Faculty Bibliography

Mucosal-associated invariant T cells (MAIT) are innate T cells restricted by major histocompatibility related molecule 1 (MR1) presenting riboflavin metabolite ligands derived from microbes. Specificity to riboflavin metabolites confers MAIT cells a broad array of host-protective activity against gram-negative and -positive bacteria, mycobacteria, and fungal pathogens. MAIT cells are present at low levels in the peripheral blood of neonates and gradually expand to relatively abundant levels during childhood. Despite no anti-viral activity, MAIT cells are depleted early and irreversibly in HIV infected adults. Such loss or impaired expansion of MAIT cells in HIV-positive children may render them more susceptible to common childhood illnesses and opportunistic infections. In this study we evaluated the frequency of MAIT cells in perinatally HIV-infected children, their response to antiretroviral treatment and their associations with HIV clinical status and related innate and adaptive immune cell subsets with potent antibacterial effector functions. We found HIV+ children between ages 3 to 18 years have significantly decreased CD8+ MAIT cell frequencies compared to uninfected healthy children. Remarkably, CD8 MAIT levels gradually increased with antiretroviral therapy, with greater recovery when treatment is initiated at a young age. Moreover, diminished CD8+ MAIT cell frequencies are associated with low CD4:CD8 ratios and elevated sCD14, suggesting a link with HIV disease progression. Last, CD8+ MAIT cell levels tightly correlate with other antibacterial and mucosa-protective immune subsets, namely, neutrophils, innate-like T cells, and Th17 and Th22 cells. Together these findings suggest that low frequencies of MAIT cells in HIV positive children are part of a concerted disruption to the innate and adaptive immune compartments specialized in sensing and responding to pathogenic or commensal bacteria.

From the use of antiretroviral therapy to prevent mother-to-child transmission to the possibility of HIV cure hinted at by the Mississippi baby experience, paediatric HIV infection has been pivotal to our understanding of HIV pathogenesis and management. Daily medication and indefinite antiretroviral therapy is recommended for children infected with HIV. Maintenance of life-long adherence is difficult and the incidence of triple-class virological failure after initiation of antiretroviral therapy increases with time. This challenge shows the urgent need to define novel strategies to provide long-term viral suppression that will allow safe interruption of antiretroviral therapy without viral rebound and any associated complications. HIV-infected babies treated within a few days of birth have a unique combination of a very small pool of integrated viruses, a very high proportion of relatively HIV resistant naive T cells, and an unparalleled capacity to regenerate an immune repertoire. These features make this group the optimum model population to investigate the potential efficacy of immune-based therapies. If successful, these investigations could change the way we manage HIV infection.

OBJECTIVES: Some perinatally infected children do not regain normal CD4 T-cell counts despite suppression of HIV-1 plasma viremia by antiretroviral therapy (ART). The frequency, severity and significance of these discordant treatment responses remain unclear. DESIGN: We examined the persistence of CD4 lymphocytopenia despite virologic suppression in 933 children (>/=5 years of age) in the USA, Latin America and the Caribbean. METHODS: CD4 T-cell trajectories were examined and Kaplan-Meier methods used to estimate median time to CD4 T-cell count at least 500 cells/mul. RESULTS: After 1 year of virologic suppression, most (99%) children achieved a CD4 T-cell count of at least 200 cells/mul, but CD4 T-cell counts remained below 500 cells/mul after 1 and 2 years of virologic suppression in 14 and 8% of children, respectively. Median times to first CD4 T-cell count at least 500 cells/mul were 1.29, 0.78 and 0.46 years for children with less than 200, 200-349 and 350-499 cells/mul at the start of virologic suppression. New AIDS-defining events occurred in nine children, including four in the first 6 months of virologic suppression. Other infectious and HIV-related diagnoses occurred more frequently and across a wide range of CD4 cell counts. CONCLUSION: ART improved CD4 cell counts in most children, but the time to CD4 cell count of at least 500 cells was highly dependent upon baseline immunological status. Some children did not reach a CD4 T-cell count of 500 cells/mul despite 2 years of virologic suppression. AIDS-defining events occurred in 1% of the population, including children in whom virologic suppression and improved CD4 T-cell counts were achieved.

The EPIICAL (Early-treated Perinatally HIV-infected Individuals: Improving Children's Actual Life with Novel Immunotherapeutic Strategies) project arises from the firm belief that perinatally infected children treated with suppressive antiretroviral therapy (ART) from early infancy represent the optimal population model in which to study novel immunotherapeutic strategies aimed at achieving ART-free remission. This is because HIV-infected infants treated within 2-3 months of life have a much reduced viral reservoir size, and rarely show HIV-specific immunity but preserve normal immune development. The goal of EPIICAL is the establishment of an international collaboration to develop a predictive platform using this model to select promising HIV therapeutic vaccine candidates, leading to prioritisation or deprioritisation of novel immunotherapeutic strategies. To establish this platform, the EPIICAL Consortium aims to: develop predictive models of virological and immunological dynamics associated with response to early ART and to treatment interruption using available data from existing cohorts/studies of early-treated perinatally HIV-infected children; optimise methodologies to better characterise immunological, virological and genomic correlates/profiles associated with viral control; test novel immunotherapeutic strategies using in vivo proof-of-concept (PoC) studies with the aim of inducing virological, immunological and transcriptomic correlates/profiles equivalent to those defined by the predictive model. This approach will strengthen the capacity for discovery, development and initial testing of new therapeutic vaccine strategies through the integrated efforts of leading international scientific groups, with the aim of improving the health of HIV-infected individuals.

Background: Regulatory T cells (Tregs) mediate immune tolerance during autoimmune disease and chronic infections. During HIV infection, Tregs may act either beneficially to curb immune activation or pathologically to suppress HIV-specific immune responses. Previous reports of Tregs during chronic HIV have conflicting results with higher or lower levels compared to controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation. Helios is a recently identified transcription factor that marks natural Tregs with suppressive activity. Moreover FOXP3+Helios+ CD4 T cells do not produce the cytokine IL-17, and have been called "bona fide" Tregs. We sought to identify these bona fide Tregs in vertically infected HIV positive children. Methodology: We evaluated Treg levels by flow cytometry in the peripheral blood of 60 children from Bomu Hospital in Mombasa, Kenya. The cohort included age-matched children between 3 to 12 years old in the following categories: HIV negative (HIV-), HIV positive antiretroviral therapy naive (ART-), and HIV positive on antiretroviral therapy (ART+). Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from each subject. Thawed PBMCs were stained for surface antibodies CD3, CD4, CD25, CD38, CD45RO, and intracellular transcription factors FOXP3 and Helios. All statistical analysis was performed with GraphPad Prism software using Mann-Whitney or Spearman's correlation tests. Results: HIV+ children (ART- and ART+) expressed higher levels of FOXP3 and Helios in CD4 T cells compared with HIV- controls (ART-: p=0.0012, ART+: p=0.0057). FOXP3+Helios+ expression inversely correlated with the percent of CD4 T cells (p<0.0001, r= -0.4883), despite nearly normal CD4 levels in ART+ children. As previously reported, HIV infected children had higher immune activation as measured by CD38+HLA-DR+ expression on CD8+ T c!

Background: Abnormal newborn screens for dysfunctional fatty acid oxidation (FAO) are more common in HIV-exposed uninfected (HEU) neonates than the general population and may be related to in utero exposure to HIV and/or combination antiretroviral therapy (cART). Normal FAO is necessary for normal growth and development. Disordered FAO can result in hypoglycemia, myopathy and liver injury. Methodology: We analyzed serum acylcarnitine profiles (ACP), as a measure of FAO, in 522 HEU neonates (age 0-7 days) enrolled in the SMARTT study of the Pediatric HIV/AIDS Cohort Study (PHACS). We estimated the prevalence of abnormal ACP and evaluated associations of abnormal ACP with in utero exposure to cART and antiretroviral (ARV) drug classes in logistic regression models, adjusting for maternal demographic characteristics and substance use. We also evaluated associations of abnormal ACP with clinical laboratory parameters (lactate, glucose, creatine kinase (CK) and alanine aminotransferase (ALT)) and measures of neurodevelopment and growth through 3 years of age. Results: Of 522 neonates, 84 (17%) had abnormal ACP, with most abnormal profiles characterized by generalized or long-chain specific acylcarnitine elevations. Maternal alcohol exposure (adjusted odds ratio (aOR)=2.55, 95% confidence interval (CI): 1.21, 5.37, p=0.01) and, as expected, lower gestational age at birth (aOR=1.21 per week lower, 95% CI: 1.07, 1.36, p<0.01) were associated with higher odds of having an abnormal ACP. In analyses adjusted for alcohol exposure and gestational age, in utero exposure to a protease inhibitor (PI) was associated with higher odds of having an abnormal ACP (aOR= 2.35, 95% CI: 0.96, 5.76, p=0.06) while exposure to a non-nucleoside reverse transcriptase inhibitor (NNRTI) (aOR=0.23, 95% CI: 0.07, 0.80, p=0.02) was associated with lower odds. ALT levels were higher in those with abnormal vs. normal ACP (geometric mean 18.7 U/L vs. 14.9 U/L, p<0.01,), but no differences in lactate, glucose or CK were observed. ACP!

We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium liver stages in rodent malarias and in vitro in P. falciparum. Since clinically relevant levels are better achieved in the non-human-primate model, and since Plasmodium knowlesi is an accepted animal model for the study of liver stages of malaria as a surrogate for P. falciparum infection, we investigated the antimalarial activity of these drugs on Plasmodium knowlesi liver stages in rhesus macaques. We demonstrate that TMP-SMX and TMP-SMX+LPV-RTV (in combination), but not LPV-RTV alone, inhibit liver stage parasite development. Because drugs that inhibit the clinically silent liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts.

While exploring the effects of aerosol IFN-gamma treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-gamma-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-gamma induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-gamma suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

Plasmodium vivax malaria causes significant morbidity and mortality worldwide, and only one drug is in clinical use that can kill the hypnozoites that cause P. vivax relapses. HIV and P. vivax malaria geographically overlap in many areas of the world, including South America and Asia. Despite the increasing body of knowledge regarding HIV protease inhibitors (HIV PIs) on P. falciparum malaria, there are no data regarding the effects of these treatments on P. vivax's hypnozoite form and clinical relapses of malaria. We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remains untested. Separately, although other antifolates have been tested against hypnozoites, the antibiotic trimethoprim sulfamethoxazole, commonly used in HIV infection and exposure management, has not been evaluated for hypnozoite-killing activity. Since Plasmodium cynomolgi is an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we investigated the antimalarial activity of these drugs on Plasmodium cynomolgi relapsing malaria in rhesus macaques. Herein, we demonstrate that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested. Because HIV and malaria geographically overlap, and more patients are being managed for HIV infection and exposure, understanding HIV drug impact on malaria infection is important.