Composition of Gut Microbiota of Children and Adolescents With Perinatal Human Immunodeficiency Virus Infection Taking Antiretroviral Therapy in Zimbabwe.

BACKGROUND: Human immunodeficiency virus (HIV) infection causes impairment of the gastrointestinal barrier, with substantial depletion of CD4+ T cells in the gut. Antiretroviral therapy (ART) restores CD4+ counts and may have beneficial effects on gut microbiota in adults. Little is known about effect of long-term ART on gut microbiome in HIV-infected children. We investigated composition of gut microbiota in HIV-infected and -uninfected children and assessed associations between gut microbiota and patient characteristics. METHODS: In a cross-sectional study, rectal swabs were collected from 177 HIV-infected and 103 HIV-uninfected controls. Gut microbial composition was explored using 16S ribosomal ribonucleic acid sequencing. RESULTS: Human immunodeficiency virus-infected children had significantly lower alpha-diversity and higher beta-diversity compared to HIV-uninfected. No association was observed between microbiome diversity and CD4+ T-cell count, HIV viral load, or HIV-associated chronic lung disease. We found enriched levels of Corynebacterium (P < .01), Finegoldia (P < .01), and Anaerococcus (P < .01) in HIV-infected participants and enrichment of Enterobacteriaceae (P = .02) in participants with low CD4+ counts (<400 cells/mm3). Prolonged ART-treatment (≥10 years) was significantly associated with a richer gut microbiota by alpha diversity. CONCLUSIONS: Human immunodeficiency virus-infected children have altered gut microbiota. Prolonged ART may restore the richness of the microbiota closer to that of HIV-uninfected children.

Leflunomide inhibition of BK virus replication in renal tubular epithelial cells.

The immunomodulatory drug leflunomide is frequently used for treating polyomavirus-associated nephropathy, yet its antiviral mechanism is unclear. We characterized the effects of the active leflunomide metabolite A771726 (LEF-A) on the polyomavirus BK (BKV) life cycle in human renal tubular epithelial cells. LEF-A at 10 microg/ml reduced the extracellular BKV load by 90% (IC(90)) but with significant host cytostatic effects. BKV genome replication, late protein expression, and virion assembly and release were inhibited with visible disruption of the nuclear replication architecture. Both host cell and antiviral effects were largely reversed by uridine addition, implicating nonspecific pyrimidine depletion as the major anti-BKV mechanism of leflunomide.

Effect of Once-Weekly Azithromycin vs Placebo in Children With HIV-Associated Chronic Lung Disease: The BREATHE Randomized Clinical Trial.

Importance: HIV-associated chronic lung disease (HCLD) in children is associated with small airways disease, is common despite antiretroviral therapy (ART), and is associated with substantial morbidity. Azithromycin has antibiotic and immunomodulatory activity and may be effective in treating HCLD through reducing respiratory tract infections and inflammation. Objective: To determine whether prophylactic azithromycin is effective in preventing worsening of lung function and in reducing acute respiratory exacerbations (AREs) in children with HCLD taking ART. Design, Setting, and Participants: This double-blind, placebo-controlled, randomized clinical trial (BREATHE) was conducted between 2016 and 2019, including 12 months of follow-up, at outpatient HIV clinics in 2 public sector hospitals in Malawi and Zimbabwe. Participants were randomized 1:1 to intervention or placebo, and participants and study personnel were blinded to treatment allocation. Participants included children aged 6 to 19 years with perinatally acquired HIV and HCLD (defined as forced expiratory volume in 1 second [FEV1] z score < -1) who were taking ART for 6 months or longer. Data analysis was performed from September 2019 to April 2020. Intervention: Once-weekly oral azithromycin with weight-based dosing, for 48 weeks. Main Outcomes and Measures: All outcomes were prespecified. The primary outcome was the mean difference in FEV1 z score using intention-to-treat analysis for participants seen at end line. Secondary outcomes included AREs, all-cause hospitalizations, mortality, and weight-for-age z score. Results: A total of 347 individuals (median [interquartile range] age, 15.3 [12.7-17.7] years; 177 boys [51.0%]) were randomized, 174 to the azithromycin group and 173 to the placebo group; 162 participants in the azithromycin group and 146 placebo group participants had a primary outcome available and were analyzed. The mean difference in FEV1 z score was 0.06 (95% CI, -0.10 to 0.21; P = .48) higher in the azithromycin group than in the placebo group, a nonsignificant difference. The rate of AREs was 12.1 events per 100 person-years in the azithromycin group and 24.7 events per 100 person-years in the placebo groups (hazard ratio, 0.50; 95% CI, 0.27 to 0.93; P = .03). The hospitalization rate was 1.3 events per 100 person-years in the azithromycin group and 7.1 events per 100 person-years in the placebo groups, but the difference was not significant (hazard ratio, 0.24; 95% CI, 0.06 to 1.07; P = .06). Three deaths occurred, all in the placebo group. The mean weight-for-age z score was 0.03 (95% CI, -0.08 to 0.14; P = .56) higher in the azithromycin group than in the placebo group, although the difference was not significant. There were no drug-related severe adverse events. Conclusions and Relevance: In this randomized clinical trial specifically addressing childhood HCLD, once-weekly azithromycin did not improve lung function or growth but was associated with reduced AREs; the number of hospitalizations was also lower in the azithromycin group but the difference was not significant. Future research should identify patient groups who would benefit most from this intervention and optimum treatment length, to maximize benefits while reducing the risk of antimicrobial resistance. Trial Registration: ClinicalTrials.gov Identifier: NCT02426112.

