Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control

Elite controllers (ECs) represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC) that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune–recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.     

Circulating Mucosal Associated Invariant T Cells Are Activated in Vibrio cholerae O1 Infection and Associated with Lipopolysaccharide Antibody Responses

Background

Mucosal Associated Invariant T (MAIT) cells are innate-like T cells found in abundance in the intestinal mucosa, and are thought to play a role in bridging the innate-adaptive interface.

Methods

We measured MAIT cell frequencies and antibody responses in blood from patients presenting with culture-confirmed severe cholera to a hospital in Dhaka, Bangladesh at days 2, 7, 30, and 90 of illness.

Results

We found that MAIT (CD3⁺CD4⁻CD161^hiVα7.2⁺) cells were maximally activated at day 7 after onset of cholera. In adult patients, MAIT frequencies did not change over time, whereas in child patients, MAITs were significantly decreased at day 7, and this decrease persisted to day 90. Fold changes in MAIT frequency correlated with increases in LPS IgA and IgG, but not LPS IgM nor antibody responses to cholera toxin B subunit.

Conclusions

In the acute phase of cholera, MAIT cells are activated, depleted from the periphery, and as part of the innate response against V. cholerae infection, are possibly involved in mechanisms underlying class switching of antibody responses to T cell-independent antigens.     

Follow-Up Study of Hypervariable Region Sequences of the Hepatitis C Virus (HCV) Genome in an Infant with Delayed Anti-HCV Antibody Responses

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti-HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti-HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.     

ITPA Polymorphisms Are Associated with Hematological Side Effects during Antiviral Therapy for Chronic HCV Infection

Background/ObjectiveGenetic polymorphisms in the inosine triphosphatase (ITPA) gene have been associated with the protection from early ribavirin(RBV)-induced hemolytic anemia among patients with chronic hepatitis C virus (HCV) infection. The aim of the present study was to investigate the association between the functional ITPA variants and hematological side effects during antiviral therapy with pegylated interferon (PegIFN) and RBV.ResultsIn total, 213 patients were included. The predicted ITPase activity was normal among 152 (71%) patients; 61 (29%) patients had ITPase deficiency. By multivariable linear regression, RBV dose in mg per kilogram (Beta 0.09, 95%CI 0.04–0.13, p<0.001) and normal ITPase activity (Beta 0.89, 95%CI 0.64–1.14, p<0.001) were associated with more Hb decline at week 4 of treatment. Patients with normal ITPase activity underwent more dose adjustments of RBV than patients with ITPase deficiency (19(13%) vs 1(2%),p = 0.014) and received erythropoietin more frequently (12 (8%) vs 0 (0%),p = 0.024).ConclusionGenetic variants in the ITPA gene protected against RBV treatment-induced anemia among Caucasian patients with chronic HCV infection. Patients with normal ITPase activity underwent more dose reductions of RBV and received erythropoietin more frequently.     

A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non-Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second-Generation Test

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non-fused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.     

The Broad Neutralizing Antibody Responses after HIV-1 Superinfection Are Not Dominated by Antibodies Directed to Epitopes Common in Single Infection

HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women’s Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.     

Genetic Variations in ADIPOQ Gene Are Associated with Chronic Obstructive Pulmonary Disease

Background

Adiponectin is reported to be related to the development of chronic obstructive pulmonary disease (COPD). Genetic variants in the gene encoding adiponectin (ADIPOQ) have been reported to be associated with adiponectin level in several genome–wide linkage and association studies. However, relatively little is known about the effects of ADIPOQ gene variants on COPD susceptibility. We determined the frequencies of single-nucleotide polymorphisms (SNPs) in ADIPOQ in a Chinese Han population and their possible association with COPD susceptibility.

Methods

We conducted a case–control study of 279 COPD patients and 367 age- and gender-distribution-matched control subjects. Seven tagging SNPs in ADIPOQ, including rs710445, rs16861205, rs822396, rs7627128, rs1501299, rs3821799 and rs1063537 were genotyped by SNaPshot. Association analysis of genotypes/alleles and haplotypes constructed from these loci with COPD was conducted under different genetic models.

Results

The alleles or genotypes of rs1501299 distributed significantly differently in COPD patients and controls (allele: P = 0.002, OR = 1.43 and 95%CI = 1.14–1.79; genotype: P = 0.008). The allele A at rs1501299 was potentially associated with an increased risk of COPD in all dominant model analysis (P = 0.009; OR: 1.54; 95%CI: 1.11–2.13), recessive model analyses (P = 0.015; OR: 1.75; 95% CI: 1.11–2.75) and additive model analyses (P = 0.003; OR: 2.11; 95% CI: 1.29–3.47). In haplotype analysis, we observed haplotypes AAAAACT and GGACCTC had protective effects, while haplotypes AGAACTC, AGGCCTC, GGAACTC, GGACACT and GGGCCTC were significantly associated with the increased risk of COPD.

Conclusions

We conducted the first investigation of the association between the SNPs in ADIPOQ and COPD risk. Our current findings suggest that ADIPOQ may be a potential risk gene for COPD. Further studies in larger groups are warranted to confirm our results.     

Two Distinct Chronic Obstructive Pulmonary Disease (COPD) Phenotypes Are Associated with High Risk of Mortality

Rationale

In COPD patients, mortality risk is influenced by age, severity of respiratory disease, and comorbidities. With an unbiased statistical approach we sought to identify clusters of COPD patients and to examine their mortality risk.

Methods

Stable COPD subjects (n = 527) were classified using hierarchical cluster analysis of clinical, functional and imaging data. The relevance of this classification was validated using prospective follow-up of mortality.

Results

The most relevant patient classification was that based on three clusters (phenotypes). Phenotype 1 included subjects at very low risk of mortality, who had mild respiratory disease and low rates of comorbidities. Phenotype 2 and 3 were at high risk of mortality. Phenotype 2 included younger subjects with severe airflow limitation, emphysema and hyperinflation, low body mass index, and low rates of cardiovascular comorbidities. Phenotype 3 included older subjects with less severe respiratory disease, but higher rates of obesity and cardiovascular comorbidities. Mortality was associated with the severity of airflow limitation in Phenotype 2 but not in Phenotype 3 subjects, and subjects in Phenotype 2 died at younger age.

Conclusions

We identified three COPD phenotypes, including two phenotypes with high risk of mortality. Subjects within these phenotypes may require different therapeutic interventions to improve their outcome.     

Dengue Virus Neutralizing Antibody Levels Associated with Protection from Infection in Thai Cluster Studies

Background

Long-term homologous and temporary heterologous protection from dengue virus (DENV) infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs) have not been clearly associated with protection from infection.

Methodology/Principal Findings

Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004–2007), cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009–2012), clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age ≥6 months were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been “susceptible” on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered “non-susceptible.” Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT). Seventeen “susceptible” (six DENV-1, five DENV-2, and six DENV-4), and 32 “non-susceptible” (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4) subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC) curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate “susceptible” from “non-susceptible” subjects.

Conclusions/Significance

PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant for both dengue epidemiology studies and vaccine development efforts.     

High,Broad, Polyfunctional,and Durable T Cell Immune Responses Induced in Mice by a Novel Hepatitis C Virus (HCV) Vaccine Candidate (MVA-HCV) Based on Modified Vaccinia Virus Ankara Expressing the Nearly Full-Length HCV Genome

A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4⁺ T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8⁺ T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8⁺ T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8⁺ T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV.     

Leukotrienes Are Upregulated and Associated with Human T-Lymphotropic Virus Type 1 (HTLV-1)-Associated Neuroinflammatory Disease

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB₄ and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB₄ and LTC₄ positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4⁺ and CD8⁺ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.     

Serum IgG Antibody Levels to Periodontal Microbiota Are Associated with Incident Alzheimer Disease

Background

Periodontitis and Alzheimer disease (AD) are associated with systemic inflammation. This research studied serum IgG to periodontal microbiota as possible predictors of incident AD.

Methods

Using a case-cohort study design, 219 subjects (110 incident AD cases and 109 controls without incident cognitive impairment at last follow-up), matched on race-ethnicity, were drawn from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a cohort of longitudinally followed northern Manhattan residents aged >65 years. Mean follow-up was five years (SD 2.6). In baseline sera, serum IgG levels were determined for bacteria known to be positively or negatively associated with periodontitis (Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans Y4, Treponema denticola, Campylobacter rectus, Eubacterium nodatum, and Actinomyces naeslundii genospecies-2). In all analyses, we used antibody threshold levels shown to correlate with presence of moderate-severe periodontitis.

