HIV infection causes systemic immune inflammation, and increases the risk for cardiovascular (CVD) disease even among those on virologically suppressive anti-retroviral treatment (ART). We performed a biostatistical analysis and screen of candidate cellular and plasma biomarkers for association with carotid artery intima-media thickness (CIMT), independent of traditional CVD risk factors such as age, gender, systolic blood pressure (SBP), lipid levels, smoking and diabetes. We conducted a multi-stage analysis based on a cross-sectional study of CVD risk in HIV-infected subjects age >45 years on ART for >6 months. The goal of this analysis was to identify candidate cellular and plasma biomarkers of CIMT in HIV-1 infected adults. We further sought to determine if these candidate biomarkers were independent of traditional CVD risk factors previously identified in HIV negative adults. High-resolution B-mode ultrasound images of the right common carotid common artery (CCA) were obtained. Plasma soluble inflammatory mediators, cytokines and chemokines were detected. Monocytes were defined by CD14/CD16 expression, and CD8+ T-cell activation by CD38/HLA-DR expression. Subjects were a median of 49.5 years old, 87% male, had a CIMT of 0.73 mm, FRS of 6%, a median viral load of 48 copies/mL, and CD4+ T cell count of 479 cells/μL. Soluble VCAM-1, and expansion of CD14dimCD16− monocytes each associated with higher CIMT independently of age and SBP. These factors are distinct components of a shared atherogenic process; 1) vascular endothelial molecular expression and 2) vascular monocytes that enter into the vascular endothelium and promote atherosclerotic plaque.
HIV; Carotid intima-media; CIMT; Cardiovascular disease; Framingham risk score; Biomarker; Screen; Regression; CD14; Monocytes; VCAM-1; Cytokines
Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.
Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.
miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.
The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.
Congenital diaphragmatic hernia (CDH) represents a spectrum of lung hypoplasia and consequent pulmonary hypertension is an important cause of postnatal morbidity and mortality. We studied biomarkers at the maternal-fetal interface to understand factors associated with the persistence of pulmonary hypertension.
Maternal and cord blood samples from fetuses with CDH and unaffected controls were analyzed using a human 39plex immunoassay kit. Cellular trafficking between the mother and the fetu was quantified using quantitative real-time PCR for non-shared alleles. Biomarker profiles were then correlated with CDH severity based on the degree of pulmonary hypertension.
Cord blood levels of epidermal growth factor, platelet-derived growth factor, and several inflammatory mediators increased significantly as the severity of CDH increased, while maternal levels growth factors and mediators decreased significantly with CDH severity. Maternal cells were increased in fetuses with severe CDH compared to controls, with elevated levels of the chemokine CXCL-10 in patients with the highest trafficking.
Patients with CDH demonstrate pro-inflammatory and chemotactic signals in fetal blood at the time of birth. Since some of these molecules have been implicated in the development of pulmonary hypertension, prenatal strategies targeting specific molecular pathways may be useful adjuncts to current fetal therapies.
Identifying antibodies to HLA (HLA-Abs) by solid phase assays is used to screen blood donors to mitigate TRALI risk. Various cutoffs for detection assays have been proposed in the literature, however, these do not take into consideration lot-to-lot variability of commercially available assays.
Samples from 93 non-transfused males were tested using five different lots of a multiplex bead-based HLA-Ab detection kit. A subset of 17 samples was tested on five days using a single lot. An additional 96 samples from donations with varied HLA-Ab levels were tested using kits from two different lots. Results were reported as an NBG (normalized background) ratio.
For the 93 non-transfused donors, NBG values generated using the reference lot were significantly higher than those obtained with three of the four comparator lots. However, for the 96 samples with low, moderate, and higher level HLA-Abs, Class-I values were 1.4 times lower and Class-II values were 1.2 times lower using the reference versus comparator lot. For class-I antibodies the between lot SD was 1.36 (CI:1.19–1.60), while the between day SD was 1.27 (CI:1.08–1.52). Similarly, for class II antibodies the between lot SD was 0.81 (CI:0.70–0.95), while the between day SD was 0.50 (CI:0.43–0.60).
