Micronutrient and Nutritional Status of HIV-Exposed and HIV-Unexposed Malawian Infants in the First Year of Life: Assessment of Ferritin, Vitamin A, and D Status and Its Association with Growth.

Breastfed Malawian infants from Human Immunodeficiency Virus (HIV)-uninfected and HIV-infected women who received antiretroviral therapy were followed until 12 months of age, allowing us to evaluate plasma levels of ferritin, vitamin A (as retinol-binding protein, RBP), and vitamin D (25(OH)D) at six months, as well as nutritional status and growth between six and 12 months. Ferritin and RBP levels were adjusted for inflammation. The study included 88 infants, 63 of whom were part of a recent cohort (2019-2021) that included 49 HIV-exposed but uninfected (HEU) and 14 HIV-unexposed and uninfected (HUU) infants, as well as 25 infants (all HEU) from an earlier cohort (2008-2011). No differences were observed between HEU and HUU infants regarding micronutrient levels, anthropometric indexes, growth, and rates of stunting, being underweight, or wasting. HEU infants from the earlier cohort, when compared to more recent HEU infants, had significantly worse anthropometric measures at six months and inferior growth between six and twelve months. Overall, ferritin deficiency involved 68.6% of infants, while vitamin A and vitamin D deficiency involved 8% and 1.2% of infants, respectively. Micronutrient deficiencies were not associated with HIV exposure, cohort, stunting, being underweight, or wasting. At six months, stunting, being underweight, and wasting involved 25.0%, 2.7% and 2.8% of infants, respectively, with no differences related to HIV exposure. Ferritin deficiency at six months was associated with inferior subsequent growth. In this small observational study conducted in Malawian infants, no major nutritional gap was observed between HIV-exposed and HIV-unexposed infants, though the study highlighted specific nutritional deficiencies that deserve attention. High rates of stunting and ferritin deficiency were observed in the first year of life in Malawian infants, irrespective of maternal HIV status; a significant association between ferritin deficiency and worse subsequent growth was found. Vitamin A and vitamin D deficiencies were much less frequent. Based on the data observed, nutritional interventions should give priority to the correction of ferritin deficiency and chronic undernutrition.

HIV-exposed infants with EBV infection have a reduced persistence of the immune response to the HBV vaccine.

BACKGROUND: In sub-Saharan African countries Epstein Barr virus (EBV) infection occurs in early childhood. We aim to investigate the factors associated with EBV acquisition and the impact of EBV infection on the humoral response to HBV vaccination in infants born from HIV-positive, antiretroviral-treated mothers in Malawi. METHODS: A total of 149 HIV-exposed infants were included in this longitudinal study. EBV anti-VCA IgG were measured using an ELISA assay. The EBV seroconversion was correlated with the maternal viro-immunological conditions, with infant growth and immunological vulnerability, and with the humoral response to the HBV vaccine. RESULTS: No infant was EBV-positive at 6 months (n. 52 tested). More than a third of infants (49/115 or 42.6 %) on study beyond 6 months seroconverted at 12 months. At 24 months, out of 66 tested infants, only 13 remained EBV-uninfected, while 53 (80.3 %) acquired EBV infection, rising the total proportion of EBV seroconversion to 88.7 % (102/115 infants). EBV seroconversion was significantly associated with a low maternal educational status but had no impact on infant growth or vulnerability to infections. Reduced HBsAb levels and accelerated waning of antibodies were associated with early EBV seroconversion. CONCLUSIONS: We found a heterogeneous timing of acquisition of EBV with the majority of infants born from HIV + mothers acquiring infection after 6 months. Anti-HBs levels were lower and appeared to wane faster in infants acquiring EBV infection.

Dried blood spots for the quantitative evaluation of IgG isotypes and correlation with serum samples in HIV-exposed uninfected (HEU) infants.

BACKGROUND: The determination of IgG levels and their subclasses can provide clinically relevant information on the status of the immune system. Here we determined the sensitivity and reproducibility of the quantification of IgG subclasses from Dried Blood Spots (DBS) in Malawian uninfected infants exposed to HIV (HEU). METHODS: Sixty paired samples of serum and DBS from HEU infants were used. Samples were collected from 1, 6, and 24-month old infants. IgGs concentrations from both serum and DBS were analyzed by BN ProSpec Siemens assay, using a different setting for sample dilutions. The reproducibility of the DBS method was tested on 10 samples run twice, starting from the DBS extraction process. To assess the systematic, proportional, and random differences, we computed the Passing-Bablok regression, and the Bland-Altman analysis to estimate the total mean bias between the two tests. RESULTS: The IgG isotypes concentrations from serum and DBS showed significant differences in all the comparisons. Generally, the DBS method underestimated IgG subclasses' values showing a recovery range between 51.2% and 77.6%. Passing Bablok regression on age-based groups showed agreement for IgG, IgG1, and IgG2, but not for IgG3 and IgG4. The mean bias obtained with the Bland Altman test varied largely depending on IgG isotypes (-0.02-2.21 g/l) Coefficient of variation <7.0% was found in the repeated tests for IgG, IgG1, IgG3, and IgG4, while it was 12.4% for IgG2. CONCLUSIONS: Varying degrees of differences were seen in the IgGs measurement in the two different matrices. In IgGs analysis, the DBS method offers promise for population-based research, but the results should be carefully evaluated and considered as a relative value since they are not equivalent to the serum concentrations.

