Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection

Size: px
Start display at page:

Download "Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection"

Transcription

1 JOURNAL OF VIROLOGY, Feb. 2003, p Vol. 77, No X/03/$ DOI: /JVI Copyright 2003, American Society for Microbiology. All Rights Reserved. Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection Uma Malhotra, 1,2 Sarah Holte, 3 Tuofu Zhu, 4,5 Elizabeth Delpit, 4 Claire Huntsberry, 1 Alessandro Sette, 6 Raj Shankarappa, 7 Janine Maenza, 1,3 Lawrence Corey, 1,3,4 and M. Juliana McElrath 1,3,4 * Program in Infectious Diseases, Clinical Research Division, 1 and Program in Biostatistics, Public Health Sciences Division, 3 Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, and Department of Medicine, 2 Department of Laboratory Medicine, 4 and Department of Microbiology, 5 University of Washington School of Medicine, Seattle, Washington 98195; Epimmune, San Diego, California ; and Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania 7 Received 20 June 2002/Accepted 18 November 2002 Mounting evidence points to a role for CD4 T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n 28) or early (n 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%, P 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%, P 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN- ) secretion at multiple subsequent time points. Env-specific CD4 T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN- -secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur. Helper activities of antigen-specific CD4 T cells mediate control of many viral infections (2, 5, 21), and they appear critical in maintaining human immunodeficiency virus type 1 (HIV-1) suppression. This is best exemplified by the frequent detection of HIV-1 Gag-specific CD4 T-helper (Th) cells in untreated persons termed long-term nonprogressors (LTNP), who are infected for more than 12 years but do not develop HIV-1 disease (27). By contrast, most untreated persons with recent seroconversion or with chronic HIV-1 infection have low to undetectable levels of HIV-1-specific Th cells, particularly when measured in vitro by antigen-induced lymphoproliferation (19, 24). However, recent studies indicate that Th responses are enhanced in patients who substantially suppress viral replication with use of antiretroviral therapy (ART). This occurs predominantly in patients who initiate treatment soon after acquisition of infection (19, 27) and in those with chronic * Corresponding author. Mailing address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., D3-100, Seattle, WA Phone: (206) Fax: (206) kd@u.washington.edu. infection who receive prolonged ART (4, 11). The newly identified HIV-1-specific Th cells following therapy may represent clonotypes that were induced with initial infection but are present at a frequency too low to detect, or they may arise from naïve CD4 T cells as a result of immune reconstitution following suppression of viremia. One goal of this study was to clarify this issue by monitoring longitudinally patients with primary (acute or early) HIV-1 infection. The Th responses found to occur in LTNP and treated patients are predominantly directed against Gag epitopes (19, 27). In addition, persons who develop Gag-specific HLA- DR13-restricted responses are more likely to maintain viral suppression (20). However, little is known about the role of Th responses to epitopes expressed by other HIV-1 genes and how these responses evolve in early infection and following treatment. Such findings could support efforts to improve immunity against HIV-1 infection by augmenting the Th responses. We were particularly interested in investigating Env-specific Th responses for several reasons. Helper epitopes have been identified within the Env protein (3, 14, 17), and CD4 T-cell lines and clones recognizing Env epitopes can exhibit cytolytic 2663

2 2664 MALHOTRA ET AL. J. VIROL. activities (23). Also, we observed that in persons presenting with acute HIV-1 infection prior to seroconversion, Env-specific responses were often detected. By contrast, Env-specific responses were less frequently detected later in infection (26). In this study, we explored several hypotheses to understand the induction and evolution of the HIV-1 Env-specific helper response following acute infection. First, we sought to determine whether Env-specific responses preferentially emerge early in acute infection and whether delay in treatment leads to greater loss of these responses. Secondly, we explored the possibilities that the autologous viral strains may mutate within the Th epitopic region from immune pressure or that the env sequence of the wild-type strains may be considerably divergent from the env sequence expressed by the proteins used in the assays. It is well recognized that the env gene is highly variable among infected individuals and undergoes marked intraindividual diversification over the course of infection. Longitudinal studies of the evolution of the C2-V5 region of the env gene in HIV-1-infected subjects reveal a divergence rate of about 1% per year from the founder strain (29). Lastly, we considered that the detection of Env-specific Th cells can be limited by the sensitivity of the assay employed, if the memory precursor frequencies are low. Thus, responses were examined by several complementary methods (i.e., lymphoproliferation-assay, gamma interferon (IFN- ) secretion by enzyme-linked immunospot (ELISPOT) assay, and intracellular cytokine staining (ICS) by flow cytometry). MATERIALS AND METHODS Study population. Subjects with HIV-1 infection were recruited and enrolled at the University of Washington (UW) Primary Infection Clinic (PIC). The UW and Fred Hutchinson Cancer Research Center (FHCRC) human subjects review boards approved the study, and volunteers provided written consent prior to participation. The duration of infection was defined as the time elapsed since the onset of clinical signs and/or symptoms suggestive of acute retroviral syndrome. Determination of the exact time of exposure to HIV-1 and acquisition of infection is frequently not possible, and the time interval from exposure to appearance of symptoms can vary. Thus, the duration of infection was defined as the time since the onset of clinical signs and/or symptoms suggestive of acute retroviral syndrome. If symptoms were absent, the duration of infection was estimated as the midpoint between the last previous documented negative enzyme immunoassay and the recent positive confirmatory test. Some patients at various time points following diagnosis elected to receive combination ART with nucleoside reverse transcriptase inhibitors and either a protease inhibitor or a nonnucleoside reverse transcriptase inhibitor. Patients underwent clinical evaluation and venipuncture for virological studies weekly for the first month, then every 4 weeks for 1 year, and then every 3 months thereafter. Anticoagulated blood for immunological studies was obtained 1 or 2 times prior to treatment and then approximately every 1 to 3 months thereafter. Virological and T-cell subset analyses. The amount of HIV-1 RNA in plasma was determined by quantitative branched-chain DNA (bdna; Chiron, Emeryville, Calif.) and ultrasensitive reverse transcriptase-pcr (RT-PCR) (Roche Molecular Systems, Branchburg, N.J.) assays. Levels were expressed as copies/ milliliter, and the lower levels of sensitivity were 500 copies/ml (for the bdna assay) and 50 copies/ml (for the RT-PCR assay) (7). We report the viral levels measured by the bdna assay that were 500 copies/ml and those measured by the ultrasensitive RT-PCR assay when the result by bdna assay was 500 copies/ml. Absolute blood CD4 T-cell count was measured by consensus flow cytometry methodology. HLA typing. Class II alleles at the DRB1 and DQB1 loci were identified by PCR using sequence-specific primers (SSP) (Micro SSP DNA typing kit; One Lambda, Inc., Canoga Park, Calif.). The Puget Sound Blood Center Immunogenetics Laboratory, Seattle, Wash., performed these studies according to the manufacturer s instructions. LPA. Lymphocyte proliferation assays (LPA) were performed as previously described (19). In brief, fresh peripheral blood mononuclear cells (PBMC) were plated at 10 5 cells/well in quadruplicate wells containing no antigen, 5 g of baculovirus-expressed recombinant HIV-1 MN gp160 and HIV-1 LAI p24 per ml, and 0.15 g of baculovirus control protein (Protein Sciences, Meriden, Conn.) per ml. On day 6, cells were pulsed with 1 Ci of [ 3 H]thymidine (Perkin-Elmer Life Sciences, Boston, Mass.), and they were washed and harvested 18 h later. The stimulation index (S.I.) was calculated as the mean counts per minute of stimulated cultures divided by mean counts per minute of unstimulated cultures. Responses with S.I. 5.0 were considered positive, based on comparisons in our laboratory with in vitro lymphoproliferation to these proteins by PBMC from HIV-1- uninfected controls. The net counts per min (mean counts per min in stimulated cultures minus mean counts per min in unstimulated cultures) for all positive responses was 1,000 cpm. Analysis of cytokine production by ELISA. PBMC (2 million/ml) were incubated in 24-well plates in the presence of either HIV-1 MN gp160 (5 g/ml), baculovirus control protein (0.15 g/ml), or peptides (2 g/ml). The secretion levels of interleukin-4 (IL-4), IL- 5, and IL-10 in cell culture supernatants harvested after 3 days and those of IFN- in cell culture supernatants harvested after 5 days were measured by enzyme-linked immunosorbent assay (ELISA) (Woburn, Boston, Mass.), according to the manufacturer s instructions. The optimal time points for the analysis of individual cytokines were determined in experiments measuring the kinetics of their production and were 5 days for IFN- and 3 days for IL-4, IL-5, and IL-10. All samples, standards, and controls were run in duplicate, and the lower limit of detection of the assays was 0.4 pg/ml. A level of cytokine secretion in antigen-stimulated cultures exceeding two times the unstimulated cultures was considered a positive response. Synthetic HIV-1 peptides. Synthetic overlapping peptides corresponding to gene products from the HIV-1 subtype B (MN) were obtained through the NIH AIDS Research and Reference Reagent Program (Bethesda, Md.). Most were 20 amino acids in length, with 10-amino-acid overlaps between sequential peptides. The peptides were reconstituted at a concentration of 2 mg/ml in 100% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) and were used at a final concentration of 2 g/ml, unless stated otherwise. The final concentration of dimethyl sulfoxide never exceeded 1%. CD4 T-cell cloning, epitope mapping, and CTL activity. CD4 T-cell clones were derived as described previously (33) by bulk culture with HIV-1 MN gp160 at 5 g/ml. On day 14, positively selected CD4 T cells were plated at 0.5 cells/well in 96-well plates with gamma-irradiated allogeneic PBMC and Epstein- Barr virus-transformed B-lymphoblastoid cell lines (B-LCL) as feeders, OKT3 (Ortho Diagnostic System, Raritan, N.J.), and recombinant IL-2. After 2 weeks, cells demonstrating growth and Env-specific proliferation in a [ 3 H]thymidine incorporation assay were further expanded into CD4 T-cell clones. Epitope mapping and specific HLA restriction analyses were performed by using autologous or HLA- mismatched allogeneic B-LCL pulsed with peptides as stimulator cells in proliferative and ELISPOT assays as previously described (20). Clones were screened for HIV-1-specific cytotoxic T-lymphocyte (CTL) activity by 51 Cr release assay (19) by using autologous B-LCL pulsed with the whole protein or the recognized peptide as targets. Results were expressed as percent specific lysis (23). Spontaneous lysis was less than 20% of maximal lysis. Antibody blocking of lymphoproliferation. Monoclonal antibodies (MAbs) recognizing HLA DR (L243), HLA DP (B7/221), and HLA DQ (SPV-L3) framework determinants were used to inhibit major histocompatibility complex class II-restricted lymphoproliferation as previously described (15). The supernatants were generated from hybridoma cell lines secreting L243 and B7/221 (American Type Culture Collection, Rockville, Md.), and SPV-L3 (provided by H. Yssel, DNAX Research Institute, Palo Alto, Calif.) and used at a 1:4 final dilution. These concentrations inhibit proliferation of CD4 T-cell clones restricted at relevant class II loci by 80% and at irrelevant HLA by 15% (15). IFN- ELISPOT assays. ELISPOT assays were performed to detect HIV-1- specific IFN- -secreting cells as previously described (20). In standard assays, CD8 T-cell-depleted PBMC were plated (10 5 cells/well) in 96-well hydrophobic polyvinylidene difluoride-backed plates (Millipore, Bedford, Mass.) precoated with 50 l of10 g of anti-ifn- MAb (1-D1K, mouse immunoglobulin G1; Mabtech, Nacka, Sweden) per ml. In stimulated ELISPOT assays, antigenspecific cell lines were first generated from previously cryopreserved PBMC cultured in the presence of 5 g of HIV-1 MN gp160 per ml for 10 days and then were depleted of CD8 T cells and plated as described above. HIV-1-specific peptides and control peptides (a pool of five irrelevant peptides derived from actin and a highly conserved region of HLA class I -chain precursor) were added to the wells at a final concentration of 2 g/ml. Plates were incubated overnight at 37 Cin5%CO 2 and then developed. Spot-forming cells (SFC) were counted by using the Immunospot (Cellular Technology Ltd., Cleveland, Ohio) optical reader and are expressed per 10 5 input cells. The number of Env-specific IFN- -secreting cells was calculated by subtracting the SFC in the negative-

