Synthesis, characterization, in vitro antimycobacterial and cytotoxicity evaluation, DFT calculations, molecular docking and ADME studies of new isomeric benzimidazole-1,2,3-triazole-quinoline hybrid mixtures.

New benzimidazole-1,2,3-triazole-quinoline hybrids and their intermediates, differing in substitutions at the C-2 and/or C6 positions of the benzimidazole ring, were successfully synthesized in 55---80 % yields, with the C6-substituted ones forming as inseparable tautomeric mixtures. The synthesized compounds were fully characterised by FT-IR, 1D- and 2D-NMR, and HRMS. In-depth NMR analysis and DFT molecular calculations showed that the tautomeric mixtures formed in a ratio of almost 1:1 ratio (cis and trans), except for 5 g, where the ratio is 1:2. In vitro antimycobacterial activity evaluation against the H37Rv strain of Mycobacterial tuberculosis was undertaken on all synthesized compounds, and a selected number were further screened for their cytotoxicity on TZM-bl cell lines. Hybrid compounds showed excellent MIC90 activities ranging from 1.07 to 8.66 µM and were all more efficacious than the first-line reference drug, ethambutol (MIC90 = 9.54 µM). In particular, hybrid compounds 5b (MIC90 = 1.54 µM, CC50 = 58.89 µM and % cell viability = 14.07), 5d (MIC90 = 2.08 µM, CC50 = 0.27 µM, and % cell viability = 149.50 %) and 5 g (MIC90 = 1.49 µM, CC50 = 4.62 µM and % cell viability = 44.03) were the most promising. Significantly, 5b and 5 g were over six times more efficacious than ethambutol but exhibited cytotoxicity towards TZM-bl cell-lines compared to 5d, which was over four times more active than ethambutol. The physical combination (mimicking combination therapy) of individual pharmacophoric components making up 5 g were less active, indicating the synergistic effect of hybridization. In addition, more than 60 % of all the synthesized hybrids showed better activity than their respective pharmacophoric components. In silico ADME studies of the hybrids revealed favourable physico-chemical properties, while molecular modeling studies suggested binding interactions with Val 61, Gly 62, Glu 65, Ala 66, and Phe 69 amino acid in a reported similar manner to bedaquiline, an approved quinoline-based anti-TB drug.

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load.

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.

Anti-HIV Activity and Immunomodulatory Properties of Fractionated Crude Extracts of Alternaria alternata.

Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug toxicity, is essential to eradicating HIV. This study investigated the potential of fractionated crude extracts of Alternaria alternata as immunomodulatory or anti-HIV drug candidates. Solid-phase extraction (SPE) was used to fractionate A. alternata PO4PR2 using three different columns: MAX (Mixed-mode, strong Anion-eXchange), MCX (Mixed-mode, strong Cation-eXchange), and HLB (Hydrophilic-Lipophilic Balance) with methanol gradient methods (5%, 45%, and 95%). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to assess the cell viability and cytotoxicity of the fractionated crude extract A. alternata PO4PR2 in the TZM-bl cell lines. This was followed by a luciferase-based antiviral assay to assess the antiviral activity of A. alternata PO4PR2. A time of addition (TOA) assay was performed to ascertain the mechanism of inhibition employed by the fractionated crude extract of A. alternata PO4PR2 in the HIV life cycle. The p24 titer was determined using an ELISA, while a luciferase-based antiviral assay was used to evaluate the HIV percentage inhibition for different HIV-1 replication cycles. The TOA assay was established using antiviral drugs that target different sites in the HIV replication cycle. These included maraviroc, azidothymidine, raltegravir, and amprenavir. The immunomodulatory effect of the fractionated crude extracts on CD4+ T cells was measured by a flow cytometric analysis, for which fluorochrome-labelled monoclonal antibodies were used as markers for activation (CD38 and HLA-DR) and exhaustion (PD-1). The MCX fraction demonstrated a more significant anti-HIV inhibition than that of the fractions generated in other columns, with an IC50 of 0.3619 µg/mL, an HIV inhibition of 77%, 5% HLB (IC50: 0.7232 µg/mL; HIV inhibition of 64%), and 5% MAX (IC50: 5.240 µg/mL; HIV inhibition of 67%). It was evident from the time of addition data that the crude extract and the 5% MCX fraction inhibited viral binding (68%), reverse transcription (75%), integration (98%), and proteolysis (77%). It was shown that A. alternata (the MCX fraction) have a significant inhibitory effect on reverse transcription (75% HIV inhibition) and integration (100% HIV inhibition). The 5% MCX (p = 0.0062), 5% HLB (p = 0.0269), and 5% MAX (p = 0.0117) fractionated A. alternata crude extracts had low levels of CD4+ T cell (CD38 + HLA-DR+) activation compared to those of the AZT treatment, while CD4+ T cell activation was insignificant. The 5% MAX and HLB A. alternata fractions may possess immunomodulatory compounds with less anti-HIV-1 activity. A. alternata could be a key source of innovative anti-HIV drugs with immunomodulatory characteristics.

