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Cytotoxic T lymphocytes, or CD8+ T cells, play an important role in the control of replication of HIV. Inducing effective and durable HIV-specific CD8+ T cell responses are, therefore, a major objective in prophylactic and curative strategies for HIV infection. To evaluate such strategies, reliable immunological assays are needed that measure the capacity of CD8+ T cells to exert their effector functions and control viremia. Classical immunological assays such as interferon-γ (IFN-γ) enzyme-linked immunospot or intracellular cytokine staining measure the production of one or several effector molecules but do not actually show suppression of viral replication. Perhaps unsurprisingly, these assays do not correlate with either prevention of infection or lower viral set-points after infection. Therefore, more relevant assays are needed which directly measure the viral inhibitory activity (VIA) of CD8+ T cells and are more likely to predict success or failure of different immune interventions. The present review discusses the methodology of the VIA in detail as well as the practical implications of the several variations that have been described. We then go onto discuss existent literature on the relationship between VIA and HIV control, give an overview of examples where VIA has been induced or boosted in vivo or in vitro, and finally discuss observed associations between VIA and other immunological parameters. We conclude that while VIA is complex and laborious, it provides functional information about CD8+ T cells that no other assay can deliver., (Copyright: © 2019 Permanyer.)

Objectives: The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398., Methods: Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied., Results: We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost., Conclusions: These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Aim: Cationic lipids (Lipofectamine™ [Invitrogen, Merelbeke, Belgium] and 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and polymers (jetPEI™ and in vivo-jetPEI™ [Polyplus-transfection, Illkirch, France]) were evaluated for their potential to deliver mRNA to monocyte-derived dendritic cells., Materials & Methods: Lipoplexes and polyplexes, containing mRNA encoding GFP or Gag protein, were incubated with human monocyte-derived dendritic cells and transfection efficiencies were assessed by flow cytometry., Results: Lipofectamine was by far the most efficient in mRNA delivery, therefore it was used in further experiments. Incubation of monocyte-derived dendritic cells isolated from HIV-1-positive donors with mRNA encoding Gag protein complexed to Lipofectamine resulted in 50% transfection. Importantly, coculture of these Gag-transfected dendritic cells with autologous T cells induced an over tenfold expansion of IFN-γ- and IL-2-secreting CD4(+) and CD8(+) T cells., Conclusion: Cationic lipid-mediated mRNA delivery may be a useful tool for therapeutic vaccination against HIV-1. This approach can be applied to develop vaccination strategies for other infectious diseases and cancer.

Background: The immune system exerts a diversifying selection pressure on HIV through cellular, humoral and innate mechanisms. This pressure drives viral evolution throughout infection. A better understanding of the natural immune pressure on the virus during infection is warranted, given the clinical interest in eliciting and sustaining an immune response to HIV which can help to control the infection. We undertook to evaluate the potential of the novel HIV-induced, monocyte-derived factor visfatin to modulate viral infection, as part of the innate immune pressure on viral populations., Results: We show that visfatin is capable of selectively inhibiting infection by R5 HIV strains in macrophages and resting PBMC in vitro, while at the same time remaining indifferent to or even favouring infection by X4 strains. Furthermore, visfatin exerts a direct effect on the relative fitness of R5 versus X4 infections in a viral competition setup. Direct interaction of visfatin with the CCR5 receptor is proposed as a putative mechanism for this differential effect. Possible in vivo relevance of visfatin induction is illustrated by its association with the dominance of CXCR4-using HIV in the plasma., Conclusions: As an innate factor produced by monocytes, visfatin is capable of inhibiting infections by R5 but not X4 strains, reflecting a potential selective pressure against R5 viruses.

