses. Most severely affected are the white blood cells known as T lymphocytes, particularly the CD4+ cells, but to loss of the specific CD4+ cells that recognize the infection in question. If this theory is correct, then those individuals who develop an opportunistic condition after their total CD4+ cell counts return to normal might be missing the specific cells that respond to the microbe causing the condition. The researchers wanted to test this theory in HIV patients with a brain tumor called primary central nervous system lymphoma (PCNS lymphoma). The Epstein-Barr virus (EBV), which causes mononucleosis in the general population, has been shown to be a cause of PCNS lymphoma in people with AIDS.For several years, scientists have speculated that susceptibility to a given opportunistic infection might be due not simply to low total CD4+ cell counts prior to lymphoma diagnosis and three had normal CD4+ cell counts, but CD4 responses specifically against EBV were absent or very low in all six patients before they were diagnosed with PCNS lymphoma. The researchers also studied a comparison group of cohort participants with comparable CD4+ cell counts but no PCNS lymphoma, and found that 13/16 of those participants did have CD4 responses to EBV.The researchers studied patients who developed PCNS lymphoma while enrolled in the Swiss HIV Cohort, an ongoing study that has enrolled more than 14,000 people. A large cohort was needed to address this question because PCNS lymphoma is uncommon, and indeed only six patients with a confirmed diagnosis were identified. Because they had been followed as part of the cohort study, these patients had given blood samples that could be tested in retrospect. Three of these patients had low CD4+ cells, rather than a given level of total CD4+ cells, is needed to prevent PCNS lymphoma. Because only a small number of cases were identified, this must be considered a preliminary result. Given the rarity of PCNS lymphoma, however, especially in people receiving HAART, it seems unlikely that a larger cohort will be available in the near future to provide a more definitive conclusion. Based on this result, it may be useful to perform similar studies of other opportunistic infections. If a “gap” in the CD4+ cell response can be shown to increase the risk of a specific condition, it may become appropriate to test specific CD4 responses before deciding to discontinue preventive treatment as CD4+ cell counts increase on HAART.These results support the idea that the action of EBV-specific CD4http://dx.doi.org/10.1371/journal.pmed.0040096.Please access these Web sites via the online version of this summary at Perspective by Mark Jacobson, MDRead the accompanying Swiss Cohort Study Web site contains information on related research projectsThe HIV InSite includes resources on HIV immunology and opportunistic infectionsThe UCSF Center for HIV Information's  The use of antiretroviral therapy (ART) has dramatically changed the course of HIV infection. ART-induced immune reconstitution (IR) is reflected by the fact that the incidence of opportunistic infections as well as several AIDS-defining malignancies decreases with treatment [The cardinal feature of HIV infection is depletion of CD4patients ,2. Immunreatment –7. Specireatment ,9.+ T cell counts to levels ≥500/μl blood is used by some to define complete IR [+ T cell immunity in patients developing PCNS lymphoma. Here, the existence of a large cohort of HIV-infected individuals allowed us to study, in a controlled setting, predisease virus-specific CD4+ T cell function in rare individuals who developed PCNS lymphoma.An increase of CD4plete IR . Among i+ T cell counts and HIV viral loads  was matched as available with two or three HIV-positive patients who had not developed PCNS lymphoma (“matched controls”) for: (i) age ± 5 y, (ii) sex, (iii) history of intravenous drug abuse, (iv) use of ART, (v) time of documented HIV positivity, (vi) duration of follow-up, and (vii) the closest available match of median and range of CD4al loads . In bothhttp://www.sigmaaldrich.com). When using B cell lysate as the source of antigen, CD8+ T cells were depleted using anti-CD8 magnetic beads  following the manufacturer's protocol. Depletion was controlled for by FACScan-based enumeration, and was ≥98% complete (unpublished data).Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation through a density gradient on Histopaque-1077  were precoated overnight with 2 μg/ml of anti-IFN-γ mAb 1-D1K , and washed six times with sterile PBS containing 1% FCS before use. After washing, 30 μl of R10 culture medium was added to each well to avoid drying of the membrane, and 100,000 viable cells per well added in 100 μl of R10. The EBV-specific CD4+ T cell response was assessed using as the source of antigen a set of peptides consisting of 33 HLA II-restricted optimal epitopes [+ T cell-depleted samples, EBV-infected B cell lysates  [+ T cell depletion) [Virus-specific interferon-γ (IFN-γ) production was determined by ELISPOT analyses as described ,12. Brieepitopes ,12 and, sys.com) . For eacpletion) ,12, and 2 before being developed. After washing with PBS, 100 μl of biotinylated anti-IFN-γ mAb 7-B6–1  was added and plates incubated for 1 h at room temperature. Plates were washed again and incubated with a 1:2,000 dilution of streptavidin-coupled alkaline phosphatase  for 1 h at room temperature in the dark. After further washing, IFN-γ production was detected as spots after 10–20 min incubation with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate . The color reaction was stopped by washing plates, and plates were air-dried before counting with an AID ELISPOT Reader Unit . Results are expressed as spot-forming cells (SFC) per million input cells. Thresholds for positive responses were determined as the mean plus three standard deviations of negative control wells.Plates were incubated for 16 h at 37 °C with 5% COhttp://www.genbio.com).EBV-VCA, and EBNA1-specific IgG antibody levels were determined using commercially available ELISA kits designed to detect IgG antibody bound to antigen-coated microtiter plates, following the manufacturer's protocol .Viral DNA quantification was performed in serum or plasma samples using routine TaqMan real-time PCR technique  was examined with an exact Mantel-Haenszel test of conditional independence. As sensitivity analysis, the cut-off of 0 SFCs was replaced by 100. Also, the patient's outcome was modelled using exact conditional logistic regression with the logarithm of the patient's median (or mean) measurement of the EBV-specific CD4+ T cell response  as a covariate. Samples from HIV-negative volunteers were nonmatched, and comparisons were performed with Fisher's exact test. The Wilcoxon test was used to quantitatively compare median EBV-specific CD4+ T cell responses in HIV-negative controls with median CD4+ T cell responses in HIV-positive matched controls. All statistical tests were two-sided and performed at the 5% significance level. Statistical analyses were performed with SAS v9.1 software .If an individual patient's SFC measurements were 0 at more than 50% of available time points or, equivalently, if the patient's median over all available time points was 0, an EBV-specific CD4In a search of the Swiss HIV Cohort Study database, six HIV-positive individuals who developed PCNS lymphoma were identified (cases 1–6). Diagnosis was established via biopsy in cases 1, 2, 3, and 5, and via imaging, detection of EBV in cerebrospinal fluid, and exclusion of alternate diagnoses in cases 4 and 6. EBV-specific antibodies were detected in all PCNS lymphoma patients more than 2.5 years prior to diagnosis of lymphoma, and in all matched controls. The search criteria applied for selection of control patients are described in the Methods, and patient characteristics are summarized in + T cell-mediated IFN-γ production was assessed covering a range of 0.5–4.7 y prior to diagnosis of lymphoma. The n-number of time points at which functional assays were performed in each patient is listed in + T cell-response was detected  . Absolute CD4+ T cell counts in samples tested from each of the controls were ≤ absolute CD4+ T cell counts of samples tested from cases, thus excluding the possibility that the observed disparities were driven by differences in CD4+ T cell counts rather than by pathogen-specific CD4+ T cell function. The maximum SFC measurement at each available time point for each case and matched control patient is listed in Three of the six PCNS lymphoma patients fulfilled IR criteria (cases 1–3) . EBV-spe+ T cell-mediated IFN-γ production in 11 randomly selected HIV-negative blood donors . The median EBV-specific CD4+ T cell-response in HIV-positive and HIV-negative controls was 403 (range 0–905) and 130 (range 40–840), respectively, and there was no evidence of a difference in these responses (p = 0.47).We also assessed the EBV-specific CD4d donors B, right.+ T cell activity from specifically decreased anti-EBV responses in the participants who developed PCNS lymphoma, we tested the CMV-specific CD4+ T cell-response in CMV-positive cases (n = 4), and compared it to CMV-positive controls (n = 4). Responses were similar, with medians of 540 SFC/106 PBMCs  and 280 SFC/106 PBMCs , respectively. In CMV-negative cases and controls no CMV-specific CD4+ T cell response was detected.Aiming at distinguishing generally depressed CD4To further test for the specificity of a possible immune deficit, CMV viral loads were assessed longitudinally in these same CMV-positive cases and controls. In each patient four to 11 samples were tested . CMV DNA was detected in only two of 29 versus one of 29 samples in CMV-positive patients versus control participants, respectively. Thus, based on circulating DNA-levels, CMV was well controlled in both cases and controls.