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We have determined the frequencies of human leucocyte antigen (HLA)-B*57:01, HLA-B*35:05, HLA-C*04 and HLA-C*08 in healthy individuals of South African Indian (SAI) ethnicity (n = 50) and South African mixed (SAM) ancestry (n = 50) using real-time allele-specific polymerase chain reaction (AS-PCR) assay. HLA-B*57:01 associates with immune hypersensitivity reaction (IHR) in individuals exposed to abacavir (ABC), while nevirapine (NVP) IHR associates with HLA-B*35:05, HLA-C*04 and HLA-C*08. Real-time AS-PCR assays typically use less DNA, are more cost-effective and rapid compared with conventional genotyping methods, such as sequence-based typing (SBT). The assay was developed using samples of known HLA class I genotype and subsequently applied to the SAI and SAM samples. HLA-B*57:01 was detected in SAM and SAI populations at frequencies of 8.0% and 12.0%, respectively, while HLA-B*35:05 was not found in SAI individuals, but was present in 6.0% of SAM individuals. HLA-C*04 was detected in 22.0% and 24.0% of SAM and SAI individuals, respectively, while 10.0% and 8.0% of SAM and SAI individuals, respectively, were HLA-C*08 positive. This study reports the development of a novel real-time AS-PCR assay to identify HLA class I alleles associated with ABC and NVP IHR and has established the frequencies of these alleles present in healthy SAI and SAM populations. Using South African demographic data, our hypothetical analysis suggests that a substantial number of individuals would benefit from the assay.

Understanding the link between vaccine immunogenicity and efficacy is currently a major focus in HIV research. Consequently, recent developments in the HIV-1 vaccine field have led to a closer look at immune responses to known efficacious vaccines. We undertook a study to explore clinical predictors of vaccine efficacy following recombinant hepatitis B (rHBV) vaccination in a cohort of HIV-uninfected, hepatitis B virus naïve women living in a peri-urban setting in Cape Town. Our aim was to define host biological risk factors associated with lack of vaccine uptake. We found a significant association (p=0.009) between body mass index (BMI) and lack of vaccine-specific IgG titre (

This study examines sequential lymph nodes from 13 drug-naive patients before and after 24 weeks of highly active antiretroviral therapy (HAART). A multipronged approach was used to study changes in HIV-1 RNA in each paired lymph node in relation to tissue architecture and frequency of naive T cells. After 24 weeks, all patients showed significant suppression of plasma viral load and 12 of 13 showed concordant viral suppression in the lymph node (p = 0.001). Using in situ hybridization and quantitative image analysis, we showed that HIV-1 RNA was reduced to below detectable levels (two copies per cell) in follicular dendritic cell (FDC) and mononuclear cell pools. Independent immunohistochemical analysis of lymph node sections revealed that 5 of 13 patients displayed increased FDC networks and 6 of 13 showed no change and all patients showed increases in tissue-resident CD4+ cells. All lymph node biopsies at 24 weeks showed increased proportions of CD4+ and CD8+ cells coexpressing the naive markers CD45RA and CD62L when compared with baseline values. Significant correlations existed between viral load suppression and loss of activated CD8+ T cells after 24 weeks in both lymph node and blood, which was mirrored by significantly lowered frequencies of activated peripheral Gag peptide/MHC tetramer+ CD8+ cells. Overall, these data show that a potent and successful treatment strategy that significantly suppresses and removes FDC-resident HIV-1 results in improvements in lymphoid architecture and by so doing provides the structures available for increased numbers of naive cells to interact with cognate antigen. In addition, our article shows that suppression of HIV-1 replication results in diminished frequencies of peripherally activated antigen-specific CD8+ cells.

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77–85) and reverse transcriptase (amino acids 464–472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50–1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.