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null) (NSG) and NOD/SCID/IL2Rγ(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

The cyclotriazadisulfonamide (CADA) compounds are a new class of specific CD4-targeted HIV entry inhibitors. The in vitro anti-HIV activity of CADA was shown to correlate with its ability to specifically downmodulate cell surface expression of the CD4 receptor in human cells. Here, we evaluated its potential as an anti-HIV microbicide. CADA exerted a clear CD4 receptor downregulating effect in dendritic cells (DC) and subsequently inhibited HIV-1(BaL) replication in DC/T cell co-cultures. The compound proved to be active against a variety of clinical isolates belonging to the HIV-1 subtypes A, B, C, D, F, G, H, AE and O. Furthermore, it prevented human T cells from being infected with the laboratory-adapted strains X4 HIV-1(NL4.3) and R5 SIV(mac251). Flow cytometric analysis demonstrated a significant and dose-dependent downregulation of CD4 on macaque PBMCs. In addition, the compound exerted a marked anti-SIV(mac251) activity in these cells from simian origin. The combination of CADA with cellulose acetate phthalate (CAP) resulted in a synergistic inhibition of HIV-1 and SIV infection. Finally, gel formulated CADA proved to preserve the CD4 downmodulating and antiviral activity of this compound when formulated as a microbicide gel. Thus, our data suggest that CADA may have potential as a broad-spectrum anti-HIV microbicide drug candidate. The preservation of the activity of gel formulated CADA will make it now feasible for testing this unique entry inhibitor in non-human primates, not only as a single drug but also in a synergistic conjunction with other anti-HIV compounds.

Objective: To study the requirements for HIV transfer between dendritic cells (DC) and CD4 T cells, using an in vitro model, combined with flow cytometry., Methods: Immature DC and macrophages (MA) were generated from monocytes. After infection, DC or MA were cultured alone or with purified CD4 T cells. Intracellular HIV was measured, using (1) the monocyte (MO)-tropic AD8 HIV, endowed with enhanced green fluorescent protein (EGFP); and (2) intracellular staining of laboratory HIV strains and clones from primary isolates., Results: (1) Clone AD8-EGFP infected DC and MA with equal efficiency, but the virus was preferentially transferred from DC to autologous T cells. (2) DC were more productively infected with R5/NSI, as compared to X4/SI, HIV, but both HIV phenotypes were easily transmitted to autologous T4 cells. (3) HIV-infected DC transferred the virus to T cells across a semi-permeable membrane, if the T cells were in contact with non-infected DC. (4) Co-culture of T cells with autologous non-infected DC induced T-cell activation. HIV-infected DC selectively increased HLA-DR on T cells and HLA-DR (+) T cells were preferential targets for HIV transfer. (5) Resting Ba-L-infected CD4 T cells were able to transmit the virus 'inversely' to co-cultured DC., Conclusion: HIV transfer between monocyte-derived dendritic cells and autologous CD4 T cells was directly demonstrated using flow cytometry. The transfer proceeded in both directions, depended on cellular contact and was associated with partial T-cell activation. This model, representing relevant in vivo targets of HIV, is useful to further investigate interactions between HIV, DC and T cells, without the need for primary ex vivo DC.

Immune parameters were compared in four groups of Ugandan subjects: HIV-and HIV+ adult patients with active pulmonary TB (HIV- PTB n = 38; HIV+ PTB n = 28), patients with HIV infection only (n = 26) and PPD+ healthy controls (n = 25). Compared with healthy controls, CD4 and CD8 T cells from patients with HIV and/or PTB expressed more activation markers (HLA-DR, CD38); their CD8 T cells expressed more CD95 (pre-apoptosis) and less CD28 (co-stimulatory receptor). Peripheral blood mononuclear cells (PBMC) of patients with either HIV or PTB were impaired in interferon-gamma (IFN-γ) production upon antigenic stimulation. PTB (with or without HIV) was characterized by monocytosis, granulocytosis, increased transforming growth factor-beta 1 production and PPD-induced apoptosis. In vivo CD4 T cell depletion, in vitro increased spontaneous CD4 T cell apoptosis and defects in IFN-γ responses upon mitogenic stimulation were restricted to HIV+ subjects (with or without PTB). Overlapping and distinctive immune alterations, associated with PTB and HIV, might explain mutual unfavourable influences of both diseases. [ABSTRACT FROM AUTHOR]