+ T cell counts <50/μl). Intriguingly, since the introduction of ART, PCNS lymphoma has also been reported in individuals with CD4+ T cell counts over 200/μl or even over 500/μl [Although PCNS lymphoma can develop in HIV-negative individuals, it much more often occurs in severely immunosuppressed HIV-infected persons  undetectable CMV replication suggest that CMV-specific immunity was adequate in both cases and controls.Here we observed—irrespectively of absolute CD4+ T cell function irrespective of absolute CD4+ T cell counts likely represents a more general phenomenon. Case reports and a small case-control study have reported clinically significant CMV and Pneumocystis jiroveci infection in individuals with elevated absolute CD4+ T cell counts, and pathogen-specific CD4+ T cell deficiency has been suggested to persist in these patients [+ T cells is consistent with a recent report showing that during acute simian immunodeficiency virus infection, up to 60% of all CD4+ T memory cells throughout the body are irreversibly lost [+ T cells with specificity for a particular pathogen. Such thorough depletion may then be largely unresponsive to long-term control of HIV replication by ART and subsequent replenishment of absolute CD4+ T cell numbers.The lack of EBV-specific CD4patients –20. The + T cell effector function and/or help for cytotoxic CD8+ T cells may provide an immunological basis for development of PCNS lymphoma in HIV-infected individuals [Absence of EBV-specific CD4ividuals –23. LargFigure S1Shown are progressors (cases) before diagnosis (left) and matched control participants (right). Each dot represents the maximum SFC result for a given participant at a given time point. Overlapping dots are distributed on the x-axis to remain separated.(32 KB PDF)Click here for additional data file.Table S1(33 KB DOC)Click here for additional data file.Table S2(64 KB DOC)Click here for additional data file.


Why this decline is slow is not understood. One potential explanation is that the low average rate of homeostatic proliferation or immune activation dictates the pace of a “runaway” decline of memory CD4+ T cells, in which activation drives infection, higher viral loads, more recruitment of cells into an activated state, and further infection events. We explore this hypothesis using mathematical models.The asymptomatic phase of HIV infection is characterised by a slow decline of peripheral blood CD4+ memory T cell loss. Instead it predicts the rapid attainment of a stable set point, so other mechanisms must be invoked to explain the slow decline in CD4+ cells.Using simple mathematical models of the dynamics of T cell homeostasis and proliferation, we find that this mechanism fails to explain the time scale of CD4+ T cell activation and proliferation drive HIV production and vice versa cannot explain the pace of depletion during chronic HIV infection. We summarize some alternative mechanisms by which the CD4+ memory T cell homeostatic set point might slowly diminish. While none are mutually exclusive, the phenomenon of viral rebound, in which interruption of antiretroviral therapy causes a rapid return to pretreatment viral load and T cell counts, supports the model of virus adaptation as a major force driving depletion.A runaway cycle in which elevated CD4 Using a simple mathematical model, Andrew Yates and colleagues show that a runaway cycle of T cell activation and infection cannot explain the slow rate of CD4 decline during chronic HIV infection. + T cells that recognize infection and enable other cells of the immune system to respond against it. Following rapid loss in the first few weeks of HIV infection, the number of CD4+ cells in the blood falls slowly, often over a period of ten years or longer. The exact mechanisms by which CD4+ cell depletion occurs are not fully understood.The AIDS virus causes disease by inactivating the body's immune responses. Most severely affected are the white blood cells known as T lymphocytes, particularly the CD4+ cell loss. In this description, CD4+ cells (like water in a sink) are constantly being eliminated by HIV (the drain), while the body is constantly replacing them with new ones (the tap). Over time, the tap cannot keep up with the drain, and CD4 counts begin to drop, leaving the body