Aviremic Subjects Research Articles

HIV and age underlie specific patterns of brain abnormalities and cognitive changes in high functioning patients.

Findings on the influence of age and HIV on brain and cognition remain equivocal, particularly in aviremic subjects without other age or HIV-related comorbidities. We aimed to (a) examine the effect of HIV status and age on structural brain measurements and cognition, and (b) apply the machine learning technique to identify brain morphometric and cognitive features that are most discriminative between aviremic subjects with HIV on stable combination antiretroviral therapy (cART) and healthy controls. Fifty-three HIV-seropositive patients and 62 healthy controls underwent neuropsychological testing (executive functions, attention, memory, learning, psychomotor speed, fluency) and volumetric MRI scans. Voxel-based morphometry, ANCOVAs, machine learning, and multivariate regression were conducted to determine the between group differences in terms of relationship of HIV status, age, and their interaction on neurocognitive and structural brain measures. Volume and gray matter (GM) thickness of the caudate, parahippocampus, insula, and inferior frontal gyrus were smaller in seropositive subjects in comparison with healthy controls (HC). They also performed worse in complex attention and cognitive fluency tasks. Support vector machine (SVM) analysis revealed that the best between-groups classification accuracy was obtained based on cognitive scores encompassing complex attention and psychomotor speed, as well as volumetric measures of white matter and total gray matter; third, fourth, and lateral ventricles; amygdala; caudate; and putamen. Both voxel-based morphometry (VBM) and regression analysis yielded that HIV and aging independently increase brain vulnerability and cognitive worsening. Patients with HIV on effective cART demonstrate smaller volumetric measures and worse cognitive functioning relative to seronegative individuals. There is no interaction between HIV infection and aging. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Association of Toll-like receptor 3 and Toll-like receptor 9 single-nucleotide polymorphisms with hepatitis C virus persistence among Egyptians.

Toll-like receptors (TLRs) give the innate immune system a considerable specificity for a large range of pathogens. TLR3 detects dsRNA of viruses while TLR9 recognizes bacterial and viral unmethylated CpG motifs. This study examined whether there is a potential association between single-nucleotide polymorphisms (SNPs) in the TLR3.rs3775290 (c.1377C/T), TLR9.rs5743836 (-1237T→C) and TLR9.rs352140 (G2848A) genes and HCV infection among Egyptian patients and healthcare workers (HCWs). We enrolled 546 subjects (409 HCWs and 137 patients) divided into four groups: group 1 included 265 seronegative, aviremic subjects; group 2 included 25 seronegative, viremic subjects; group 3 included 87 subjects with spontaneously resolved HCV infection; and group 4 included 169 chronic HCV patients. All subjects were genotyped for TLR3.rs3775290, TLR9.rs5743836 and TLR9.rs352140 SNPs by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. TLR3.rs3775290 "CC" genotype was associated with chronic HCV infection, where there was a significantly greater frequency of this genotype among chronic patients when compared to subjects with spontaneously resolved infection (63.9% vs. 51.9%; p = 0.033; OR = 1.639 and 95% CI = 0.94-2.84). However, this SNP did not correlate with the HCV RNA load among the chronic subjects (p > 0.05). There was no significant difference in TLR9.rs5743836 and TLR9.rs352140 genotype distribution between groups (p > 0.05). Lack of association between the three SNPs was found, as the three SNPs are located on two different chromosomes. In conclusion, the TLR3.rs3775290 "CC" genotype was associated with HCV chronicity, while the TLR9 gene may not play a major role in HCV infection.

Ultra-Sensitive HIV-1 Latency Viral Outgrowth Assays Using Humanized Mice.