3 VOL. 77, 2003 Env-SPECIFIC T-HELPER CELLS IN HIV-1 INFECTION 2665 control wells from those in the antigen-stimulated wells. The positive and negative ELISPOT assay responses were determined by using a combination of standard statistical methods. Specifically, the numbers of SFC per well were assumed to follow a simple Poisson model, and the criterion for a positive assay was based on an exact binomial test statistic. Additionally, the number of SFC was always 20 per 10 5 cells for a response designated positive. Twelve HIV-1- uninfected donors served as negative controls, and no false positives were observed for either the gp160 protein or the peptide-stimulated ELISPOT assays based on these criteria. Intracellular immunofluorescent staining for IFN- production. ICS for IFN- -producing antigen-specific CD4 T cells was performed as previously described (6, 31, 32) with modifications. PBMC (2 million/ml) were incubated with HIV-1 MN gp160 (final concentration, 5 g/ml) or peptide (final concentration, 2 g/ml) in the presence of anti-cd49d (Beckman Coulter, Miami, Fla.) and anti- CD28 (BD Biosciences, San Jose, Calif.) purified MAbs (final concentration, 1.0 g/ml) at 37 C. At 2 h brefeldin A (Sigma) was added (final concentration, 10 g/ml), and incubation continued for an additional 4 h. The cells were fixed and permeabilized and then stained with fluorochrome-labeled MAbs (intracellular IFN- fluorescein isothiocyanate, CD69 PE, CD4 PerCP-Cy5.5; BD Biosciences) at room temperature for 30 min, washed, and fixed in 2% paraformaldehyde. Analysis was performed within 24 hr with a FACScalibur with Cell Quest software (BD Biosciences). Class II peptide binding assays. Peptide-binding assays were performed as described previously (6, 30, 31). Specifically, purified human HLA class II molecules (5 to 500 nm) were incubated with various unlabeled peptide inhibitors and1to10nm 125 I-radiolabeled probe peptide for 48 h in phosphate-buffered saline containing 0.05% Nonidet P-40 in the presence of a protease inhibitor cocktail. Class II peptide complexes were separated from free peptide by gel filtration on TSK200 columns (catalog no ; TosoHaas, Montgomeryville, Penn.), and the fraction of bound peptide was calculated as previously described (30). The titer of each DR preparation was determined in the presence of a fixed amount of the appropriate 125 I-radiolabeled peptide to determine the concentration of class II molecules necessary to bind 10 to 20% of the total radioactivity. Inhibitor peptides were typically tested at concentrations ranging from 120 g/ml to 1.2 ng/ml in two to four completely independent experiments. Under conditions where the concentration of label is less than that of MHC and the 50% inhibitory concentration (IC 50 ) is greater than or equal to the concentration MHC, the measured IC 50 values are reasonable approximations of true K d values. Peptides were classified as binders for each HLA-DR molecule for which the binding capacity was 1,000 nm. TCR V usage by multiplex PCR, TCR sequencing, and longitudinal analysis of clone transcripts. To assess T-cell receptor (TCR) V usage, total RNA (2 g) from CD4 T-cell clones ( cells) was reverse transcribed, and five aliquots of the cdna were amplified for 30 PCR cycles with five multiplex primer sets consisting of a total of 25 5 V -specific sense primers and the 3 C specific antisense primer (1). The amplified products were electrophoresed on 2% agarose gels. Direct sequencing of the PCR product was performed by using the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems Inc., Foster City, Calif.) and the specific 5 V and 3 C primers. Once characterized, the clone was tracked in stored or fresh specimens over time using total RNA from 2 million CD4 T-cell-enriched PBMC. Primers specific for the V in the originally isolated clone and C were utilized in the PCRs. Two elongations of 10 cycles were performed in separate tubes, initiated by either the fluorescent dye-labeled J or the clonotype-specific primer. The two reaction products from each amplified DNA were mixed and loaded in a lane and electrophoresed under denaturing conditions on a 373A DNA sequencer (Applied Biosystems), and the fluorescent profiles were analyzed by GeneScan software (25) through the FH- CRC Shared Resources Facilities. Viral diversity at Th epitopes. The level of amino acid variation within each of the epitopes was analyzed by examining 244 full-length HIV-1 subtype B env sequences obtained from the Los Alamos HIV sequence database. The putative amino acid sequences derived from nucleic acid sequences were aligned and analyzed by using the programs Clustal W (32) and Genetic Data Environment (GDE) (United Kingdom Human Genome Mapping Project Resource Center; Amino acid variability was assessed by using the computer program Seqvar, which was kindly provided by Andrew Rambaut (University of Oxford, Oxford, United Kingdom). The program estimates the level of variability at each amino acid position in the sequence alignment by using Shannon entropy. To examine the relative levels of variability over the entire gp160 and to highlight the location of the Th epitopes, a moving average of the estimated variability over three consecutive amino acid positions was plotted. To examine the variability at each amino acid position within the six Env Th epitopes, the site-by-site variability was plotted. Virus DNA sequencing. CD14 monocytes, resting CD4 T lymphocytes, and activated CD4 T lymphocytes were purified from PBMC by negative selection, using magnetic bead depletion followed by fluorescent-activated cell sorting. Genomic DNA was isolated from purified cells by using the QIAamp tissue kit (Qiagen, Valencia, Calif.). Viral RNA was isolated from plasma by using the Qiagen viral RNA kit, according to the manufacturer s protocol, and reverse transcribed to cdna. HIV-1 env gp120 sequences were amplified from cellular DNA and cdna by using a nested PCR and the following primers: gp120 outer PE0 and PE2 and gp120 inner PE1 and P2 (34). Multiple independent PCR products generated from target sources containing 20 to 200 copies of HIV-1 DNA or cdna (purified cells or plasma) were cloned and sequenced (34). Six to 17 clone sequences were aligned by using Clustal W (32). Quantitative PCR detection of TCRBV CDR3 in PBMC and T-cell clones. Genomic DNA was isolated from PBMC by using the QIAamp DNA blood minikit as recommended by the manufacturer (Qiagen). Clone transcripts in the DNA were quantitated by real-time PCR by using Perkin-Elmer ABI Prism 7700 sequence detector as previously described, with modifications (10, 28). Briefly, 3 g of genomic DNA was amplified in the presence of 0.5 M forward (5 -GTT CCCGCTAGGAACCTG-3 ) and reverse (5 -GGGTTGGAGTCGGCTGCT- 3 ) primers and a 50 nm dt-fam/tamra fluorogenic probe (5 -TCCAGGT GAGCCAGGCCATCACTA-3 ) (Synthegen, Houston, Tex.). The primers were designed to amplify the TCRBV CDR3 region. After 2 min of incubation at 50 C and 2 min of denaturation at 95 C, 45 cycles (95 C for 20 s and 60 C for 60 s) of amplification were performed. Samples were coamplified with two sets of standards. For the first standard, human placental DNA (Sigma) was used to amplify the -globin gene in a serial dilution (100,000, 10,000, 1,000, 100, and 10 copies/ reaction) to calculate the total number of cells. To make a clone-specific standard, clones were spiked into 1 million HIV-negative unrelated PBMC to create a serial dilution (300,000, 100,000, 30,000, 10,000, 3,000, 1,000, 300, 100, 30, and 10 copies/reaction). The results were analyzed by using Applied Biosystems sequence detection system (version 1.6.3). Statistical methods. Longitudinal data were analyzed by using generalized estimating equations to accommodate for possible correlation due to repeated data from the same individual (8). Continuous data were analyzed by using linear regression models, and the probability of responses was determined by using logistic regression models (8). The criterion for a positive ELISPOT assay was based on an exact binomial test statistic, with the numbers of SFC per well assumed to follow a simple Poisson model. RESULTS Study population. Ninety-two HIV-1-infected subjects enrolled in the study. Most were Caucasian males reporting unprotected sex with another male as their predominant risk factor for acquiring infection (Table 1). The subjects were seen at a median of 55 days following the appearance of symptoms; 28 were enrolled within 30 days of symptoms. The majority experienced clinical symptoms during acute infection (Table 1). Twenty-nine subjects remained untreated, and 63 subjects received ART for a median duration of 805 days (range, 168 to 1,659). Therapy was initiated after a median interval of 93 days (range, 9 to 2,608 days) after the appearance of symptoms. Eleven subjects initiated therapy within 30 days, 36 subjects did so between 30 and 365 days, and 16 subjects initiated therapy more than 365 days after the appearance of symptoms (Table 2). HIV-1-specific LP responses during acute infection and following treatment. To define the specificities of Th responses induced in HIV-1 infection, we prospectively analyzed Envand Gag-specific lymphoproliferative (LP) responses among 62 untreated subjects with primary infection, including 33 subjects assessed prior to treatment. We accrued 124 total observations from a mean of two time points and used generalized estimating equations to control for repeated measures for the same subject. Overall, there was a low probability (19%) of detecting HIV-1-specific LP responses in the untreated patients (Table 2). When present, the responses were more likely to be Gag

