[Show abstract][Hide abstract] ABSTRACT: A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.
[Show abstract][Hide abstract] ABSTRACT: We used bone marrow/liver/thymus (BLT) humanized mice to establish the effect of semen on vaginal HIV infection and on the efficacy of topically applied maraviroc. Our results demonstrate that vaginal transmission of cell-free HIV occurs efficiently in the presence of semen and that topically applied maraviroc efficiently prevents HIV transmission in the presence of semen. We also show that semen has no significant effect on the transmission of transmitted/founder viruses or cell-associated viruses.
[Show abstract][Hide abstract] ABSTRACT: The switching thresholds of magnetophoretic transistors for sorting cells in microfluidic environments are characterized. The transistor operating conditions require short 20–30 mA pulses of electrical current. By demonstrating both attractive and repulsive transistor modes, a single transistor architecture is used to implement the full write cycle for importing and exporting single cells in specified array sites.
[Show abstract][Hide abstract] ABSTRACT: Importance:
Approximately 240,000 children become infected with HIV annually, the majority via breastfeeding. Despite daily exposure to virus in breast milk, most infants breastfed by HIV-positive women do not acquire HIV. The low risk of breastfeeding-associated HIV transmission is likely due antiviral factors in breast milk. It is well-documented that breast milk of HIV-negative women can inhibit HIV infection. Here, we demonstrate, for the first time, that breast milk of HIV-positive mothers (non-transmitters and transmitters) inhibits HIV transmission. We also demonstrate that breast milk can prevent multiple routes of HIV acquisition and that this activity is unique to human milk. Collectively, our results support current guidelines which recommend that HIV-positive women in resource-limited settings exclusively breastfeed in combination with infant or maternal antiretroviral therapy.
Journal of Virology 08/2015; DOI:10.1128/JVI.01702-15 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.
[Show abstract][Hide abstract] ABSTRACT: Nef is a multifunctional HIV-1 protein critical for progression to AIDS. Humans infected with nef(-) HIV-1 have greatly delayed or no disease consequences. We have contrasted nef(-) and nef(+) infection of BLT humanized mice to better characterize Nef's pathogenic effects.
Mice were inoculated with CCR5-tropic HIV-1JRCSF (JRCSF) or JRCSF with an irreversibly inactivated nef (JRCSFNefdd). In peripheral blood (PB), JRCSF exhibited high levels of viral RNA (peak viral loads of 4.71 × 10(6) ± 1.23 × 10(6) copies/ml) and a progressive, 75% loss of CD4(+) T cells over 17 weeks. Similar losses were observed in CD4(+) T cells from bone marrow, spleen, lymph node, lung and liver but thymocytes were not significantly decreased. JRCSFNefdd also had high peak viral loads (2.31 × 10(6) ± 1.67 × 10(6)) but induced no loss of PB CD4(+) T cells. In organs, JRCSFNefdd produced small, but significant, reductions in CD4(+) T cell levels and did not affect the level of thymocytes. Uninfected mice have low levels of HLA-DR(+)CD38(+)CD8(+) T cells in blood (1-2%). Six weeks post inoculation, JRCSF infection resulted in significantly elevated levels of activated CD8(+) T cells (6.37 ± 1.07%). T cell activation coincided with PB CD4(+) T cell loss which suggests a common Nef-dependent mechanism. At 12 weeks, in JRCSF infected animals PB T cell activation sharply increased to 19.7 ± 2.9% then subsided to 5.4 ± 1.4% at 14 weeks. HLA-DR(+)CD38(+)CD8(+) T cell levels in JRCSFNefdd infected mice did not rise above 1-2% despite sustained high levels of viremia. Interestingly, we also noted that in mice engrafted with human tissue expressing a putative protective HLA-B allele (B42:01), JRCSFNefdd exhibited a substantial (200-fold) reduced viral load compared to JRCSF.