Response to the conjugate pneumococcal vaccine (PCV13) in patients with chronic lymphocytic leukemia (CLL).

Pneumococcal (PC) vaccination is recommended for patients with chronic lymphocytic leukemia (CLL). However, response to vaccines has been investigated in a small series of CLL patients. We analyzed the antibody response and outcomes of 112 CLL patients who received the 13-valent pneumococcal conjugate vaccine (PCV13). An immune response was defined by a twofold increase in the PC-IgG levels assessed by ELISA. The median age of patients was 68 years, 23.2% showed IgG levels ≤ 400 mg/L, 6.3% progressive disease, 52% unmutated IGHV. Twenty-two (19.6%) patients were treatment-naïve and 90 (80.4%) previously treated (40.2% front-line chemoimmunotherapy; ibrutinib first/advanced-line, 9.8%/21.4%; idelalisib advanced-line, 8.9%). Nine (8%) patients developed an immune response, eight treatment-naive, and one on front-line ibrutinib. No responses were observed in patients previously treated with chemoimmunotherapy. Age ≥ 60 years (p = 0.007), IgG levels < 400 mg/L (p < 0.0001), prior treatment (p < 0.0001), and signs of disease progression (p = 0.04) were associated with a lower response rate. Pneumonia-free survival was significantly shorter in patients with clinical signs of progressive disease (HR, 8.39), prior pneumonia (HR, 7.03), and TP53 disruption (HR, 2.91). In conclusion, our results suggest that vaccination should be offered at diagnosis to CLL patients with early stage and stable disease who have better resources for an effective immune response.

Transcriptome Profiling of Human Monocyte-Derived Macrophages Upon CCL2 Neutralization Reveals an Association Between Activation of Innate Immune Pathways and Restriction of HIV-1 Gene Expression.

Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.

Markers of microbial translocation during pregnancy: differences among HIV+ women of African and European provenance.

INTRODUCTION: Microbial translocation (MT) markers are indicators of HIV-related immune activation, but reference values are mostly derived from European or North American populations and could be substantially different in populations living in developing countries. Here we evaluate possible differences in MT markers levels in HIV+ pregnant women of different geographical provenance. METHODOLOGY: This study is nested within an observational study of pregnant women with HIV in Italy. Women were dichotomized on the basis of provenance in two groups of European (n = 14) and African (n = 26) origin. Soluble CD14, lipopolysaccharide-binding protein (LBP) and intestinal-fatty acid binding protein (I-FABP) were measured in plasma samples collected between the first and second trimester of pregnancy. RESULTS: Demographic and viroimmunological characteristics were similar between groups, although European women were more commonly smokers and HCV-coinfected. Irrespective of origin, LBP plasma levels were positively correlated with I-FABP (r = 0.467, p = 0.004) and sCD14 levels (r = 0.312 p = 0.060). Significantly higher levels of sCD14 (1885 vs. 1208 ng/mL, p = 0.005) LBP (28.5 vs. 25.3 µg/mL, p = 0.050) and I-FABP (573.4 vs. 358.2 pg/mL, p = 0.002) were observed in European compared with African women. A multivariable linear regression analysis, adjusted for smoking and HCV coinfection confirmed the association between sCD14 levels and women provenance (p = 0.03). CONCLUSIONS: Our observations indicate significant differences in soluble markers among women of different provenance. In the design and analysis of studies evaluating MT markers, population-specific reference values should be considered.

Microbial translocation and T cell activation are modified by direct-acting antiviral therapy in HCV-infected patients.

