BACKGROUND:Virus-specific CD8+ T-cells play an important part in HIV cure/remission in adults, yet their role in paediatric immune control is limited by tolerogenic early-life immunity. Very-early ART initiation, while effective in restricting viral reservoir size, also prevents antigenic exposure and, thereby, the induction of HIV-specific CD8+ T-cell responses. Analytical treatment interruption (ATI) is an established tool in cure/remission studies for assessing time to viral rebound and viral setpoint off ART yet the impact of ATI on HIV-specific immune responses and plasma viral load (pVL) remains understudied in paediatric populations. METHODS:We evaluated a historical randomised cohort of early ART-treated children in South Africa. Infants with HIV were randomized to either Arm-1, receiving an initial course of ART followed by three short, pVL-guided treatment interruptions, or Arm-2, receiving continuous ART. Both arms then underwent an extended ATI in the second year of life. Virological outcomes, viral sequencing, and HIV-specific T-cell responses were assessed longitudinally. RESULTS:During the extended ATI, Arm-1 participants, following multiple treatment interruptions, demonstrated a lower peak and set-point pVL compared to Arm-2, alongside an early induction of HIV-specific CD8+ T-cell responses. One Arm-1 participant achieved undetectable pVL for 1.5 months during the extended ATI, before losing immune control associated with viral escape within dominant Gag CD8+ T-cell epitopes. CONCLUSIONS:Short-term viral exposure from serial ATIs is associated with the induction and/or boosting of HIV-specific CD8+ T-cell responses and enhanced immune control of HIV in early ART-treated children.

BackgroundElite controllers can spontaneously control HIV-1 infection without antiretroviral treatment but remain at risk of developing non-AIDS-related conditions. The adaptive immune system is key in mediating spontaneous viral control; however, the innate immune response remains understudied. We assessed the quality of the innate immune responses by evaluating the phenotype and function of antigen-presenting cells (APCs) in South African adults living with HIV (PWH).MethodologyA total of 73 black South Africans were included in this study. Of these, 55 were living with HIV and included 16 individuals with spontaneous viral control (PWHEC), 20 HIV progressors (PWHPROG), and 19 individuals suppressed on ART (PWHART). Eighteen individuals without HIV infection (PWOHHIV-) served as the control group. Monocyte subsets, T cell and monocyte activation and the production of tumour necrosis factor-alpha (TNF-α), interferon-alpha (IFN-α), and interleukin-1 beta (IL-1β) by monocytes, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells were analyzed using multicolour flow cytometry following stimulation with toll-like receptor (TLR)4 (LPS), TLR7/8 (CL097), and TLR9 (CpG-ODN2216) ligands. Plasma biomarkers, soluble CD14 (sCD14), and D-dimer were assessed using enzyme-linked immunosorbent assay.ResultsOur findings show a reduced expression of CD86 on monocytes of PWHEC (p=0.04) compared to PWOHHIV-. A reduced frequency of the classical monocyte (CD14+CD16) subset in PWHEC (p=0.02) and PWHPROG (p=0.05) compared to PWOHHIV-. TNF-α and IL-1β production was lower in monocytes and mDCs of PWHEC compared to PWOHHIV- post-stimulation with TLR4, and TLR7/8 (all p<0.05). Increased sCD14 levels in PWHEC compared to PWOHHIV- (p=0.01) indicate persistent immune activation, whereas increased D-dimer levels in PWHPROG compared to PWHART (p=0.01) and PWHEC (p=0.04) suggest higher inflammation in PWHPROG.ConclusionPWHEC exhibits similar immune responses as other PWH including PWHPROG, their innate immune profiles are characterized by lower levels of monocyte activation, reduced levels of classical monocytes, reduced capacity to produce pro-inflammatory cytokines, and elevated biomarkers associated with unfavourable disease outcomes. These findings highlight the need for continuous monitoring and potential therapeutic interventions to mitigate chronic inflammation in PWHEC. Furthermore, it expands our understanding of complex innate immune cell responses in PWHEC.