Phagocytosis and oxidative burst in whole-blood granulocytes were assessed by flow cytometry with Phagotest and Bursttest kits, respectively. Seventy individuals were included in this study: 15 healthy, normal donors, 18 human immunodeficiency virus (HIV) type 1 (HIV-1)-seropositive patients, 19 patients with pulmonary tuberculosis (TB), and 18 patients co-infected with Mycobacterium tuberculosis and HIV-1 (TB-HIV). Granulocyte phagocytosis was assessed by incubating whole blood with fluorescence-labelled Escherichia coli and measuring the proportion of granulocytes with ingested bacteria and the capacity (fluorescence intensity) of each cell to phagocytose E. coli . The percentage of granulocytes converting nonfluorescent dihydrorhodamine to fluorescent rhodamine 123 on production of reactive oxygen intermediates (ROIs) and the mean channel shift were assessed as a measure of oxidative burst. No differences in the proportion of granulocytes that were capable of phagocytosing or producing ROIs in response to E. coli were observed between any of the study groups. Phagocytosis was significantly enhanced in granulocytes from HIV-1-infected individuals. On the other hand, granulocytes from individuals infected with M. tuberculosis alone or in combination with HIV-1 had a significantly reduced capacity to phagocytose E. coli and to produce ROIs in response to E. coli as an agonist. These results provide evidence that granulocytes from individuals with pulmonary TB with or without concomitant infection with HIV-1 have an impaired ability to phagocytose and to undergo oxidative burst, possibly contributing to the enhanced susceptibility to opportunistic infections in these patients.

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

The generation of polyfunctional CD8+ T cells, in response to vaccination or natural infection, has been associated with improved protective immunity. However, it is unclear whether the maintenance of polyfunctionality is related to particular cellular phenotypic characteristics. To determine whether the cytokine expression profile is linked to the memory differentiation stage, we analyzed the degree of polyfunctionality of HIV-specific CD8+ T cells within different memory subpopulations in 20 antiretroviral therapy-naive HIV-1–infected individuals at ∼34 wk postinfection. These profiles were compared with CMV-specific CD8+ T cell responses in HIV-uninfected control subjects and in individuals chronically infected with HIV. Our results showed that the polyfunctional abilities of HIV-specific CD8+ T cells differed according to their memory phenotype. Early-differentiated cells (CD45RO+CD27+) exhibited a higher proportion of cells positive for three or four functions (p < 0.001), and a lower proportion of monofunctional cells (p < 0.001) compared with terminally differentiated (TD; CD45RO−CD27−) HIV-specific CD8+ T cells. The majority of TD HIV-specific CD8+ T cells were monofunctional (median 69% [interquartile range: 57–83]), producing predominantly CD107a or MIP1β. Moreover, proportions of HIV-specific monofunctional CD8+ T cells positively associated with proportions of TD HIV-specific CD8+ T cells (p = 0.019, r = 0.54). In contrast, CMV-specific CD8+ T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Overall, these data show that the polyfunctional abilities of HIV-specific CD8+ T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses.

HIV-specific early-differentiated cells of a central memorylike phenotype (CD45RO+CD27+) exhibited a higher proportion of cells positive for three or four functions (p

BACKGROUND: Sputum Mycobacterium tuberculosis (Mtb) culture is commonly used to assess response to antibiotic treatment in individuals with pulmonary tuberculosis (TB). Such techniques are constrained by the slow growth rate of Mtb, and more sensitive methods to monitor Mtb clearance are needed. The goal of this study was to evaluate changes in plasma cytokines in patients undergoing treatment for TB as a means of identifying candidate host markers associated with microbiologic response to therapy. METHODS: Twenty-four plasma cytokines/chemokines were measured in 42 individuals diagnosed with active pulmonary TB, 52% were HIV co-infected. Individuals, undergoing a 26-week standard TB treatment, were followed longitudinally over 18 months and measurements were associated with HIV status and rates of sputum culture conversion. RESULTS: Plasma concentrations of interferon-inducible protein-10 (IP-10) and vascular endothelial growth factor (VEGF) were significantly reduced upon TB treatment, regardless of HIV status. By the end of treatment, IP-10 concentrations were significantly lower in HIV negative individuals when compared to HIV-positive individuals (p = 0.02). Moreover, in HIV negative patients, plasma VEGF concentrations, measured as early as 2-weeks post TB treatment initiation, positively correlated with the time of sputum conversion (p = 0.0017). No significant changes were observed in other studied immune mediators. CONCLUSIONS: These data suggest that VEGF plasma concentration, measured during early TB treatment, could represent a surrogate marker to monitor sputum culture conversion in HIV uninfected individuals.