Nef expression was necessary for both systemic T cell activation and substantial CD4(+) T cell loss from blood and tissues. JRCSFNefdd infection did not activate CD8(+) T cells or reduce the level of CD4(+) T cells in blood but did result in a small Nef-independent decrease in CD4(+) T cells in organs. These observations strongly support the conclusion that viral pathogenicity is mostly driven by Nef. We also observed for the first time substantial host-specific suppression of HIV-1 replication in a small animal infection model.
[Show abstract][Hide abstract] ABSTRACT: Since the onset of the HIV epidemic, there has been a shift from a deadly diagnosis to the management of a chronic disease. This shift is the result of the development of highly effective drugs that are able to suppress viral replication for years. The availability of these regimens has also shifted the neurocognitive pathology associated with infection from potentially devastating to a much milder phenotype. As the disease outcome has changed significantly with the availability of antiretroviral therapy, there is an opportunity to re-evaluate the currently available models to address the neurocognitive pathology seen in suppressed patients. In the following, we seek to summarize the current literature on humanized mouse models and their utility in understanding how HIV infection leads to changes in the central nervous system (CNS). Also, we identify some of the unanswered questions regarding HIV infection of the CNS as well as the opportunities and limitations of currently existing models to address those questions. Finally, our conclusions indicate that the earlier humanized models used to study HIV infection in the CNS provided an excellent foundation for the type of work currently being performed using novel humanized mouse models. We also indicate the potential of some humanized mouse models that have not been used as of this time for the analysis of HIV infection in the brain.
Journal of NeuroVirology 11/2014; 21(3). DOI:10.1007/s13365-014-0299-6 · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary antibody deficiencies present clinically as reduced or absent plasma antibodies without another identified disorder that could explain the low immunoglobulin levels. Bone marrow-liver-thymus (BLT) humanized mice also exhibit primary antibody deficiency or hypogammaglobulinemia. Comprehensive characterization of B cell development and differentiation in BLT mice revealed other key parallels with primary immunodeficiency patients. We found that B cell ontogeny was normal in the bone marrow of BLT mice but observed an absence of switched memory B cells in the periphery. PC-KLH immunizations led to the presence of switched memory B cells in immunized BLT mice although plasma cells producing PC- or KLH- specific IgG were not detected in tissues. Overall, we have identified the following parallels between the humoral immune systems of primary antibody deficiency patients and those in BLT mice that make this in vivo model a robust and translational experimental platform for gaining a greater understanding of this heterogeneous array of humoral immunodeficiency disorders in humans: (i) hypogammaglobulinemia; (ii) normal B cell ontogeny in bone marrow; and (iii) poor antigen-specific IgG response to immunization. Furthermore, the development of strategies to overcome these humoral immune aberrations in BLT mice may in turn provide insights into the pathogenesis of some primary antibody deficiency patients which could lead to novel clinical interventions for improved humoral immune function.