ABSTRACT Phenotypic changes and functional impairment of natural killer (NK) cells occur early in HIV-1 infection. Antiretroviral therapy (ART) effectively restores CD4+ T cell counts and suppresses HIV-1 to undetectable levels. The role and efficacy of immediate ART initiation in mitigating NK cell aberrations remain to be elucidated comprehensively. This study hypothesized that HIV-1 infection negatively influences NK cell evolution and that early ART initiation restores these perturbations. Blood samples were collected longitudinally from five acutely HIV-1 infected men who have sex with men in Nairobi, Kenya. Participants were immediately initiated on ART after HIV-1 diagnosis. Blood samples were drawn pre-infection and at sequential bi-weekly post-infection time points. Peripheral blood mononuclear cells were stained with panel NK cells surface markers to assess HIV-induced phenotypic changes by flow cytometry. Some cells were also stimulated overnight with K562 cell line, IL-2, and IL-15 and stained for flow cytometry functionality. HIV-1 infection was associated with significant reductions in the production of IFN-γ ( P = 0.0264), expression of CD69 ( P = 0.0110), and expression of NK cell inhibitory receptor Siglec7 ( P = 0.0418). We observed an increased NK cell degranulation ( P = 0.0100) and an upregulated expression of cell exhaustion marker PD-1 ( P = 0.0513) at post-infection time points. These changes mainly were restored upon immediate initiation of ART, except for Siglec7 expression, whose reduced expression persisted despite ART. Some HIV-associated changes in NK cells may persist despite the immediate initiation of ART in acute HIV-1 infections. Our findings suggest that understanding NK cell dynamics and their restoration after ART can offer insights into optimizing HIV-1 treatment and potentially slowing disease progression. IMPORTANCE Natural killer (NK) cells play a crucial role in controlling of HIV-1 replication and progression to disease. Perturbations of their functionality may therefore result in deleterious disease outcomes. Previous studies have demonstrated reduced NK cell functionality in chronic HIV-1 infection that positively correlated to HIV-1 viral load. This may suggest that control of HIV-1 viremia in acute HIV-1 infection may aid in enhancing NK cell response boosting the inate immunity hence effective control of viral spread and establishment of viral reservoir. Antiretroviral therapy (ART) effectively supresses HIV-1 viremia to undectable levels and restores CD4+ T cell counts. Our study highlights the significant role of early ART initiation in mitigating NK cell disruptions caused by acute HIV-1 infection. Our results suggest that early initiation of ART could have benefits beyond suppressing viral load and restoring CD4+ T cell counts. In addition, it could boost the innate immunity necessary to control disease progression.

BACKGROUND:HIV type 1 ((human immunodeficiency virus) HIV-1) elite controllers (ECs) are a rare subset of people living with HIV-1 (PLWH) who control viral replication in the absence of antiretroviral treatment (ART) and may provide a model for a functional cure. We investigated the role of natural killer (NK) cells in HIV-1 ECs from South Africa. METHODS:Phenotypic (CD69, CD38, CD57, PD-1), functional (CD107a, IFN-γ (inferferon gamma)), and nutrient transporter profiles (glucose transporter 1, CD98) of NK cells from ECs (n = 20), viremic progressors (VPs; n = 19), PLWH on ART (n = 20), and people without HIV-1 (PWOH; n = 21) were analyzed using flow cytometry. The Kruskal-Wallis test and followed by the Mann-Whitney U test were used to determine differences among the study groups. The Spearman rank correlation coefficient was used to determine significant associations. RESULTS:Compared with the other study groups, the percentage of CD69-expressing NK cells was higher in ECs, whereas the percentage of CD38-expressing NK cells was higher in VPs. Percentages of CD69 + CD38 - NK cells were elevated in ECs compared with VPs ( P = 0.003), but were not different to PLWH on ART and PWOH. Differentiation, exhaustion, and metabolic profiles were not different in ECs compared with PLWH on ART and PWOH; however, NK cell function was lower than in PWOH. CONCLUSIONS:These findings demonstrate that NK cells from ECs have an activated, mature profile with low levels of immune exhaustion and a reduced metabolic phenotype suggesting functional competence. This insight could inform the development of novel immunotherapeutic strategies for treating HIV-1.

Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen–antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.

Abstract Currently, there are point-of-care (POC) tests for the detection of HIV and Mycobacterium tuberculosis (M.tb), however none can detect these pathogens simultaneously. We developed a colorimetric multiplex HIV and active TB diagnostic technology, using HIV-1 p24 and M.tb CFP10, ESAT6 and pstS1 antigens immobilized in high-binding 96 well plates or on epoxy-coated microarray slides. These antigens were used to bind corresponding antibodies in human serum at concentrations commonly found in infected individuals. This was followed by the addition of HRP conjugated secondary antibodies using either 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) or 3,3',5,5'-tetramethyl Benzidine (TMB) substrates. The sensitivity of the assay in the plates was compared to epoxy-coated slides and the latter had a better limit of detection, averaging 815-fold lower than the plates. Also, the TMB substrate produced better sensitivity than ABTS. Furthermore, we studied the specificity of the antigen-antibody reactions for this technology and determined that it was highly specific. Lastly, we investigated the stability of our technology at different pH, temperature, and storage conditions and found that it was optimal at pH 7.4, 25 ˚C, and dry storage for 1 to 90 days. Thus, the developed diagnostic technology has potential for the detection of HIV and TB co-infection at POC.

HIV nonprogression despite persistent viremia is rare among adults who are naive to antiretroviral therapy (ART) but relatively common among ART-naive children. Previous studies indicate that ART-naive pediatric slow progressors (PSPs) adopt immune evasion strategies similar to those described in natural hosts of SIV. However, the mechanisms underlying this immunophenotype are not well understood. In a cohort of early-treated infants who underwent analytical treatment interruption (ATI) after 12 months of ART, expression of PD-1 on CD8+ T cells immediately before ATI was the main predictor of slow progression during ATI. PD-1+CD8+ T cell frequency was also negatively correlated with CCR5 and HLA-DR expression on CD4+ T cells and predicted stronger HIV-specific T lymphocyte responses. In the CD8+ T cell compartment of PSPs, we identified an enrichment of stem-like TCF-1+PD-1+ memory cells, whereas pediatric progressors and viremic adults had a terminally exhausted PD-1+CD39+ population. TCF-1+PD-1+ expression on CD8+ T cells was associated with higher proliferative activity and stronger Gag-specific effector functionality. These data prompted the hypothesis that the proliferative burst potential of stem-like HIV-specific cytotoxic cells could be exploited in therapeutic strategies to boost the antiviral response and facilitate remission in infants who received early ART with a preserved and nonexhausted T cell compartment.