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC(50) values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants., Author Summary An important role for the polyfunctional T-cell fraction of anti-HIV CD8 responses during chronic HIV infection has previously been suggested. This study characterized the role of polyfunctional T-cells directed against the transmitted/founder virus in the selection of viral escape mutants during acute HIV-1 infection within a unique cohort of individuals recruited within 3 weeks from the onset of symptoms at the time when the virus load was still declining. For the first time, the sequences of the transmitted/founder virus isolated from each patient were used. Interestingly, polyfunctionality was not found to be a pre-requisite for selection of escape mutations. A novel significant correlation is found between the order of appearance of escape mutations in different epitope sequences and both the magnitude of the CD8+ T-cell responses and the degree of entropy of the individual epitopes. A high proportion of the T-cells participating in the total response produced MIP-1β, suggesting that mechanisms not limited to the killing of infected cells might play a relevant role in early infection. This highlights the importance of measuring the quality of the CD8+ lymphocyte response and the sequence of the transmitted virus isolates to better understand the mechanisms of control of HIV replication during acute infection.

In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae . Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8 + T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.

Background: Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection. Methodology: FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution. Principal Findings: HIV infected individuals had significantly higher frequencies of CD4 + FOXP3 + T cells (median of 8.11%; range 1.33%–26.27%) than healthy controls (median 3.72%; range 1.3–7.5%; P=0.002), despite having lower absolute counts of CD4 + FOXP3 + T cells. There was a significant positive correlation between the frequency of CD4 + FOXP3 + T cells and viral load (rho=0.593 P=0.003) and a significant negative correlation with CD4 count (rho= 20.423 P=0.044). 48% of our patients had CD4 counts below 200 cells/ml and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%–26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4 + T cells following antigenic or other stimulation. Conclusions/Significance: FOXP3 expression in the CD4 + T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.

Mycobacterium bovis BCG is considered an attractive live bacterial vaccine vector. In this study, we investigated the immune response of baboons to a primary vaccination with recombinant BCG (rBCG) constructs expressing the gag gene from a South African HIV-1 subtype C isolate, and a boost with HIV-1 subtype C Pr55(gag) virus-like particles (Gag VLPs). Using an interferon enzyme-linked immunospot assay, we show that although these rBCG induced only a weak or an undetectable HIV-1 Gag-specific response on their own, they efficiently primed for a Gag VLP boost, which strengthened and broadened the immune responses. These responses were predominantly CD8+ T cell-mediated and recognised similar epitopes as those targeted by humans with early HIV-1 subtype C infection. In addition, a Gag-specific humoral response was elicited. These data support the development of HIV-1 vaccines based on rBCG and Pr55(gag) VLPs.

Objectives To describe the baseline demographic data, clinical characteristics and HIV-incidence rates of a cohort at high risk for HIV infection in South Africa as well as the challenges experienced in establishing and maintaining the cohort. Methodology/Principle Findings Between August 2004 and May 2005 a cohort of HIV-uninfected women was established for the CAPRISA 002 Acute Infection Study, a natural history study of HIV-1 subtype C infection. Volunteers were identified through peer-outreach. The cohort was followed monthly to determine HIV infection rates and clinical presentation of early HIV infection. Risk reduction counselling and male and female condoms were provided. After screening 775 individuals, a cohort of 245 uninfected high-risk women was established. HIV-prevalence at screening was 59.6% (95% CI: 55.9% to 62.8%) posing a challenge in accruing HIV-uninfected women. The majority of women (78.8%) were self-identified as sex-workers with a median of 2 clients per day. Most women (95%) reported more than one casual sexual partner in the previous 3 months (excluding clients) and 58.8% reported condom use in their last sexual encounter. Based on laboratory testing, 62.0% had a sexually transmitted infection at baseline. During 390 person-years of follow-up, 28 infections occurred yielding seroincidence rate of 7.2 (95% CI: 4.5 to 9.8) per 100 person-years. Despite the high mobility of this sex worker cohort retention rate after 2 years was 86.1%. High co-morbidity created challenges for ancillary care provision, both in terms of human and financial resources. Conclusions/Significance Challenges experienced were high baseline HIV-prevalence, lower than anticipated HIV-incidence and difficulties retaining participants. Despite challenges, we have successfully accrued this cohort of HIV-uninfected women with favourable retention, enabling us to study the natural history of HIV-1 during acute HIV-infection. Our experiences provide lessons for others establishing similar cohorts, which will be key for advancing the vaccine and prevention research agenda in resource-constrained settings.