There is inter-lot variability in the tested HLA detection assay as well as significant bias between lots. It may be reasonable to develop a new cutoff when a new lot is obtained.
TRALI; anti-HLA antibodies; anti-HLA screening assay; lot-to-lot variability
CD4+ T cells are critically important in HIV infection, being both the primary cells infected by HIV and likely playing a direct or indirect role in helping control virus replication. Key areas of interest in HIV vaccine research are mechanisms of viral escape from the immune response. Interestingly, in HIV infection it has been shown that peptide sequence variation can reduce CD4+ T cell responses to the virus, and small changes to peptide sequences can transform agonist peptides into antagonist peptides.
We describe, at a molecular level, the consequences of antagonism of HIV p24-specific CD4+ T cells. Antagonist peptide exposure in the presence of agonist peptide caused a global suppression of agonist-induced gene expression and signaling molecule phosphorylation. In addition to down-regulation of factors associated with T cell activation, a smaller subset of genes associated with negative regulation of cell activation was up-regulated, including KFL-2, SOCS-1, and SPDEY9P. Finally, antagonist peptide in the absence of agonist peptide also delivered a negative signal to T cells.
Small changes in p24-specific peptides can result in T cell antagonism and reductions of both T cell receptor signaling and activation. These changes are at least in part mediated by a dominant negative signal delivered by antagonist peptide, as evidenced by up-regulation of negative regulatory genes in the presence of agonist plus antagonist stimulation. Antagonism can have dramatic effects on CD4+ T cell function and presents a potential obstacle to HIV vaccine development.
TCR; Cell signaling; Peptide antagonism; HIV
(See the article by Delwart et al, on pages 875–85, and see the editorial commentary by Katz, on pages 867–9.)
Background. There have been anecdotal reports of influenza viremia since the 1960s. We present an assessment of the prevalence of seasonal and 2009 H1N1 influenza viremia (via RNA testing) in blood donor populations using multiple sensitive detection assays.
Methods. Several influenza RNA amplification assays, including transcription-mediated amplification (TMA) and 2 reverse-transcription polymerase chain reaction (RT-PCR) assays, were evaluated and used to test donor samples. Retrospective samples from 478 subjects drawn at sites with high influenza activity were tested. Prospective samples were collected from 1004 blood donors who called their donation center within 3 days of donation complaining of influenza-like illness (ILI). The plasma collected on the day of donation for these subjects was tested.
Results. Of the repository samples, 2 of 478 plasma samples were initially reactive but not repeat reactive by influenza TMA. Of blood donors reporting ILI symptoms postdonation, 1 of 1004 samples was TMA initially reactive but not repeat reactive; all samples were nonreactive by RT-PCR testing.
Conclusions. Targeting blood donor populations most likely to have influenza infection, we failed to detect influenza RNA in 1482 donor samples, with most tested by 3 different RNA assays. Seasonal influenza does not appear to pose a significant contamination threat to the blood supply.
Among 127 HIV-infected women, the magnitude of HDLc increases after HAART initiation predicted the magnitude of concurrent decreases in inflammation biomarkers. After HAART initiation, changes in LDLc and inflammation were unrelated. In the same population, predicted risk of coronary heart disease based upon levels of standard clinical risk factors was similar before and after HAART treatment. Thus, it remains unknown whether short-term treatment-related changes in standard risk factors may appreciably change risk of CVD.
lipids; HAART; HIV infection; inflammation
West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.