4 2666 MALHOTRA ET AL. J. VIROL. TABLE 1. Clinical and demographic profile of the 92 study participants with acute and early HIV- 1 infection Characteristic Value Mean age (range) (yr)...33 (20 58) Gender (no. of subjects) Male...91 Female... 1 Race (no. of subjects) White...83 Black... 2 Hispanic... 4 Other... 3 Risk behavior associated with HIV-1 acquisition (no. of subjects) a MSM a...83 MSM and IDU... 4 Heterosexual... 2 Heterosexual and IDU... 1 Unknown... 2 Median duration of infection at entry (range) (days)...55 (3 187) No. of subjects (%) with symptomatic acute infection...75 (82%) a MSM, men who have sex with men; IDU, injection drug use. TABLE 2. Probability of detecting Gag- or Env-specific Th proliferative responses among untreated and treated subjects stratified by duration of infection prior to treatment Antigen No treatment (n 62) Probability of detecting response among subjects with: Treatment beginning after duration of infection of (in days): 30 (n 11) (n 36) 365 (n 16) Gag Env Gag or Env FIG. 1. HIV-1 Gag and Env-specific Th responses of 62 untreated subjects with acute or early HIV-1 infection. All assays were performed with fresh PBMC. Each symbol represents the measurement for an individual subject at enrollment. The net counts per minute for the positive Env-specific responses ranged from 1,111 to 7,696 (median, 3,479), and for the positive Gag-specific responses, they ranged from 1,128 to 26,956 (median, 3,917). specific than Env specific (16 versus 5%). However, closer inspection revealed distinct differences in the kinetics of Envand Gag-specific proliferative responses (Fig. 1). When all measurements taken from specimens within the first year of infection were taken into consideration, the probability of identifying Env-specific proliferative responses was significantly higher among subjects with acute infection ( 30 DPI) rather than early infection ( 30 DPI) (21 versus 0.5%, P 0.001) (Fig. 1). By contrast, Gag-specific proliferative responses were detected throughout acute and early infection (Fig. 1), and the probability of detecting these among subjects 30 DPI was similar to the probability for those 30 DPI (13 versus 17%, P 0.64). Next, we determined whether the duration of infection prior to therapy had an impact on the ability to mount a detectable Env- or Gag-specific proliferative response following suppression of viremia with ART ( 24 weeks). The 63 treated subjects were stratified into three groups according to the duration of infection prior to treatment initiation: within 30 days, between 30 and 365 days, and more than 365 days (Table 2). As in the untreated group, Gag-specific proliferative responses predominated over Env-specific responses following treatment, and the probabilities of detecting Gag-specific responses were comparable among the three groups (Table 2). Of note, several subjects who initiated therapy even after the first year of infection developed Gag-specific responses, but none of these subjects developed detectable Env-specific responses (Table 2). Moreover, none of the four subjects with Env-specific proliferative responses at the time of enrollment demonstrated these responses again during a mean of 10 (range 3 to 18) subsequent follow-up visits despite the early initiation of ART. Antiviral activities of Env-specific Th responses. We sought to determine why early Env-specific Th responses are not preserved by patients who initiate early ART. Detailed studies were conducted with stored specimens from subject 1212, who enrolled 11 days after infection and who manifested an early Env-specific Th proliferative response (Fig. 2). On presentation with acute HIV-1 infection, subject 1212 had an initial viral load in plasma of 6 million copies/ml and a CD4 T-cell count of 755 cells/mm 3 (Fig. 2A). Within 5 days (16 DPI) plasma HIV-1 RNA spontaneously declined more than one log to 102,045 copies/ml, and ART was then initiated. Plasma viremia decreased further to 500 copies/ml by 12 weeks and 50 copies/ml by 28 weeks of therapy, and CD4 T-cell counts remained stable (Fig. 2A). Studies of the Th responses revealed an Env-specific LP response (S.I. of 8) at day 11, which was lost by day 16 and undetectable at all subsequent 18 time points examined over 120 weeks of therapy (Fig. 2B). However, Gag-specific LP responses, absent at two time points (11 and 16 DPI) prior to initiation of therapy, were detected intermittently after therapy (Fig. 2B). To understand the antiviral function and fate of the Envspecific responses in subject 1212, we first established Envspecific CD4 T-cell clones by using cryopreserved PBMC obtained from 11 DPI and then mapped their MHC class II-restricted epitopes by LP assays. Seven (no. 1 to 7) of 12 clones recognized the highly conserved C3 region of gp120 PI20-PKISFEPIPIHYCAPAGFAI (Fig. 2C), and the responses were further mapped to the 12-mer SP12-

5 VOL. 77, 2003 Env-SPECIFIC T-HELPER CELLS IN HIV-1 INFECTION 2667 FIG. 2. Characterization of Env-specific Th responses of subject (A) Effect of combination ART on plasma viremia (RNA copies/ milliliter) and CD4 T-cell count (no. of cells/cubic millimeter) for subject The subject presented on day 11, and treatment was initiated on day 16 of infection. (B) Effects of ART on HIV-1-specific Th responses measured by lymphoproliferation and indicated by S.I. All assays were performed with fresh PBMC. (C) Env- specific CD4 T-cell clones established from peripheral blood taken on day 11 of infection. Clones map to epitopes in gp120 and gp41 (clones 1 to 7, PI20; clone 8, DP20; and clone 9, VD20). (D) Mapping of immunodominant epitope region PI20 by ELISPOT assay. (E) Env- specific clones exhibit cytotoxic activity when incubated with autologous EBV-transformed lymphoblastoid cell lines pulsed with a specific peptide at an E:T ratio of 20 to 1. (F) Secretion of IFN-, IL-4, and IL-10 by Env-specific CD4 T-cell clones after stimulation with the envelope or control peptide (clone 1, PI20; clone 9, VD20; clone 10, RD20; clone 11, NN20; and clone 12, TF20). A level of cytokine secretion in antigen-stimulated cultures exceeding two times that of the unstimulated cultures was considered a positive response.

6 2668 MALHOTRA ET AL. J. VIROL. TABLE 3. The binding capacity (IC 50 ) of Th epitope regions for common DRB1, DRB3, DRB4, and DRB5 alleles a IC 50 of Th epitope region for indicated allele Env peptide DRB1* DRB3* 0101 DRB4* 0101 DRB5* 0101 DP ,667 3,032 5,833 3,846 5,414 PI , , VD ,371 3,462 1, , a Peptides are classified as a binder for a DR molecule if the binding capacity is 1,000 nm (shown in bold);, IC 50 20,000 nm. SFEPIPIHYCAP in an IFN- ELISPOT assay (Fig. 2D). In LP assays, clone no. 8 proliferated in response to a peptide in the C1 region of gp120 (DP20-DTEVHNVWATQACVPTDPNP) and clone no. 9 recognized a highly conserved region in gp41 (VD20-VWGIKQLQARVLAVERYLKD) (Fig. 2C). In addition, the Env-specific CD4 T-cell clones (no. 10 to 12) recognized three additional epitope regions: gp120 RD20- RDKMQKEYALLYKLDIVSID; gp120 NN20-NFTDNAKT IIVHLNESVQIN; and gp41 TF20-TKAKRRVVQREKRAA IGALF (data not shown). Thus, the acute Env-specific Th response was broad, recognizing at least six distinct epitopes throughout the protein. To determine the class II molecules responsible for presenting the HIV-1 epitopes to the CD4 T-cell clones, LP assays were performed in the presence and absence of anti-dp, anti- DQ, and anti-dr antibodies. Peptide-specific proliferation of clones no. 1 and no. 9 was inhibited more than 99% by anti-dr antibody, in contrast to 15% inhibition with the anti-dp and anti-dq antibodies, indicating HLA-DR as the restriction element (data not shown). Further restriction analysis by lymphoproliferation revealed promiscuous binding to multiple HLA-DR molecules (data not shown), and fine mapping to a distinct HLA-DR molecule was not possible for these clones. HLA restriction analyses could not be performed with clones no. 8, 10, 11, and 12 due to limited availability of the clones. The affinity of three of the Env-specific Th epitopes (PI20, DP20, and VD20) to various HLA-DR molecules was determined by using IC 50 binding assays (Table 3). Of note, the two epitope regions PI20 and VD20 each bound 7 of the 12 common DRB1, 3, 4, and 5 molecules tested at an IC 50 of 1,000 nm, indicating their promiscuous affinity for multiple class II molecules. To determine if the Env-specific CD4 T cells from subject 1212 were capable of antiviral activities, clones no. 1, 8, and 9, recognizing PI20, DP20, and VD20, respectively, were assessed for lysis of autologous B-LCL pulsed with their specific peptide epitopes. These three clones demonstrated 10 to 39% greater cytotoxicity of targets expressing the Env epitope in contrast to the control peptide at an E:T ratio of 20 to 1 (Fig. 2E). In addition, clones (1, 9, 10, 11, and 12) were examined for their ability to secrete helper and antiviral cytokines in response to stimulation with their putative Env epitope. Culture supernatants were tested for secretion of IL-4 and IL-10 (72-h harvest), and IFN- (120-h harvest) to determine the Th profile. The clones 1 and 9, recognizing PI20 and VD20, respectively, secreted high levels of IFN- (608- to 1,564-fold over background), IL-4 (53- to 854-fold), and IL-10 (176 to 265-fold) indicating a Th0 profile (Fig. 2F). The clones 10, 11, and 12, recognizing RD20, NN20, and TF20, respectively, secreted predominantly IFN- (39- to 160-fold) and lower levels of IL-4 (6- to 16-fold) and IL-10 (4- to 30-fold) (Fig. 2F). Thus, the earliest Env-specific responses in subject 1212 detected at day 11 of infection recognized epitopes in gp120 and gp41, were capable of HIV-1- specific cytolytic activities, and demonstrated potential for antiviral function through secretion of IFN-. Evolutionary changes in Th epitopes in subject Since the HIV-1 envelope consistently exhibits intrahost and interhost variability, we anticipated that mutations in sequences spanning the Th epitopes, particularly those lying within hypervariable regions, could occur over time. We also considered the possibility that there may be significant divergence between the autologous epitopes and the sequences of the HIV-1 MN strain upon which the screening peptides were based. To address these issues, we first mapped the position of the epitopes on the variability plot for the entire gp160 protein. Two of the epitopes (RD20 and NN20) were present within regions of the HIV-1 genome that exhibit a high level of variability, while the others were in regions of lower variability (Fig. 3A). This was also evident in a site-by-site analysis of variability within each of these epitopes (Fig. 3B). A comparison of the MN-derived peptide sequences to the autologous sequences (Fig. 3C) revealed a three- and four-amino-acid difference between the sequences within the RD20 and NN20 epitopes, respectively. A one-amino-acid difference was noted in the DP20 autologous and MN sequences, while the TF20 and SP12 autologous sequences were identical to the MN sequences. Next we examined the possibility that the loss of Th responses following acute infection was due to the evolution of variant sequences leading to a loss of stimulation and Th-cell responsiveness over time. Multiple sequences encoding the five gp120 epitopes (but not the gp41 epitope) were analyzed from samples collected at 16 (mean of 25 clones) and 683 (mean of 27 clones) DPI from subject We found minimal heterogeneity and change over time in the RD20 and NN20 autologous sequences, even though these epitopes reside in regions of high variability. However, we found several variants in the relatively conserved sequences of DP20 and TF20 from blood monocytes sampled at the first time point but fewer variants in the samples from 683 DPI. Thus, the paucity of envelopespecific immune responses at the later time points in this subject was not explained by viral escape within epitopes recognized during acute infection. Longitudinal tracking of Env-specific Th responses in subject To determine if Env-specific Th cells persisted despite the inability to proliferate to the cognate epitope, we