The advent of T-cell assay methodologies that are amenable to high throughput coupled with the availability of large libraries of overlapping peptides have revolutionized the fields of vaccine efficacy testing and cellular immune response assessment. Since T-cell assay performance is critically dependent upon the quality and specificity of the stimulating peptides, assurance of high-quality and reliable input peptides is an important aspect of assay validation. Herein, we demonstrate that individual peptides from large human immunodeficiency virus (HIV)-based peptide library sets obtained directly from two independent custom peptide suppliers contained contaminating peptides capable of giving false-positive results, which were consistent with nominal antigen-specific CD8 + T-cell responses. In-depth investigation of the cellular response in terms of responding CD8 + T-cell frequency and human leukocyte antigen (HLA) restriction led to the conclusion that one set of HIV type 1 (HIV-1)-derived peptides was contaminated with a peptide from human cytomegalovirus (HCMV), which is commonly used in cellular immunology research applications. Analytical characterization of the original stock of the suspect HIV-1 peptide confirmed the presence of ∼1% by weight of the HCMV peptide. These observations have critical implications for quality assurance (QA) and quality control (QC) of peptides used in clinical trials where cellular immune-based assays are important end-point determinants. We propose a simple schema of biological QA/QC protocols to augment the standard biochemical QA/QC analyses as a means to circumvent this and other problems that can affect cellular immune-based assay outcome and interpretation.

Cytotoxic T lymphocyte (CTL) responses are thought to be essential for the control of HIV-1 replication in vivo and immunogens that elicit CTL responses are currently a major focus of HIV vaccine research. Here we investigated two aspects of the CTL response to HIV-1 subtype C that are important for vaccine design and efficacy monitoring. First, we assessed the relationship between the CTL response and sequence diversity, using a robust statistical method. While peptides that were most frequently recognized by the CTL response in Nef and p24 tended to be conserved, this was not the case for p17 where epitope recognition coincided with highly variable regions. Second, we investigated the relationship between observed and predicted CTL responses, given the HLA genotype of infected individuals. Only 52% of the Nef peptides and 64% of the Gag peptides that elicited a CTL response contained sequence motifs thought to be required for binding by the HLA-A or -B alleles found in the corresponding patient. In a comparable subtype B dataset a much higher proportion of the peptides that elicited a CTL response were consistent with the patient HLA genotype (96% and 83% for Nef and Gag, respectively). We demonstrate that this difference between subtypes C and B is likely to result from a combination of a tendency for HLA alleles common in Southern African populations to be poorly characterized, as well as a tendency for sequence motifs associated with HLA recognition to be overspecified for sequence variation found in the B clade. Our results suggest that knowledge of HLA binding motifs is likely to be biased toward certain populations and subtypes. This can have important implications for understanding immune escape and predicting vaccine efficacy in the context of populations primarily infected with non-B subtype HIV-1.

Several HIV-1 subtype C-specific gag- and/or nef-based vaccines are currently intended for clinical trial in southern Africa. Here we provide sequences of 64 gag and 45 nef genes sampled in Malawi, Zambia, Zimbabwe, and South Africa and assess the degree of southern African HIV-1 diversity that will confront these vaccines. Whereas reasonable phylogenetic evidence exists for geographical clustering of subtype C gag and nef sequences from various other parts of the world, there is little evidence of similar population founder effects in the southern African epidemic. The entire breadth of subtype C diversity is represented in the southern African genes suggesting there may be no advantage in producing region- or country-specific subtype C vaccines. We do not, however, find much evidence of intersubtype recombination in the Southern African genes, implying that the likelihood of vaccine failure due to the emergence of intersubtype recombinants is probably low.

OBJECTIVES Southwest Tanzania is affected by an HIV-1 epidemic consisting of subtypes A, C, and D, and their recombinant forms. This study was designed to assess whether the Gag- and Nef-specific T-cell response is biased towards recognizing the infecting subtype. METHODS The infecting subtypes were characterized with a Multi-hybridization assay that discriminates between subtypes A, C and D. The interferon-gamma ELISPOT assay was used to detect the Gag- and Nef-specific T-cell responses in freshly isolated peripheral blood mononuclear cells in 56 seropositive patients. To study the HIV-specific T-cell responses, isolate-based Gag and Nef peptide sets representative of the locally occurring subtypes were used. The results were analysed at the total protein and single peptide level. RESULTS In the study population, 35% were infected with a pure C subtype, 24% and 23% with ACD or AC recombinant forms, respectively. The total magnitude (P < 0.01) and breadth (P < 0.01) of the Gag-specific T-cell response detected with the subtype C-Gag peptide set was significantly greater than that detected with either the subtype A-Gag or D-Gag peptide sets. No significant difference was observed in the Nef-specific response. In 85% of responses targeting the most immunodominant Gag epitopes with subtype-specific sequence differences, the best recognized epitope variant corresponded to the infecting subtype. CONCLUSIONS The Gag-specific T-cell response had a preference for recognizing peptides related to the infecting subtype.