More than a decade after West Nile virus (WNV) entered North America, and despite a significant increase in reported cases during the 2012 and 2013 seasons, no treatment or vaccine for humans is available. Although antiviral T cells contribute to the control of WNV, little is known about their regulation during acute infection. We analyzed the expression of Tim-3 and PD-1, two recently identified T cell negative immune checkpoint receptors, over the course of WNV infection. Symptomatic WNV+ donors exhibited higher frequencies of Tim-3+ cells than asymptomatic subjects within naïve/early differentiated CD28+/–CD57–CD4+ and differentiated CD28–CD57–CD8+ T cells. Our study links Tim-3-expression on T cells during acute WNV infection with the development of symptomatic disease, suggesting Tim-3 and its ligands could be targeted therapeutically to alter anti-WNV immunity and improve disease outcome.
Rates of insulin resistance are increased in HIV-infected patients on stable antiretroviral therapy (ART). Such increase may partially be due to HIV-induced immune dysregulation involving monocytes (MO) and its subsets.
Materials and Methods
Cross-sectional analysis of 141 HIV-infected subjects age ≥ 40 years on stable ART. Homeostatic model assessment–insulin resistance (HOMA-IR) and rates of metabolic syndrome were calculated. Subjects were classified by fasting glucose and oral glucose tolerance test (OGTT) into clinical diabetes categories. Multi-parametric flow cytometry was used to determine MO subset percentages: [classical (CD14++CD16−), intermediate (CD14++CD16+), non-classical (CD14low/+CD16++), and a recently identified fourth (CD14low/+CD16−) ‘transitional’ MO subset] and percentage of activated (CD38+HLA-DR+) CD8 T cells. Absolute levels of cells were calculated using clinical CBC and T cell subset data. Multiple plasma soluble biomarkers were assessed by Luminex technology.
Median age 50 years, CD4 count (percent) 505 cells/µL (29%), and 89% male. Total MO (r = −0.23, p = 0.006) and classical and non-classical MO subsets correlated negatively with CD4 percent. No correlations were seen with CD4 count as absolute values. Log-total MO and log-classical MO predicted HOMA-IR independently of HIV immuno-virologic and diabetes risk factors (β = 0.42, p = 0.02 and β = 0.35, p = 0.02, respectively) and were increased in subjects with metabolic syndrome (p = 0.03 and p = 0.05 respectively). Total and/or subset MO levels correlated with multiple soluble plasma biomarkers including CRP, IL-6, MMP-9, MPO, SAA, SAP and tPAI-1, with tPAI-1 independently predicting HOMA-IR (β = 0.74, p<0.001).
MO levels increase with worsening HIV immune dysregulation as assessed by CD4 percent. CD4 percent may provide additional information about MO and metabolic risk in this population beyond absolute values. MO, and specifically classical MO, may contribute to insulin resistance and metabolic syndrome during chronic HIV infection. Multiple soluble plasma biomarkers including tPAI-1 increase with increase in MO. Levels of tPAI-1 independently predict the development of insulin resistance.
It is thought that HLA antibodies might contribute to the pathogenesis of transfusion-related acute lung injury (TRALI). Many methods exist to detect HLA antibodies, including ELISA, flow cytometry, and multiplex bead-based assays, as well as the older lymphocytotoxicity assay. The sensitivity of each of these testing platforms varies, and it is not obvious how to compare HLA antibody results obtained on different platforms. This issue has become increasingly important as some blood centers have begun HLA antibody apheresis donor screening as a TRALI risk-reduction measure.
STUDY DESIGN AND METHODS
525 serum samples were selected from 7,841 donors in the Leukocyte Antibody Prevalence Study (LAPS) repository based on risk for the development of HLA antibodies, using the number of pregnancies as the risk factor. There were 81 males, and the pregnancy history of the 444 female donors was 0 (n=187), 1 (n=67), or 2+ pregnancies (n=190). Replicate frozen serum aliquots were sent to four different assay manufacturers blinded for HLA Ab detection using five different assays.
