In depth analysis of the HIV-specific CD8 T cell response in seropositives exposed to a mixed subtype epidemic

The importance of HLA class I restricted CD8 T cell response in the control of HIV infection is generally accepted, yet few studies have shown a correlation of the CD8 T cell response with markers of HIV disease progression. Disease progression is dependent on several factors such as the virus load at set-point, rarely occurring mutations in the HIV co-receptor genes or the HLA class I alleles expressed by an HIV infected individual. It has been shown previously that particular HLA class I alleles are associated with a low plasma viral load. However, a relationship between these "protective\u27; HLA class I alleles and an efficient HIV-specific CD8 T cell response has not yet been demonstrated. In order to study the mechanism underlying the beneficial effect of "protective\u27; HLA class I alleles, 53 HIV-1 seropositive individuals from a high-risk cohort in southwest Tanzania were HLA-typed and the plasma viral load and CD4 counts were determined. The recognition of CD8 T cell epitopes within Gag, Nef and Env was analysed using a gamma interferon ELISPOT assay and freshly isolated peripheral blood mononuclear cells (PBMC). Gag and Nef were frequent targets of the HIV-specific CD8 T cell response with a median recognition of 3 Gag, 2 Nef and 1 Env epitopes per individual. The HLA class I alleles B5801, B8101 and B0702 were associated with a low median plasma viral load. At the same time expression of these correlated with a broader recognition of Gag epitopes (P = 0.0035), if compared with all other common HLA class I alleles. Further analysis of the Gag-specific T cell response revealed an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = -0.36, P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (aa001-aa075 and aa248-aa500) was strongly associated with the maintenance of a low viral load, indicating that this pattern of HIV-specific CD8 T cell recognition is important for the control of viral replication during the chronic phase of HIV infection. In the second part of the present work, recognition of Gag and Nef variant epitopes was analysed using HIV-1 subtype A, C and D derived peptides. 83% of the Gag-specific responses were detected with subtype C derived peptides, whereas only 51% and 57% of responses were detected with subtype A or D peptides, respectively. The superiority of the subtype C Gag peptides for detecting CD8 T cell responses may in part reflect the predominance of subtype C and C-containing recombinant HIV-1 strains within the studied cohort. Nonetheless, screening for CD8 T cell responses with any one subtype-specific peptide set detected fewer responses and underestimated the breadth and the magnitude of responses within individuals, compared to the combined peptide sets from the three subtypes. Cross-subtype recognition for the 16 most frequently recognized peptides was identical only when the peptide sequences were invariant; in 9 of these 16 peptides, diminished recognition was the result of subtype-related sequence variation, and a frame-of-epitope effect diminished or abrogated recognition in 4 peptides. A minimal set of 15 frequently recognized Gag and Nef peptides was then designed and tested with cells from 50 HIV seropositive individuals and elicited responses in 47 of them, at a mean frequency of 715 SFC/106 peripheral blood mononuclear cells. Therefore incomplete cross-recognition between subtypes A, C, and D can be partially overcome using a defined peptide set representing frequently recognized epitopes, and potentially, by adjusting epitope frame within peptides. These strategies can help to define optimal vaccine epitopes. In the third part of the present work, the Gag-specific CD8 T cell response of individuals infected with a single HIV strain was compared with the response observed in individuals infected with multiple HIV-1 strains. Breadth of Gag epitope recognition and cross-recognition of subtype-specific peptide pools was enhanced in multiply infected study subjects, whereas CD8 T cell recognition of Nef or Env appeared to be unaffected. Therefore the increased viral diversity in individuals infected with multiple HIV-1 strains affects the Gag-specific T cell response. As a consequence inclusion of multiple Gag variants in a HIV vaccine is likely to enhance vaccine-induced CD8 T cell responses and cross-recognition of HIV epitopes.Es ist allgemein akzeptiert, daß die HLA Klasse I restringierte CD8 T Zellantwort bei der Kontrolle der Virusreplikation während einer HIV Infektion eine entscheidene Rolle spielt. Allerdings konnten nur wenige Studien eine Korrelation zwischen der