susceptible to the infections that define AIDS.For over a decade, researchers have used a “tap and drain” analogy to describe CD4+ cells that are activated in response to invading microbes (such as HIV) are highly susceptible to infection with HIV, and following infection these cells may produce many new copies of HIV before dying. Therefore, one popular explanation for CD4+ cell loss is the “runaway” hypothesis, in which CD4+ cells infected by HIV produce more virus particles, which activate more CD4+ cells that in turn become infected, leading to an ongoing cycle of CD4+ cell activation, infection, HIV production, and cell destruction. The authors wanted to investigate whether this hypothesis can explain why CD4+ cell levels fall slowly.CD4+ cells are produced and eliminated), the authors show that, if the “runaway” hypothesis were correct, then CD4+ cells would fall to low levels over a few months, not over several years. They therefore conclude that the “runaway” hypothesis cannot explain the slow pace of CD4+ cell depletion in HIV infection.Using a mathematical model (a series of equations to describe the processes by which CD4+ cells. Because of its simplicity, the model used in this study is convincing in its rejection of the runaway hypothesis, but a more detailed analysis will be needed to tell us precisely what the slow process is. One possibility raised by the authors is that this process is the slow adaptation of the virus itself over the course of infection. Specifically identifying this process will provide a key insight into the nature of HIV disease and indicate potential new approaches to therapy.These results show that a slow process must be active in the HIV-induced depletion of CD4http://dx.doi.org/10.1371/journal.pmed.0040177.Please access these Web sites via the online version of this summary at Perspective on this article by Rob J. de BoerRead the related HIV InSite Knowledge Base includes information on HIV immunologyUniversity of California, San Francisco's National Institute of Allergy and Infectious Diseases provides AIDS fact sheets and brochures that cover a variety of topicsThe  In the acute phase, virus appears to preferentially and efficiently target HIV-specific [+ T cells that reside in mucosal tissue [+ cells in the gut [+ cells. Studies in HIV-infected humans mirror these observations [+ T cell compartments is slowly eroded over a time scale of several years. What causes this slow loss is not known.Untreated HIV infection in humans is characterised by extensive depletion of CD4specific ,2 and efl tissue . Studiesrvations –7. Follo+ T cell activation and proliferation markers are observed in blood [There is considerable loss of uninfected cells in the chronic phase –12, suggin blood –22, and in blood .+ T cell compartments.This increase in IA and immunopathology may be a direct result of the immune cell interactions with HIV. An alternative hypothesis is that it is a secondary effect—the acute mucosal lymphopaenia causes the relocation of immunogenic gut antigens and systemic responses . In eith+ turnover and the slow decline in the blood remains unclear. The average rates of division and death (turnover) of all CD4+ subpopulations are increased, and these rates are fast compared to the overall rate of T cell depletion. These two key observations are often restated in terms of a “tap and drain” analogy [+ T cell numbers. Yet we still need to explain the mechanism underlying this imbalance, or the failure of T cell homeostasis.Nevertheless, the causal link between the increased CD4 analogy  in which+ T cells. Specifically, can this mechanism provide the slow time scale for decline of peripheral blood CD4+ memory T cells? In this paper we use a simple mathematical model of T cell dynamics to address this question. We begin by constructing a model of memory CD4+ T cell maintenance in health, and discuss how it changes in the presence of HIV.The increased susceptibility of activated or proliferating T cells to infection  suggests+ T cell pool comprises naive (antigen-inexperienced) cells and heterogeneous memory (antigen-experienced) populations. The mechanisms by which memory cells are maintained are not completely defined [+ T cells results in compensatory homeostatic proliferation [The CD4 defined , but sur defined . In uninferation . This obferation , or at tferation .