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.

Antibody Response to the SARS-CoV-2 Vaccine and COVID-19 Vulnerability during the Omicron Pandemic in Patients with CLL: Two-Year Follow-Up of a Multicenter Study.

High morbidity and mortality due to COVID-19 were described in the pre-vaccination era in patients with chronic lymphocytic leukemia (CLL). To evaluate COVID-19 morbidity after the SARS-CoV-2 vaccine, we carried out a prospective study in 200 CLL patients. The median age of patients was 70 years; 35% showed IgG levels ≤ 550 mg/dL, 61% unmutated IGHV, and 34% showed TP53 disruption. Most patients, 83.5%, were previously treated, including 36% with ibrutinib and 37.5% with venetoclax. The serologic response rates to the second and third dose of the vaccine were 39% and 53%, respectively. With a median follow-up of 23.4 months, 41% of patients experienced COVID-19, 36.5% during the Omicron pandemic, and 10% had subsequent COVID-19 events. Severe COVID-19 requiring hospitalization was recorded in 26% of patients, and 4% died. Significant and independent factors associated with the response to the vaccine and vulnerability to COVID-19 were age (OR: 0.93; HR: 0.97) and less than 18 months between the start of targeted agents and vaccine (OR: 0.17; HR: 0.31). TP53 mutation and ≥two prior treatments also emerged as significant and independent factors associated with an increased risk of developing COVID-19 (HR: 1.85; HR: 2.08). No statistical difference in COVID-19 morbidity was found in patients with or without antibody response to the vaccine (47.5% vs. 52.5%; p = 0.21). Given the persistent risk of infection due to the continuous emergence of SARS-CoV-2 variants, our results support the importance of new vaccines and protective measures to prevent and mitigate COVID-19 in CLL patients.

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.

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.

IgG abnormalities in HIV-positive Malawian women initiating antiretroviral therapy during pregnancy persist after 24 months of treatment.

OBJECTIVES: Hypergammaglobulinemia and anomalies in the IgG subclass distribution are common in HIV-infected individuals and persist even after many years of antiretroviral therapy (ART). The aim of this study was to investigate the IgG profile and dynamics in pregnant HIV-infected Malawian women in the Option B era. METHODS: Thirty-seven treatment-naive women received ART from the third trimester of pregnancy to 6 months post delivery (end of the breastfeeding period). ART continuation (group C) or interruption (group I) was then decided on the basis of the CD4+ cell count at enrolment (>350 or ≤350/µl). Total IgG and IgG subclasses were determined in maternal serum using a nephelometric assay at baseline and at 6 and 24 months postpartum. RESULTS: At enrolment, 36/37 women had IgG levels >15g/l and there was a predominance of the IgG1 isotype (more than 90%) in parallel with underrepresentation of IgG2 (5.0%). After 6 months of ART, both groups showed a significant median decrease in total IgG (-3.1g/l in group I, -3.5g/l in group C) and in IgG1 (-4.0g/l and -3.6g/l, respectively), but only a modest recovery in IgG2 levels (+0.16 in group I, +0.14g/l in group C). At month 24, hypergammaglobulinemia was still present in 73.7% of women in group C, although a significant reduction was observed in total IgG level and in IgG1 and IgG3 subclasses (p<0.0001 in all cases). IgG2 levels did not show any significant change. In group I at 24 months, total IgG and IgG subclasses had returned to levels comparable to those at baseline. CONCLUSIONS: The beneficial effects of 24 months of ART appear to be limited in the B-cell compartment, with an incomplete reduction of total IgG levels and no recovery of IgG2 depletion. A short ART period did not have significant effects on IgG abnormalities in women who interrupted treatment.

Levels of bone markers in a population of infants exposed in utero and during breastfeeding to tenofovir within an Option B+ programme in Malawi.