Parameters of immune activation /differentiation were studied in a group of newly diagnosed HIV and HIV+ pulmonary tuberculosis (TB) patients. Compared with controls, HLA-DR expression on both CD4 and CD8 T cells from the HIV- TB patients was approximately doubled; HLA-DR on T cells from the HIV+ group was tripled. The monocytes from both groups of patients expressed abnormally high levels of the Fcγ receptors I and III. Serum levels of tumour necrosis factor-alpha (TNF-α), neopterin and β2-microglobulin were increased in HIV- and even more so in HIV+ TB patients. The expression of HLA-DR on T cell subsets and of FcγR on monocytes correlated with each other, but not with serum activation markers. This pattern of non-specific activation during TB infection may be associated with enhanced susceptibility to HIV infection. [ABSTRACT FROM AUTHOR]

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen-presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage-related fashion. CD28-CD8+ T cells preferentially expressed CD57 and CD11 b, but lacked CD26 and IL- 2Rα. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co-stimulation with cell-bound anti-CD3 and B7. Nevertheless, when stimulated with plate-fixed anti-CD3, CD8+ T cells from HIV-infected subjects proliferated normally, and normal levels of IL-2Rα and transferrin-receptor could be induced on CD28-CD8+ T cells from the patients. In addition, stimulation with plate-fixed anti-CD3 induced proliferative responses in highly purified CD28-CD8+ T cells from both HIV- and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti-CD3 redirected assay, was predominantly exerted by CD28-CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down-regulation and were slightly hyporesponsive to B7 co-stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co-stimulation via B7. CD28- CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7-independent stimulation. [ABSTRACT FROM AUTHOR]

The expression and co-expression profiles of functionally important monocyte surface markers were compared between control and HIV+ individuals using combined physical gating and dim CD4 expression to delineate the monocytes. The FcγRII (CD32), the MHC class II antigen HLA-DR and the adhesion molecules CD11a (LFA-1α), CD18 and CD54 (ICAM-1) showed an unimodal distribution. Of these markers, CD11a and HLA-DR were up-regulated in the HIV+ subjects compared with controls. The expression levels of the adhesion molecules correlated with each other in both patients and controls. The CD11b (CR3-α), CD14, FcγRI, and FcγRIII markers were bimodally distributed. Compared with controls, monocytes from seropositives contained fewer CD14bright+ cells, an equal proportion of FcγRIbright+ cells, but twice as many FcγRIII+ cells. The expression level of FcγRI and CD11b within their brightly positive subset increased as CD4T cells decreased. Both in patients and controls., co-expression of bright CD11b, CD14 and FcγRI was shown, whereas the FcγRIII+ cells were negative or dim positive for the former triad. We conclude that the expression of two FcγR (I and III), of the adhesion molecules CD11a and CD11b and of HLA-DR showed particular alterations on monocytes from HIV+ subjects. The relationship of these phenotypic observations with altered cytokine profiles and altered monocyte function is discussed. [ABSTRACT FROM AUTHOR]