+ T cell homeostasis that distinguishes between resting cells and cells that have received homeostatic division signals and so are transiently activated  compete for homeostatic division signals and receive them at an average rate a. The mean interdivision time of memory CD4+ T cells in healthy humans (1/a) has been estimated using deuterated glucose labelling and is approximately 15 d for effector memory CD4+ T cells, 48 d for central memory cells [a increases) under lymphopaenic conditions, as observed experimentally in mice (unpublished data). We assume that the death rate of resting memory CD4+ T cells (δ) is low [a(x) and δ(x). For the simulations we show below, we used the linear forms a(x) = a(1 − x/κ) and δ(x) = δ0 (x/κ), where κ is a constant that scales the size of the memory T cell population. With our choice of parameters, in a “full” memory CD4+ compartment the mean time between divisions is 60 d, decreasing to 30 d under lymphopaenic conditions, and the average lifetime of resting cells is 400 d.We assume that resting memory CD4ry cells , or 26 dry cells . Earlierry cells . We repr) is low ,37 and dy) and then return to the resting state at rate r, dividing once as they do so. The inverse of r is the average time for transit through the cell cycle from a resting state, which we choose to be 24 h, or r = 1 d−1 [+ memory cells are in an activated or dividing state y at any time in spleen and blood [μ) given a and r.In our model, cells that have received a homeostatic division signal enter an actively dividing state  determines the time scale for homeostatic turnover or recovery from lymphopaenia. Even if we conservatively use the slowest estimates of memory turnover rates and we neglect the increase in homeostatic proliferation observed under lymphopaenia, our basic conclusion is unchanged. We describe it in the following section.Our key observation here is that the rate + homeostasis  are more susceptible to HIV infection than resting cells (x). We also assume that infection of the homeostatically dividing cells occurs at a rate proportional to both the average viral infectivity p, a parameter that represents the efficiency of infection, and the viral load, which we take to be proportional to the number of infected cells at any time (z). Infected cells have a short lifetime . For no+ T cell numbers (x + y + z) decline to a set-point which is inversely related to p  cells make the dominant contribution to increased T cell turnover during HIV infection, which decreases rapidly and substantially when viral replication and new infection is blocked by ART [This model does not include immune-activated cells, which may be recruited from naive or memory CD4d by ART . This re+ compartments, rapid proliferation, and death, either through activation-induced cell death or by cytopathic infection. Immune-activated cells may also return to a resting memory state. In other words, we argue that the chronic IA picture is essentially of a form similar to Chronic T cell activation comprises several processes—recruitment from naive or resting memory CD4a*) per memory CD4+ T cell in untreated HIV infection is not known. The death rate γ1 has been estimated for acute LCMV infections [−1 for antigen-specific CD4+ T cells. This estimate may be reduced under conditions of chronic stimulation as in HIV infection. We choose the rate of return to resting memory γ2 to be 10% of the effector cell death rate. However, our results are insensitive to the choice of γ1 and γ2.The average rate of antigen or bystander activation  as might be expected intuitively (simulation results not shown). In a*, is smaller than the homeostatic activation rate a. In this case we see that the approach to a steady state is still too rapid to explain the progression of chronic HIV infection. The key point here is that in the presence of low average rates of IA, homeostatic activation provides a lower bound on the average time between T cell activation events and so determines the time scale of equilibration of the memory CD4+ T cell compartment. Thus other mechanisms must be invoked to explain the slow failure of homeostasis in HIV infection.When Resting cells may be infected—potentially with lower susceptibility—and generate virus upon activation. Including latent infection and assuming that these cells behave as normal resting memory cells, our conclusions are unchanged. Latently infected cells may be required to account for viral “rebound” after cessation of ART see .