Background. Mechanisms underlying an association between human immunodeficiency virus (HIV) or antiretroviral therapy (ART) during pregnancy with risk of preterm delivery (PTD) and small-for-gestational-age (SGA) remain unclear. We explored the association between cellular immune activation and PTD or SGA in women with HIV initiating ART during or before pregnancy. Methods. Women with HIV enrolled at median 15 weeks' gestation, were analyzed for immune markers, and matched on ART initiation timing (15 women initiated pre- and 15 during pregnancy). There were 30 PTD (delivery <37 weeks), 30 SGA (weight for age <= 10th percentile) cases, and 30 controls (term, weight for gestational age >25th percentile) as outcomes. Lymphocytes, monocytes, and dendritic cell populations and their activation status or functionality were enumerated by flow cytometry. Results. PTD cases initiating ART in pregnancy showed decreased CD8(+) T cell, monocyte, and dendritic cell activation; increased classical (CD14(+)CD16(-)) and intermediate (CD14(+)CD16(+)) monocyte frequencies; and decreased inflammatory monocytes (CD14(dim)CD16(+)) compared with SGA cases and term controls (all P<.05). Allowing for baseline viral load, the immune markers remained significantly associated with PTD but only in women initiating ART in pregnancy. Lower monocyte activation was predictive of PTD. TLR ligand-induced interferon-alpha and macrophage inflammatory protein-1 beta levels in monocytes were significantly lower in PTD women initiating ART in pregnancy. Conclusion. Low immune activation, skewing toward anti-inflammatory monocytes, and lower monocyte cytokine production in response to TLR ligand stimulation were associated with PTD but not SGA among women initiating ART in, but not before, pregnancy, suggesting immune anergy to microbial stimulation as a possible underlying mechanism for PTD in women initiating ART in pregnancy.

: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leukocyte antigen class I (HLA-I) molecules modulating natural killer cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here, we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits natural killer cells in individual encoding for this genotype.

: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits NK cells in individual encoding for this genotype.

HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C*03:04 and disinhibits NK cells in individual encoding for this genotype. Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Natural killer (NK) cells are an important component of the innate immune system, able to rapidly respond to target cells without prior sensitization. The effector function of NK cells to stimuli are determined by the interplay between activating and inhibitory receptors [1], including killer immunoglobulin receptors (KIRs). KIRs bind to human leukocyte antigen class I (HLA-I) molecules to monitor HLA-I expression levels on cells, and the affinity of these KIR/HLA-I interactions is dependent on the sequence of the HLA-I presented peptide [2, 3]. KIR/HLA-I compound genotypes have been associated with the speed of HIV-1 disease progression [4], and selection of viral sequence polymorphisms associated with specific KIR or KIR/HLA-I compound genotypes in HIV-1 infection have been reported [5-7], indicating KIR+ NK cell-mediated immune selection pressure. We previously showed selection of two minor HIV-1 sequence polymorphisms in a cohort of 392 clade C HIV-1 infected South Africans and demonstrated that these mutations within p24 Gag were associated with combined expression of KIR2DL3 and HLA-C*03:04 genotypes and mediated escape from NK cell responses through enhanced engagement of inhibitory KIR2DL3 [8]. Here we investigated another sequence polymorphism at position 84 of p24 Gag (TGag84V) associated with the combined HLA-C*03:04/KIR2DL2 genotype in this cohort. While 42 infected individuals expressing both KIR2DL2 and HLA-C*03:04 all harbored viruses encoding for the major variant threonine (T) at amino acid position 84 of HIV-1 Gag, the minor variant valine (V) at position 84 was not present in the KIR2DL2/HLA-C*03:04 individuals yet was present in 10% (35/350) of individuals not encoding for KIR2DL2 and HLA-C*03:04 (see section A of Supplementary Material; Fig 1A , http://links.lww.com/QAD/B855, P=0.02, Fischer’s Exact test). These data suggest a significant advantage for HIV-1 to encode for TGag84 in KIR2DL2/HLA-C*03:04 individuals. The TGag84V polymorphism is located within the C-terminal region of the HLA-C*03:04-restricted peptide RSLYNTVATL (RSTL10). The major variant (RSLYNTVATL (RSTL10)) and the minor variant (RSLYNTVAVL (RSVL10)) peptides equally stabilized HLA-C*03:04 expression (method described in section B of Supplementary Material , http://links.lww.com/QAD/B855) on transfected TAP-deficient 721.221 cells (221-TAPko-HLA-C*03:04) (T [mean 2.2 ± 0.20 SD] to V [mean 2.3± 0.26SD], P=0.8), and resulted in an up to 3-fold higher expression of HLA-C*03:04 compared to non-peptide-pulsed control cells (Fig 1B). These HLA-C*03:04-stabilization data demonstrated that the TGag84V polymorphism identified within HIV-1 p24 Gag did not impact the ability of the respective peptides to bind to HLA-C*03:04. Next, the RSTL10 and RSVL10 peptides were assessed for their ability to mediate KIR2DL2-binding to HLA-C*03:04, using a KIR2DL2-Fc fusion construct, as previously described [8], (see also section C of Supplementary Material , http://links.lww.com/QAD/B855). The HLA-C*03:04-presented GAVDPLLAL (GAL) peptide derived from the human importin-α-1 subunit (residues 204-212) [9], is known to bind KIR2DL2 [9] and was used as positive control, whereas the variant GAVDPLLKL (GKL) peptide which stabilizes HLA-C*03:04 at similar levels but does not bind KIR2DL2 [9] was Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. used as a negative control. Results from 5 independent KIR2DL2-Fc-binding assays consistently demonstrated weaker binding of KIR2DL2-Fc to HLA-C*03:04 presenting the minor variant peptide RSVL10, while the major variant peptide RSTL10 mediated significantly higher binding of KIR2DL2-Fc to HLA-C*03:04 (Fig 1C, P=0.008, Mann Whitney test). Higher KIR2DL2-Fc-binding furthermore had significant functional consequences for KIR2DL2 NK cells, as demonstrated by increased inhibition of CD107a expression as a read-out for NK cell activation [10], (Supplementary Figure 1 , http://links.lww.com/QAD/B855), of KIR2DL2 primary NK cells following co-incubation of 221-TAPko-HLA-C*03:04 cells pulsed with the major variant RSTL10 peptide compared to the minor variant RSVL10 peptide (method described in section D of Supplementary Material , http://links.lww.com/QAD/B855) (Fig 1D, P=0.007, Mann Whitney test, N = 8). In summary, the results from these KIR2DL2/HLA-C*03:04 binding assays and functional assays using KIR2DL2 primary NK cells demonstrate that the minor VGag84 HIV-1 variant that was circulating in KIR2DL2/HLA-C*03:04 negative individuals, but not detected in individuals encoding for KIR2DL2/HLA-C*03:04, mediated significantly reduced binding of KIR2DL2 to HLA-C*03:04, resulting in stronger activation of primary KIR2DL2 NK cells. Previous studies have reported minor HIV-1 sequence polymorphisms selected in individuals expressing specific KIRs or KIR/HLA-I combinations that enhanced binding of inhibitory KIRs and inhibit KIR NK cells, consistent with KIR NK cell-mediated selection of viral escape variants [3, 6-8]. Here we report a sequence polymorphism within HIV-1 Gag (TGag84V) for which the major variant (TGag84) was encoded in all individuals expressing KIR2DL2/HLA-C*03:04, while the minor variant (VGag84) was detected in some individuals not encoding for this KIR/HLA compound genotype. The combined KIR2DL2/HLA-C*03:04 genotype is frequent in the studied population of HIV-1 clade C infected individuals from South Africa (42 of 392 individuals, 10.7%), and these observations are consistent with the selection and potential accumulation of the TGag84 variant over time, similar to what has been described for HLA-B*51restricted epitope TAFTIPSI in different populations [11]. The current study was a cross sectional analysis in chronically HIV-1 clade C infected individuals in which the timing of HIV-1 infection was not known. It was therefore not possible to determine whether the TGag84 polymorphism was indeed selected in KIR2DL2/HLA-C*03:04 individuals infected with the minor VGag84 variant; and the fact that less than 10% of circulating viruses in these 392 individuals encoded for VGag84 suggests that most individuals were initially infected with the TGag84 polymorphism. Longitudinal studies with accurate timing of primary infection will be required to determine whether the TGag84 variant is indeed selected in KIR2DL2/HLA-C*03:04 individuals infected with the minor VGag84 variant, leading to the accumulation of the TGag84 variant over time. In summary, these data suggest potential accumulation of sequence polymorphisms that reduce recognition by KIR NK cells over time, eventually representing the major variant in the circulating HIV-1 strains, similar to the enrichment of HIV-1 variants selected in response to CD8T-cell-mediated immune pressure [11]. Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