Among the assays with different manufacturer designated cutoffs, the flow cytometry and multiplex bead based-assays (Luminex) typically resulted in a larger proportion of HLA Ab positive samples compared with ELISA based assays. Capitalizing upon having quantitative results from five different assays on the same group of samples, latent variable analysis was used to derive a new set of cutoffs. These cutoffs, termed consensus cutoffs, yielded similar sensitivities across test platforms, thereby increasing concordance amongst assays. Assay agreement was higher in ever pregnant females than in males and never pregnant females.
Different assays resulted in varied positivity rates when the manufacturer’s suggested cutoffs were used, demonstrating that care needs to be taken when comparing clinical outcomes data generated using different HLA antibody assays and testing platforms. The method used here, involving latent variable analysis, presents one possible approach to calculating comparable cutoffs that result in broad agreement across assays with respect to positivity designation.
We examined serum lipids in association with carotid artery intima-media thickness (CIMT) in HIV-infected and HIV-uninfected women.
In 2003–4, among 1827 Women’s Interagency HIV Study participants, we measured CIMT and lipids (high-density lipoprotein cholesterol [HDL-c], low-density lipoprotein cholesterol [LDL-c], total cholesterol [TC], non-HDL-c). A subset of 520 treated HIV-infected women had pre-1997 lipid measures. We used multivariable linear regression to examine associations between lipids and CIMT.
In HIV-uninfected women, higher TC, LDL-c and non-HDL-c were associated with increased CIMT. Among HIV-infected women, associations of lipids with CIMT were observed in treated but not untreated women. Among the HIV-infected women treated in 2003–4, CIMT was associated both with lipids measured a decade earlier in infection, and with late lipid measurements.
Among HIV-infected women, hyperlipidemia is most strongly associated with subclinical atherosclerosis in treated women. Among treated women, the association appeared strongest early in the disease course.
cardiovascular diseases; carotid arteries; HAART; HIV; lipids
Trauma and transfusion can both alter immunity, and while transfusions are common among traumatically injured patients, few studies have examined their combined effects on immunity.
STUDY DESIGN AND METHODS
We tracked the plasma levels of 41 immunomodulatory proteins in 56 trauma patients from time of injury up to 1 year later. In addition, a murine model was developed to distinguish between the effects of transfusion and underlying injury and blood loss.
Thirty-one of the proteins had a statistically significant change over time after traumatic injury, with a mixed early response that was predominantly anti-inflammatory followed by a later increase in proteins involved in wound healing and homeostasis. Results from the murine model revealed similar cytokine responses to humans. In mice, trauma/hemorrhage caused early perturbations in a number of the pro- and anti-inflammatory mediators measured, and transfusion blunted early elevations in IL-6, IL-10, MMP-9, and IFN-γ. Transfusion caused or exacerbated changes in MCP-1, IL-1α, IL-5, IL-15, and soluble E-selectin. Finally, trauma/hemorrhage alone increased KC and IL-13.
This work provides a detailed characterization of the major shift in the immunological environment in response to trauma and transfusion and clarifies which immune mediators are affected by trauma/hemorrhage and which by transfusion.
Allogeneic Transfusion; Trauma/Hemorrhage; Mouse model; Cytokines; Immune Dysregulation
HLA antibodies are a possible cause of transfusion-related acute lung injury (TRALI), and fluorescent bead assays are often used for antibody detection. Serum is the manufacturer’s recommended sample, but plasma may be easier to obtain for studies of HLA antibody prevalence and TRALI case investigations.
STUDY DESIGN AND METHODS
Specimens were obtained from 44 multiparous females positive for HLA antibodies by lymphocytotoxicity testing at least 13 years prior, and from 1,000 contemporary blood donors. Screening tests were performed using a Luminex-based assay. In addition to comparing results obtained with paired plasma and serum samples, the effects of storage at 4 °C for one week and of multiple freeze-thaw cycles were evaluated.