Introduction The biological mechanisms underlying HIV risk in younger women is unclear. HIV is primarily transmitted across the genital mucosa and preferentially infects CD4 + T-cells. We investigated the influence of asymptomatic sexually transmitted infections (STIs) and bacterial vaginosis (BV) on CD4 + T-cell activation and inflammation in the genital tracts of adolescents from South Africa. Methods Cervical cytobrush mononuclear cells were isolated from 148 adolescents (16–22 years) from Cape Town, and expression of T-cell activation and proliferation markers (CD38, HLA-DR, Ki67, CCR5) was measured by FACs. Adolescents were screened for BV (Nugent) and STIs ( C. trachomatis , N. gonnorhoea, T. vaginalis , M. genitalium , HSV-2) by PCR. For comparison, 11 HIV-negative adult women were included. Concentrations of 48 cytokines, chemokines and growth factors were measured in matching menstrual cups by Luminex. Results Adolescents (median 18 years; IQR 17–20) had significantly higher frequencies of activated CD4 + T-cells (CD38 + , HLADR + , CD38 + HLADR + : each p C. trachomatis positive. Adolescents with an STI, despite these being asymptomatic, had higher frequencies of activated and proliferating T-cells compared to those with no STI/BV (CD4 + CD38 + HLADR + : p = 0.047; CD4 + Ki67 + : p = 0.020). Women positive for chlamydia had significantly higher frequencies of CD4 + CD38 + T-cells (p = 0.006). Women with both STIs and BV had the most pronounced increase in CD4 + T-cell activation (CD38 + : p = 0.002; CD38 + HLADR + : p = 0.001; Ki67+: p = 0.002). Higher cervical T-cell activation marker expression was directly associated with increased genital cytokine profiles. Conclusion Heightened levels of genital immune activation and inflammation found in South African adolescent females, partly due to the presence of asymptomatic STIs and BV could increase their risk for HIV infection.

Introduction South African adolescent females are at high risk of HIV acquisition, disproportionate to their sexual behaviour. We hypothesised that biological changes associated with puberty may influence this susceptibility. Methods This study was conducted in two South African sites, the Desmond Tutu Youth Centre, Masiphumele, Cape Town and the Perinatal HIV Research Centre, Soweto, Johannesburg. Cytokines were measured by Luminex. Sexual risk behaviour, contraceptive use and the prevalence of sexually transmitted infections (STIs) [ C. trachomatis (CT), N. gonorrhoeae (NG), T. vaginalis , M. genitalium , HSV-2, syphilis], bacterial vaginosis (BV) and candida were assessed in each women. Results The prevalence of STIs or BV was 71% in Cape Town and 54% in Johannesburg, with 47% of Cape Town and 42% of Johannesburg women having BV. The CT prevalence in Cape Town [62/148 (42%)] was substantially higher than Johannesburg [26/149 (17%); p Conclusion An alarmingly high STI and BV prevalence was found in these at risk populations, indicating a need for improved preventative strategies. In young women, BV caused a greater degree of inflammation than STIs and its effective management requires further investigation. This work was supported by a Strategic Health Innovation Partnerships (SHIP) grant from the South African Medical Research Council and European and Developing Countries Clinical Trials Partnership (EDCTP). The cohort was supported by grants from the EDCTP. SLB was supported by the HIV Vaccine Trials Network, the Fogarty Foundation and the South African Medical Research Council (MRC). No pharmaceutical grants were received in the development of this study.

The definition of immune correlates of protection in HIV-1 infection is pivotal to the design of successful vaccine candidates and strategies. Although significant methodological and conceptual strides have been made in our understanding of HIV-specific cellular immunity, we have not yet defined those parameters that have a role in controlling the spread of HIV infection. This review discusses the basis of our understanding of HIV-specific cellular immunity and identifies its shortcomings. Furthermore, potential protective characteristics will be proposed that may ultimately be required for an effective vaccine designed to stimulate cellular immunity against HIV-1.