HIV-spezifischen CD8 T Zellantwort und dem Krankheitsverlauf nachweisen. Der Krankheitsverlauf hängt von verschiedenen Faktoren ab, darunter die Viruslast nach der Primaervirämie, selten vorkommenden Mutationen in den HIV Korezeptorgenen, oder den HLA Klasse I Allelen. So geht beispielsweise die Expression bestimmter HLA Klasse I Allele mit einer niedrigen Plasma Viruslast einher. Eine Verbindung zwischen solchen "protektiven\u27; HLA Klasse I Allelen und einer effizienten HIV-spezifischen CD8 T Zellantwort konnte jedoch bisher noch nicht nachgewiesen werden. Zur Beantwortung des zugrundeliegenden Mechanismus, der mit "protektiven" HLA Allelen verbunden ist, wurden 53 HIV infizierte Frauen einer Hochrisikokohorte im Südwesten Tansanias bezüglich des HLA Typs, der Viruslast im Plasma und der CD4 T Zellzahl charakterisiert. Gleichzeitig wurde die Gag-, Nef- und Env-spezifische CD8 T Zell Antwort unter Verwendung eines Interferon gamma ELISPOT Assays bestimmt. Am häufigsten fanden sich in den Studienteilnehmerinnen CD8 T Zellantworten gegen Epitope in Gag und Nef, weniger häufig gegen Epitope in Env (Median: 3 Gag, 2 Nef und 1 Env erkannte Epitope/Individuum). Die HLA Klasse I Allele B5801, B8101 und B0702 waren mit einer niedrigen Viruslast assoziiert. Gleichzeitig korrelierte die Expression von einem oder zwei dieser Allele mit einer breiteren Erkennung von Gag Epitopen (P =0,0035, Median:4 erkannte Epitope/Individuum) verglichen mit anderen HLA Klasse I Allelen (Median: 2 erkannte Epitopen/Individuum). Die weitere Analyse der Gag-spezifischen CD8 T Zell Antwort offenbarte ein inverses lineares Verhätnis zwischen der Anzahl der erkannten Gag Epitope und der Viruslast im Plasma (R =-0,36 ; P = 0,0016). Des weiteren war die Erkennung von multiplen Epitopen in 2 Regionen des Gag Proteins (as001-as075 und as 248-500, GagR1R3) stark mit einer dauerhaften Kontrolle der Virusreplikation nach Serokonversion assoziiert. Zusammenfassend lassen diese Ergebnisse darauf schließen, daß die Erkennung multipler Epitope im Gag Protein, ganz besonders in GagR1R3, wichtig für die Kontrolle der Virusreplikation während der chronischen Phase der HIV Infektion ist. Im zweiten Teil der vorliegenden Arbeit wurde die Erkennung von HIV-1 Subtyp A-, C- und D-spezifischen Gag und Nef Epitopvarianten durch CD8 T Zellen untersucht. 83% der Gag-spezifischen Epitopantworten wurden mit den Subtyp C Peptiden detektiert, während lediglich 51% und 57% der Gag-spezifischen Epitopantworten mit den Subtyp A beziehungsweise Subtyp D Peptiden detektiert wurden. Die erhöhte Frequenz Subtyp C-spezifischer Antworten könnte zum Teil mit dem vorherrschenden Vorkommen Subtyp C und Subtyp C-beinhaltenender rekombinanter HIV-I Stämme in Südwest Tanzania zusammenhängen. Dennoch würde die Anzahl der Gag-spezifischen Epitopantworten durch die ausschließliche Verwendung von Subtyp C-spezifischen Peptiden unterschätzt. Die Erkennung von Subtyp-spezifischen Peptidevarianten durch CD8 T Zellen war lediglich für Peptide gleicher Aminosäuresequenz identisch. Für 9 der 16 am häufigsten erkannten Peptide konnte eine abgeschwächte Erkennung von Varianten des selben Peptids mit Aminosäuresequenzunterschieden erklärt werden. Für 4 Peptide konnte eine abgeschwächte Erkennung von Peptidvarianten mit einer unterschiedlichen Positionierung des Epitops innerhalb der Peptidvarianten erklärt werden. Aufgrund dieser Ergebnisse wurde ein minimales Set aus 15 der am häufigsten erkannten Peptiden zusammengestellt Dieses bestand vor allem aus Subtyp C-spezifischen, aber auch aus Subtyp A- und D-spezifischen Peptiden und wurde anschließend mit peripheren mononuklären Blutzellen ("peripheral blood mononuclear cells", PMBC) von 50 HIV infizierten Personen auf Erkennung getestet. Dieser Peptidpool wurde mit einer durchschnittlichen Frequenz von 715 SFC/106 PBMC von 47 dieser Personen erkannt. Zusammenfassend könnte diese Strategie dazu beitragen, optimale CD8 T Zell Epitopevarianten zu identifizieren, die in HIV Impfungen beinhaltet sein sollten. Im dritten Teil der vorliegenden Arbeit wurde die Gag-spezifische CD8 T Zell Antwort von Studienteilnehmerinnen verglichen, die mit einem oder mehreren verschiedenen HIV-1 Stämmen infiziert waren. Eine Mehrfachinfektion führt dazu, daß mehr Gagepitope erkannten werden. Darüber hinaus war auch die gleichzeitige Erkennung von subtypspezifischen Peptiden in diesen Studienteilnehmerinnen besser ausgeprägt. ... Für einen potentiellen HIV Impfstoff lässt sich aus der vorliegenden Arbeit schließen, daß es entscheidend sein kann, eine effiziente CD8 T Zellantwort zu induzieren. Vor allem sollten hierbei Gag-spezifische Epitopevarianten verschiedener Subtypen berücksichtigt werden