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin.

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

Azithromycin versus placebo for the treatment of HIV-associated chronic lung disease in children and adolescents (BREATHE trial): study protocol for a randomised controlled trial.

BACKGROUND: Human immunodeficiency virus (HIV)-related chronic lung disease (CLD) among children is associated with substantial morbidity, despite antiretroviral therapy. This may be a consequence of repeated respiratory tract infections and/or dysregulated immune activation that accompanies HIV infection. Macrolides have anti-inflammatory and antimicrobial properties, and we hypothesised that azithromycin would reduce decline in lung function and morbidity through preventing respiratory tract infections and controlling systemic inflammation. METHODS/DESIGN: We are conducting a multicentre (Malawi and Zimbabwe), double-blind, randomised controlled trial of a 12-month course of weekly azithromycin versus placebo. The primary outcome is the mean change in forced expiratory volume in 1 second (FEV1) z-score at 12 months. Participants are followed up to 18 months to explore the durability of effect. Secondary outcomes are FEV1 z-score at 18 months, time to death, time to first acute respiratory exacerbation, number of exacerbations, number of hospitalisations, weight for age z-score at 12 and 18 months, number of adverse events, number of malaria episodes, number of bloodstream Salmonella typhi infections and number of gastroenteritis episodes. Participants will be followed up 3-monthly, and lung function will be assessed every 6 months. Laboratory substudies will be done to investigate the impact of azithromycin on systemic inflammation and on development of antimicrobial resistance as well as impact on the nasopharyngeal, lung and gut microbiome. DISCUSSION: The results of this trial will be of clinical relevance because there are no established guidelines on the treatment and management of HIV-associated CLD in children in sub-Saharan Africa, where 80% of the world's HIV-infected children live and where HIV-associated CLD is highly prevalent. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02426112 . Registered on 21 April 2015.

A wide range of medium-sized, highly cationic, alpha-helical peptides show antiviral activity against herpes simplex virus.

Ten highly cationic, alpha-helical peptides were synthesized and tested for antiviral activity against herpes simplex virus 1 and 2 (HSV-1 and HSV-2). Several of the peptides were found to exhibit antiviral activity. The peptides affinity for heparan sulfate (HS) increased with the number of cationic residues. Net charge could be decisive for the anti-HSV-1 activity, while secondary structure of the peptides seems more important for the anti-HSV-2 activity. The peptides were able to inhibit the entry of HSV-1 into the host cell, probably by blocking HS at the cell surface. HSV plaque formation was inhibited in a dose-dependent manner when cells were exposed to the peptides prior to the addition of virus. Lower inhibition activity was observed when the virus was allowed to attach to the cell surface before the addition of peptide. However, the plaque size was smaller compared to the untreated control, indicating that the peptides may also interfere with cell-to-cell spread of the virus. The two most potent antiviral peptides exhibited synergy with acyclovir against HSV.

No detectable Chlamydia pneumoniae and cytomegalovirus DNA in leukocytes in subjects with echolucent and echogenic carotid artery plaques.