With more than 6 million HIV positive individuals—the highest of any country in the world—South Africa is the epicentre of the HIV/AIDS pandemic. In much of sub-Saharan Africa, especially South Africa, the HIV/AIDS epidemic is growing fastest in teenagers, especially young girls (see sidebar) [1]. Hence, any future vaccination strategy in this setting will, of necessity, have to target young adolescents prior to sexual debut. The current candidate HIV vaccines that are being considered for efficacy trials are likely to act principally by reducing viral loads, hence reducing both transmission to sexual partners and progression to AIDS [2]. One of the limitations to conducting HIV vaccine trials is the dearth of data on important HIV disease parameters such as viral load, immune responses, and disease progression in African adolescents. While there is observational research currently being conducted in the United States on adolescents, such as the Reaching for Excellence in Adolescent Care and Health (REACH) study [3], there is a need for information on set point, HIV transmission, and disease progression in adolescents from Africa, even if only to confirm whether the disease process in African adolescents differs from those in developed countries. The Joint United Nations Programme on HIV/AIDS notes that children, including infants and adolescents, should be eligible for enrolment in HIV preventive vaccine trials, both as a matter of equity and because young adolescents and children are at high risk of HIV infection [4]. However, no distinction is drawn between adolescent participation with parental consent and the autonomous participation of adolescents. This distinction is particularly important in sub-Saharan Africa, where it is often difficult or impossible to obtain parental consent. The Centre for the AIDS Programme of Research in South Africa (CAPRISA), which is funded by the US National Institutes of Health, recently proposed a follow-up study of adolescents and young adults with acute HIV infection to obtain the essential information on subtype C viral set point, prognostic viral load measurements, and disease outcome—all important biological outcome markers in HIV vaccine efficacy trials. The study proposed to enrol, as one of its groups, adolescents as young as 14 years old from a prenatal and family planning clinic, where those who volunteered could do so without parental consent when parents or legal guardians were not available. Three ethics committees supported the enrolment of this cohort, although a fourth did not. As a result, the study protocol was amended to include participants only above the age of 18. Several works have highlighted the ethico-legal challenges implicit in enrolling adolescents in South African HIV vaccine trials [5–7]. However, the enrolment of autonomous adolescents in crucial HIV observational studies and in other types of sensitive non-HIV research in South Africa is equally problematic. We present the ethico-legal challenges, as well as the scientific and social grounds, to justify the autonomous participation of adolescents in HIV observational studies—such as the CAPRISA study—as well as in future HIV vaccine trials in South Africa and much of the rest of sub-Saharan Africa. We argue that the same reasoning applies to other types of sensitive non-HIV research involving adolescents. We also suggest how conflicts between restrictive laws and flexible ethics guidelines should be addressed.

The European Commission (EC) has strong commitments and recognises the need to continue to ensure that HIV/AIDS research efforts receive global attention. The EC is facing this challenge in a global context and has made substantial investments together with European Developing Countries Clinical Trial Partnership (EDCTP) to formulate a program for the accomplishment of a scientific strategic plan promoting the European/African HIV vaccine development approach. The EC and EDCTP has convened a number of meetings by experts in basic and clinical virology, immunology, epidemiology, as well as industrial and regulatory representatives. The remit of the committee of experts was to define (1) objective criteria for selection of HIV candidates; (2) to determine criteria for selection of sites for clinical trials in Europe and Africa. The resulting consensus paper will guide the EC and EDCTP in developing HIV vaccine strategy and recommendations.

Defining viral dynamics in natural infection is prognostic of disease progression and could prove to be important for vaccine trial design as viremia may be a likely secondary end point in phase III HIV efficacy trials. There are limited data available on the early course of plasma viral load in subtype C HIV-1 infection in Africa. Plasma viral load and CD4+ T cell counts were monitored in 51 recently infected subjects for 9 months. Individuals were recruited from four southern African countries: Zambia, Malawi, Zimbabwe, and South Africa and the median estimated time from seroconversion was 8.9 months (interquartile range, 5.7-14 months). All were infected with subtype C HIV-1 and median viral loads, measured using branched DNA, ranged from 3.82-4.02 log10 RNA copies/ml from 2-24 months after seroconversion. Viral loads significantly correlated with CD4+ cell counts (r=-0.5, p0.0001; range, 376-364 cells/mm3) and mathematical modeling defined a median set point of 4.08 log10 (12 143 RNA copies/ml), which was attained approximately 17 months after seroconversion. Comparative measurements using three different viral load platforms (bDNA, Amplicor, and NucliSens) confirmed that viremia in subtype C HIV-1-infected individuals within the first 2 years of infection did not significantly differ from that found in early subtype B infection. In conclusion, the course of plasma viremia, as described in this study, will allow a useful baseline comparator for understanding disease progression in an African setting and may be useful in the design of HIV-1 vaccine trials in southern Africa.