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo.

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27(-) phenotype was associated with active TB in HIV(-) (p = 0.0003) and HIV(+) (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV(-) subjects, MTB-specific CD4 T cell populations from HIV(+) TB-asymptomatic subjects were often dominated by CD27(-) cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression

Serological Protection 5-6 Years Post Vaccination Against Yellow Fever in African Infants Vaccinated in Routine Programmes.

Introduction: Although effective live attenuated yellow fever (YF) vaccines have been available for over 9 decades sporadic outbreaks continue to occur in endemic regions. These may be linked to several factors including epidemiological factors such as vector and intermediate host distribution or vaccine coverage and efficacy. The World Health Organization's research priorities include gathering systematic evidence around the potential need for booster vaccination with YF vaccine whether this follows full or fractional doses in children. Knowledge on the longevity of response to YF vaccine and the implications of this response needs to be consolidated to guide future vaccination policy. Methods: We measured anti-YF IgG by microneutralization assay in a group of 481 African infants who had received YF vaccine as part of routine EPI programmes, to explore serological protection from YF 5-6 years post YF vaccination, as well as the effect of co variates. Findings: Notably, 22.2% of the cohort had undetectable antibody concentrations, with another 7.5% revealing concentrations below the threshold of seropositivity of 0.5 IU/mL. Sex, season, country and time since vaccination did not affect the longevity of antibody concentration or having antibody concentrations above a defined threshold. Conclusion: Roughly 30% of children in this cohort did not demonstrate anti-yellow fever antibody concentrations above the defined threshold of protection, with 20% having no demonstrable antibody. Knowledge on the longevity of response to YF vaccine and the implications needs to be consolidated to guide future vaccination policy

The TAM-TB Assay—A Promising TB Immune-Diagnostic Test With a Potential for Treatment Monitoring