The Duffy Antigen Receptor for Chemokines (DARC)-null trait, common among persons of African descent and associated with lower absolute neutrophil counts (ANCs), may be linked to increased risk to certain infections including HIV-1 but the underlying causes are poorly understood. We hypothesized that DARC-null-linked neutropenia may negatively impact neutrophil immunoregulatory modulation of other immune cells such as natural killer (NK) and CD8+ T cells leading to altered phenotype, functionality and homeostatic activity of these immune cells. HIV-1 uninfected (n = 20) and HIV-1 chronically infected (n = 19) participants were assessed using multi-parametric flow cytometry to determine NK and CD8+ T cell counts, phenotypic profiles, and cytokine production and degranulation. Annexin V and carboxyfluorescein succinimidyl ester (CFSE) staining were used to examine NK cell survival and NK cell and CD8+ T cell proliferation respectively. Participants were genotyped for the DARC-null polymorphism using allelic discrimination assays and ANCs were measured by full blood count. In HIV uninfected individuals, a reduction of total NK cell counts was noted in the absence of DARC and this correlated with lower ANCs. HIV uninfected DARCnull subjects displayed a less mature NK cell phenotype. However, this did not translate to differences in NK cell activation or effector functionality by DARC state. Whilst HIV-1 infected subjects displayed NK cell profiling that is typical of HIV infection, no differences were noted upon DARC stratification. Similarly, CD8+ T cells from HIV infected individuals displayed phenotypic and functional modulation that is characteristic of HIV infection, but profiling was unaffected by the DARC-null variant irrespective of HIV status. Overall, the data suggests that the DARC-null polymorphism and lower ANCs does not impede downstream cytolytic cell priming and functionality.

: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits NK cells in individual encoding for this genotype.

Neutrophils are well-recognized for their pathogen killing mechanisms and disorders of neutrophil count and function are associated with recurrent infections. The Duffy Antigen Receptor for Chemokines (DARC)-null genotype is predominant in sub-Saharan African ancestry populations and is the major genetic determinant of benign ethnic neutropenia which has been associated with increased risk of Human Immunodeficiency Virus (HIV)-1 acquisition and mother-to-child transmission. However, the impact of DARC-null-linked neutropenia on HIV disease progression remains controversial. While the DARC-null genotype is associated with low numbers of circulating neutrophils, the effects of the polymorphism on neutrophil functions is unknown. We investigated the impact of the DARC-null trait and lower absolute neutrophil counts (ANCs) on key neutrophil effector functions [proteolytic activity within the phagosome following Fc receptor-mediated phagocytosis, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation] in 20 HIV negative and 22 HIV-1 chronically infected black South Africans. Phagosome maturation was measured by flow cytometry following Fc-mediated uptake of IgG opsonized beads; ROS production was measured by chemi-luminescence after activation of neutrophils with phorbol 12-myristate 13-acetate (PMA). Activated neutrophils were also visualized by fluorescent microscopy for NET quantification. Study subjects were genotyped for the DARC trait using TaqMan allelic discrimination assays and ANCs were measured by full blood count. As expected, the DARC-null polymorphism was highly prevalent in our participant cohort (69%) and was strongly associated with lower ANCs in uninfected (p = 0.0007) and HIV-1 infected (p = 0.03) subjects. We observed enhanced proteolytic activity within the phagosome in the absence of DARC at 10 min (p = 0.05 and p = 0.009) and 60 min (p = 0.05 and p = 0.07) in uninfected and HIV-1 infected subjects, respectively. ROS was unaffected by DARC trait irrespective of HIV status. Furthermore, formation of NETs was reduced in neutrophils from DARC-null subjects (p = 0.04) following prolonged in vitro stimulation, but only in HIV-1 infected subjects. The data indicate differential neutrophil function in the absence of DARC that may be moderately modulated by HIV-1 infection but overall, the data suggest that DARC-null trait is not deleterious to neutrophil effector functions in African populations.

