IFNγ-response to different <i>Mtb</i>-antigens in patients enrolled for the study.

<p>IFNγ-response to different <i>Mtb</i>-antigens was evaluated by short-term stimulation of whole blood in <i>Mtb</i>-infected patients with or without HIV infection. IFNγ from day-1 supernatants were evaluated by commercial ELISA in response to HBHA (A), QFT-IT antigen (B) and Mitogen (C). The square represents LTBI group, the triangle with the bottom tip represents HIV-LTBI group, the dot represents active TB group and the triangle with the top tip represents HIV-TB group. The data are shown after the subtraction of the results obtained in the unstimulated samples. Horizontal lines indicate the median of IFNγ production. Dotted lines indicate the cut-off for either QFT-IT or Mitogen as indicated by the suppliers and for HBHA antigen as for previously shown results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183846#pone.0183846.ref024" target="_blank">24</a>;<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183846#pone.0183846.ref025" target="_blank">25</a>]. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; QFT-IT: QuantiFERON-TB Gold (QFT^®) in tube; IFN: interferon; IU: International Unit.</p

Immune characterization of the HBHA-specific response in Mycobacterium tuberculosis-infected patients with or without HIV infection

<div><p>Introduction</p><p>RD1-based Interferon-γ Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with <i>Mycobacterium tuberculosis</i> (<i>Mtb)</i> containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI).</p><p>Methods</p><p>49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)γ release was evaluated by ELISA whereas cytokine profile [IFNγ, tumor necrosis (TNF)α, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry.</p><p>Results</p><p>Among LTBI individuals, HBHA stimulation induced IFNγ release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4⁺ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8⁺ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4⁺ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations.</p><p>Conclusions</p><p>These results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control.</p></div

IFNγ profile of CD4⁺ and CD8⁺ T-cells in response to different stimuli.

<p>The IFNγ cytokine profile of CD4⁺ and CD8⁺ T-cells was analyzed by ICS. In particular, we evaluated the “total IFNγ response” and then calculated the proportion of this response within the total cytokine response (IFNγ and/or TNFα and/or IL2). PBMC were stimulated overnight with HBHA (A, B), RD1 proteins (C, D), CMV (E, F) and SEB (G, H) and analyzed within the CD4⁺ and CD8⁺ T-cells by flow cytometry. A-H: The bar graphs show the proportion of IFNγ-expressing cells within the CD4⁺ and CD8⁺ T-cells over the total responding T-cells in the different groups analyzed. The horizontal lines represent the median. Statistical analysis was performed using Mann-Whitney test and p value was considered significant if < 0.05. A, C, E, G) Proportion of total IFNγ-producing CD4⁺ T-cells in response to each stimulus; B, D, F, H) Proportion of total IFNγ-producing CD8⁺ T-cells in response to each stimulus. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; RD: region of difference; CMV: cytomegalovirus; SEB: staphylococcal enterotoxin B; IFN: interferon.</p

Analysis of polyfunctional and monofunctional response to different antigens of CD4⁺ and CD8⁺ T-cell subsets in patients enrolled for the study.

<p>The graphs show the frequency of polyfunctional (more than one cytokine) and monofunctional (one cytokine) response, evaluated by flow cytometry, to HBHA (A, B), RD1 proteins (C, D) and CMV (E, F) of CD4⁺ and CD8⁺ T-cell subsets in the <i>Mtb</i>-infected patients with or without HIV infection. A positive cytokine response was defined as at least twice the background. A frequency of any cytokine-producing T-cells (IFNγ and/or TNFα and/or IL2) of at least 0.03% was considered as a positive CD4 and CD8 T-cell response. The horizontal lines represent the median; filled symbols represent polyfunctional T-cells, open symbols represent monofunctional T-cells. Statistical analysis was performed using the Mann-Whitney test and p value was considered significant if < 0.05. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection; HBHA: heparin-binding haemagglutinin; RD: region of difference; CMV: cytomegalovirus.</p

Flow chart of the patients enrolled for the study.

<p>Forty-nine <i>Mtb</i>-infected subjects were prospectively enrolled for the study. Twenty-two patients out of the total were HIV-uninfected, for 13 of these a diagnosis of active TB was made and 9 resulted LTBI. Among the remaining 27 HIV-infected patients, all naïve to ART, 12 were diagnosed as HIV-TB and 15 resulted HIV-LTBI. Footnotes: HIV: human immunodeficiency virus; TB: tuberculosis; LTBI: latent TB infection.</p

VLC patients: RH of various endpoints from fitting a Cox regression analysis.

<p>VLC patients: RH of various endpoints from fitting a Cox regression analysis.</p

Characteristics of patients—VLC group: CD4 count less than 200 or AIDS.

<p>Characteristics of patients—VLC group: CD4 count less than 200 or AIDS.</p

Kaplan Meier curves of the probability of reaching the different end-points according to the PI/r component of the initial cART regimen in 1362 HIV positive LC patients.

<p>Kaplan Meier curves of the probability of reaching the different end-points according to the PI/r component of the initial cART regimen in 1362 HIV positive LC patients.</p

The presence of resistance-associated mutations in reverse transcriptase gene is associated with cerebrospinal fluid HIV-1 escape: a multicentric retrospective analysis

Higher risk of cerebrospinal fluid escape (CVE) has been associated with the use of specific antiretroviral (ARV) classes, such as protease inhibitors. We assessed whether archived resistance-associated mutations (RAMs) can mediate this relationship by identifying patients treated with incompletely active antiretroviral regimens. A retrospective multicentric study on 282 adult people with HIV on antiretroviral therapy (ART) and available historical plasma genotype resistance testing (HGRT) for reverse transcriptase (RT) and protease genes between 2001 and 2021. The odds ratio for demographic, clinic-, and ART-related variables and CVE was estimated by multivariable modeling. HGRT-adjusted central nervous system effectiveness penetration (CPE) score was computed in modeling the risk. Median age, plasma VL, and CD4 count were 49 years, <50?copies/mL, and 310?cells/µL. CVE was detected in 51 participants (17.0%). No difference in CVE prevalence was observed according to ART type, number of ARVs or ARV classes. Participants with CVE had more frequently plasma (52.9% vs. 32.1%, p?=?0.005) and CSF RAMs in RT (n?=?63, 57.1% vs. 28.6%, p?=?0.029), but not in protease gene. The presence of plasma RAMs in RT associated with increased odds of CVE in adjusted analyses (aOR 3.9, p

Characteristics of patients—LC group: CD4 ≤350 cells/mm³ or AIDS.

<p>Characteristics of patients—LC group: CD4 ≤350 cells/mm³ or AIDS.</p