+ compartments from the naive population. Naive CD4+ T cells show very little homeostatic turnover under normal conditions and are likely to be replenished largely by thymic output, which may be significantly impaired in HIV infection [The results we describe above are robust to adding small influx terms into either resting or activated memory CD4nfection . Furthernfection . The rolnfection . Howeverp allows comparisons to be drawn with other measurements of virus infectivity in the literature. In our model the key quantity is the rate of infection per activated CD4+ T cell, or pz, where z is the number or density of productively infected cells. In models used extensively elsewhere in the HIV literature  to be in the range 0–0.2 d−1 for 100 < p < 1,000 and for all levels of IA. Steady state numbers of cells at around 50% of healthy numbers correspond to values of pz of approximately 0.1 d−1, in reasonable agreement with these studies.The predicted dependence of steady state pool size with example, ), the cor et al.  estimatePerelson  estimatep), average death rates will remain higher than healthy levels during the return to a higher homeostatic set point. This prediction of elevated cell death even during the recovery of cell numbers arises because in our model proliferating cells are more susceptible to death than are resting cells, as has been noted previously in other HIV modelling studies [Qualitatively, we note that our model predicts that even after inhibition of viral replication with ART  the probability of productive infection upon encounter between a virion and a susceptible T cell, and (ii) the dependence of plasma virion density on infected cell numbers. The first may change over the course of an infection through a switch in coreceptor usage [+ T cells ex vivo over time. We note that this measure of fitness is not necessarily related to virus diversity. Clinical evidence is conflicting in this regard, and the association between diversity and progression is unclear [+ T cells.Increasing the efficiency of infection, ur model  and 6, sor usage ,53 or pe unclear –56. A fuv in the model above) over time [Impaired T cell help and/or CTL exhaustion may decrease the rate of recognition and killing of infected cells by CTLs , which is likely to be an increasing function of the viral load and/or p. If reservoirs of latently infected cells persist during treatment, retaining a representative memory of the pretreatment virus population, the virus adaptation model predicts that allowing viral replication to restart will drive the memory CD4+ numbers quickly—on a time scale of weeks—to the pretreatment set point determined by p0, a*, and the remaining (constant) homeostatic parameters. Other models predict the rapid return to the previous set point only if the homeostatic parameters remain constant during treatment—that is, if there is not significant recovery of CTL function or repair of lymphoid architecture or T cell regenerative capacity while virus replication is suppressed.All of these models seek to explain the imbalance between the average rates of division and death across the CD4t levels . Assume Clearly the virus adaptation model is not the whole story. Assuming treatment effectively abrogates the production of virus, this model predicts the full reconstitution of the T cell memory compartment during sustained treatment, which is not usually observed. This discrepancy between the virus adaptation model and the observations implies an additional role for lasting impairment to homeostatic mechanisms in HIV infection, perhaps from attrition of homeostatic resources or through differentiation (senescence or exhaustion).+ T cell pool to reject the idea of slow runaway decline in HIV infection, in which homeostatic compensation and/or IA drives infection, higher viral loads, more recruitment of cells into an activated state, and further infection events—and intuitively should lead to erosion of the memory CD4+ compartment. We argue that this mechanism fails to explain the slow decline of this cell population in the chronic phase of HIV infection. The model indicates that the memory CD4+ T cell pool will reach a dynamic equilibrium or set point within weeks or months of the end of the acute infection, a time scale that is dictated by the average time between homeostatic proliferation events. However, the “runaway” model may well be an appropriate description of depletion in the early stages of infection, and particularly among responding HIV-specific cells. Our study highlights how understanding memory CD4+ T cell dynamics in chronic HIV infection requires a quantitative description of T cell homeostasis in health, as well as knowledge of how HIV affects the turnover and differentiation of T cell subsets.In this paper we have used a model of self-renewing memory CD4The ordinary differential equation models used in this study were simulated and analysed using Mathematica .Text S1+ T cell population, in the form of slow differentiation into an exhausted or less responsive phenotype induced by chronic exposure to HIV antigens or inflammatory signals, can induce a slow decline in total cell numbers.We illustrate with an extension to the model how adding heterogeneity to the memory CD4(303 KB PDF)Click here for additional data file.