BACKGROUND: Microbial translocation from the gut lumen has been involved in the pathogenesis of liver damage in hepatitis C virus (HCV) infection. AIM: To investigate the impact of direct-acting antiviral treatment on microbial translocation and T-cell activation, in patients with hepatitis C-related liver disease. METHODS: We enrolled two groups of HCV-infected patients undergoing direct-acting antiviral treatment: patients with fibrosis ≥F3 according to Metavir (Group ≥F3); patients with hepatitis C recurrence after liver transplantation and Metavir ≥F2 (Group Liver Transplantation + ≥F2). All patients were treated with direct-acting antivirals based on ongoing guidelines. Surrogate biomarkers of microbial translocation (plasma concentrations of soluble-CD14, lipopolysaccharide-binding protein and intestinal fatty acid-binding protein) were evaluated at baseline, at first month, at the end of treatment and 3 months later. T-cell activation was measured by expression of CD38+ HLA-DR at the same time points, only in Group ≥F3. RESULTS: There were 32 patients in Group ≥F3 and 13 in Group LT + ≥F2. At baseline, levels of soluble-CD14 and lipopolysaccharide-binding protein were significantly higher in both groups vs healthy controls. Baseline soluble-CD14 correlated with glutamic-oxalacetic transaminase (r = 0.384, P = 0.009) and glutamic-pyruvic transaminase (r = 0.293, P = 0.05). A significant decrease in plasma levels of surrogate microbial translocation biomarkers was observed during and after treatment in the two groups although values were not normalised. In Group ≥F3, CD38+ HLADR+ T-cell expression was significantly decreased by direct-acting antiviral treatment. Relapsers (9%) showed higher soluble-CD14 levels at baseline. CONCLUSION: Surrogate microbial translocation markers and T cell activation are increased in HCV-infected patients with liver fibrosis and decrease during direct-acting antiviral treatment.

Reduced Plasma Levels of sCD14 and I-FABP in HIV-infected Patients with Mesalazine-treated Ulcerative Colitis.

BACKGROUND: Microbial translocation (MT) is a shared feature of HIV infection and inflammatory bowel disease (IBD). AIMS: This study was conducted to assess the impact of IBD (and particularly ulcerative colitis, UC) on plasma markers of MT and immune activation in HIV+ subjects. METHODS: A cross-sectional study was conducted in 3 groups of patients: HIV+/UC+(group HIV/UC); HIV+/UC- (group HIV); HIV-/UC+(group UC). Plasma levels of soluble CD14 (sCD14), intestinal fatty acid-binding protein (I-FABP), and endotoxin core antibodies (endoCAB) were measured as plasma markers of MT. Inflammation and immune activation were evaluated by measuring plasma levels of IL-6, IL-21, TNF-alpha, and high-sensitivity C-reactive protein (hs-CRP). T- and B-cells subpopulations were characterized by FACS analysis. RESULTS: Seven patients were enrolled in group HIV/UC, 9 in HIV, and 10 in UC. All HIV-positive patients had plasma values of HIV-1 RNA<37 copies/mL for at least 12 months and good immunological recovery. All patients with UC were treated with oral mesalazine. Markers of MT, immune activation, and inflammation were not increased in subjects with HIV/UC. In fact, they had lower levels of I-FABP (p=0.001) and sCD14 (p=0.007) when compared to other patients groups. Positive correlations were found between I-FABP and sCD14 (r=.355, p=0.076). Frequency of T- and B-cell subsets did not differ among groups. CONCLUSIONS: Our results suggest that UC does not worsen MT, inflammation, or immune activation in HIV-infected subjects. The anti-inflammatory activity of chronic mesalazine administration on intestinal mucosa may contribute to this finding.

Drug resistance mutations 18 months after discontinuation of nevirapine-based ART for prevention of mother-to-child transmission of HIV in Malawi.

OBJECTIVES: The objective of this study was to determine the prevalence of drug resistance mutations among HIV-positive women in Malawi 18 months after discontinuing nevirapine-based ART for the prevention of mother-to-child transmission. PATIENTS AND METHODS: HIV-infected antiretroviral-naive (except for single-dose nevirapine) pregnant Malawian women receiving a nevirapine-based triple antiretroviral regimen from Week 25 of gestation until 6 months of breastfeeding were included in this analysis. Drug resistance was assessed in HIV-DNA 24 months post-partum and at baseline (before the initiation of treatment). In patients with resistance, the presence of mutations was also evaluated in the corresponding plasma samples. RESULTS: Seven out of 42 (16.7%) women studied had archived drug resistance at Month 24 [six cases had NNRTI-associated mutations and two cases the M184I mutation]. In four cases, resistance mutations were already present at baseline (all NNRTI mutations). In three cases, there was an emergence of 'new' resistance (also present in the plasma in one case). Of the 35 women without resistance mutations at Month 24, only one subject had resistance mutations at baseline. Baseline resistance was significantly more common among women with mutations at 24 months compared with those harbouring a WT virus (4/7 versus 1/35, P < 0.001). CONCLUSIONS: Among women who had discontinued drugs 6 months post-partum, only 3/42 (7.1%) had accumulated new resistance mutations in HIV-DNA 2 years after delivery. These findings are reassuring in terms of the safety of the Option B strategy for the prevention of HIV mother-to-child transmission.

Evaluation of HIV-1 integrase inhibitors on human primary macrophages using a luciferase-based single-cycle phenotypic assay.

Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients.