P02-07. High Concentrations of Interleukin-15 and Low Concentrations of CCL5 in Breast Milk are Associated with Protection against Postnatal HIV Transmission

Background: Natural variations in IL-15 concentration have not been investigated for an association with an immune-protection against HIV. Given IL-15's central role in anti-HIV immunity, we hypothesized that higher concentrations of IL-15 in breast milk may protect against postnatal mother-to-child HIV transmission. Methods: In a case-control study nested within a clinical trial in Zambia, we compared IL-15 concentrations in breast milk of 22 HIV-infected women who transmitted HIV to their infants through breastfeeding with those of 72 who did not, as well as 18 HIV-uninfected women. Breast milk HIV RNA quantity, sodium, CXCL12, CCL5, and IL-8 concentrations were measured as well as maternal plasma HIV RNA concentrations and CD4 cell count. We used logistic regression modeling to adjust for potential confounders. Results: Higher concentrations of IL-15 in breast milk (adjusted odds ratio [AOR]: 0.01 per log10 pg/ml increase, 95% confidence interval [CI]: <0.001 to 0.3) were associated with protection against postnatal HIV transmission in univariate analysis and after adjusting for maternal CD4 cell counts, breast milk HIV RNA, CCL5, CXCL12, and IL-8 concentrations. Breast milk IL-15 concentration correlated with breast milk sodium, the other cytokines and HIV RNA concentration. It was inversely correlated with infant birth weight and tended to be higher in 1 week than in 1 month post-partum samples. Breast milk CCL5 concentrations were associated with increased risk of HIV transmission (AOR: 12.7 95% CI: 1.6 to 102.0) in adjusted analysis. Breast milk CXCL12 and IL-8 concentrations were not independently associated with transmission. Conclusion: High concentration of IL-15 were associated with a protection against breastfeeding HIV transmission after adjusting for other pro-inflammatory cytokines, HIV RNA in breast milk, and maternal CD4 cell count. These results corroborate a protective role of IL-15-mediated cellular immunity against HIV transmission during breastfeeding. They are informative for vaccination studies using IL-15 as an adjuvant

Shared and Distinct Features of Human Milk and Infant Stool Viromes.

Infants acquire many of their microbes from their mothers during the birth process. The acquisition of these microbes is believed to be critical in the development of the infant immune system. Bacteria also are transmitted to the infant through breastfeeding, and help to form the microbiome of the infant gastrointestinal (GI) tract; it is unknown whether viruses in human milk serve to establish an infant GI virome. We examined the virome contents of milk and infant stool in a cohort of mother-infant pairs to discern whether milk viruses colonize the infant GI tract. We observed greater viral alpha diversity in milk than in infant stool, similar to the trend we found for bacterial communities from both sites. When comparing beta diversity, viral communities were mostly distinguishable between milk and infant stool, but each was quite distinct from adult stool, urine, and salivary viromes. There were significant differences in viral families in the infant stool (abundant bacteriophages from the family Siphoviridae) compared to milk (abundant bacteriophages from the family Myoviridae), which may reflect significant differences in the bacterial families identified from both sites. Despite the differences in viral taxonomy, we identified a significant number of shared viruses in the milk and stool from all mother-infant pairs. Because of the significant proportion of bacteriophages transmitted in these mother-infant pairs, we believe the transmission of milk phages to the infant GI tract may help to shape the infant GI microbiome

Deregulation of HDAC5 by Viral Interferon Regulatory Factor 3 Plays an Essential Role in Kaposi's Sarcoma-Associated Herpesvirus-Induced Lymphangiogenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis.IMPORTANCE Several lines of evidences indicate that KSHV infection of LECs induces pathological lymphangiogenesis and that the results resemble KS-like spindle morphology. However, the underlying molecular mechanism remains unclear. Here, we demonstrated that KSHV vIRF3 is readily detected in over 40% of various KS lesions and functions as a potent prolymphangiogenic factor by blocking the phosphorylation-dependent cytosolic translocation of HDAC5, which in turn modulates global gene expression in LECs. Consequently, vIRF3-HDAC5 interaction contributes to virus-induced lymphangiogenesis. The results of this study suggest that KSHV vIRF3 plays a crucial role in KSHV-induced malignancy

Alterations to the Gastrointestinal Microbiome Associated with Methamphetamine Use among Young Men who have Sex with Men.

Methamphetamine (MA) use is a major public health problem in the United States, especially among people living with HIV (PLWH). Many MA-induced neurotoxic effects are mediated by inflammation and gut microbiota may play a role in this process, yet the effects of MA on the microbiome have not been adequately explored. Therefore, we performed 16S rRNA gene sequencing on rectal swab samples from 381 men who have sex with men, 48% of whom were PLWH and 41% of whom used MA. We compared microbiome composition between MA users and non-users while testing for potential interactions with HIV and controlling for numerous confounders using inverse probability of treatment weighting. We found that MA use explained significant variation in overall composition (R2 = 0.005, p = 0.008) and was associated with elevated Finegoldia, Parvimonas, Peptoniphilus, and Porphyromonas and reduced Butyricicoccus and Faecalibacterium, among others. Genera including Actinomyces and Streptobacillus interacted with HIV status, such that they were increased in HIV+ MA users. Finegoldia and Peptoniphilus increased with increasing frequency of MA use, among others. In summary, MA use was associated with a microbial imbalance favoring pro-inflammatory bacteria, including some with neuroactive potential and others that have previously been associated with poor HIV outcomes

Restriction of HIV-1 Genotypes in Breast Milk Does Not Account for the Population Transmission Genetic Bottleneck That Occurs following Transmission

BACKGROUND. Breast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established. METHODOLOGY/PRINCIPAL FINDINGS. We compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue. CONCLUSIONS/SIGNIFICANCE. Despite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.National Institute of Child Health and Human Development; National Institutes of Health (R01 HD 39611, R01 HD 40777); International Maternal Pediatric Adolescent AIDS Clinical Trials Group (U01 AI068632-01); National Institutes of Health Cellular, Biochemical; Molecular Sciences Training Program Grant (T 32 067587

Differential Effects of Early Weaning for HIV-Free Survival of Children Born to HIV-Infected Mothers by Severity of Maternal Disease

BACKGROUND. We previously reported no benefit of early weaning for HIV-free survival of children born to HIV-infected mothers in intent-to-treat analyses. Since early weaning was poorly accepted, we conducted a secondary analysis to investigate whether beneficial effects may have been hidden. METHODS. 958 HIV-infected women in Lusaka, Zambia, were randomized to abrupt weaning at 4 months (intervention) or to continued breastfeeding (control). Children were followed to 24 months with regular HIV PCR tests and examinations to determine HIV infection or death. Detailed behavioral data were collected on when all breastfeeding ended. Most participants were recruited before antiretroviral treatment (ART) became available. We compared outcomes among mother-child pairs who weaned earlier or later than intended by study design adjusting for potential confounders. RESULTS. Of infants alive, uninfected and still breastfeeding at 4 months in the intervention group, 16.1% who weaned as instructed acquired HIV or died by 24 months compared to 16.0% who did not comply (p=0.98). Children of women with less severe disease during pregnancy (not eligible for ART) had worse outcomes if their mothers weaned as instructed (RH=2.60 95% CI: 1.06-6.36) compared to those who continued breastfeeding. Conversely, children of mothers with more severe disease (eligible for ART but did not receive it) who weaned early had better outcomes (p-value interaction=0.002). In the control group, weaning before 15 months was associated with 3.94-fold (95% CI: 1.65-9.39) increase in HIV infection or death among infants of mothers with less severe disease. CONCLUSION. Incomplete adherence did not mask a benefit of early weaning. On the contrary, for women with less severe disease, early weaning was harmful and continued breastfeeding resulted in better outcomes. For women with more advanced disease, ART should be given during pregnancy for maternal health and to reduce transmission, including through breastfeeding. TRIAL REGISTRATION. Clinical trials.gov NCT00310726National Institute of Child Health and Human Development (NICHD); National Institutes of Health (R01 HD 39611, R01 HD 40777

The Mucosae-Associated Epithelial Chemokine (MEC/CCL28) Modulates Immunity in HIV Infection

BACKGROUND. CCL28 (MEC) binds to CCR3 and CCR10 and recruits IgA-secreting plasma cells (IgA-ASC) in the mucosal lamina propria (MLP). Mucosal HIV-specific IgA are detected in HIV-infection and exposure. The CCL28 circuit was analyzed in HIV-infected and-exposed individuals and in HIV-unexposed controls; the effect of CCL28 administration on gastrointestinal MLP IgA-ASC was verified in a mouse model. METHODOLOGY/FINDINGS. CCL28 was augmented in breast milk (BM) plasma and saliva of HIV-infected and –exposed individuals; CCR3+ and CCR10+ B lymphocytes were increased in these same individuals. Additionally: 1) CCL28 concentration in BM was associated with longer survival in HIV vertically-infected children; and 2) gastro-intestinal mucosal IgA-ASC were significantly increased in VSV-immunized mice receiving CCL28. CONCLUSIONS. CCL28 mediates mucosal immunity in HIV exposure and infection. CCL28-including constructs should be considered in mucosal vaccines to prevent HIV infection of the gastro-intestinal MLP via modulation of IgA-ASC.Istituto Superiore di Sanita' "Programma Nazionale di Ricerca sull' AIDS"; DG Right to Health and Solidarity Policy; EMPRO and AVIP EC WP6 Projects; Japan Health Science Foundation; National Institutes of Child Health and Human Development (HD 39611, HD 40777

Breast milk and in utero transmission of HIV-1 select for envelope variants with unique molecular signatures

Additional file 5: Figure S5. Representative CD4 infectivity curves using Affinofile cells for IUT (top) and BMT (bottom) maternal–infant pairs. Affinofile cells were induced to generate a 100-fold range of CD4 surface density (ABS/cell) and infected with 2000 IU pseudotyped virus. Percent infection was measured as the percent luciferase relative to infected and maximally induced Affinofile cells. Data shown are representative curves among 3–4 experimental replicates

4E10-Resistant HIV-1 Isolated from Four Subjects with Rare Membrane-Proximal External Region Polymorphisms

Human antibody 4E10 targets the highly conserved membrane-proximal external region (MPER) of the HIV-1 transmembrane glycoprotein, gp41, and has extraordinarily broad neutralizing activity. It is considered by many to be a prototype for vaccine development. In this study, we describe four subjects infected with viruses carrying rare MPER polymorphisms associated with resistance to 4E10 neutralization. In one case resistant virus carrying a W680G substitution was transmitted from mother to infant. We used site-directed mutagenesis to demonstrate that the W680G substitution is necessary for conferring the 4E10-resistant phenotype, but that it is not sufficient to transfer the phenotype to a 4E10-sensitive Env. Our third subject carried Envs with a W680R substitution causing variable resistance to 4E10, indicating that residues outside the MPER are required to confer the phenotype. A fourth subject possessed a F673L substitution previously associated with 4E10 resistance. For all three subjects with W680 polymorphisms, we observed additional residues in the MPER that co-varied with position 680 and preserved charged distributions across this region. Our data provide important caveats for vaccine development targeting the MPER. Naturally occurring Env variants described in our study also represent unique tools for probing the structure-function of HIV-1 envelope