7 VOL. 77, 2003 Env-SPECIFIC T-HELPER CELLS IN HIV-1 INFECTION 2669 FIG. 3. Genetic variability in regions encoding Th epitopes and the evolutionary changes in autologous viral sequences. (A) Amino acid variability in Th epitopes. Entropy measurements of amino acid variability mapped across the gp160 coding sequence by using 244 full-length subtype B sequences obtained from the Los Alamos HIV sequence database are shown. The thick blue line indicates the V1-to-V5 variable region, and the thick red line denotes the six Th epitopes recognized by patient The full gene plot illustrates a moving average of variability at adjoining three-amino-acid windows. Two of the epitopes (RD20 and NN20) mapped to regions of high variability, while the other epitopes mapped to more conserved regions in the protein. (B) Site-by- site analysis of variability. The variability at each amino acid position within the six Th epitopes was examined by plotting the site-by-site variability. (C) Evolutionary changes at the Th gp120 epitopes in autologous viral sequences sampled at 16 and 683 DPI from patient Identity to amino acid in the consensus sequence is indicated by a dot. Numbers at the ends of the sequences indicate the proportion of clones encoding the given variant. Compartments sampled included monocytes, resting CD4 T cells, activated CD4 T cells, and plasma.

8 2670 MALHOTRA ET AL. J. VIROL. FIG. 4. Longitudinal tracking of Env-specific Th cells from subject PBMC from six time points between 16 DPI and 27 months posttreatment (weeks 0, 24, 40, 78, 104, and 120 posttreatment) were tested in a batch analysis for lymphoproliferation (S.I. are shown) and IFN- secretion by ELISA in response to stimulation with peptide PI20 for 120 h. PBMC from these time points were also stimulated with the peptide for 10 days, and IFN- ELISPOT assays were performed with CD8-depleted PBMC poststimulation. Env-specific IFN- SFC were detected by stimulated ELISPOT only. repeated the assays measuring lymphoproliferation and IFN- secretion in a batch analysis using PBMC from six time points (weeks 0 [16 DPI], 24, 40, 78, 104, and 120 posttreatment). The Env peptide PI20 was used to stimulate these cells, since the findings above indicated that it was recognized by the majority of clones and remained conserved over the period of study. Neither peptide-specific lymphoproliferation nor IFN- secretion (by ELISA) was detected at any of the six time points (Fig. 4). An overnight IFN- ELISPOT assay and IFN- ICS by flow cytometry (data not shown) were also unable to detect responses, with lower levels of sensitivity in our laboratory of and 0.01%, respectively. Finally, we hypothesized that the Env-specific Th cells were present at such low frequency that they could not be detected by the conventional assays described above. Thus, PBMC from the six time points were first amplified by in vitro stimulation with the peptide PI20 for 10 days and then assessed qualitatively for IFN- secretion in an ELISPOT assay. After stimulation, IFN- -secreting cells were detected in the expanded T-cell population at weeks 40, 78, 104, and 120 with levels ranging from 73 to 980 SFC/100,000 input cells (Fig. 4). By contrast, 20 IFN- SFC/10 5 cells were detected in similarly stimulated PBMC from nine HIV-1-seronegative subjects. Subsequent experiments confirmed that the T cells secreting IFN- in these assays were within the CD8 T-cell-depleted PBMC fraction (data not shown). Longitudinal tracking of Env P120-specific clonal transcripts. To determine if the Env PI20-specific Th responses elicited following viral suppression with ART represented newly acquired Th cells or the original clonal response detected at day 11, the PBMC from day 16 of infection and week 24 posttreatment were examined for the presence of clone transcripts identical to those found among Env-specific Th clones established at day 11. Using multiplex RT-PCR to amplify the TCRBV family, we found that six of the seven Th clones recognizing Env PI20 at day 11 used BV13 (Fig. 5A). Analysis of the rearranged VDJ region of five of the BV13 clones revealed four unique sequences, with clones 1 and 2 sharing an identical sequence (Fig. 5B). To determine if this common clonotype persisted, cdna from CD4 T cells isolated from day 16 and week 24 were amplified by RT-PCR with clonotypic primers based on the dominant sequence. The clonal transcripts were observed at both the early and later time points (Fig. 5C). By contrast, these transcripts were not detected in CD4 T cells from a different HIV-1-infected subject (no. 1243) (Fig. 5C). Of note, the clonal transcripts were not detected by real-time quantitative PCR performed with DNA, the lower limit of detection of which was 1/10,000 cells. Thus, these studies demonstrate that Env-specific CD4 T cells elicited during acute infection persist at low frequencies. Envelope-specific IFN- secretion in subjects receiving delayed ART. To test the hypothesis that Env-specific Th cells may be induced and maintained even in subjects who delay initiating ART until after the acute infection stage, we examined the Env-specific Th responses of seven such subjects. Four subjects initiated treatment during the first year, two during the second year, and one during the fourth year of infection (Table 4). The subjects were selected based on the availability of cryopreserved PBMC samples for ELISPOT assays. None of these subjects demonstrated Env-specific proliferative responses prior to initiation of treatment, and the probability of an Env-specific proliferative response after treatment was low (13%), for a mean of 10 posttreatment visits (total, 70 observations). The PBMC (CD8 depleted) from these subjects were tested for the presence of IFN- SFC recognizing the whole gp160 protein and Env 20-mer peptide pools after a median duration of 12 months (range, 8 to 24 months) of ART. No responses were detected by standard assays (data not shown). However, Env-specific SFC were detected by stimulated assays in six of the seven subjects (Table 4). Responses to the gp160 protein (median, 64 SFC/10 5 cells) were identified for four of six subjects, and responses to the peptide pools (82 SFC/10 5 cells) were found for all six subjects initiating therapy within the first 2 years (Table 4). No IFN- SFC were detected for the one subject with advanced disease (CD4 T-cell count, 61/ mm 3 ) who had delayed therapy by approximately 4 years. No positive responses to the Env protein or peptides were identified for 12 HIV-1-uninfected control subjects (Table 4). Thus, despite a delay in therapy of up to 2 years, Env-specific IFN- -secreting Th cells were detected for all subjects by stimulated ELISpot assays but not by standard assays. DISCUSSION We have detailed for the first time Th responses to HIV-1, notably Env-specific Th cells, within 5 days of infection. These helper activities, associated with cytotoxicity and antiviral cytokine secretion, represent one of the earliest indicators of acquired immunity in HIV-1 infection. With this study we provide clear evidence that HIV-1-specific Th proliferative responses can occur in primary infection, although the proba-

9 VOL. 77, 2003 Env-SPECIFIC T-HELPER CELLS IN HIV-1 INFECTION 2671 FIG. 5. Longitudinal tracking of Env-specific clonal transcripts from subject (A) Analysis of TCRBV expression in clone 1212 no. 1 by multiplex RT-PCR and agarose gel electrophoresis. The outside lane (M) contains the molecular weight markers (100-bp ladder). The arrowhead indicates the single distinct band visualized in this gel. The size and location of the band indicate that the clone expressed TCRBV13. Clones 2, 3, 4, 5, and 6 also expressed TCRBV13 (data not shown). (B) Direct DNA sequencing was performed with the amplified products from clones 1, 2, 3, 4, and 5 by using the TCRBC primer. Four unique TCRBV CDR sequences were detected, with clones 1 and 2 sharing identical sequences. Nucleotide mismatches between the CDR sequences for clones 1 and 2 versus clones 3, 4, and 5 are indicated in bold. (C) Detection of clone 1212 no. 1 transcripts before and after treatment. RT-PCR was performed with CD4 -enriched PBMC from weeks 0 and 24 by using primers specific for the V in the clone and C. On each of the amplified DNA samples, two elongations of 10 cycles were performed, initiated by either the fluorescent dye-labeled J or the clonotype primer. The two reaction products were electrophoresed, and the fluorescent profiles were analyzed by GeneScan software. Shown are histograms for spectratyping analysis of CDR3 lengths with the fragment length depicted on the x axis and fluorescent intensity on the y axis. Priming with the J primer depicts a single clonal expansion in clone 1, polyclonal profile in CD4 -enriched PBMC from subject 1212 at weeks 0 (16 DPI) and 24 posttreatment and from subject Priming with the clonotypic primer depicts the presence of the clone transcripts in clone 1, CD4 -enriched PBMC from subject 1212 at time points week 0 (16 DPI) and week 24 posttreatment, but not in the CD4 -enriched PBMC from subject The negative control (no cdna) sample showed no amplification with either J or clonotypic primers. bility of detecting them is low (19%). We demonstrate that Gag-specific LP responses may occur in untreated subjects throughout primary infection (acute, 13%; early, 17%). By contrast, Env-specific Th proliferative responses are more likely to be detected during acute (21%) than during early infection (0.5%) for untreated subjects. Even when using flowcytometric detection of antigen- induced IFN- production, responses to Env determinants are rarely detected in most persons with established HIV-1 infection (26). Thus, some of the earliest Th responses in HIV-1 infection may be directed to epitopes within the HIV-1 envelope, but these become rapidly undetectable by conventional assays measuring lymphoproliferation or IFN- secretion within days after infection. Hence, the role of Env-specific Th responses as an important mediator of T-cell help may be underappreciated because of the brief period of detectability and the need to employ sensitive techniques capable of detecting low frequencies of memory precursors. Within the first year of infection, once ART is initiated, HIV-1 Env- and Gag-specific proliferative responses become more prominent, with threefold increases in the probability of measuring a response by lymphoproliferation. Gag-specific responses climb even if ART is initiated later than 1 year after infection, but this is not the case with Env-specific responses. Some of our findings may help explain why Env-specific proliferative responses do not reemerge under the same conditions. When we evaluated one patient, subject 1212, within 11 days of infection and 2 years later, we did not observe evidence for viral escape within the Env Th epitopic region, even within variable regions of the envelope protein. However, we acknowledge that additional epitopes within the hypervariable regions of the envelope may be recognized initially, and these were not identified in our study, since responses were screened for recognition of epitopes only within the HIV-1 MN laboratory strain. The Th responses targeting epitopes in these hypervariable regions may be the ones that apply immune pressure to viral replication and promote escape mutations. Longitudinal studies to assess the recognition of additional envelope epitopes within the regions where sequence variation has occurred over time are in progress. These regions may be more likely to contain Th epitopes that were recognized in early infection and in which escape mutations have occurred. Additionally, subject 1212 initiated treatment early during acute infection and the rapid control of virus replication may have impeded viral diversification and the emergence of immune escape variants. Besides, minority escape variants replicating at a low