In vitro anti-HIV, cytotoxicity and nutritional analysis of Trianthema portulacastrum L. (Aizoaceae).

The development of antiretroviral therapy has brought a tremendous relief to the world as it minimizes mortality, reduces HIV transmission, and suppresses progression in infected patients. However, the orthodox antiretroviral therapy is faced with limitations which have necessitated a continuous search for more novel plant-based antiviral compounds, which can bypass the existing barriers created by drug resistance and target more viral proteins. Despite the edibility and enormous pharmacological benefits of T. portulacastrum, little is known about its nutrient profiles and potential use as a natural source of antiviral drug. This study focuses on the full feed analysis and anti-HIV potential of two biotypes of T. portulacastrum. Ethanolic extracts of both biotypes of T. portulacastrum (T01 and T02) had significant inhibitory effects on the level of replication of the HIV-1. Both extracts induced the inhibition of at least 50% of the HIV-1 viral load at considerably low IC50 values of 1.757 mg/mL (T01) and 1.205 mg/mL (T02) which is comparable to the AZT standard. The protein composition ranged between 8.63-22.69%; fat (1.84-4.33%); moisture (7.89-9.04%); fibre (23.84-49.98%); and carbohydrate content (38.54-70.14%). Mineral contents of tested T. portulacastrum varied considerably in different parts of the plant. Nitrogen N mineral ranged between 13.8-36.3 mg/g; sodium Na (2.0-14.0 mg/g); potassium K (14.0-82.0 mg/g); magnesium Mg (2.8-7.1 mg/g); calcium Ca (9.1-24.7 mg/g); phosphorus P (1.3-3.6 mg/g); iron Fe (193.5-984.0 ppm); zinc Zn (42.5-96.0 ppm); manganese Mn (28.5-167.5 ppm); and copper Cu (2.0-8.5 ppm). These mineral values are comparable or higher than values quoted for common vegetables, suggesting that T. portulacastrum is a nutrient-dense vegetable that could provide alternative sources of antiviral nutrients to HIV-infected individuals. Further studies are recommended to unravel key metabolites responsible for high nutrient profiles and antiretroviral effects in T. portulacastrum.

HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703.

The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade-infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401-restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401-restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703.

Developments in Exploring Fungal Secondary Metabolites as Antiviral Compounds and Advances in HIV-1 Inhibitor Screening Assays.

The emergence of drug-resistant Human Immunodeficiency Virus-1 strains against anti-HIV therapies in the clinical pipeline, and the persistence of HIV in cellular reservoirs remains a significant concern. Therefore, there is a continuous need to discover and develop new, safer, and effective drugs targeting novel sites to combat HIV-1. The fungal species are gaining increasing attention as alternative sources of anti-HIV compounds or immunomodulators that can escape the current barriers to cure. Despite the potential of the fungal kingdom as a source for diverse chemistries that can yield novel HIV therapies, there are few comprehensive reports on the progress made thus far in the search for fungal species with the capacity to produce anti-HIV compounds. This review provides insights into the recent research developments on natural products produced by fungal species, particularly fungal endophytes exhibiting immunomodulatory or anti-HIV activities. In this study, we first explore currently existing therapies for various HIV-1 target sites. Then we assess the various activity assays developed for gauging antiviral activity production from microbial sources since they are crucial in the early screening phases for discovering novel anti-HIV compounds. Finally, we explore fungal secondary metabolites compounds that have been characterized at the structural level and demonstrate their potential as inhibitors of various HIV-1 target sites.

Epigenetic Induction of Secondary Metabolites Production in Endophytic Fungi Penicillium chrysogenum and GC-MS Analysis of Crude Metabolites with Anti-HIV-1 Activity.

The continuous burden of human immunodeficiency virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated four endophytic fungal isolates from a medicinal plant, Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate, and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Penicillium chrysogenum P03MB2 showed anti-HIV activity with an IC50 of 0.6024 µg/mL compared to untreated fungal crude extract (IC50 5.053 µg/mL) when treated with sodium butyrate. The profile of secondary metabolite compounds from the bioactive, partially purified extracts were identified by gas chromatography-mass spectrometry (GC-MS), and more bioactive compounds were detected in treated P. chrysogenum P03MB2 fractions than in untreated fractions. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro (13.64%), cyclotrisiloxane, hexamethyl (8.18%), cyclotetrasiloxane, octamethyl (7.23%), cyclopentasiloxane, decamethyl (6.36%), quinoline, 1,2-dihydro-2,24-trimethyl (5.45%), propanenitrile (4.55%), deca-6,9-diene (4.55%), dibutyl phthalate (4.55%), and silane[1,1-dimethyl-2-propenyl)oxy]dimethyl (2.73%) were the most abundant compounds. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with stronger anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites which can be developed into therapeutic compounds.

Cationic Chitosan Derivatives for the Inactivation of HIV-1 and SARS-CoV-2 Enveloped Viruses.

Cationic chitosan derivatives have been widely studied as potential antimicrobial agents. However, very little is known about their antiviral activity and mode of action against enveloped viruses. We investigated the ability of hydroxypropanoic acid-grafted chitosan (HPA-CS) and N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) to inactivate enveloped viruses like the human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The membrane-disrupting potential of the chitosan derivatives was initially investigated in a hemolysis assay. At 1.0 mg/mL, about 80% hemolysis was observed for the cationic chitosan derivatives, which was significant when compared to almost no membrane-disrupting activity by the unmodified chitosan. Virus inhibition was evaluated using the luciferase-based antiviral assay against the HIV-1 NL4.3 virus (400 TCID). The IC50 of HPA-CS was 4.109 mg/mL, while the HTCC showed a higher antiviral activity at an IC50 = 0.225 mg/mL. For practical application, the antiviral efficacies of the HTCC-coated and uncoated nonmedical masks were evaluated for SARS- CoV-2 virus capture. The coated masks demonstrated an almost excellent performance with nearly 100% viral inhibition compared to less than 60% inhibition by the uncoated masks. Molecular docking predictions suggest that the HTCC polymers interact with the viral spike protein, blocking the coronavirus interaction with the target host cell's angiotensin-converting enzyme 2 cellular receptors.

Influence of Gag-protease-mediated replication capacity on disease progression in individuals recently infected with HIV-1 subtype C.

HLA class I-mediated selection of immune escape mutations in functionally important Gag epitopes may partly explain slower disease progression in HIV-1-infected individuals with protective HLA alleles. To investigate the impact of Gag function on disease progression, the replication capacities of viruses encoding Gag-protease from 60 individuals in early HIV-1 subtype C infection were assayed in an HIV-1-inducible green fluorescent protein reporter cell line and were correlated with subsequent disease progression. Replication capacities did not correlate with viral load set points (P = 0.37) but were significantly lower in individuals with below-median viral load set points (P = 0.03), and there was a trend of correlation between lower replication capacities and lower rates of CD4 decline (P = 0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (P = 0.04), but host HLA-B-specific polymorphisms were associated with higher viral load set points (P = 0.01). Further, polymorphisms associated with host-specific protective HLA alleles were linked with higher viral load set points (P = 0.03). These data suggest that transmission or early HLA-driven selection of Gag polymorphisms results in reduced early cytotoxic T-lymphocyte (CTL) responses and higher viral load set points. In support of the former, 46% of individuals with nonprotective alleles harbored a Gag polymorphism exclusively associated with a protective HLA allele, indicating a high rate of their transmission in sub-Saharan Africa. Overall, HIV disease progression is likely to be affected by the ability to mount effective Gag CTL responses as well as the replication capacity of the transmitted virus.

Secondary metabolites produced by endophytic fungi, Alternaria alternata, as potential inhibitors of the human immunodeficiency virus.

Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungi were isolated from Sclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata (strain ID PO4PR1, PO4PR2, and PO2PL1) of the fifteen isolate's crude extracts showed anti-HIV-1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170 µg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2 ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile (16,67%); Pyrrolol[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4-dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2-Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds.

Impact of HLA in mother and child on disease progression of pediatric human immunodeficiency virus type 1 infection.

A broad Gag-specific CD8(+) T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8(+) T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8(+) T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fisher's exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8(+) T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants.

Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8+ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8+ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4+ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8+ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.

Detection of HIV type 1 gag-specific CD4(+) T cell responses in acutely infected infants.

Multiple HIV-1-specific cytokine and proliferative responses by CD4(+) T cells have not been studied in acutely infected infants. Using an intracellular cytokine staining assay, 34 untreated clade C HIV-1-infected infants (2-102 days old) were assessed for IFN-gamma, 28/34 for IL-2, and 26/34 for TNF-alpha responses to all HIV-1 proteins. Responses were detected in 29%, 36%, and 15% of infants, respectively. Twelve of the original 34 infants were then studied longitudinally for 14 months to determine the effect of viral load on IFN-gamma Gag-specific responses: seven infants were treated for 1 year, stopped treatment, and resumed when CD4% was < 20 and five infants were treated only when the CD4% was <20. Following treatment cessation, there was an immediate increase in viral load followed by an increase in the magnitude of CD4(+) Gag-specific responses. Despite this, the majority of infants (54%) had to restart treatment by 24 months of age, indicating that the immune responses were antigen driven but not associated with protection. Among untreated infants HIV-specific CD4(+) responses were detected sporadically indicating a dysfunctional immune response in the face of constant exposure to high levels of viremia.

High frequency of rapid immunological progression in African infants infected in the era of perinatal HIV prophylaxis.

OBJECTIVES: To determine the natural history of HIV infection following peripartum single-dose nevirapine (sd-NVP) prophylaxis in a resource-limited country, and to assess implications for antiretroviral therapy (ART) roll-out programmes. METHODS: Infants of HIV-infected mothers in KwaZulu-Natal, South Africa, were tested on days 1 and 28 to detect intrauterine (IU) and intrapartum (IP) infection. Infant follow-up included monthly viral load and CD4 cell measurement. ART was initiated at infant CD4 cell% < or = 20%. RESULTS: In 740 infants born to 719 HIV-infected women, mother-to-child transmission (MTCT) was 10.3% (69% IU, 31% IP). Median viral load was higher in mothers of infants infected IP than IU (279 000 versus 86 600 copies/ml; P = 0.039) and lower in mothers of uninfected infants (median 26 750 copies/ml; P < 0.001). Peak viraemia was higher in infants infected IP than IU (5 160 000 versus 984 000 copies/ml; P < 0.001). Median viral load at birth in IU-infected infants (155 000 copies/ml) fell 1.4 log to 6510 copies/ml by day 5 and was beneath the detection limit using dried blood spot analysis in 38% of infants. CD4 cell% declined rapidly, to < or = 20% in 70% and < or = 25% in 85% [current World Health Organization (WHO) criteria for initiating ART] of infants by 6 months. CONCLUSIONS: MTCT was reduced by sd-NVP through an effect on IP transmission. Where MTCT occurred despite NVP, two-thirds of transmissions arose IU; IP-infected babies were born to mothers with very high viral load. Disease progression was particularly rapid, 85% infants meeting WHO criteria for ART within 6 months. These findings argue for more effective MTCT-prevention programmes in resource-limited countries.

Preservation HIV-1-specific IFNγ+ CD4+ T-cell responses in breakthrough infections after exposure to tenofovir gel in the CAPRISA 004 microbicide trial.

The Centre for the AIDS Program of Research in South Africa 004 trial demonstrated reduction of sexual HIV-1 acquisition in women using a vaginal microbicide containing tenofovir. A better understanding of the consequences of antiretroviral-containing microbicides for immune responses in individuals with intercurrent HIV-1 infection is needed for future trials combining the use of microbicides with HIV-1 vaccines. Investigation of immune responses in women who acquired HIV-1 although using tenofovir gel showed significantly higher (P = 0.01) Gag-specific IFNγ+ CD4+ T-cell responses. The use of tenofovir-containing gel around the time of infection can modulate HIV-1 immunity, and these immunological changes need to be considered in future trials combining vaccines and microbicides.