Tuberculosis (TB) epidemiology is changing in Western and Central Europe due to the rise in immigration and refugees fleeing high-TB-burden areas of war and devastation. The change in local demography and the lack of sensitive and specific TB diagnostic and monitoring tools, especially for cases of childhood TB, leads to either missed cases or over-treatment of this group. Here we present a promising new diagnostic approach, the T cell activation marker (TAM)-TB assay, and its performance in a case of extra-pulmonary TB occurring in a 16 year old refugee from Afghanistan. This assay is based on the characterization of 3 activation markers (CD38, HLA-DR, and Ki67) and one maturation marker (CD27) on M. tuberculosis-specific CD4 T cells. It was performed at time-points TO (10 days), T1 (1 month), T2 (6 months), and T3 (12 months) post-treatment initiation. All markers were able to detect active tuberculosis (aTB) within this patient at T0 and reverted to a healthy/LTBI phenotype at the end of treatment. Tantalizingly, there was a clear trend toward the healthy/LTBI phenotype for the markers at T1 and T2, indicating a potential role in monitoring anti-TB treatment in the future. This assay may therefore contribute to improved TB diagnostic algorithms and TB treatment monitoring, potentially allowing for individualization of TB treatment duration in the future

Cytokine response to selected MTB antigens in Ghanaian TB patients, before and at 2 weeks of anti-TB therapy is characterized by high expression of IFN-gamma and Granzyme B and inter- individual variation

Background: There has been a long held belief that patients with drug-susceptible TB are non-infectious after two weeks of therapy. Recent microbiological and epidemiological evidence has challenged this dogma, however, the nature of the Mtb-specific cellular immune response during this period has not been adequately investigated. This knowledge could be exploited in the development of immunological biomarkers of early treatment response. Methods: Cellular response to four Mtb infection phase-dependent antigens, ESAT-6/CFP-10 fusion protein and three DosR encoded proteins (Rv1733c, Rv2029c, Rv2628) were evaluated in a Ghanaian TB cohort (n=20) before and after 2 weeks of anti TB therapy. After 6-days in vitro stimulation, Peripheral blood mononuclear cell (PBMC) culture supernatant was harvested and the concentration of IFN-gamma, Granzyme B, IL-10, IL-17, sIL2R alpha and TNF-alpha were determined in a 6-plex Luminex assay. Frequencies of IFN-gamma + CD4 and CD8 T cells were also determined in an intracellular cytokine assay. Results: All antigens induced higher levels of IFN-gamma, followed by Granzyme B, TNF-alpha and IL-17 and low levels of IL-10 and sIL-2R-alpha in PBMC before treatment and after 2 weeks of treatment. Median cytokine levels of IFN-gamma, Granzyme B, IL-17 and sIL-2R-alpha increased during week two, but it was significant for only Rv1733-specific production of Granzyme B (P = 0.013). The median frequency of antigen specific IFN-gamma + CD4 T cells increased at week two; however, only the increase in the ESAT-6/CFP-10-specific response was significant (P = 0.0008). In contrast, the median frequency of ESAT-6/CFP-10-specific IFN-gamma + CD8 T cell responses declined during week two (P = 0.0024). Additionally, wide inter-individual variation with three distinct patterns were observed; increase in all cytokine levels, decrease in all cytokine levels and fluctuating cytokine levels after 2 weeks of treatment. Conclusion: The second week of effective chemotherapy was characterized by a general increase in cytokine response to Mtb-specific antigens suggestive of an improvement in cellular response with therapy. However, the wide inter-individual variation observed would limit the utility of cytokine biomarkers during this period

The TAM-TB Assay—A Promising TB Immune-Diagnostic Test With a Potential for Treatment Monitoring

Tuberculosis (TB) epidemiology is changing in Western and Central Europe due to the rise in immigration and refugees fleeing high-TB-burden areas of war and devastation. The change in local demography and the lack of sensitive and specific TB diagnostic and monitoring tools, especially for cases of childhood TB, leads to either missed cases or over-treatment of this group. Here we present a promising new diagnostic approach, the T cell activation marker (TAM)-TB assay, and its performance in a case of extra-pulmonary TB occurring in a 16 year old refugee from Afghanistan. This assay is based on the characterization of 3 activation markers (CD38, HLA-DR, and Ki67) and one maturation marker (CD27) on M. tuberculosis-specific CD4 T cells. It was performed at time-points T0 (10 days), T1 (1 month), T2 (6 months), and T3 (12 months) post-treatment initiation. All markers were able to detect active tuberculosis (aTB) within this patient at T0 and reverted to a healthy/LTBI phenotype at the end of treatment. Tantalizingly, there was a clear trend toward the healthy/LTBI phenotype for the markers at T1 and T2, indicating a potential role in monitoring anti-TB treatment in the future. This assay may therefore contribute to improved TB diagnostic algorithms and TB treatment monitoring, potentially allowing for individualization of TB treatment duration in the future