10 2672 MALHOTRA ET AL. J. VIROL. TABLE 4. Envelope-specific IFN- -secreting Th cells in HIV-1-infected subjects after delayed ART a Subject no. Duration of infection (days) Data collected at inception of treatment Viral load (RNA copies/ml) Duration of No. of IFN- SFC/10 5 input cells CD4 T-cell treatment count/mm 3 (days) Gp160 protein Peptide pools , b , , , , , ,376 38, Uninfected Uninfected Uninfected Uninfected 1 2 ES0000 Uninfected Uninfected 4 0 JM000 Uninfected 0 0 JV000 Uninfected Uninfected 0 ND c 1959 Uninfected 17 ND 1137 Uninfected 2 ND 1242 Uninfected 0 ND a HIV-1-uninfected subjects were not treated, and their viral loads and CD4 T-cell counts were not measured. b The Env-specific immune response of subject 1291 mapped to Env PI20, the dominant epitope region recognized by subject Subjects 1212 and 1291 share the DRB1*0701 haplotype. c ND, not done. rate may not be easily identified in virus strains isolated from genomic DNA. It is also important to consider that the contemporaneous CD8 cytotoxic-t-cell response during acute infection may exert the predominant immune pressure and that HIV-1 may escape within these class I-restricted epitopes. Recent studies have demonstrated that HIV-1-specific Th cells are preferentially infected in comparison to other memory CD4 T cells and that this selection occurs in acute as well as in chronic infection (9). The early evolution of Env-specific Th cells during the phase of high-level viremia may render these cells even more susceptible to either activation-induced apoptosis or death as a consequence of direct viral infection. Hence, here we explored the possibility that the Env-specific Th cells either were deleted from the memory pool or were reduced to such low frequency that they were no longer detectable by conventional assays. CD4 T cells specific for the immunodominant Env epitope PI20 were not detected posttreatment by lymphoproliferation, ex vivo IFN- ELISPOT, or ICS assays. However, PI20-specific IFN- -secreting cells were detected following one cycle of in vitro stimulation with gp160. Moreover, TCR clonotypes targeting the immunodominant gp120 epitope region PI20, while not detected by PCR amplification of DNA, were detected by RT-PCR with GeneScan analysis, the latter having severalfold-greater sensitivity than the former. Since no evidence of viral diversification in these epitopic regions was found, these findings indicate that the precursor frequency of Env-specific memory CD4 T cells may be profoundly low despite control of viremia with ART initiated as early as 16 DPI. Of note, during the first 24 weeks after initiation of ART, Env-specific Th cells were not detected in the PBMC by conventional assays to measure antigen-specific lymphoproliferation (LPA) or ex vivo IFN- secretion (ELISPOT and ICS). Furthermore, antigen-specific IFN- SFC were not detected during this time by ELISPOT assays performed with the PBMC after a round of in vitro stimulus with gp160. During these phases of high and declining viremia, the antigen- specific cells are likely of the effector phenotype and die upon in vitro stimulation. Alternately, viremia may have a suppressive effect on the proliferation and function of all Th cells. This effect may be global, as evidenced by impaired memory responses not only to HIV-1 but also to a variety of recall antigens during acute and early infection (24). Inhibition of virusspecific proliferation has also been reported in association with high levels of viremia for subjects who undergo interruptions of ART, with restoration of proliferative responses on resumption of ART and control of virus replication (22). In six of our seven subjects who had delayed initiation of ART we found demonstrable IFN- SFC by stimulated ELISPOT specific for either the Env protein or peptide pools. Our data support the notion that HIV-1-specific Th cells may persist despite long treatment delays, albeit at very low frequencies ( to 0.01%), and thus are not identified by standard ELISPOT, LP, and ICS assays but require amplification of precursors in vitro for the detection of IFN- -secreting cells. Admittedly, this is an unconventional strategy for detecting antigen-specific T cells and provides a qualitative assessment of functional memory cells. Further studies are needed to determine whether there is a critical threshold of functional memory CD4 T cells that must be maintained to contribute to the control of viral replication by helper activities. Nevertheless, the mere presence of the Th cells indicates that they may be capable of in vivo boosting with therapeutic HIV-1 vaccination regimens. The emergence of Env-specific Th responses after ART may represent the restoration of the proliferative capacity and amplification of the memory

Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to

Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to Class II tetramer staining Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to PE were combined with dominant HIV epitopes (DRB1*0101-DRFYKTLRAEQASQEV, DRB1*0301- PEKEVLVWKFDSRLAFHH,

More information

Innate and Cellular Immunology Control of Infection by Cell-mediated Immunity

Innate and Cellular Immunology Control of Infection by Cell-mediated Immunity Innate & adaptive Immunity Innate and Cellular Immunology Control of Infection by Cell-mediated Immunity Helen Horton PhD Seattle Biomedical Research Institute Depts of Global Health & Medicine, UW Cellular

More information

Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4

Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4 Human Immunodeficiency Virus Type-1 Myeloid Derived Suppressor Cells Inhibit Cytomegalovirus Inflammation through Interleukin-27 and B7-H4 Ankita Garg, Rodney Trout and Stephen A. Spector,,* Department

More information

Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific

Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific SUPPLEMENTARY FIGURE LEGEND Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific CD4 + T cells correlates with intracellular CTLA-4 levels. (a) Comparative CTLA-4 levels

More information

Micropathology Ltd. University of Warwick Science Park, Venture Centre, Sir William Lyons Road, Coventry CV4 7EZ

Micropathology Ltd. University of Warwick Science Park, Venture Centre, Sir William Lyons Road, Coventry CV4 7EZ www.micropathology.com info@micropathology.com Micropathology Ltd Tel 24hrs: +44 (0) 24-76 323222 Fax / Ans: +44 (0) 24-76 - 323333 University of Warwick Science Park, Venture Centre, Sir William Lyons

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Complete but curtailed T-cell response to very-low-affinity antigen Dietmar Zehn, Sarah Y. Lee & Michael J. Bevan Supp. Fig. 1: TCR chain usage among endogenous K b /Ova reactive T cells. C57BL/6 mice

More information

Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design Pratima Kunwar 1,7, Natalie Hawkins 2, Warren L. Dinges 1,3, Yi Liu 5, Erin E. Gabriel

More information

Therapeutic Immunization with Autologous DC Pulsed with Autologous Inactivated HIV-1 Infected Apoptotic Cells

Therapeutic Immunization with Autologous DC Pulsed with Autologous Inactivated HIV-1 Infected Apoptotic Cells Therapeutic Immunization with Autologous DC Pulsed with Autologous Inactivated HIV-1 Infected Apoptotic Cells Sharon A. Riddler, MD, MPH University of Pittsburgh May 2008 Slide 1 HIV and DC Vaccines During

More information

Supplementary Data 1. Alanine substitutions and position variants of APNCYGNIPL. Applied in

Supplementary Data 1. Alanine substitutions and position variants of APNCYGNIPL. Applied in Supplementary Data 1. Alanine substitutions and position variants of APNCYGNIPL. Applied in Supplementary Fig. 2 Substitution Sequence Position variant Sequence original APNCYGNIPL original APNCYGNIPL

More information

Chronic HIV-1 Infection Frequently Fails to Protect against Superinfection

Chronic HIV-1 Infection Frequently Fails to Protect against Superinfection Chronic HIV-1 Infection Frequently Fails to Protect against Superinfection Anne Piantadosi 1,2[, Bhavna Chohan 1,2[, Vrasha Chohan 3, R. Scott McClelland 3,4,5, Julie Overbaugh 1,2* 1 Division of Human

More information

PBMC from each patient were suspended in AIM V medium (Invitrogen) with 5% human

PBMC from each patient were suspended in AIM V medium (Invitrogen) with 5% human Anti-CD19-CAR transduced T-cell preparation PBMC from each patient were suspended in AIM V medium (Invitrogen) with 5% human AB serum (Gemini) and 300 international units/ml IL-2 (Novartis). T cell proliferation

More information

Supporting Information

Supporting Information Supporting Information Chapuis et al. 10.1073/pnas.1113748109 SI Methods Selection of Patients, Targets, Isolation, and Expansion of Melanoma- Specific CTL Clones. Patients were HLA-typed, and their tumors

More information

Why are validated immunogenicity assays important for HIV vaccine development?

Why are validated immunogenicity assays important for HIV vaccine development? Why are validated immunogenicity assays important for HIV vaccine development? There is a need to compare immunogenicity of products in the pipeline, when similar or different in class when developed by

More information

DEBATE ON HIV ENVELOPE AS A T CELL IMMUNOGEN HAS BEEN GAG-GED

DEBATE ON HIV ENVELOPE AS A T CELL IMMUNOGEN HAS BEEN GAG-GED DEBATE ON HIV ENVELOPE AS A T CELL IMMUNOGEN HAS BEEN GAG-GED Viv Peut Kent Laboratory, University of Melbourne, Australia WHY ENVELOPE? Env subject to both humoral and cellular immune responses Perhaps

More information

MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All

MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All MATERIALS AND METHODS Antibodies (Abs), flow cytometry analysis and cell lines Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All other antibodies used

More information

Supplementary Data Table of Contents:

Supplementary Data Table of Contents: Supplementary Data Table of Contents: - Supplementary Methods - Supplementary Figures S1(A-B) - Supplementary Figures S2 (A-B) - Supplementary Figures S3 - Supplementary Figures S4(A-B) - Supplementary

More information

Supplementary Fig. 1: Ex vivo tetramer enrichment with anti-c-myc beads

Supplementary Fig. 1: Ex vivo tetramer enrichment with anti-c-myc beads Supplementary Fig. 1: Ex vivo tetramer enrichment with anti-c-myc beads Representative example of comparative ex vivo tetramer enrichment performed in three independent experiments with either conventional

More information

Rapid perforin upregulation directly ex vivo by CD8 + T cells is a defining characteristic of HIV elite controllers

Rapid perforin upregulation directly ex vivo by CD8 + T cells is a defining characteristic of HIV elite controllers Rapid perforin upregulation directly ex vivo by CD8 + T cells is a defining characteristic of HIV elite controllers Adam R. Hersperger Department of Microbiology University of Pennsylvania Evidence for

More information

Diagnostic Methods of HBV and HDV infections

Diagnostic Methods of HBV and HDV infections Diagnostic Methods of HBV and HDV infections Zohreh Sharifi,ph.D Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Hepatitis B-laboratory diagnosis Detection

More information

Alessandra Franco MD PhD UCSD School of Medicine Department of Pediatrics Division of Allergy Immunology and Rheumatology

Alessandra Franco MD PhD UCSD School of Medicine Department of Pediatrics Division of Allergy Immunology and Rheumatology Immunodominant peptides derived from the heavy constant region of IgG1 stimulate natural regulatory T cells: identification of pan- HLA binders for clinical translation Alessandra Franco MD PhD UCSD School

More information

Supplemental Information. T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism

Supplemental Information. T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism Immunity, Volume 33 Supplemental Information T Cells Enhance Autoimmunity by Restraining Regulatory T Cell Responses via an Interleukin-23-Dependent Mechanism Franziska Petermann, Veit Rothhammer, Malte

More information

HIV-1 acute infection: evidence for selection?