Of 42 evaluable subjects with HLA antibodies documented >13 years earlier, only 1 showed loss of detectable antibodies, with 39 (93%) positive in the screening assay for class I and 24 (57%) positive in the screening assay for HLA class II antibodies. In 968 evaluable contemporary donors, 291 screened positive for HLA class I and 206 for HLA class II antibodies using a low assay cut-off. Screening test concordance using paired plasma and serum samples was high, particularly for subjects with higher level antibodies. Refrigeration of samples for one week did not significantly affect assay results, while repeated freeze-thaw cycles caused a decrement in signal level.
Serum and plasma samples gave concordant results in the majority of cases, particularly for specimens with higher-level antibodies. High-level HLA antibodies were present in most individuals for over 13 years.
Chronic infection by HIV increases the risk of cardiovascular disease (CVD) despite effective antiretroviral therapy (ART). The mechanisms linking HIV to CVD have yet to be fully elucidated. High plasma levels of the pro-inflammatory cytokine IL-6, which may be triggered by IL-1β, is a biomarker of CVD risk in HIV-negative adults, and of all-cause mortality in HIV disease. Monocytes play a pivotal role in atherosclerosis, and may be major mediators of HIV-associated inflammation. We therefore hypothesized that monocytes from HIV-infected adults would display high inflammatory responses. Employing a 10-color flow cytometry intracellular cytokine staining assay, we directly assessed cytokine and chemokine responses of monocytes from the cryopreserved peripheral blood of 33 chronically HIV-1 infected subjects. Participants were 45 years or older, on virologically suppressive ART and at risk for CVD. This group was compared to 14 HIV-negative subjects matched for age and gender, with similar CVD risk. We simultaneously detected intracellular expression of IL-1β, IL-6, IL-8 and TNF in blood monocytes in the basal state and after stimulation by triggers commonly found in the blood of treated, chronically HIV-infected subjects: lipopolysaccharide (LPS) and oxidized low-density lipoprotein (oxLDL). In the absence of stimulation, monocytes from treated HIV-infected subjects displayed a high frequency of cells producing IL-1β (median 19.5%), compared to low levels in HIV-uninfected persons (0.9% p<0.0001). IL-8, which is induced by IL-1β, was also highly expressed in the HIV-infected group in the absence of stimulation, 43.7% compared to 1.9% in HIV-uninfected subjects, p<0.0001. Strikingly, high basal expression of IL-1β by monocytes predicted high IL-6 levels in the plasma, and high monocyte IL-6 responses in HIV-infected subjects. Hyper-inflammatory IL-1β enriched monocytes may be a major source of IL-6 production and systemic inflammation in HIV-infected adults, and may contribute to the risk for all-cause mortality and cardiovascular disease in treated HIV infection.
Inflammation and hemostasis perturbation may be involved in vascular complications of HIV infection. We examined atherogenic biomarkers and subclinical atherosclerosis in HIV-infected adults before and after beginning highly-active antiretroviral therapy (HAART).
In the Women's Interagency HIV Study (WIHS), 127 HIV-infected women studied pre- and post-HAART were matched to HIV-uninfected controls. Six semi-annual measurements of soluble CD14, tumor necrosis factor (TNF)-alpha, soluble interleukin (IL)-2 receptor, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, D-dimer, and fibrinogen were obtained. Carotid artery intima-media thickness (CIMT) was measured by B-mode ultrasound.
Relative to HIV-uninfected controls, HAART-naïve HIV-infected women had elevated levels of soluble CD14 (1945 vs 1662 ng/mL, Wilcoxon signed rank P<0.0001), TNF-alpha (6.3 vs 3.4 pg/mL, P<0.0001), soluble IL-2 receptor (1587 vs 949 pg/mL, P<0.0001), IL-10 (3.3 vs 1.9 pg/mL, P<0.0001), MCP-1 (190 vs 163 pg/mL, P<0.0001) and D-dimer (0.43 vs 0.31 µg/mL, P<0.01). Elevated biomarker levels declined after HAART. While most biomarkers normalized to HIV-uninfected levels, in women on effective HAART, TNF-alpha levels remained elevated compared to HIV-uninfected women (+0.8 pg/mL, P=0.0002). Higher post-HAART levels of soluble IL-2 receptor (P=0.02), IL-6 (P=0.05), and D-dimer (P=0.03) were associated with increased CIMT.
Untreated HIV infection is associated with abnormal hemostasis (e.g., D-dimer), and pro-atherogenic (e.g., TNF-alpha) and anti-atherogenic (e.g., IL-10) inflammatory markers. HAART reduces most inflammatory mediators to HIV-uninfected levels. Increased inflammation and hemostasis are associated with subclinical atherosclerosis in recently treated women. These findings have potential implications for long-term risk of cardiovascular disease in HIV-infected patients, even with effective therapy.
antiretroviral therapy; cardiovascular diseases; cytokines; hemostasis; HIV; inflammation
From early in the HIV epidemic it was appreciated that many inflammatory markers such as neopterin and TNF-α were elevated in patients with AIDS. With the advent of modern technology able to measure a broad array of cytokines, we now know that from the earliest points of infection HIV induces a cytokine storm. This review will focus on how cytokines are disturbed in HIV infection and will explore potential therapeutic uses of cytokines. These factors can be used directly as therapy during HIV infection, either to suppress viral replication or prevent deleterious immune effects of infection, such as CD4+ T cell depletion. Cytokines also show great promise as adjuvants in the development of HIV vaccines, which would be critical for the eventual control of the epidemic.
HIV; cytokine; chemokine; immune activation; vaccine
Prior to the identification of hepatitis C virus (HCV), transfusion-transmission was common. Viral transmission in subjects with a known date of infection allows the study of the immune responses to acute HCV infection. We analysed 39 soluble immune factors in serum samples from subjects with transfusion-transmitted HCV. Dynamic expression kinetics of interferon gamma-induced protein 10 (IP-10), tumour necrosis factor-alpha and interleukin (IL)-10 were observed during acute HCV infection. Serum IP-10 was the only analyte that was significantly elevated in HCV resolvers compared with uninfected controls. In individuals who progressed to chronic HCV elevated levels of IP-10 and IL-10 coincided with first significant alanine aminotransferase elevation and remained elevated during the first year of acute HCV infection. In addition to monitoring lack of reduction in viral load, serum levels of IP-10 and IL-10 expression during acute HCV infection may be useful biomarkers to predict the progress to chronic HCV.
The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.
Recent-infection testing assays/algorithms (RITAs) have been developed to exploit the titer and avidity of HIV antibody evolution following seroconversion for incidence estimation. The Vitros Anti-HIV 1+2 assay (Ortho-Clinical Diagnostics) was approved by the FDA to detect HIV-1 and HIV-2 infections. We developed a less-sensitive (LS) and an avidity-modified version of this assay to detect recent HIV infection. Seroconversion panels (80 subjects, 416 samples) were tested to calculate the mean duration of recent infection (MDR) for these assays. A panel from known long-term (2+ years) HIV-infected subjects on highly active antiretroviral therapy (HAART) (n = 134) and subjects with low CD4 counts (AIDS patients [n = 140]) was used to measure the false-recent rate (FRR) of the assays. Using a signal-to-cutoff ratio of 20 and the LS-Vitros assay gave a RITA MDR of 215 days (95% confidence interval [95% CI], ±65 days) and using an avidity index (AI) of 0.6 gave an MDR of 170 days (±44 days), while a combination of the two assays yielded a MDR of 146 days (±38.6) and an FRR of 8%. Misclassifying subjects with known long-term infection as recently infected occurred in 14% of AIDS patients and 29% (95% CI, 22, 38) of HAART subjects and 3% (95% CI, 0.8, 7.2) and 42% (95% CI, 33, 51), respectively, for the LS- and avidity-modified Vitros assays, with a misclassification rate of 15% (95% CI, 11, 20) overall using a dual-assay algorithm. Both modified Vitros assays can be used to estimate the length of time since seroconversion and in calculations for HIV incidence. Like other RITAs, they are subject to high FRR in subjects on HAART or with AIDS.