In the current quest for a complete cure for HIV/AIDS, highly sensitive HIV-1 latency detection methods are critical to verify full viral eradication. Until now, the in vitro quantitative viral outgrowth assays (qVOA) have been the gold standard for assessing latent HIV-1 viral burden. However, these assays have been inadequate in detecting the presence of ultralow levels of latent virus in a number of patients who were initially thought to have been cured, but eventually showed viral rebound. In this context, new approaches utilizing in vivo mouse-based VOAs are promising. In the murine VOA (mVOA), large numbers of CD4+ T cells or PBMC from aviremic subjects are xenografted into immunodeficient NSG mice, whereas in the humanized mouse-based VOA (hmVOA) patient CD4+ T cell samples are injected into BLT or hu-hematopoetic stem cells (hu-HSC) humanized mice. While latent virus could be recovered in both of these systems, the hmVOA provides higher sensitivity than the mVOA using a fewer number of input cells. In contrast to the mVOA, the hmVOA provides a broader spectrum of highly susceptible HIV-1 target cells and enables newly engrafted cells to home into preformed human lymphoid organs where they can infect cells in situ after viral activation. Hu-mice also allow for both xenograft- and allograft-driven cell expansions with less severe GvH providing a longer time frame for potential viral outgrowth from cells with a delayed latent viral activation. Based on these advantages, the hmVOA has great potential in playing an important role in HIV-1 latency and cure research.

The induction of CD80 and apoptosis on B cells and CD40L in CD4+ T cells in response to seasonal influenza vaccination distinguishes responders versus non-responders in healthy controls and aviremic ART-treated HIV-infected individuals

BackgroundStudies have shown that HIV infection is associated with an impaired influenza vaccine response. We examined the role of cellular phenotypes and function in influenza vaccine responsiveness in healthy controls and aviremic HIV-infected subjects on antiretroviral treatment (ART). Methods16 healthy controls and 26 ART+ aviremic HIV+ subjects were enrolled in the current study. Blood was collected at pre-vaccination (D0), and on days 7–10 (D7) and 14–21 (D14) following the 2013–2014 seasonal influenza vaccine administrations. Subjects were classified as responders if neutralizing titers against H1N1 virus increased ⩾4-fold at D14 compared to D0. A serial analysis of B and CD4+ T cell frequencies and activation was performed on D0 and D7 by flow cytometry. Results9 of 26 (34.6%) HIV-infected individuals and 7 of 16 (43.8%) healthy controls were classified as responders to influenza vaccines. Total B cell apoptosis (annexin V) was increased on D7 post-vaccination in non-responders but not in responders among both controls and HIV+ subjects. Surface CD80 expression on memory B cells and intracellular CD40L expression on memory CD4+ T cells were induced on D7 in responders of controls but not in non-responders. The CD80 and CD40L induction was not demonstrable in HIV-infected subjects regardless of responders and non-responders. Memory CD4+ T cell cycling tended to increase on D7 in the four study groups but did not achieve significance. All the other parameters were indistinguishable between responders and non-responders, regardless of HIV-infection status. ConclusionThe perturbation of activation and apoptotic induction on B cells or CD4+ T cells after seasonal influenza vaccination in non-responders and HIV-infected subjects may help understand the mechanism of impaired vaccine responsiveness.

Maraviroc Reduces Arterial Stiffness in PI-Treated HIV-infected Patients.

The Δ32-CCR5 deletion of the CCR5 receptor is protective toward coronary artery pathology and myocardial infarction. Maraviroc (MVC), a CCR5 antagonist, was recently introduced in the therapy of HIV infection; we evaluated whether this drug could modulate the atherosclerotic burden in aviremic PI-treated HIV-positive individuals who underwent MVC intensification. Thus, the effect of MVC on intima media thickness, arterial stiffness, metabolic parameters, pro-inflammatory cytokines, endothelial dysfunction, and microbial traslocation markers was analyzed in 6 aviremic PI-treated HIV-positive individuals and were compared to those obtained in 9 additional aviremic PI-treated subjects that were enrolled retrospectively from our outpatients cohort. MVC intensification resulted in a significant reduction in intima media thickness, pulse wave velocity and triglycerides compared to baseline. Notably, MVC was also associated with a significant reduction of IL-6, microbial translocation indexes, sICAM and sVCAM; these changes were maintained throughout the 6 months of MVC intensification. No significant modifications were observed in CD4 counts, HIV viral load, and cholesterolemia. Results herein support a role of CCR5 antagonists in reducing the cardiovascular risk in HIV-infection. The hampering of inflammation, microbial translocation and the improvement of endothelial function could justify the protective role of CCR5 antagonists on atherosclerotic burden.

Key differences in B cell activation patterns and immune correlates among treated HIV-infected patients versus healthy controls following influenza vaccination

BackgroundThere is increasing recognition of the role of B cell dysfunction in HIV pathogenesis, but little is known about how these perturbations may influence responses to vaccinations. MethodsHealthy controls (n=16) and antiretroviral therapy (ART)-treated aviremic HIV-infected subjects (n=26) receiving standard-of-care annual influenza vaccinations were enrolled in the present study. Total bacterial 16S rDNA levels were assessed by quantitative polymerase chain reactions in plasma. Serologic responses were characterized by ELISA, hemagglutination inhibition assay (HI), and microneutralization, and cell-mediated responses were assessed by ELISPOT (antigen-specific IgG+ antibody-secreting cells (ASCs)) and flow cytometry at pre-vaccination (D0), day 7–10 (D7) and day 14–21 (D14) post-vaccination. ResultsDecreased peripheral CD4+ T cell absolute counts and increased frequencies of cycling and apoptotic B cells were found at baseline in HIV-infected subjects relative to healthy controls. In healthy controls, post-vaccination neutralizing activities were related to the frequencies of vaccine-mediated apoptosis and cycling of B cells, but not to CD4+ T cell counts. In patients, both baseline and post-vaccination neutralizing activities were directly correlated with plasma level of bacterial 16S rDNA. However, overall vaccine responses including antibody titers and fold changes were comparable or greater in HIV-infected subjects relative to healthy controls. ConclusionB cell function correlates with measures of recall humoral immunity in response to seasonal influenza vaccination in healthy controls but not in ART-treated patients.

Differential distribution of IL28B.rs12979860 single-nucleotide polymorphism among Egyptian healthcare workers with and without a hepatitis C virus-specific cellular immune response.

The CC genotype of the interleukin (IL)-28B.rs12979860 gene has been associated with spontaneous hepatitis C virus (HCV) clearance and treatment response. The distribution and correlation of an IL28B.rs12979860 single-nucleotide polymorphism (SNP) with HCV-specific cell-mediated immune (CMI) responses among Egyptian healthcare workers (HCWs) is not known. We determined this relationship in 402 HCWs who serve a patient cohort with ~85% HCV prevalence. We enrolled 402 HCWs in four groups: group 1 (n = 258), seronegative aviremic subjects; group 2 (n = 25), seronegative viremic subjects; group 3 (n = 41), subjects with spontaneously resolved HCV infection; and group 4 (n = 78), chronic HCV patients. All subjects were tested for an HCV-specific CMI response using an ex-vivo interferon-gamma (IFNγ) ELISpot assay with nine HCV genotype-4a overlapping 15-mer peptide pools corresponding to all of the HCV proteins. All subjects were tested for IL28B.rs12979860 SNP by real-time PCR. An HCV-specific CMI was demonstrated in ~27% of the seronegative aviremic HCWs (group 1), suggesting clearance of infection after low-level exposure to HCV. The frequency of IL28B.rs12979860 C allele homozygosity in the four groups was 49%, 48%, 49%, and 23%, while that of the T allele was 14%, 16%, 12 and 19%, respectively, suggesting differential distributions among subjects with different HCV status. As reported, IL28B.rs12979860 predicted the outcome of HCV infection (p < 0.05), but we did not find any relationship between the IL28B genotypes and the outcome of HCV-specific CMI responses in the four groups (p > 0.05). The data show differential IL28B.rs12979860 genotype distribution among Egyptian HCWs with different HCV status and could not predict the outcome of HCV-specific CMI responses.

Genotypic tropism testing in proviral DNA to guide maraviroc initiation in aviremic subjects: 48-week analysis of the PROTEST study.

IntroductionIn a previous interim 24-week virological safety analysis of the PROTEST study [1], initiation of Maraviroc (MVC) plus 2 nucleoside reverse-transcriptase inhibitors (NRTIs) in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA was associated with low rates of virological failure. Here we present the final 48-week analysis of the study.MethodsPROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910) carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL) <50 c/mL on stable ART during the previous 6 months, requiring an ART change due to toxicity, with no antiretroviral resistance to the ART started, and R5 HIV by proviral DNA genotypic tropism testing (defined as a G2P FPR >10% in a singleton), initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week)*100.ResultsTropism results were available from 141/175 (80.6%) subjects screened: 87/141 (60%) were R5 and 74/87 (85%) were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62%) subjects, NNRTIs in 27 (36%) and integrase inhibitors (INIs) in 1 (2%). The main reasons for treatment change were dyslipidemia (42%), gastrointestinal symptoms (22%), and liver toxicity (15%). MVC was given alongside TDF/FTC in 40 (54%) subjects, ABC/3TC in 30 (40%), AZT/3TC in 2 (3%) and ABC/TDF in 2 (3%). Sixty-two (84%) subjects maintained VL<50 c/mL through week 48, whereas 12 (16%) discontinued treatment: two (3%) withdrew informed consent, one (1%) had a R5→X4 shift in HIV tropism between the screening and baseline visits, one (1%) was lost to follow-up, one (1%) developed an ART-related adverse event (rash), two (3%) died due to non-study-related causes (1 myocardial infarction at week 0 and 1 lung cancer at week 36), and five (7%) developed protocol-defined virological failure, although two of them regained VL<50 c/mL with the same MVC regimen (Table 1). ConclusionsInitiation of MVC plus 2 NRTIs in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA is associated with low rates of virological failure up to one year.

Subclinical Atherosclerosis and Markers of Immune Activation in HIV-Infected Children and Adolescents

HIV-infected adults display increased cardiovascular disease, probably driven by inflammation and immune activation. These relationships have not been addressed in vertically HIV-infected children and adolescents, a population at very high risk for long-term non-AIDS complications. Carotid intima media thickness (IMT) was measured in a cohort of HIV-infected children and adolescents and healthy controls. C-reactive protein and markers of immune activation (CD38⁺HLA-DR⁺) and immune senescence (CD28⁻CD57⁺) were determined. One hundred fifty HIV-infected patients and 150 controls were included, 64.8% female. IMT was thicker in HIV-infected patients (0.434 mm ± 0.025 vs. 0.424 mm ± 0.018, P < 0.001). After adjustment by age, sex, body mass index, and smoking status, HIV infection was independently associated with thicker IMT (odds ratio, 2.28; 95% confidence interval: 1.25 to 4.13; P = 0.007). Among HIV-related variables, a low CD4 nadir was related to an increased IMT. Although HIV-infected subjects presented higher frequencies of activated CD4⁺ and CD8⁺ T cells (P = 0.002 and P = 0.087, respectively), no relation was found between IMT and inflammation, immune activation, or senescence. Structural changes of the vasculature present early in vertically HIV-infected subjects as well as immune activation and senescence. These patients should be carefully monitored for the prompt detection and early treatment of cardiovascular disease.

Hepatitis C Viremia and the Risk of Chronic Kidney Disease in HIV-Infected Individuals

The role of active hepatitis C virus (HCV) replication in chronic kidney disease (CKD) risk has not been clarified. We compared CKD incidence in a large cohort of HIV-infected subjects who were HCV seronegative, HCV viremic (detectable HCV RNA), or HCV aviremic (HCV seropositive, undetectable HCV RNA). Stages 3 and 5 CKD were defined according to standard criteria. Progressive CKD was defined as a sustained 25% glomerular filtration rate (GFR) decrease from baseline to a GFR < 60 mL/min/1.73 m2. We used Cox models to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). A total of 52 602 HCV seronegative, 9508 HCV viremic, and 913 HCV aviremic subjects were included. Compared with HCV seronegative subjects, HCV viremic subjects were at increased risk for stage 3 CKD (adjusted HR 1.36 [95% CI, 1.26, 1.46]), stage 5 CKD (1.95 [1.64, 2.31]), and progressive CKD (1.31 [1.19, 1.44]), while HCV aviremic subjects were also at increased risk for stage 3 CKD (1.19 [0.98, 1.45]), stage 5 CKD (1.69 [1.07, 2.65]), and progressive CKD (1.31 [1.02, 1.68]). Compared with HIV-infected subjects who were HCV seronegative, both HCV viremic and HCV aviremic individuals were at increased risk for moderate and advanced CKD.