HIV-1 acute infection: evidence for selection? HIV-1 acute infection: evidence for selection? ROLLAND Morgane University of Washington Cohort & data S6 S5 T4 S4 T2 S2 T1 S1 S7 T3 DPS (days post symptoms) 3 (Fiebig I) 7 (Fiebig I) 13 (Fiebig V) 14 (Fiebig

More information

Detailed step-by-step operating procedures for NK cell and CTL degranulation assays

Detailed step-by-step operating procedures for NK cell and CTL degranulation assays Supplemental methods Detailed step-by-step operating procedures for NK cell and CTL degranulation assays Materials PBMC isolated from patients, relatives and healthy donors as control K562 cells (ATCC,

More information

Determinants of Immunogenicity and Tolerance. Abul K. Abbas, MD Department of Pathology University of California San Francisco

Determinants of Immunogenicity and Tolerance. Abul K. Abbas, MD Department of Pathology University of California San Francisco Determinants of Immunogenicity and Tolerance Abul K. Abbas, MD Department of Pathology University of California San Francisco EIP Symposium Feb 2016 Why do some people respond to therapeutic proteins?

More information

Supplemental Materials and Methods Plasmids and viruses Quantitative Reverse Transcription PCR Generation of molecular standard for quantitative PCR

Supplemental Materials and Methods Plasmids and viruses Quantitative Reverse Transcription PCR Generation of molecular standard for quantitative PCR Supplemental Materials and Methods Plasmids and viruses To generate pseudotyped viruses, the previously described recombinant plasmids pnl4-3-δnef-gfp or pnl4-3-δ6-drgfp and a vector expressing HIV-1 X4

More information

Citation for published version (APA): Von Eije, K. J. (2009). RNAi based gene therapy for HIV-1, from bench to bedside

Citation for published version (APA): Von Eije, K. J. (2009). RNAi based gene therapy for HIV-1, from bench to bedside UvA-DARE (Digital Academic Repository) RNAi based gene therapy for HIV-1, from bench to bedside Von Eije, K.J. Link to publication Citation for published version (APA): Von Eije, K. J. (2009). RNAi based

More information

staining and flow cytometry

staining and flow cytometry Detection of influenza virus-specific T cell responses by intracellular by cytokine intracellular staining cytokine staining and flow cytometry Detection of influenza virus-specific T cell responses and

More information

HIV Diagnostic Testing

HIV Diagnostic Testing In The name of God HIV Diagnostic Testing By : Dr. Shahzamani PhD of Medical virology Purpose of HIV Testing To identify asymptomatic individuals To diagnose HIV infection in those who practice high risk

More information

Supplementary Figure 1. Using DNA barcode-labeled MHC multimers to generate TCR fingerprints

Supplementary Figure 1. Using DNA barcode-labeled MHC multimers to generate TCR fingerprints Supplementary Figure 1 Using DNA barcode-labeled MHC multimers to generate TCR fingerprints (a) Schematic overview of the workflow behind a TCR fingerprint. Each peptide position of the original peptide

More information

MHC Tetramers and Monomers for Immuno-Oncology and Autoimmunity Drug Discovery

MHC Tetramers and Monomers for Immuno-Oncology and Autoimmunity Drug Discovery MHC Tetramers and Monomers for Immuno-Oncology and Autoimmunity Drug Discovery Your Partner in Drug Discovery and Research MHC Tetramer Background T-Cell Receptors recognize and bind to complexes composed

More information

Lecture 6. Burr BIO 4353/6345 HIV/AIDS. Tetramer staining of T cells (CTL s) Andrew McMichael seminar: Background

Lecture 6. Burr BIO 4353/6345 HIV/AIDS. Tetramer staining of T cells (CTL s) Andrew McMichael seminar: Background Lecture 6 Burr BIO 4353/6345 HIV/AIDS Andrew McMichael seminar: Background Tetramer staining of T cells (CTL s) 1. Vβ 19: There are 52 T cell receptor (TCR) Vβ gene segments in germ line DNA (See following

More information

RAISON D ETRE OF THE IMMUNE SYSTEM:

RAISON D ETRE OF THE IMMUNE SYSTEM: RAISON D ETRE OF THE IMMUNE SYSTEM: To Distinguish Self from Non-Self Thereby Protecting Us From Our Hostile Environment. Innate Immunity Acquired Immunity Innate immunity: (Antigen nonspecific) defense

More information

Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-10. Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald,

Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-10. Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald, Human and mouse T cell regulation mediated by soluble CD52 interaction with Siglec-1 Esther Bandala-Sanchez, Yuxia Zhang, Simone Reinwald, James A. Dromey, Bo Han Lee, Junyan Qian, Ralph M Böhmer and Leonard

More information

JOURNAL OF VIROLOGY, May 2000, p Vol. 74, No. 9. Copyright 2000, American Society for Microbiology. All Rights Reserved.

JOURNAL OF VIROLOGY, May 2000, p Vol. 74, No. 9. Copyright 2000, American Society for Microbiology. All Rights Reserved. JOURNAL OF VIROLOGY, May 2000, p. 4127 4138 Vol. 74, No. 9 0022-538X/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved. Anti-Human Immunodeficiency Virus Type 1 (HIV-1)

More information

HD1 (FLU) HD2 (EBV) HD2 (FLU)

HD1 (FLU) HD2 (EBV) HD2 (FLU) ramer staining + anti-pe beads ramer staining a HD1 (FLU) HD2 (EBV) HD2 (FLU).73.11.56.46.24 1.12 b CD127 + c CD127 + d CD127 - e CD127 - PD1 - PD1 + PD1 + PD1-1 1 1 1 %CD127 + PD1-8 6 4 2 + anti-pe %CD127

More information

DATA SHEET. Provided: 500 µl of 5.6 mm Tris HCl, 4.4 mm Tris base, 0.05% sodium azide 0.1 mm EDTA, 5 mg/liter calf thymus DNA.

DATA SHEET. Provided: 500 µl of 5.6 mm Tris HCl, 4.4 mm Tris base, 0.05% sodium azide 0.1 mm EDTA, 5 mg/liter calf thymus DNA. Viral Load DNA >> Standard PCR standard 0 Copies Catalog Number: 1122 Lot Number: 150298 Release Category: A Provided: 500 µl of 5.6 mm Tris HCl, 4.4 mm Tris base, 0.05% sodium azide 0.1 mm EDTA, 5 mg/liter

More information

Potential cross reactions between HIV 1 specific T cells and the microbiome. Andrew McMichael Suzanne Campion

Potential cross reactions between HIV 1 specific T cells and the microbiome. Andrew McMichael Suzanne Campion Potential cross reactions between HIV 1 specific T cells and the microbiome Andrew McMichael Suzanne Campion Role of the Microbiome? T cell (and B cell) immune responses to HIV and Vaccines are influenced

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1: Cryopreservation alters CD62L expression by CD4 T cells. Freshly isolated (left) or cryopreserved PBMCs (right) were stained with the mix of antibodies described

More information

NK mediated Antibody Dependent Cellular Cytotoxicity in HIV infections

NK mediated Antibody Dependent Cellular Cytotoxicity in HIV infections NK mediated Antibody Dependent Cellular Cytotoxicity in HIV infections Amy Chung Dr. Ivan Stratov Prof. Stephen Kent ADCC process consists of Target cell QuickTime and a TIFF (Uncompressed) FcγR decompressor

More information

Immune Responses and Viral Replication in Long-Term Inapparent Carrier Ponies Inoculated with Equine Infectious Anemia Virus

Immune Responses and Viral Replication in Long-Term Inapparent Carrier Ponies Inoculated with Equine Infectious Anemia Virus JOURNAL OF VIROLOGY, July 2000, p. 5968 5981 Vol. 74, No. 13 0022-538X/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved. Immune Responses and Viral Replication in Long-Term

More information

Nature Medicine: doi: /nm.2109

Nature Medicine: doi: /nm.2109 HIV 1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs Christoph C. Carter, Adewunmi Onafuwa Nuga, Lucy A. M c Namara, James Riddell IV, Dale Bixby, Michael

More information

Progressive Telomere Shortening of Epstein-Barr Virus Specific Memory T Cells during HIV Infection: Contributor to Exhaustion?

Progressive Telomere Shortening of Epstein-Barr Virus Specific Memory T Cells during HIV Infection: Contributor to Exhaustion? BRIEF REPORT Progressive Telomere Shortening of Epstein-Barr Virus Specific Memory T Cells during HIV Infection: Contributor to Exhaustion? Debbie van Baarle, 1 Nening M. Nanlohy, 1 Sigrid Otto, 1 Fiona

More information

Effector mechanisms of cell-mediated immunity: Properties of effector, memory and regulatory T cells

Effector mechanisms of cell-mediated immunity: Properties of effector, memory and regulatory T cells ICI Basic Immunology course Effector mechanisms of cell-mediated immunity: Properties of effector, memory and regulatory T cells Abul K. Abbas, MD UCSF Stages in the development of T cell responses: induction

More information

A VACCINE FOR HIV BIOE 301 LECTURE 10 MITALI BANERJEE HAART

A VACCINE FOR HIV BIOE 301 LECTURE 10 MITALI BANERJEE HAART BIOE 301 LECTURE 10 MITALI BANERJEE A VACCINE FOR HIV HIV HAART Visit wikipedia.org and learn the mechanism of action of the five classes of antiretroviral drugs. (1) Reverse transcriptase inhibitors (RTIs)

More information

HIV-1 p24 ELISA Pair Set Cat#: orb54951 (ELISA Manual)

HIV-1 p24 ELISA Pair Set Cat#: orb54951 (ELISA Manual) HIV-1 p24 ELISA Pair Set Cat#: orb54951 (ELISA Manual) BACKGROUND Human Immunodeficiency Virus ( HIV ) can be divided into two major types, HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1 is related to

More information

Decreased EBNA-1-specific CD8 T cells in patients with Epstein Barr virus-associated

Decreased EBNA-1-specific CD8 T cells in patients with Epstein Barr virus-associated Decreased EBNA-1-specific CD8 T cells in patients with Epstein Barr virus-associated nasopharyngeal carcinoma Mark H. Fogg a, Lori J. Wirth b, Marshall Posner b, and Fred Wang a,1 a Department of Medicine,

More information

Selection on the Human Immunodeficiency Virus Type 1 Proteome following Primary Infection

Selection on the Human Immunodeficiency Virus Type 1 Proteome following Primary Infection JOURNAL OF VIROLOGY, Oct. 2006, p. 9519 9529 Vol. 80, No. 19 0022-538X/06/$08.00 0 doi:10.1128/jvi.00575-06 Copyright 2006, American Society for Microbiology. All Rights Reserved. Selection on the Human

More information

Cover Page. The handle holds various files of this Leiden University dissertation.