OBJECTIVES: No data are available on bone metabolism in infants exposed to tenofovir during breastfeeding. We investigated bone metabolism markers in the first year of life in infants from mothers who received tenofovir, lamivudine and efavirenz during pregnancy and 12 months of breastfeeding in a national Option B+ programme in Malawi. METHODS: Serum samples collected at 6 and 12 months in tenofovir-exposed infants and in a small sample of tenofovir-unexposed infants from the same clinical centre were analysed in batches for levels of bone-specific alkaline phosphatase (BAP; marker of bone formation) and of C-terminal telopeptide of type I collagen (CTX; marker of bone resorption). RESULTS: Overall, 136 tenofovir-exposed infants were evaluated. No infant had at either timepoint CTX values above the upper normal limit, while most of them had at 6 and 12 months levels of BAP above the upper normal limit for the age range. Levels of bone markers showed no differences by gender and no association with growth parameters. Tenofovir-unexposed and -exposed children had similar mean levels of bone markers at 6 months (CTX: 0.62 versus 0.55 ng/mL, P = 0.122; BAP: 384 versus 362 U/L, P = 0.631). CONCLUSIONS: No significant association between treatment with tenofovir and CTX or BAP levels was found. The high levels of BAP, coupled to the normal levels observed for CTX, might reflect primarily skeletal growth. Potential negative effects of prolonged exposure to tenofovir through breastfeeding cannot however be excluded and longitudinal studies that evaluate bone mineralization status in children enrolled in Option B+ programmes are warranted.

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.

Viro-immunological response and emergence of resistance in HIV-infected women receiving combination antiretroviral regimens for the prevention of mother-to-child transmission in Malawi.

OBJECTIVES: To identify factors associated with detectable viral load and the emergence of drug resistance in a cohort of HIV-infected pregnant women in Malawi receiving antiretroviral combination regimens for the prevention of mother-to-infant transmission. METHODS: The study included 260 treatment-naive women who had received a three-drug nevirapine-based regimen from week 25 of gestational age until 6 months after delivery. HIV RNA was determined at month 6 and drug resistance was assessed if viral load was >50 copies/mL. Attendance at the scheduled follow-up visits was used as an indirect measure of treatment adherence. RESULTS: The rate of detectable HIV RNA at 6 months was 9.6% (25/260). The only significant predictor of this occurrence was the presence of ≥1 missed visit during follow-up (P = 0.012). Resistance was assessed in 19 of these women: 7 (37%) had a wild-type virus and the other 12 (63%) had resistance-associated mutations (nucleoside reverse transcriptase inhibitor, 7/12; non-nucleoside reverse transcriptase inhibitor, 11/12). Three of 12 cases (25%) in which mutations were detected had a viral load <1000 copies/mL. The emergence of resistance was not correlated with the presence of baseline mutations in either plasma or archived DNA. CONCLUSIONS: In this cohort of women, detectable HIV RNA 6 months post-partum was infrequent and associated with low adherence to the treatment programme. Mutations were present in 63% of the women with detectable viral load at 6 months who had samples available for resistance testing. The impact of resistance on treatment re-initiation in women discontinuing drugs after the risk of transmission has ceased can be limited.

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.

Determinants of virologic and immunologic outcomes in chronically HIV-infected subjects undergoing repeated treatment interruptions: the Istituto Superiore di Sanita-Pulsed Antiretroviral Therapy (ISS-PART) study.

BACKGROUND: Factors influencing the outcome of structured treatment interruptions (STIs) in HIV chronic infection are not fully elucidated. METHODS: In ISS-PART, 273 subjects were randomly assigned to arm A (137 assigned to continuous highly active antiretroviral therapy [HAART]) and arm B (136 assigned to 5 STIs of 1, 1, 2, 2, and 3 months' duration, each followed by 3 months of therapy). Main outcome measures were the proportion of subjects with a CD4 count >500 cells/mm3, the rate of virologic failure, and the emergence of resistance at 24 months. RESULTS: The proportion of subjects with a CD4 count >500 cells/mm3 was higher in arm A than in arm B (86.5% vs. 69.1%; P = 0.0075). Pre-HAART CD4 cell count and male gender were independent predictors of a CD4 count >500 cells/mm3 in arm B. The overall risk of virologic failure was not increased in arm B; however, it was higher in the 38 subjects who had resistance mutations in the rebounding virus. Archived mutations at baseline and the use of a regimen that included an unboosted protease inhibitor (PI), compared with nonnucleoside reverse transcriptase inhibitor-based HAART, independently predicted the emergence of plasma mutations during STI (P = 0.002 for DNA mutations and P = 0.048 for PI-based HAART). CONCLUSIONS: Our results suggest that patients with preexisting mutations and treated with unboosted PI-based HAART should not be enrolled in studies of time-fixed treatment interruptions, being at higher risk of developing plasma mutations during STI and virologic failure at therapy reinstitution.