Results

Mean age was 72 years (SD 6.9) for controls, and 79 years (SD 4.6) for cases (p<0.001). Non-Hispanic Whites comprised 26%, non-Hispanic Blacks 27%, and Hispanics 48% of the sample. In a model adjusting for baseline age, sex, education, diabetes mellitus, hypertension, smoking, prior history of stroke, and apolipoprotein E genotype, high anti-A. naeslundii titer (>640 ng/ml, present in 10% of subjects) was associated with increased risk of AD (HR = 2.0, 95%CI: 1.1–3.8). This association was stronger after adjusting for other significant titers (HR = 3.1, 95%CI: 1.5–6.4). In this model, high anti-E. nodatum IgG (>1755 ng/ml; 19% of subjects) was associated with lower risk of AD (HR = 0.5, 95%CI: 0.2–0.9).

Conclusions

Serum IgG levels to common periodontal microbiota are associated with risk for developing incident AD.     

Cirrhosis,Liver Transplantation and HIV Infection Are Risk Factors Associated with Hepatitis E Virus Infection

Background

Acute and chronic hepatitis E have been associated with high mortality and development of cirrhosis, particularly in solid-organ recipients and patients infected by human immunodeficiency virus. However, data regarding the epidemiology of hepatitis E in special populations is still limited.

Aims

Investigate seroprevalence and possible factors associated with HEV infection in a large cohort of immunosuppressed patients.

Methods

Cross-sectional study testing IgG anti-HEV in serum samples from 1373 consecutive individuals: 332 liver-transplant, 296 kidney-transplant, 6 dual organ recipients, 301 non-transplanted patients with chronic liver disease, 238 HIV-infected patients and 200 healthy controls.

Results

IgG anti-HEV was detected in 3.5% controls, 3.7% kidney recipients, 7.4% liver transplant without cirrhosis and 32.1% patients who developed post-transplant cirrhosis (p<0.01). In patients with chronic liver disease, IgG anti-HEV was also statistically higher in those with liver cirrhosis (2% vs 17.5%, p<0.01). HIV-infected patients showed an IgG anti-HEV rate of 9.2%, higher than those patients without HIV infection (p<0.03). Multivariate analysis showed that the factors independently associated with anti-HEV detection were liver cirrhosis, liver transplantation and HIV infection (OR: 7.6, 3.1 and 2.4). HCV infection was a protective factor for HEV infection (OR: 0.4).

Conclusions

HEV seroprevalence was high in liver transplant recipients, particularly those with liver cirrhosis. The difference in anti-HEV prevalence between Liver and Kidney transplanted cases suggests an association with advanced liver disease. Further research is needed to ascertain whether cirrhosis is a predisposing factor for HEV infection or whether HEV infection may play a role in the pathogeneses of cirrhosis.     

Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice

Background

With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.

Methodology/Principal Findings

A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.

Conclusions/Significance

In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.     

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer,Productive Infection,and Replication and Their Requirement for HCV Receptors

Hepatitis C virus (HCV) infection is believed to begin with interactions between cell-free HCV and cell receptors that include CD81, scavenger receptor B1 (SR-B1), claudin-1 (CLDN1), and occludin (OCLN). In this study, we have demonstrated that HCV spreading from infected hepatocytes to uninfected hepatocytes leads to the transfer of HCV and the formation of infection foci and is cell density dependent. This cell-cell contact-mediated (CCCM) HCV transfer occurs readily and requires all these known HCV receptors and an intact actin cytoskeleton. With a fluorescently labeled replication-competent HCV system, the CCCM transfer process was further dissected by live-cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and posttransfer. Importantly, the CCCM HCV transfer leads to productive infection of target cells. Taken together, these results show that CCCM HCV transfer constitutes an important and effective route for HCV infection and dissemination. These findings will aid in the development of new and novel strategies for preventing and treating HCV infection.     

Hepatitis C Virus (HCV) Infection May Elicit Neutralizing Antibodies Targeting Epitopes Conserved in All Viral Genotypes

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.