PLoS ONE 10/2014; 9(10):e108663. DOI:10.1371/journal.pone.0108663 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gastrointestinal (GI) track represents an important battlefield where pathogens first try to gain entry into a host. It is also a universe where highly diverse and ever changing inhabitants co-exist in an exceptional equilibrium without parallel in any other organ system of the body. The gut as an organ has its own well-developed and fully functional immune organization that is similar and yet different in many important ways to the rest of the immune system. Both a compromised and an overactive immune system in the gut can have dire and severe consequences to human health. It has therefore been of great interest to develop animal models that recapitulate the key aspects of the human condition to better understand the interplay of the host immune system with its friends and its foes. However, reconstitution of the GI tract in humanized mice has been difficult and highly variable in different systems. A better molecular understanding of the development of the gut immune system in mice has provided critical cues that have been recently used to develop novel humanized mouse models that fully recapitulate the genesis and key functions of the gut immune system of humans. Of particular interest is the presence of human gut-associated lymphoid tissue (GALT) aggregates in the gut of NOD/SCID BLT humanized mice that demonstrate the faithful development of bona fide human plasma cells capable of migrating to the lamina propria and producing human IgA1 and IgA2.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Antiretroviral therapy (ART) improves the quality of life for HIV infected individuals. However, ART is currently a lifelong commitment because HIV persists during treatment despite being suppressed below detection. If therapy is stopped, the HIV reappears. A concerted effort is ongoing to develop new eradication therapies to prevent virus rebound, but there are challenges to be overcome. Our work is a major step forward in this process. We measured persistent HIV throughout the body during ART using bone marrow/liver/thymus (BLT) humanized mice, a model validated to study HIV persistence. HIV infected BLT mice were treated with tenofovir, emtricitabine and raltegravir. Despite documented tissue penetration by these drugs, we found that HIV expression persists in cells isolated from all the tissues analyzed (bone marrow, thymus, spleen, lymph nodes, liver, lung, intestines and peripheral blood cells). We therefore complemented ART with an immunotoxin that specifically kills HIV expressing cells while leaving other cells untouched. Our results demonstrate a dramatic reduction in persistent HIV throughout the body resulting from the killing of virus producing cells. Thus, our study provides new insights into the locations of HIV persistence during ART and a demonstration that persistent HIV can be successfully targeted inside the body.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Elite controllers or suppressors (ES) are HIV-1-infected patients who maintain undetectable viral loads without antiretroviral therapy. The mechanism of control remains unclear, but the HLA-B*57 allele is overrepresented in cohorts of these patients. However, many HLA-B*57 patients develop progressive disease, and some studies have suggested that infection with defective viruses may be the cause of the lack of high levels of virus replication and disease progression in ES. We therefore performed a comprehensive comparative in vivo and in vitro characterization of viruses isolated from well-defined ES. For this purpose, we first performed full-genome sequence analysis and in vitro fitness assays on replication-competent isolates from HLA-B*57 ES and HLA-B*57 chronic progressors (CPs). Under our experimental conditions, we found that isolates from ES and CPs can replicate in vitro. However, since inherently these assays involve the use of unnaturally in vitro-activated cells, we also investigated the replication competence and pathogenic potential of these HIV isolates in vivo using humanized BLT mice. The results from these analyses demonstrate that virus isolates from ES are fully replication competent in vivo and can induce peripheral and systemic CD4 T cell depletion. These results provide the first direct in vivo evidence that viral fitness does not likely determine clinical outcome in HLA-B*57 patients and that elite suppressors can control replication-competent, fully pathogenic viruses. A better understanding of the immunological bases of viral suppression in ES will serve to inform novel approaches to preventive and therapeutic HIV vaccine design.
Elite suppressors are HIV-1-infected patients who have undetectable levels of viremia despite not being on antiviral drugs. One of the most fundamental questions about this phenomenon involves the mechanism of control. To address this question, we isolated virus from elite suppressors and from HIV-1-infected patients who have the usual progressive disease course. We compared how well the isolates from the two groups of patients replicated in culture and in humanized mice. Our results suggest that elite suppressors are capable of controlling HIV-1 due to the possession of unique host factors rather than infection with defective virus.
Journal of Virology 01/2014; 88(6). DOI:10.1128/JVI.03380-13 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined.
We investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed.
Our results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a conserved property of divergent lentiviruses, it is likely to be relevant for peripheral T-cell depletion in poorly adapted primate lentiviral infections.
[Show abstract][Hide abstract] ABSTRACT: What started as a game of discovery for a child with a chemistry set has resulted in a lifetime of fulfillment and dedication to science. It is deeply rewarding to continuously let my curiosity ask questions for which there are no known answers. Equally rewarding has been the opportunity to mentor young students and postdoctorate graduates through their formative years in science.
Molecular biology of the cell 11/2013; 24(21):3275-7. DOI:10.1091/mbc.E13-07-0407 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution.
We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs[increment]-1 and LAINeffs[increment]-13). Subsequently, LAINeffs[increment]-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs[increment]-1 and LAINeffs[increment]-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs[increment]-1 and LAINeffs[increment]-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs[increment]-1 and LAINeffs[increment]-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence.