Cover Page. The handle   holds various files of this Leiden University dissertation. Cover Page The handle http://hdl.handle.net/1887/23854 holds various files of this Leiden University dissertation. Author: Marel, Sander van der Title: Gene and cell therapy based treatment strategies

More information

Supplementary Information. A vital role for IL-2 trans-presentation in DC-mediated T cell activation in humans as revealed by daclizumab therapy

Supplementary Information. A vital role for IL-2 trans-presentation in DC-mediated T cell activation in humans as revealed by daclizumab therapy Supplementary Information A vital role for IL-2 trans-presentation in DC-mediated T cell activation in humans as revealed by daclizumab therapy Simone C. Wuest 1, Jehad Edwan 1, Jayne F. Martin 1, Sungpil

More information

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4 + T Cells

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4 + T Cells Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4 + T Cells Gavin M. Mason, Sarah Jackson, Georgina Okecha, Emma Poole, J. G. Patrick Sissons, John Sinclair,

More information

Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot

Islet viability assay and Glucose Stimulated Insulin Secretion assay RT-PCR and Western Blot Islet viability assay and Glucose Stimulated Insulin Secretion assay Islet cell viability was determined by colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay using CellTiter

More information

Significance of the MHC

Significance of the MHC CHAPTER 7 Major Histocompatibility Complex (MHC) What is is MHC? HLA H-2 Minor histocompatibility antigens Peter Gorer & George Sneell (1940) Significance of the MHC role in immune response role in organ

More information

Human Immunodeficiency Virus type 1 (HIV-1) p24 / Capsid Protein p24 ELISA Pair Set

Human Immunodeficiency Virus type 1 (HIV-1) p24 / Capsid Protein p24 ELISA Pair Set Human Immunodeficiency Virus type 1 (HIV-1) p24 / Capsid Protein p24 ELISA Pair Set Catalog Number : SEK11695 To achieve the best assay results, this manual must be read carefully before using this product

More information

Bead Based Assays for Cytokine Detection

Bead Based Assays for Cytokine Detection Bead Based Assays for Cytokine Detection September 27, 2014 6 th EFIS-EJI South East European Immunology School SEEIS 2014 Timisoara, Romania The Cells of the Immune System The Immune Reaction (Th2) (Th1)

More information

Gladstone Institutes, University of California (UCSF), San Francisco, USA

Gladstone Institutes, University of California (UCSF), San Francisco, USA Fluorescence-linked Antigen Quantification (FLAQ) Assay for Fast Quantification of HIV-1 p24 Gag Marianne Gesner, Mekhala Maiti, Robert Grant and Marielle Cavrois * Gladstone Institutes, University of

More information

Supplementary Figure 1. ALVAC-protein vaccines and macaque immunization. (A) Maximum likelihood

Supplementary Figure 1. ALVAC-protein vaccines and macaque immunization. (A) Maximum likelihood Supplementary Figure 1. ALVAC-protein vaccines and macaque immunization. (A) Maximum likelihood tree illustrating CRF01_AE gp120 protein sequence relationships between 107 Envs sampled in the RV144 trial

More information

Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation Willemijn Hobo 1, Wieger Norde 1 and Harry Dolstra 2*

Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation Willemijn Hobo 1, Wieger Norde 1 and Harry Dolstra 2* Ex vivo Human Antigen-specific T Cell Proliferation and Degranulation Willemijn Hobo 1, Wieger Norde 1 and Harry Dolstra 2* 1 Department of Laboratory Medicine - Laboratory of Hematology, Radboud University

More information

CHAPTER 3 LABORATORY PROCEDURES

CHAPTER 3 LABORATORY PROCEDURES CHAPTER 3 LABORATORY PROCEDURES CHAPTER 3 LABORATORY PROCEDURES 3.1 HLA TYPING Molecular HLA typing will be performed for all donor cord blood units and patients in the three reference laboratories identified

More information

SUPPLEMENTARY INFORMATION. Divergent TLR7/9 signaling and type I interferon production distinguish

SUPPLEMENTARY INFORMATION. Divergent TLR7/9 signaling and type I interferon production distinguish SUPPLEMENTARY INFOATION Divergent TLR7/9 signaling and type I interferon production distinguish pathogenic and non-pathogenic AIDS-virus infections Judith N. Mandl, Ashley P. Barry, Thomas H. Vanderford,

More information

Application of μmacs Streptavidin MicroBeads for the analysis of HIV-1 directly from patient plasma

Application of μmacs Streptavidin MicroBeads for the analysis of HIV-1 directly from patient plasma Excerpt from MACS&more Vol 8 1/2004 Application of μmacs Streptavidin MicroBeads for the analysis of HIV-1 directly from patient plasma L. Davis Lupo and Salvatore T. Butera HIV and Retrovirology Branch,

More information

Viral Genetics. BIT 220 Chapter 16

Viral Genetics. BIT 220 Chapter 16 Viral Genetics BIT 220 Chapter 16 Details of the Virus Classified According to a. DNA or RNA b. Enveloped or Non-Enveloped c. Single-stranded or double-stranded Viruses contain only a few genes Reverse

More information

Supplementary Figure 1

Supplementary Figure 1 Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition

More information

Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay

Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Hepatitis B Antiviral Drug Development Multi-Marker Screening Assay Background ImQuest BioSciences has developed and qualified a single-plate method to expedite the screening of antiviral agents against

More information

RAISON D ETRE OF THE IMMUNE SYSTEM:

RAISON D ETRE OF THE IMMUNE SYSTEM: RAISON D ETRE OF THE IMMUNE SYSTEM: To Distinguish Self from Non-Self Thereby Protecting Us From Our Hostile Environment. Innate Immunity Adaptive Immunity Innate immunity: (Antigen - nonspecific) defense

More information

Received 29 August 2002/Accepted 3 December 2002

Received 29 August 2002/Accepted 3 December 2002 JOURNAL OF VIROLOGY, Mar. 2003, p. 3099 3118 Vol. 77, No. 5 0022-538X/03/$08.00 0 DOI: 10.1128/JVI.77.5.3099 3118.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved. Simian-Human

More information

Rapid antigen-specific T cell enrichment (Rapid ARTE)

Rapid antigen-specific T cell enrichment (Rapid ARTE) Direct ex vivo characterization of human antigen-specific CD154+CD4+ T cell Rapid antigen-specific T cell enrichment (Rapid ARTE) Introduction Workflow Antigen (ag)-specific T cells play a central role

More information

08/02/59. Tumor Immunotherapy. Development of Tumor Vaccines. Types of Tumor Vaccines. Immunotherapy w/ Cytokine Gene-Transfected Tumor Cells

08/02/59. Tumor Immunotherapy. Development of Tumor Vaccines. Types of Tumor Vaccines. Immunotherapy w/ Cytokine Gene-Transfected Tumor Cells Tumor Immunotherapy Autologous virus Inactivation Inactivated virus Lymphopheresis Culture? Monocyte s Dendritic cells Immunization Autologous vaccine Development of Tumor Vaccines Types of Tumor Vaccines

More information

Trends in molecular diagnostics

Trends in molecular diagnostics Trends in molecular diagnostics Detection of target genes of interest Quantification Infectious diseases HIV Hepatitis C & B TB / MAC Cytomegalovirus Herpes simplex Varicella zoster CT/GC HPV Profiling

More information

Direct ex vivo characterization of human antigen-specific CD154 + CD4 + T cells Rapid antigen-reactive T cell enrichment (Rapid ARTE)

Direct ex vivo characterization of human antigen-specific CD154 + CD4 + T cells Rapid antigen-reactive T cell enrichment (Rapid ARTE) Direct ex vivo characterization of human antigen-specific CD154 + CD4 + T cells Rapid antigen-reactive T cell enrichment (Rapid ARTE) Introduction Workflow Antigen (ag)-specific T cells play a central

More information

Optimizing Intracellular Flow Cytometry:

Optimizing Intracellular Flow Cytometry: Optimizing Intracellular Flow Cytometry: Simultaneous Detection of Cytokines and Transcription Factors An encore presentation by Jurg Rohrer, PhD, BD Biosciences 10.26.10 Outline Introduction Cytokines

More information

Madhav V. Dhodapkar, Joseph Krasovsky, Ralph M. Steinman, and Nina Bhardwaj

Madhav V. Dhodapkar, Joseph Krasovsky, Ralph M. Steinman, and Nina Bhardwaj Mature dendritic cells boost functionally superior CD8 + T-cell in humans without foreign helper epitopes Rapid PUBLICATION Madhav V. Dhodapkar, Joseph Krasovsky, Ralph M. Steinman, and Nina Bhardwaj Laboratory

More information

Received 4 December 2001/Accepted 29 April 2002

Received 4 December 2001/Accepted 29 April 2002 JOURNAL OF VIROLOGY, Aug. 2002, p. 8433 8445 Vol. 76, No. 16 0022-538X/02/$04.00 0 DOI: 10.1128/JVI.76.16.8433 8445.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved. The Relationship

More information

Critical Role for Alpha/Beta and Gamma Interferons in Persistence of Lymphocytic Choriomeningitis Virus by Clonal Exhaustion of Cytotoxic T Cells

Critical Role for Alpha/Beta and Gamma Interferons in Persistence of Lymphocytic Choriomeningitis Virus by Clonal Exhaustion of Cytotoxic T Cells JOURNAL OF VIROLOGY, Sept. 2001, p. 8407 8423 Vol. 75, No. 18 0022-538X/01/$04.00 0 DOI: 10.1128/JVI.75.18.8407 8423.2001 Copyright 2001, American Society for Microbiology. All Rights Reserved. Critical

More information

Alternate Antibody-Based Therapeutic Strategies To Purge the HIV Cell Reservoir

Alternate Antibody-Based Therapeutic Strategies To Purge the HIV Cell Reservoir Alternate Antibody-Based Therapeutic Strategies To Purge the HIV Cell Reservoir Giuseppe Pantaleo, M.D. Professor of Medicine Head, Division of Immunology and Allergy Executive Director, Swiss Vaccine

More information

In vitro human regulatory T cell suppression assay

In vitro human regulatory T cell suppression assay Human CD4 + CD25 + regulatory T cell isolation, in vitro suppression assay and analysis In vitro human regulatory T cell suppression assay Introduction Regulatory T (Treg) cells are a subpopulation of

More information

Cytotoxicity assays. Rory D. de Vries, PhD 1. Viroscience lab, Erasmus MC, Rotterdam, the Netherlands

Cytotoxicity assays. Rory D. de Vries, PhD 1. Viroscience lab, Erasmus MC, Rotterdam, the Netherlands Cytotoxicity assays Rory D. de Vries, PhD 1 1 Viroscience lab, Erasmus MC, Rotterdam, the Netherlands Anti-influenza immunity Humoral / CD4+ / CD8+ / NK? Function of CTL Elimination of virus-infected cells?