Wuchereria bancrofti infection is linked to systemic activation of CD4 and CD8 T cells

Background Susceptibility to HIV has been linked to systemic CD4+ T cell activation in cohorts of seronegative individuals with high HIV-exposure risk. We recently described an increased risk of HIV transmission in individuals infected with Wuchereria bancrofti, the causative agent for lymphatic filariasis, in a prospective cohort study. However, the reason for this phenomenon needs further investigation. Methodology/Principal findings Two-hundred and thirty-five HIV negative adults were tested using Trop Bio ELISA for detection of W. bancrofti infection and Kato Katz urine filtration and stool based RT-PCR for detection of soil transmitted helminths and schistosomiasis. FACS analysis of the fresh peripheral whole blood was used to measure T cell activation markers (HLA-DR, CD38), differentiation markers (CD45, CD27), markers for regulatory T cells (FoxP3, CD25) and the HIV entry receptor CCR5. Frequencies of activated HLA-DRpos CD4 T cells were significantly increased in subjects with W. bancrofti infection (n = 33 median: 10.71%) compared to subjects without any helminth infection (n = 42, median 6.97%, p = 0.011) or those with other helminths (Schistosoma haematobium, S. mansoni, Trichuris trichiura, Ascaris lumbricoides, hookworm) (n = 151, median 7.38%, p = 0.009). Similarly, a significant increase in HLA-DR(pos)CD38(pos) CD4 T cells and effector memory cells CD4 T cells (CD45RO(pos)CD27(neg)) was observed in filarial infected participants. Multivariable analyses further confirmed a link between W. bancrofti infection and systemic activation of CD4 T cells independent of age, fever, gender or other helminth infections. Conclusions/Significance W. bancrofti infection is linked to systemic CD4 T cell activation, which may contribute to the increased susceptibility of W. bancrofti infected individuals to HIV infection

Neutrophils Contribute to Severity of Tuberculosis Pathology and Recovery From Lung Damage Pre- and Posttreatment.

BACKGROUND: Despite microbiological cure, about 50% of tuberculosis (TB) patients have poor lung recovery. Neutrophils are associated with lung pathology; however, CD16/CD62L-defined subsets have not been studied in TB. Using flow cytometry, we monitored frequencies, phenotype, and function of neutrophils following stimulation with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL) and ESAT-6/CFP-10 fusion protein (EC) in relation to lung pathology. METHODS: Fresh blood from 42 adult, human immunodeficiency virus (HIV)-negative TB patients were analyzed pre- and post-therapy, with disease severity determined using chest radiography and bacterial load. Flow cytometry was used to monitor frequencies, phenotype, and function (generation of reactive oxygen species [ROS], together with CD11b, tumor necrosis factor, and interleukin 10 [IL-10] expression) of neutrophils following 2-hour stimulation with Mtb-specific antigens. RESULTS: Total neutrophils decreased by post-treatment compared to baseline (P = .0059); however, CD16brCD62Lbr (segmented) neutrophils increased (P = .0031) and CD16dimCD62Lbr (banded) neutrophils decreased (P = .038). Banded neutrophils were lower in patients with severe lung damage at baseline (P = .035). Following WCL stimulation, ROS from segmented neutrophils was higher in patients with low Mtb loads even after adjusting for sex (P = .038), whereas IL-10-expressing CD16dimCD62Llo cells were higher in patients with mild damage (P = .0397) at baseline. CONCLUSIONS: High ROS generation, low levels of banded neutrophils, and high levels of IL-10-expressing CD16dimCD62Llo neutrophils are associated with reduced lung pathology at diagnosis. Hence, neutrophils are potential early indicators of TB severity and promising targets for TB host-directed therapy