Despite growing evidence that HIV-1-specific CD4+ T helper (Th) cells may play a role in the control of viremia, discrete Th cell epitopes remain poorly defined. Furthermore, it is not known whether Th cell responses generated using vaccines based on clade B virus sequences will elicit immune responses that are effective in regions of the world where non-clade B viruses predominate. To address these issues we isolated CD4+ T cell clones from individuals with vigorous HIV-1-specific Th cell responses and identified the minimum epitopes recognized. The minimum peptide length required for induction of CD4+ T cell proliferation, IFN-γ secretion, and cytolytic activity ranged from 9 to 16 amino acids in the five epitopes studied. Cross-clade recognition of the defined epitopes was examined for variant peptides from clades A, B, C, D, and AE. Over half the variant epitopes (17 of 32) exhibited impaired recognition, defined as less than 50% of the IFN-γ secretion elicited by B clade consensus sequence. There was no evidence for antagonistic activity mediated by the variant peptides, and despite strong responses there was no escape of autologous virus from Th responses in the epitopes we studied. Abrogated recognition of variant CD4+ T cell epitopes presents a potential obstacle to vaccine development.
Perturbations of plasma IL-10, IL-12, IL-13, TNF-α, and IFN-γ were measured longitudinally in HIV-1 se-roconverting plasma donors and were compared to subjects with symptomatic primary HIV-1 infection. Control groups included uninfected patients with symptoms and risks for primary HIV-1, healthy controls, and asymptomatic plasma donors with primary HCV. IL-10, TNF-α, and IFN-γ rapidly rose in acute HIV-1 infection, while IL-13 predominated in acute HCV. Subjects with symptomatic primary HIV-1 had higher cytokine levels than asymptomatic subjects, statistically significant for TNF-α. Cytokine alterations occurred within 7 days of detectable HIV-1 viremia, emphasizing the need to study the earliest events of infection.
The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.
We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.
We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.
These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.
Transfusion related acute lung injury (TRALI) has been associated with both HLA and HNA antibodies. HNA antibody frequency, specificity, and demographic associations have not been well defined in the blood donor population.
A subset of 1171 donors (388 non-transfused males, 390 HLA antibody negative females with three or more pregnancies, and 393 HLA antibody positive females with three or more pregnancies) from a larger leukocyte antibody prevalence study (LAPS) was tested for IgG and IgM HNA antibody using a granulocyte immunofluorescence flow cytometry assay. Additional testing on selected samples included monoclonal antibody immobilization of granulocyte antigen – flow cytometry and granulocyte genotyping.
Eight samples were HNA antibody positive (prevalence 0.7% [95% CI, 0.3 - 1.3%]). Three HNA antibodies (one IgG and two IgM) were found in non-transfused males (prevalence 0.8% [95% CI, 0.2 - 2.2%]); all were pan-reactive or non-specific. One HLA antibody negative previously pregnant female had an IgG HNA antibody with HNA-1a specificity (prevalence 0.3% [95% CI, 0.01-1.4%]). Four HLA antibody positive previously pregnant females demonstrated HNA antibodies, three IgG and one IgM (prevalence 1% [95% CI, 0.3 - 2.6%]). Two of these were HNA-1a specific, one HNA-4a specific, and one non-specific.
HNA antibodies occur with low frequency in the donor population and are present in both male and female donors. Despite the implementation of TRALI reduction strategies, HNA antibodies are still present in donor blood products. Though our data do not create a case for urgent implementation of donor HNA antibody testing, future new developments for high throughput HNA antibody screening, including for HNA-3a, may warrant reconsideration.