Issue Highlights—May 2013

Issue Highlights—May 2013

ST114 IL28B SNP does not predict the outcome of Hepatitis C Virus-specific cellular immune response

The CC genotype of single nucleotide polymorphism (SNP) at the rs12979860 site of interleukin (IL)28B gene has been associated with spontaneous Hepatitis C Virus (HCV) clearance and interferon-treatment response. The role of IL28B SNP in predicting HCV-specific cell mediated immune (CMI) responses has not been investigated. We recently reported HCV-specific CMI responses in exposed uninfected healthcare workers (HCW) at high risk of infection without detecting viremia or seroconversion. The HCW at the National Liver Institute serve a patient cohort with ∼90% HCV prevalence. We investigated the HCV-specific CMI response in seronegative, aviremic HCW (n=102) and their chronic (n=36) and resolved (n=20) counterparts. We used an enzyme-linked immunospot (ELISPOT) assay to quantify interferon gamma production in response to a set of genotype 4a overlapping 15mer peptide pools covering the whole HCV genome. Also, we examined IL28B genotype of these HCW by real-time PCR. We confirmed our previous findings of HCV-specific CMI without viremia or seroconversion in more than 20% of these HCW suggesting clearance of low levels of HCV exposures. The percentage of IL28B CC allele in chronic, resolved and seronegative aviremic subjects were 31%, 45%, and 46%, while that of the TT allele was 31%, 0% and 8%, respectively. Although IL28B SNP predicted the outcome of HCV infection, we did not find any correlation between the different IL28B genotypes and the outcome of HCV-specific CMI responses in the three groups tested (p>0.05). Taken together, these data support the notion that a protective HCV vaccine targeting CMI is feasible regardless of IL28B genotype. Also, IL28B does not predict the outcome of HCV-specific CMI responses.

CD4+CD25+CD127− assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV‐1 infected viremic and aviremic subjects

Although likely pivotal, the role of regulatory T cells (Tregs) in HIV pathogenesis remains elusive. This can be partly explained by analytical issues regarding their phenotypic identification in clinical studies. Instead of intracellular FOXP3 staining, CD4+CD25+CD127- phenotype has been proposed as an alternative to identify Tregs in clinical samples. However, its use remains controversial in viremic patients. Therefore, the objective of the present study was to assess the correlation between frequencies of CD4+CD25+CD127- and CD4+CD25+FOXP3+ lymphocytes in viremic and matched aviremic HIV-infected patients. Peripheral blood was collected from HIV-1 infected patients. Eleven viremic patients (Viral Load > 40 copies/mL) were matched (age, sex, CD4+ cell number) with 8 aviremic patients under highly active antiretroviral therapy (HAART). Fresh whole blood was immediately stained to analyze by flow cytometry the correlation between CD4+CD25+CD127- and the reference phenotype CD4+CD25+FOXP3+ lymphocytes in the same tube (four color staining CD4/CD25/CD127/FOXP3 for concomitant analysis of cell surface and intracellular markers). In both groups, no significant differences were observed when comparing CD4+CD25+CD127- and CD4+CD25+FOXP3+ cell frequencies. In line, a strong correlation between CD4+CD25+CD127- and CD4+CD25+FOXP3+ lymphocyte percentages was observed in the whole patient population (r: 0.948, P < 0.001) or each group separately: aviremic (r: 0.968, P < 0.001), viremic (r: 0.9, P < 0.001). Finally, we found that most CD4+FOXP3+ cells were indeed CD25+CD127-, both in viremic and aviremic groups (88.5% and 90.9%, respectively). We observed that CD4+CD25+CD127- phenotype is a good and easy-to-perform surrogate identification strategy for FOXP3+ regulatory T cells in both viremic and aviremic HIV-1-infected subjects. Thus, it represents a useful tool for monitoring Tregs in clinical research studies based on large cohorts of patients prospectively monitored, including HIV-infected subjects.