Ascaris lumbricoides Infection and Its Relation to Environmental Factors in the Mbeya Region of Tanzania, a Cross-Sectional, Population-Based Study

Background: With one quarter of the world population infected, the intestinal nematode Ascaris lumbricoides is one of the most common infectious agents, especially in the tropics and sub-tropics. Infection is caused by oral intake of eggs and can cause respiratory and gastrointestinal problems. To identify high risk areas for intervention, it is necessary to understand the effects of climatic, environmental and socio-demographic conditions on A. lumbricoides infection. Methodology: Cross-sectional survey data of 6, 366 study participants in the Mbeya region of South-Western Tanzania were used to analyze associations between remotely sensed environmental data and A. lumbricoides infection. Non-linear associations were accounted for by using fractional polynomial regression, and socio-demographic and sanitary data were included as potential confounders. Principal Findings: The overall prevalence of A. lumbricoides infection was 6.8%. Our final multivariable model revealed a significant non-linear association between rainfall and A. lumbricoides infection with peak prevalences at 1740 mm of mean annual rainfall. Mean annual land surface temperature during the day was linearly modeled and negatively associated with A. lumbricoides infection (odds ratio (OR) = 0.87, 95% confidence interval (CI) = 0.78-0.97). Furthermore, age, which also showed a significant non-linear association (infection maximum at 7.7 years),socio-economic status (OR = 0.82, CI = 0.68-0.97),and latrine coverage around the house (OR = 0.80, CI = 0.67-0.96) remained in the final model. Conclusions: A. lumbricoides infection was associated with environmental, socio-demographic and sanitary factors both in uni-and multivariable analysis. Non-linear analysis with fractional polynomials can improve model fit, resulting in a better understanding of the relationship between environmental conditions and helminth infection, and more precise predictions of high prevalence areas. However, socio-demographic determinants and sanitary conditions should also be considered, especially when planning public health interventions on a smaller scale, such as the community level

Depletion and activation of mucosal CD4 T cells in HIV infected women with HPV-associated lesions of the cervix uteri

Background: The burden of HPV-associated premalignant and malignant cervical lesions remains high in HIV+ women even under ART treatment. In order to identify possible underlying pathophysiologic mechanisms, we studied activation and HIV co-receptor expression in cervical T-cell populations in relation to HIV, HPV and cervical lesion status. Methods Cervical cytobrush (n = 468: 253 HIV- and 215 HIV+;71% on ART) and blood (in a subset of 39 women) was collected from women in Mbeya, Tanzania. Clinical data on HIV and HPV infection, as well as ART status was collected. T cell populations were characterized using multiparametric flow cytometry-based on their expression of markers for cellular activation (HLA-DR), and memory (CD45RO), as well as HIV co-receptors (CCR5, alpha(4)beta(7)). Results Cervical and blood T cells differed significantly, with higher frequencies of T cells expressing CD45RO, as well as the HIV co-receptors CCR5 and alpha(4)beta(7)in the cervical mucosa. The skewed CD4/CD8 T cell ratio in blood of HIV+ women was mirrored in the cervical mucosa and HPV co-infection was linked to lower levels of mucosal CD4 T cells in HIV+ women (%median: 22 vs 32;p = 0.04). In addition, HIV and HPV infection, and especially HPV-associated cervical lesions were linked to significantly higher frequencies of HLA-DR+ CD4 and CD8 T cells (p-values < 0.05). Interestingly, HPV infection did not significantly alter frequencies of CCR5+ or alpha(4)beta(7)+ CD4 T cells. Conclusion The increased proportion of activated cervical T cells associated with HPV and HIV infection, as well as HPV-associated lesions, together with the HIV-induced depletion of cervical CD4 T cells, may increase the risk for HPV infection, associated premalignant lesions and cancer in HIV+ women. Further, high levels of activated CD4 T cells associated with HPV and HPV-associated lesions could contribute to a higher susceptibility to HIV in HPV infected women