Infection with HIV results in a progressive depletion of CD4+ T cells and leads to significant in vivo lymphocyte phenotype changes. In this regard, the expression of HLA-DR and CD38 on CD8+ T cells has been shown to increase dramatically with disease progression. We investigated the expression of both activation markers on CD4+ T cells in HIV-1-infected subjects at different clinical stages of infection and compared the in vivo activation of CD4+ T cells with parameters of viral activity and CD8+ T cell activation. Fresh peripheral venous blood was obtained from 54 HIV infected subjects and from 28 uninfected healthy controls. Three-colour immunophenotyping of the CD4+ T cell subset showed that the proportion of CD4+ T cells expressing HLA-DR (10% in HIV negative controls) or CD38 (62% in H IV-negative controls) was higher in asymptomatic (P < 0.05 for CD38) and symptomatic (P < 0.001 for HLA-DR and CD38) HIV-infected subjects than in controls, whereas the proportion of CD4+ T cells expressing CD45RO (54% in controls) remained relatively unchanged. Simultaneous expression of HLA-DR and CD38 on CD4 T cells increased from 2.3% in controls to 11% (P < 0.001) in asymptomatic and 22% (P < 0.001) in symptomatic HIV-infected subjects. This relative increase of CD38 and HLA-DR expression occurred mainly on CD4+ T cells co-expressing CD45RO. Changes in expression of HLA-DR and CD38 on CD4+ T cells correlated with similar changes on CD8+ T lymphocytes, with the presence of HIV antigen in the circulation, and with the disease stage of HIV infection. [ABSTRACT FROM AUTHOR]

The activity of both cytotoxic T lymphocyte (CTL) and natural killer (NK) cells were measured crosssectionally in 43 subjects seropositive for HIV, in 27 HIV- blood donors and in 24 HIV- persons from the Outpatients Clinic for sexually transmitted diseases. CTL activity was evaluated using the HL-60 cells coated with OKT3 as the targets and freshly separated peripheral blood lymphocytes as the effectors. In 20 out of 43 HIV- subjects, CTL activity was significantly enhanced in comparison to the HIV subjects. This lytic activity correlated positively with the percentages of CD3+ HLA-DR+ of CD8+ CR3- and of CD57+CD16- lymphocytes, and was greatly reduced after elimination of CD8+, of HLA-DR+ or of CD57+ cells. The median CTL activity seemed to increase from CDC group II to CDC group IV (Centers for Disease Control classification), but to return back to control levels in those patients with a history of opportunistic infections. NK function in HIV+ subjects was not significantly different from that in the blood donors, In seropositive patients, NK activity correlated positively with the percentages of both CD16+ CD57+ and of CD8+ CR3+ cells and was strongly diminished after elimination of CD16+ or of CD57+ cells. There was no significant change in NK function according to the clinical stage. The data show that circulating CD8+ HLA-DR+ Cp57+ T cells in HIV+ subjects are activated cytotoxic T cells and point to progressive (over) activation of this T cell compartment until the onset of opportunistic infections. [ABSTRACT FROM AUTHOR]

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4+ or CD8+ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8+ and CD4+ T cells from HIV-infected and HIV- subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8+ T cells from patients proliferated consistently less than controls, while responses from CD4+ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4+ and CD8+ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4+ T cells. Compared with controls, CD4+ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8+ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4+ T cells from HIV+ subjects to various costimulatory pathways are relatively intact, whereas CD8+ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8+ T cell failure might contribute to viral and neoplastic complications of HIV infection. [ABSTRACT FROM AUTHOR]

The cellular immune responses to fractionated Haemophilus ducreyi antigens, coated on latex beads, were assessed in patients with chancroid and in controls, using an in vitro lymphocyte proliferation assay. Several tractions of H. ducreyi antigen revealed stimulating activity. However, only the molecular size ranges 91-78 kD, 59 -29 kD. and 25-21 kD induced proliferation that may be specifically related to H. ducreyi infection. Lymphocytes from four HIV- patients, successfully treated for chancroid, were not stimulated by H. ducreyi antigen. In general, lymphocytes from HIV+ chancroid patients were less responsive to H. ducreyi antigen compared with those from HIV- chancroid patients. However, two HIV-infected patients showed exceptionally strong responses to high molecular weight fractions. To our knowledge this is the first report demonstrating that H. ducreyi contains specific T cell-stimulating antigens. Based on this work, further identification and purification of the T cell antigens is feasible. [ABSTRACT FROM AUTHOR]