52. Increases in CD4 Counts and Effects on HIV in Aviremic HIV-Infected Subjects Infused with Zinc Finger Nuclease (ZFN) CCR5 Modified Autologous CD4 T-Cells (SB-728-T)

52. Increases in CD4 Counts and Effects on HIV in Aviremic HIV-Infected Subjects Infused with Zinc Finger Nuclease (ZFN) CCR5 Modified Autologous CD4 T-Cells (SB-728-T)

598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3)

598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3)

Reply

Our article1 reported that viral RNA could not be detected in the peripheral blood mononuclear cells (PBMCs) of aviremic seropositive blood donors, a conclusion supported by another recent article which also concluded that there was no evidence for persistent hepatitis C virus (HCV) infection in PBMCs after clearance of viremia in plasma.2 Decreasing or disappearing anti-HCV antibody titers in aviremic subjects also argue against continued HCV protein expression.3 Whether other reservoirs of HCV RNA, such as hepatocytes, exist in aviremic subjects remains unclear with some4-6 although not all7, 8 studies finding no HCV RNA in the liver of treated sustained viral responders. We clearly acknowledged1 that in chronically viremic subjects, PBMCs could be a site of HCV replication and that in vitro mitogen plus cytokine treatment might increase PBMC-associated HCV levels.8-10 With respect to the relative sensitivities of the HCV RNA assays used, we performed two different tests for HCV RNA in PBMCs (cell-associated transcription-mediated amplification [CA-TMA] and reverse transcription nested polymerase chain reaction [RT-nPCR]). We demonstrated a sensitivity of 2-50 HCV RNAs in 5 million PBMCs for the CA-TMA assay and 15-150 HCV RNAs in 5 million cells using RT-nPCR (adjusted from a sensitivity of 1-10 HCV RNAs for RT-nPCR, because only a fraction of total PBMC RNA and derived complementary DNA is sampled).1 Pham et al. report a sensitivity for their RT-nPCR-NAH (nucleic acid hybridization) assay of <10 viral genome equivalent (vge)/mL for serum (and in their letter, <5 vge/million PBMCs). We quantified the sensitivities of our assays using serial dilutions of plasma samples of known viral loads (prequantified using a commercial viral load assay) in the presence of total cellular RNA, as well as of total RNA from the PBMC of viremic subjects and showed the actual replicate assay results.1 Pham et al. used dilutions of a plasmid without interfering cellular nucleic acids for “semiquantitative estimation” in a “preliminary experiment” whose results where not shown.10 The fraction of total complementary DNA derived from PBMCs or 250 μL serum they used as input for their nPCR was not specified.10 With regard to the total number of cell equivalents analyzed in respective assays, we indeed analyzed only 0.16 million PBMCs by RT-nPCR but in parallel, and with consistent results, also used CA-TMA on 2.5 million PBMCs,1 slightly more than the 1 million to 2 million PBMCs Pham and Michalak reported in their letter. We agree with Pham and Michalak that difference in assay sensitivity is a likely explanation for the reported discrepancy but feel that the more relevant assay is that used for detecting HCV RNA in plasma. In our case, we used an extensively validated plasma HCV RNA assay used for blood-donation screening (Transcription mediated amplification, Procleix human immunodeficieny virus-1/HCV assay, Gen-probe, with a 95% detection sensitivity of 30 HCV RNA/mL).11-13 This TMA assay was used in duplicate assays (consuming a total of 1 mL plasma) to identify a total of 69 plasma aviremic blood donors, none of whom possessed detectable PBMC-associated HCV RNA.1 Pham et al. analyzed and found HCV RNA in the PBMCs of two aviremic subjects (so classified using their RT-nPCR-NAH).10 We suspect that further testing of the plasma from these two subjects using TMA assays might have detected a low-level viremia. Also, mostly consistent with our results, Pham et al. reported in 2008 detecting HCV RNA in only one of 10 PBMCs from aviremic subjects (although stimulation with interleukin-2 plus mitogen and testing of purified immune cell subsets lead to the detection of HCV RNA in all 10 PBMCs).14 Our selection criterion for the absence of plasma viremia (based on a highly sensitive, extensively validated assay used in duplicate tests to increase sensitivity) may therefore have been more stringent than that used by Pham et al. and account for the discrepant results seen in PBMCs. Ultimately, we agree with Pham and Michalak that the question of a residual HCV reservoir in the PBMCs of truly aviremic subjects will best be resolved using the demonstrably most sensitive detection assays for HCV RNA in both plasma and PBMC. The use of the same HCV RNA quantitation standards15 by different groups will help compare assay sensitivities. Eric Delwart Ph.D.*, * Blood Systems Research Institute, Department of Laboratory Medicine, University of California, San Francisco, San Francisco CA.

Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia

We determined whether hepatitis C virus (HCV) RNA could be detected associated with peripheral blood mononuclear cells (PBMC) of seropositive blood donors who had spontaneously or therapeutically cleared their plasma viremia. Blood donor plasma viremia status was first determined with a highly sensitive transcription-mediated amplification (TMA) test performed in duplicate assays. PBMC from 69 aviremic and 56 viremic blood donors were then analyzed for the presence of HCV RNA with TMA adapted to detect viral RNA in PBMC and with a reverse transcription-nested polymerase chain reaction assay. PBMC-associated HCV RNA was detected in none of the 69 aviremic donors, including all 6 subjects with a sustained viral response following antiviral therapy. PBMC-associated HCV RNA was detected in 43 of the 56 viremic donors. The 13 viremic donors with no detectable PBMC-associated HCV RNA all had very low viral loads (6 positive only in 1 of 2 duplicate plasma TMA assays, 6 with viral loads below 100 HCV RNA copies/mL, and 1 with a viremia of 2700 HCV RNA copies/mL). The absence of detectable PBMC HCV RNA detection in all 69 aviremic donors reported here contrasts with prior studies, possibly as a result of the higher sensitivity of the TMA assay used to test for plasma viremia. Our results indicate that PBMC are unlikely to serve as a long-lived reservoir of HCV in aviremic subjects.

Immunophenotypic patterns of CD8 + T cell subsets expressing CD8αα and IL-7Rα in viremic, aviremic and slow progressor HIV-1-infected subjects

Evidence from animal models suggests that the expression of CD8αα homodimer on CD8 + T cells plays a key role in the generation of long-lived memory cells. Here, we studied the quantitative alterations of CD8 + T cell subsets expressing CD8αα, interleukin-7 receptor (IL-7Rα) and activation markers in HIV-1-infected individuals including aviremic, viremic and slow progressor subjects using eight-color flow cytometry. Compared to slow progressor subjects, expression of CD8αα was significantly reduced in aviremic and viremic patients and this reduction occurred mainly within central memory cell subsets and not in naive and effector memory compartments. Persistence of antigenemia leads to IL-7Rα loss mainly on central and pre-terminal memory CD8 + T cell subsets in viremic patients but not in slow progressor subjects. Compared to aviremic and viremic patients, slow progressor subjects had lower levels of IL-7 and reduced activated cells. The expression of CD8αα was not significantly related to IL-7Rα although negative associations were evidenced within all CD8 + T cell subsets. Collectively, these results further advance the characterization of immunophenotypic patterns of CD8 + T cell subsets expressing CD8αα/IL-7Rα and provide new insights into the ability of HIV-1 infection to alter memory cell population.

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef

Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy.

Increased natural killer cell activity in viremic HIV-1 infection.

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-gamma and TNF-alpha than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3-/CD56+/CD94+ and CD3-/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.