The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.
[Show abstract][Hide abstract] ABSTRACT: The major targets of HIV infection in humans are CD4+ T cells. CD4+ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages.
NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOMs are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4+ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4+ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo.
NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.
[Show abstract][Hide abstract] ABSTRACT: Abnormal gut-associated lymphoid tissue (GALT) in humans is associated with infectious and autoimmune diseases, which cause dysfunction of the gastrointestinal (GI) tract immune system. To aid in investigating GALT pathologies in vivo, we bioengineered a human-mouse chimeric model characterized by the development of human GALT structures originating in mouse cryptopatches. This observation expands our mechanistic understanding of the role of cryptopatches in human GALT genesis and emphasizes the evolutionary conservation of this developmental process. Immunoglobulin class switching to IgA occurs in these GALT structures, leading to numerous human IgA-producing plasma cells throughout the intestinal lamina propria. CD4(+) T cell depletion within GALT structures results from HIV infection, as it does in humans. This human-mouse chimeric model represents the most comprehensive experimental platform currently available for the study and for the preclinical testing of therapeutics designed to repair disease-damaged GALT.
[Show abstract][Hide abstract] ABSTRACT: Rectal microbicides are being developed to prevent new HIV infections in both men and women. We focused our in vivo
preclinical efficacy study on rectally-applied tenofovir. BLT humanized mice (n = 43) were rectally inoculated with either the primary isolate HIV-1JRCSF or the MSM-derived transmitted/founder (T/F) virus HIV-1THRO within 30 minutes following treatment with topical 1% tenofovir or vehicle. Under our experimental conditions, in the absence of drug treatment we observed 50% and 60% rectal transmission by HIV-1JRCSF and HIV-1THRO, respectively. Topical tenofovir reduced rectal transmission to 8% (1/12; log rank p = 0.03) for HIV-1JRCSF and 0% (0/6; log rank p = 0.02) for HIV-1THRO. This is the first demonstration that any human T/F HIV-1 rectally infects humanized mice and that transmission of the T/F virus can be efficiently blocked by rectally applied 1% tenofovir. These results obtained in BLT mice, along with recent ex vivo, Phase 1 trial and non-human primate reports, provide a critically important step forward in the development of tenofovir-based rectal microbicides.
PLoS ONE 03/2013; 8(3):e60024. DOI:10.1371/journal.pone.0060024 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Over 90% of the adult human population is chronically infected with the Epstein-Barr virus (EBV), an oncogenic herpesvirus. EBV primarily infects naïve human B cells and persists latently in memory B cells. Most individuals experience an asymptomatic infection that is effectively controlled by the adaptive immune response. However, EBV-associated lymphomas can develop in immune compromised individuals. These tumors typically express all nine EBV latent proteins (latency III). Latency III is also associated with the expression of three precursor microRNAs (miRNAs) located within the EBV BHRF1 gene locus. The role of these BHRF1 miRNAs was unclear until recent in vitro studies demonstrated that they cooperate to enhance virus-induced B cell transformation and decrease the antigenic load of virus infected cells, indicating that the BHRF1 miRNA cluster may serve as a novel therapeutic target for the treatment of latency III EBV-associated malignancies. However, to date, it is not known if BHRF1 miRNAs enhance virus-induced oncogenesis and/or immune evasion of EBV in vivo. To understand the in vivo contribution of the BHRF1 miRNA cluster to EBV infection and EBV-associated tumorigenesis, we monitored EBV infection and assessed tumor formation in humanized mice exposed to wild-type virus and a viral mutant (Δ123) that lacks all three BHRF1 miRNAs. Our results demonstrate that while the BHRF1 miRNAs facilitate the development of acute systemic EBV infection, they do not enhance the overall oncogenic potential of EBV in vivo.
Journal of Virology 03/2013; 87(10). DOI:10.1128/JVI.00281-13 · 4.44 Impact Factor