BACKGROUND: Controversy exists whether persistent Chlamydia pneumoniae or cytomegalovirus infections cause initiation or progression of atherosclerosis. C. pneumoniae DNA in peripheral blood mononuclear cells (PBMC) has been proposed to be a more reliable marker of cardiovascular risk than are C. pneumoniae antibodies. Reported prevalences of C. pneumoniae DNA among cardiovascular patients vary greatly, indicating methodological limitations. There is an increasing concern that published results may have been biased by extensive use of less specific polymerase chain reaction (PCR) technology. METHODS: C. pneumoniae DNA and cytomegalovirus DNA were determined by probe-based real-time PCR technology in PBMCs among subjects with echolucent (n=29) or echogenic (n=28) carotid artery plaques, and in controls without carotid plaques (n=38), all recruited from a population-based study. Samples were examined in multiple repeats with PCR assays targeting two different sequences of the genome for both microorganisms. RESULTS AND CONCLUSION: IgG seropositivity was frequent in all three groups, confirming previous exposure, but C. pneumoniae DNA or cytomegalovirus DNA was not detected in a single PBMC sample by means of probe-based, highly sensitive, and specific real-time PCR assays. Our results indicate that persistent C. pneumoniae or CMV infection is not a common phenomenon in subjects with carotid atherosclerosis.

Prediction of activity, synthesis and biological testing of anti-HSV active peptides.

Herpes simplex virus infections can be treated with a number of drugs, but as for all pathogens, there is a constant need for new therapies. In the search for lead compounds some peptides have proven to possess an antiviral effect, but it is still unclear what mechanisms are responsible for this effect. We wish to report on the use of principal properties of amino acids for developing quantitative structure-activity relationships (QSAR:s) as a tool for modelling peptide activity and predicting the activity of new peptides. In order to test the reliability of the method, new peptides have been designed by using multivariate methodology, synthesized and tested for a number of responses. Two of the new peptides synthesized were active at lower concentrations than experienced before regarding entry and herpes simplex virus activity, but they were not able to completely inhibit viral infection. This may reflect differences in mode of action of peptides depending on the amino acid content.

Modelling of anti-HSV activity of lactoferricin analogues using amino acid descriptors.

Herpes simplex virus (HSV) causes a number of diseases and new therapies are being pursued vigorously. Earlier studies have shown that modified peptides based on lactoferricins reduce HSV-1 and HSV-2 infection, and structure-activity studies indicate that the anti-viral activity correlates with the binding affinity for heparan sulphate and chondroitin sulphate. In this study it is shown that theoretically derived amino acid descriptors can be used to model the anti-viral activity of peptides, as well as other peptide properties, even more accurately.

Human lactoferricin is partially folded in aqueous solution and is better stabilized in a membrane mimetic solvent.

Lactoferricins are highly basic bioactive peptides that are released in the stomach through proteolytic cleavage of various lactoferrin proteins. Here we have determined the solution structure of human lactoferricin (LfcinH) by conventional two-dimensional nuclear magnetic resonance methods in both aqueous solution and a membrane mimetic solvent. Unlike the 25-residue bovine lactoferricin (LfcinB), which adopts a somewhat distorted antiparallel beta sheet, the longer LfcinH peptide shows a helical content from Gln14 to Lys29 in the membrane mimetic solvent but a nonexistent beta-sheet character in either the N- or C-terminal regions of the peptide. The helical characteristic of the LfcinH peptide resembles the conformation that this region adopts in the crystal structure of the intact protein. The LfcinH structure determined in aqueous solution displays a nascent helix in the form of a coiled conformation in the region from Gln14 to Lys29. Numerous hydrophobic interactions create the basis for the better-defined overall structure observed in the membrane mimetic solvent. The 49-residue LfcinH peptide isolated for these studies was found to be slightly longer than previously reported peptide preparations and was found to have an intact peptide bond between residues Ala11 and Val12. The distinct solution structures of LfcinH and LfcinB represent a novel difference in the physical properties of these two peptides, which contributes to their unique physiological activities.

Anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface.

Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Lactoferricin (Lfcin) is a potent antimicrobial peptide generated from the N-terminal part of LF by pepsin cleavage. In this study, we investigated the mechanisms of the anti-herpes simplex virus (anti-HSV) activity of LF and Lfcin. The results demonstrated that LF and Lfcin inhibited the entry of HSV into Vero cells. LF had no effect against HSV after the virus had entered the cells, while Lfcin exerted antiviral activity also after the initial binding of the virus to the host cell. The distribution of LF and Lfcin in the cells was investigated by immunogold-labeling and transmission electron microscope (TEM). LF was found mainly at the cell surface in cells expressing heparan sulphate. Lfcin was randomly distributed intracellularly. LF must be present at the cell surface to exert antiviral activity, while Lfcin exert its antiviral activity also when found mainly intracellularly. Both LF and Lfcin were dependent on the presence of heparan sulphate at the cell surface to exert their antiviral activity.