This longitudinal study aimed to determine the incidence and pathogenic implications of dual human immunodeficiency virus type 1 (HIV-1) infection in a cohort of female sex workers. Blood samples from 31 recently infected women were screened by use of a heteroduplex mobility assay and sequencing. The median viral load set point was 5404 copies/ mL (n = 22), which was measured by use of the bDNA assay. Within 3 months of infection, 19% (6/31) of the women were dually infected with 2 distinct HIV-1 subtype C viruses. No evidence of superinfection was detected over the course of 24 months of follow-up, indicating that the risk of dual infection is highest around the time of the initial infection. There was a significant association between dual infection and elevated viral load set point.

Characterization of human leukocyte antigen (HLA) class I restricted epitopes derived from viral pathogens is imperative for formulating therapeutic interventions, as well as for vaccine design and monitoring. Sensitive, easy and cost-effective assays that measure the frequency of antigen-specific T lymphocytes are crucial for evaluating and improving vaccines and therapies. This paper reviews the ELISPOT technique that allows for quantifying HIV-specific T lymphocytes at the single cell level from peripheral blood by detection of antigen-induced cytokine secretion. The assay can be used successfully to quantify T cell immune responses in humans infected with different pathogens and to assess T cell immunogenicity of vaccines in phase I/II and III clinical trials. This review focuses on the ELISPOT methodology and discusses how it can be standardized and potentially used by multiple international laboratories attached to clinical trial sites.

My first recollection of Prof Barry Schoub was at a National Institute for Virology (NIV) journal club in November 1994. At that time, I was a lecturer at the University of the Witwatersrand and spending two to three days a week at the AIDS Research Unit at the NIV. This was my first experience of a journal club at the NIV, and I was struck how the Director of the NIV was rolling up his sleeves and presenting a paper to the gathered staff. Through my own myopic research and academic background, which had shaped my expectations where Directors or Professors didn’t seem to become involved with equal dialogue with fellow colleagues, this first impression was to be the most lasting I have of Barry: down to earth, ready to become involved and not bound by ego. This memory of academic equality, not limited by position, has been a role model for my own involvement with leadership.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ + IL2 + TNFα + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.

To determine how a program for learning immunology could be most useful for clinicians in South Africa, we consulted with the country's 20 leading HIV-specialist pediatricians. They told us that immunology and its underlying concepts were perceived to be complex and arcane and that there was a need for immunology to be better integrated into “real-life” clinical practice. Most saw immunology as predominantly a laboratory science with little application to clinical practice; this highlighted a gap between clinical management of patients and theoretical understanding of the etiology and immunopathogenesis of disease. We speculated that one of the possible reasons for this perception and knowledge gap is that immunology is not an independent discipline within the South African medical curriculum. Instead, it is diffusely integrated into disciplines such as pathology or internal medicine and is not specifically provided for during post-degree training.

We have previously shown that total lymphoid irradiation conditioning of baboons before renal transplantation is a potent immunosuppressive strategy, resulting in quiescent T cell function and increased NK cell activity. This paper shows that proportion of cells expressing NK- associated antigens, which expand during and after total lymphoid irradiation, includes large, granular cells expressing surface antigens associated with monocytes. NK cells and monocytes from peripheral blood were detected and quantified using an array of fluorescent-labelled monoclonal antibodies and flow cytometry. During and after exposure to total lymphoid irradiation there was an increase in the frequency of large, granular CD11b(+) and CD16(+), CD56(+), CD16(+) CD38(+) cells and absolute numbers of CD14(+)CD11b(+) and CD14(+) CD16(+) mononuclear cells. This suggested that expansion of a unique population of CD14(+) monocytes, co-expressing CD16 and CD11b antigens, normally found on NK cells. Consequently, the significant increase in CD14(+) CD16(+) cells demonstrates that CD16(+) cells do not solely define NK cells in the mononuclear cell fraction isolated during and after this form of radioconditioning.