HIV-1 viremia has been shown to induce several phenotypic and functional abnormalities in natural killer (NK) cells. To assess immune defects associated with HIV viremia, we examined NK cell function, differentiation status, and phenotypic alterations based on expression of inhibitory and activating receptors on NK cells in HIV-1 subtype C chronically infected participants from Durban, South Africa. NK cell phenotypic profiles were characterized by assessing sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7), NKG2A, and NKG2C markers on frozen peripheral blood mononuclear cells from viremic, antiretroviral therapy (ART)-naive HIV-1 chronically infected participants (n = 23), HIV-1 chronically infected participants who had been on combination antiretroviral therapy (cART) for at least 12 months (n = 23) compared with healthy donors (n = 23). NK cell differentiation was assessed by measurement of killer immunoglobulin receptor (KIR) and NKG2A expression; CD57 and CD107a measurements were carried out in HIV viremic and healthy donors. All phenotypic and functional assessments were analyzed by using multicolor flow cytometry. HIV-1-infected participants displayed greater frequencies of the CD56-CD16+ (CD56negative) NK cell subset compared with healthy donors (p < .0001). Downregulation of Siglec-7 and NKG2A and upregulation of NKG2C were more pronounced in the CD56negative NK cell subset of viremic participants. The CD56negative subset demonstrated a differentiated (KIR+NKG2A-) phenotype with reduced CD57 expression and lower degranulation capacity in HIV-1-infected participants compared with healthy donors. HIV-1 infection induces the expansion of the CD56negative NK cell subset marked by altered receptor expression profiles that are indicative of impaired function and may explain the overall NK cell dysfunction observed in chronic HIV-1 infection.

Background: Tuberculosis (TB) remains a major cause of global morbidity and mortality, especially in the context of HIV coinfection because immunity is not completely restored following antiretroviral therapy (ART). The identification of immune correlates of risk for TB disease could help in the design of host-directed therapies and clinical management. This study aimed to identify innate immune correlates of TB recurrence in HIV+ ART-treated individuals with a history of previous successful TB treatment. Methods: Twelve participants with a recurrent episode of TB (cases) were matched for age, sex, time on ART, pre-ART CD4 count with 12 participants who did not develop recurrent TB in 60 months of follow-up (controls). Cryopreserved peripheral blood mononuclear cells from time-points before TB recurrence were stimulated with ligands for Toll-like receptors (TLR) including TLR-2, TLR-4, and TLR-7/8. Multicolor flow cytometry and intracellular cytokine staining were used to detect IL-1β, TNF-α, IL-12, and IP10 responses from monocytes and myeloid dendritic cells (mDCs). Results: Elevated production of IL-1β from monocytes following TLR-2, TLR-4, and TLR-7/8 stimulation was associated with reduced odds of TB recurrence. In contrast, production of IL-1β from both monocytes and mDCs following Bacillus Calmette–Guérin (BCG) stimulation was associated with increased odds of TB recurrence (risk of recurrence increased by 30% in monocytes and 42% in mDCs, respectively). Conclusion: Production of IL-1β by innate immune cells following TLR and BCG stimulations correlated with differential TB recurrence outcomes in ART-treated patients and highlights differences in host response to TB.

Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in “shock-and-kill” cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to “corner” the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.

ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter ( n = 53) and transmitter ( n = 44) mothers ( P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses ( n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers ( P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants ( n = 28) in comparison to their mothers ( P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that “consensus-like” virus sequences correspond to lower in vitro replication abilities yet appear to be preferentially transmitted suggests that viral characteristics favoring transmission are decoupled from those that enhance replicative capacity.