More information

Instructions for Use. RealStar Influenza S&T RT-PCR Kit /2017 EN

Instructions for Use. RealStar Influenza S&T RT-PCR Kit /2017 EN Instructions for Use RealStar Influenza S&T RT-PCR Kit 3.0 01/2017 EN RealStar Influenza S&T RT-PCR Kit 3.0 For research use only! (RUO) 163003 INS-163000-EN-S02 96 01 2017 altona Diagnostics GmbH Mörkenstr.

More information

Defining kinetic properties of HIV-specific CD8 + T-cell responses in acute infection

Defining kinetic properties of HIV-specific CD8 + T-cell responses in acute infection Defining kinetic properties of HIV-specific CD8 + T-cell responses in acute infection Yiding Yang 1 and Vitaly V. Ganusov 1,2,3 1 Department of Microbiology, University of Tennessee, Knoxville, TN 37996,

More information

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness CLINICAL MICROBIOLOGY REVIEWS, Oct. 2007, p. 550 578 Vol. 20, No. 4 0893-8512/07/$08.00 0 doi:10.1128/cmr.00017-07 Copyright 2007, American Society for Microbiology. All Rights Reserved. Clinical Significance

More information

Instructions for Use. RealStar Influenza Screen & Type RT-PCR Kit /2017 EN

Instructions for Use. RealStar Influenza Screen & Type RT-PCR Kit /2017 EN Instructions for Use RealStar Influenza Screen & Type RT-PCR Kit 4.0 05/2017 EN RealStar Influenza Screen & Type RT-PCR Kit 4.0 For research use only! (RUO) 164003 INS-164000-EN-S01 96 05 2017 altona

More information

Phosphate buffered saline (PBS) for washing the cells TE buffer (nuclease-free) ph 7.5 for use with the PrimePCR Reverse Transcription Control Assay

Phosphate buffered saline (PBS) for washing the cells TE buffer (nuclease-free) ph 7.5 for use with the PrimePCR Reverse Transcription Control Assay Catalog # Description 172-5080 SingleShot Cell Lysis Kit, 100 x 50 µl reactions 172-5081 SingleShot Cell Lysis Kit, 500 x 50 µl reactions For research purposes only. Introduction The SingleShot Cell Lysis

More information

EBV Infection and Immunity. Andrew Hislop Institute for Cancer Studies University of Birmingham

EBV Infection and Immunity. Andrew Hislop Institute for Cancer Studies University of Birmingham EBV Infection and Immunity Andrew Hislop Institute for Cancer Studies University of Birmingham EBV Introduction Large ds DNA virus Spread by saliva contact Lifelong infection Predominantly B-lymphotropic

More information

Multi-Virus-Specific T cell Therapy for Patients after HSC and CB Transplant

Multi-Virus-Specific T cell Therapy for Patients after HSC and CB Transplant Multi-Virus-Specific T cell Therapy for Patients after HSC and CB Transplant Hanley PJ, Krance BR, Brenner MK, Leen AM, Rooney CM, Heslop HE, Shpall EJ, Bollard CM Hematopoietic Stem Cell Transplantation

More information

Received 19 September 2007/Accepted 21 November 2007

Received 19 September 2007/Accepted 21 November 2007 JOURNAL OF VIROLOGY, Feb. 2008, p. 1723 1738 Vol. 82, No. 4 0022-538X/08/$08.00 0 doi:10.1128/jvi.02084-07 Copyright 2008, American Society for Microbiology. All Rights Reserved. Patterns of CD8 Immunodominance

More information

5. Over the last ten years, the proportion of HIV-infected persons who are women has: a. Increased b. Decreased c. Remained about the same 1

5. Over the last ten years, the proportion of HIV-infected persons who are women has: a. Increased b. Decreased c. Remained about the same 1 Epidemiology 227 April 24, 2009 MID-TERM EXAMINATION Select the best answer for the multiple choice questions. There are 60 questions and 9 pages on the examination. Each question will count one point.

More information

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES

Fayth K. Yoshimura, Ph.D. September 7, of 7 HIV - BASIC PROPERTIES 1 of 7 I. Viral Origin. A. Retrovirus - animal lentiviruses. HIV - BASIC PROPERTIES 1. HIV is a member of the Retrovirus family and more specifically it is a member of the Lentivirus genus of this family.

More information

The functional CD8 T cell response to HIV becomes type-specific in progressive disease

The functional CD8 T cell response to HIV becomes type-specific in progressive disease The functional CD8 T cell response to HIV becomes type-specific in progressive disease Sang Kyung Lee, Zhan Xu, Judy Lieberman, and Premlata Shankar Center for Blood Research and Department of Pediatrics,

More information

Principle of the FluoroSpot assay. Anti-tag mab-green. Streptavidin-Red. Detection mab-tag. Detection mab-biotin. Analyte. Analyte.

Principle of the FluoroSpot assay. Anti-tag mab-green. Streptavidin-Red. Detection mab-tag. Detection mab-biotin. Analyte. Analyte. FluoroSpot 1 The principle objective of the FluoroSpot assay is the simultaneous measurement of dual cytokine secretion at the single cell level. This is accomplished by using a mixture of monoclonal antibodies

More information

Predicting the Impact of a Nonsterilizing Vaccine against Human Immunodeficiency Virus

Predicting the Impact of a Nonsterilizing Vaccine against Human Immunodeficiency Virus JOURNAL OF VIROLOGY, Oct. 2004, p. 11340 11351 Vol. 78, No. 20 0022-538X/04/$08.00 0 DOI: 10.1128/JVI.78.20.11340 11351.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Predicting

More information

HIV-1 Subtypes: An Overview. Anna Maria Geretti Royal Free Hospital

HIV-1 Subtypes: An Overview. Anna Maria Geretti Royal Free Hospital HIV-1 Subtypes: An Overview Anna Maria Geretti Royal Free Hospital Group M Subtypes A (1, 2, 3) B C D F (1, 2) G H J K Mechanisms of HIV-1 genetic diversification Point mutations RT error rate: ~1 per

More information

Supplementary Data. Treg phenotype

Supplementary Data. Treg phenotype Supplementary Data Additional Experiment An additional experiment was performed using cryopreserved peripheral blood mononuclear cells (PBMC) derived from five renal cell carcinoma (RCC) patients [see

More information

Optimizing Intracellular Flow Cytometry:

Optimizing Intracellular Flow Cytometry: Optimizing Intracellular Flow Cytometry: Simultaneous Detection of Cytokines and Transcription Factors Presented by Jurg Rohrer, PhD, BD Biosciences 23-10780-00 Outline Introduction Cytokines Transcription

More information

Diagnostic Methods of HBV infection. Zohreh Sharifi,ph.D of Virology Research center, Iranian Blood Transfusion Organization (IBTO)

Diagnostic Methods of HBV infection. Zohreh Sharifi,ph.D of Virology Research center, Iranian Blood Transfusion Organization (IBTO) Diagnostic Methods of HBV infection Zohreh Sharifi,ph.D of Virology Research center, Iranian Blood Transfusion Organization (IBTO) Hepatitis B-laboratory diagnosis Detection of HBV infection involves

More information

HIV-1 Viral Load Real Time (RG)

HIV-1 Viral Load Real Time (RG) -1 Viral Load Real Time (RG) Real Time RT-PCR type 1 RNA quantification assay MSP Reg. pending Valdense 3616. 11700. Montevideo. Uruguay. phone (598) 2 336 83 01. Fax (598) 2 336 71 60. Info@atgen.com.uy

More information

Medical Virology Immunology. Dr. Sameer Naji, MB, BCh, PhD (UK) Head of Basic Medical Sciences Dept. Faculty of Medicine The Hashemite University

Medical Virology Immunology. Dr. Sameer Naji, MB, BCh, PhD (UK) Head of Basic Medical Sciences Dept. Faculty of Medicine The Hashemite University Medical Virology Immunology Dr. Sameer Naji, MB, BCh, PhD (UK) Head of Basic Medical Sciences Dept. Faculty of Medicine The Hashemite University Human blood cells Phases of immune responses Microbe Naïve

More information

TITLE: MODULATION OF T CELL TOLERANCE IN A MURINE MODEL FOR IMMUNOTHERAPY OF PROSTATIC ADENOCARCINOMA

TITLE: MODULATION OF T CELL TOLERANCE IN A MURINE MODEL FOR IMMUNOTHERAPY OF PROSTATIC ADENOCARCINOMA AD Award Number: DAMD17-01-1-0085 TITLE: MODULATION OF T CELL TOLERANCE IN A MURINE MODEL FOR IMMUNOTHERAPY OF PROSTATIC ADENOCARCINOMA PRINCIPAL INVESTIGATOR: ARTHUR A HURWITZ, Ph.d. CONTRACTING ORGANIZATION:

More information

COURSE: Medical Microbiology, MBIM 650/720 - Fall TOPIC: Antigen Processing, MHC Restriction, & Role of Thymus Lecture 12

COURSE: Medical Microbiology, MBIM 650/720 - Fall TOPIC: Antigen Processing, MHC Restriction, & Role of Thymus Lecture 12 COURSE: Medical Microbiology, MBIM 650/720 - Fall 2008 TOPIC: Antigen Processing, MHC Restriction, & Role of Thymus Lecture 12 FACULTY: Dr. Mayer Office: Bldg. #1, Rm B32 Phone: 733-3281 Email: MAYER@MED.SC.EDU

More information

Supporting Information

Supporting Information Supporting Information Horwitz et al. 73/pnas.35295 A Copies ml - C 3NC7 7 697 698 7 7 73 76-2 2 Days Gp2 residue G458D G459D T278A 7/36 N28 K D 28 459 A28T ID# 697 ID# 698 ID# 7 ID# 7 ID# 73 ID# 76 ID#

More information

TITLE: Development of Antigen Presenting Cells for adoptive immunotherapy in prostate cancer

TITLE: Development of Antigen Presenting Cells for adoptive immunotherapy in prostate cancer AD Award Number: W8-XWH-5-- TITLE: Development of Antigen Presenting Cells for adoptive immunotherapy in prostate cancer PRINCIPAL INVESTIGATOR: Mathias Oelke, Ph.D. CONTRACTING ORGANIZATION: Johns Hopkins

More information

The Swarm: Causes and consequences of HIV quasispecies diversity

The Swarm: Causes and consequences of HIV quasispecies diversity The Swarm: Causes and consequences of HIV quasispecies diversity Julian Wolfson Dept. of Biostatistics - Biology Project August 14, 2008 Mutation, mutation, mutation Success of HIV largely due to its ability

More information