Short communication: CD8(+) T cell polyfunctionality profiles in progressive and nonprogressive pediatric HIV type 1 infection.

Pediatric HIV-1 infection is characterized by rapid disease progression and without antiretroviral therapy (ART), more than 50% of infected children die by the age of 2 years. However, a small subset of infected children progresses slowly to disease in the absence of ART. This study aimed to identify functional characteristics of HIV-1-specific T cell responses that distinguish children with rapid and slow disease progression. Fifteen perinatally HIV-infected children (eight rapid and seven slow progressors) were longitudinally studied to monitor T cell polyfunctionality. HIV-1-specific interferon (IFN)-γ(+) CD8(+) T cell responses gradually increased over time but did not differ between slow and rapid progressors. However, polyfunctional HIV-1-specific CD8(+) T cell responses, as assessed by the expression of four functions (IFN-γ, CD107a, TNF-α, MIP-1ß), were higher in slow compared to rapid progressors (p=0.05) early in infection, and was associated with slower subsequent disease progression. These data suggest that the quality of the HIV-specific CD8(+) T cell response is associated with the control of disease in children as has been shown in adult infection.

A molecular assay for sensitive detection of pathogen-specific T-cells.

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

Immunodominant HIV-1-specific HLA-B- and HLA-C-restricted CD8+ T cells do not differ in polyfunctionality.

HIV-1 specific HLA-B-restricted CD8+ T cell responses differ from HLA-C-restricted responses in antiviral effectiveness. To investigate possible reasons for these differences, we characterized the frequency and polyfunctionality of immmunodominant HLA-B*57/B5801- and HLA-Cw*07-restricted CD8+ T cells occurring concurrently in nine study subjects assessing IFN-gamma, TNF-alpha, IL-2, MIP-1beta, and CD107a by flow cytometry and analyzed sequence variation in targeted epitopes. HLA-B*57/5801 and HLA-Cw*07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p=0.84). Possession of three or more functions correlated positively with CD4+ T cell counts (r=0.85; p=0.006) and monofunctional CD8+ T cells inversely correlated with CD4 cell counts (r=-0.79; p=0.05). There were no differences in polyfunctionality of CD8+ T cells specific to wildtype versus mutated epitopes. These results suggest that loss of polyfunctionality and increase in monofunctional HIV-1-specific CD8+ T cells are associated with disease progression independent of restricting HLA allele. Furthermore, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV-1 infection.

Immunodominant HIV-1 Cd4+ T cell epitopes in chronic untreated clade C HIV-1 infection.

BACKGROUND: A dominance of Gag-specific CD8+ T cell responses is significantly associated with a lower viral load in individuals with chronic, untreated clade C human immunodeficiency virus type 1 (HIV-1) infection. This association has not been investigated in terms of Gag-specific CD4+ T cell responses, nor have clade C HIV-1-specific CD4+ T cell epitopes, likely a vital component of an effective global HIV-1 vaccine, been identified. METHODOLOGY/PRINCIPAL FINDINGS: Intracellular cytokine staining was conducted on 373 subjects with chronic, untreated clade C infection to assess interferon-gamma (IFN-gamma) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-gamma-producing CD4+ T cell responses were detected in 261/373 (70%) subjects, with the Gag responders having a significantly lower viral load and higher CD4 count than those with no detectable Gag response (p<0.0001 for both parameters). To identify individual peptides targeted by HIV-1-specific CD4+ T cells, separate ELISPOT screening was conducted on CD8-depleted PBMCs from 32 chronically infected untreated subjects, using pools of overlapping peptides that spanned the entire HIV-1 clade C consensus sequence, and reconfirmed by flow cytometry to be CD4+ mediated. The ELISPOT screening identified 33 CD4+ peptides targeted by 18/32 patients (56%), with 27 of the 33 peptides located in the Gag region. Although the breadth of the CD4+ responses correlated inversely with viral load (p = 0.015), the magnitude of the response was not significantly associated with viral load. CONCLUSIONS/SIGNIFICANCE: These data indicate that in chronic untreated clade C HIV-1 infection, IFN-gamma-secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control.