Pre-transplant conditioning with total lymphoid irradiation (TLI)induces antigen non-specific suppressor cells in the circulation of baboons. This study aims to identify the properties and phenotypes of cells imparting suppression of mixed lymphocyte cultures (MLC) from TLI and non-TLI conditioning animals. The MLC consisted of recipient responder T cells stimulated with pooled normal mononuclear cells (MNC) with suppressor MNC, autologous to the responding cell, added at intervals after transplantation. Suppression was non-specific and MLC proliferation was down-graded by MNC isolated from long surviving animals. Suppression was mediated by a soluble product which could pass a 0.4 micron cellulose membrane in a specially designed well insert. Addition of exogenous IL-2 failed to reverse suppression, but complete reversal was achieved by treatment of suppressor MNC with L-leucyl-L leucine methyl ester. Additionally, depletion of CD11b(+) and CD38 (+)MNC from suppressor cells significantly reversed MLC inhibition. Consequently, the suppressor cells appear to express phenotypes common to monocytes and possess contra-IL-2 properties. Consequently these cells may function by skewing T cell responsiveness away from allo-antigen reactivity in vivo.

TLI has potent immunosuppressive effects, but there is concern over its possible carcinogenic properties. The aim of the study was to assess to what degree TLI may compromise natural killer (NK) and lymphokine activated killer (LAK) cell function. There was a significant increase (P less than 0.0001) in both NK-cell function (measured against K562 targets) and spontaneous LAK-cell function (measured against DAUDI targets) in fresh blood lymphocytes throughout a course of 8 x 100 cGy fractionated TLI. This may be related to a three- and sevenfold increase, respectively, in CD16+ CD8- and CD56+ CD2- cell frequencies over the same period. Mitogen-induced interleukin 2 (IL-2) synthesis from blood lymphocytes was inhibited by up to 75% with as little as 100 cGy of TLI. Expression of IL-2 receptors on fresh lymphocytes did not vary and remained low. Therefore spontaneous LAK occurrence appeared to be triggered through an IL-2-independent pathway. The in vitro addition of IL-2 verified that cells retained their ability to respond to this lymphokine resulting in greatly enhanced induced LAK function. This was most probably mediated by CD56+ cells which were found to readily express IL-2 receptors upon mitogen stimulation. In conclusion, fractionated low-dose TLI appears to enhance MHC unrestricted immune surveillance in a manner independent of IL-2 production.

Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of

This study aimed to characterize genetic features of HIV-1 subtype C envelope glycoproteins capable of eliciting cross-reactive neutralizing antibodies during natural infections. The gp160 sequences were determined for 36 HIV-1 subtype C isolates (donor viruses) from infected individuals residing in Malawi, Zimbabwe, Zambia and South Africa, whose sera displayed a range of cross-neutralizing activities against a panel of 5 subtype C and 5 subtype B viruses (panel viruses). Hierarchical clustering analysis of neutralization data of the panel viruses predicted phylogenetic relationships between subtype B and C panel viruses, suggesting some subtype-specific neutralization determinants. A similar comparison of subtype C donor viruses showed no significant correlation; however of three donor sequence pairs resolvable by phylogenetic analysis, two were also associated within the neutralization clustering dendrogram, suggesting that closely related viruses may elicit antibodies targeting common neutralization determinants. Significantly, viruses that had shorter V1–V4 loops induced antibodies that showed more neutralization breadth against the subtype C panel viruses (p=0.0135). This study indicates that that some structural features of envelope, such as shorter variable loops, may facilitate the elicitation of cross-reactive neutralizing antibodies in natural infections. Collectively these data provide some insights into design features of an envelope immunogen aimed at inducing neutralizing antibodies.

We have characterized 43 nef sequences from subtype C HIV-1-infected South Africans and compared deduced amino acid sequences with other subtypes to identify areas of conservation. Our Nef amino acid sequences were aligned with a consensus subtype B, HXB2 reference strain and a consensus subtype C sequence. All were found to be highly homologous to subtype B in the central region of Nef, but more variable at the N and C termini of the molecule. Alignment of a consensus amino acid sequence generated from South African subtype C Nef with subtypes A, B, and D underscores cross-clade conservation in the central domain of the molecule. This domain is also rich in previously described cytotoxic T lymphocyte (CTL) epitopes that are restricted by commonly found HLA molecules in the South African population.