Modeling Anti-HIV-1 HSPC-Based Gene Therapy in Humanized Mice Previously Infected with HIV-1.

Investigations of anti-HIV-1 human hematopoietic stem/progenitor cell (HSPC)-based gene therapy have been performed by HIV-1 challenge after the engraftment of gene-modified HSPCs in humanized mouse models. However, the clinical application of gene therapy is to treat HIV-1-infected patients. Here, we developed a new method to investigate an anti-HIV-1 HSPC-based gene therapy in humanized mice previously infected with HIV-1. First, humanized mice were infected with HIV-1. When plasma viremia reached >107 copies/mL 3 weeks after HIV-1 infection, the mice were myeloablated with busulfan and transplanted with anti-HIV-1 gene-modified CD34+ HSPCs transduced with a lentiviral vector expressing two short hairpin RNAs (shRNAs) against CCR5 and HIV-1 long terminal repeat (LTR), along with human thymus tissue under the kidney capsule. Anti-HIV-1 vector-modified human CD34+ HSPCs successfully repopulated peripheral blood and lymphoid tissues in HIV-1 previously infected humanized mice. Anti-HIV-1 shRNA vector-modified CD4+ T lymphocytes showed selective advantage in HIV-1 previously infected humanized mice. This new method will be useful for investigations of anti-HIV-1 gene therapy when testing in a more clinically relevant experimental setting

MicroRNA-29 family expression and its relation to antiviral immune response and viro-immunological markers in HIV-1-infected patients

Abstract BACKGROUND: Several in vitro studies suggested the microRNA-29 (miRNA-29) family is involved in regulating HIV-1 and modulating the expression of interleukin (IL)-32, an anti-HIV-1 cytokine. METHODS: To investigate the contribution of the miRNA-29 family to HIV-1 infection in vivo, we compared miRNA-29 expression in PBMC collected from 58 HIV-1-infected patients, naïve for antiretroviral therapy, and 21 gender- and age-matched HIV-1 seronegative healthy donors, using RT-Taqman assays. The relation between miRNA-29 levels and HIV-1 viro-immunological markers and the activation rate of antiviral immune response were also evaluated. In addition, we profiled miRNA-29 expression in CD4+ T lymphocytes and CD14+ monocytes collected from 5 antiretroviral treated HIV-1 infected patients. RESULTS: miRNA-29b levels were higher in HIV-1-infected patients than in the control group (p < 0.001). There were no correlations with either HIV-1 RNA levels or CD4+ T count, whereas a significant correlation was found between miRNA-29-a/c levels and integrated HIV-1 DNA (miRNA-29a: p = 0.009, r = -0.448; miRNA-29c: p = 0.029; r = -0.381). When the HIV-1-infected patients were grouped on the basis of their plasma HIV-1 RNA and CD4+ T cell count, we also found that patients expressing the lowest levels of miRNA-29c showed high viraemia, low CD4+ T cell count and high levels of integrated HIV-1 DNA. Moreover, miRNA-29b levels were correlated with those of IL-32nonα (p = 0.028; r = -0.298). Patients expressing higher levels of miRNA-29b showed lower levels of MxA, an interferon-stimulated gene, also induced by IL-32 (p = 0.006 r = -0.397). Lastly, we found that CD4+ T lymphocytes and CD14+ monocytes shared similar miRNA-29a/b/c expression patterns but the amount of miRNA-29a/b/c, IL-32 isoforms and MxA were highly variable in these two cellular subsets. CONCLUSIONS: The miRNA-29 family could influence the clinical progression of HIV-1 infection, the HIV-1 proviral load and the innate immune response against HIV-1

Soluble levels of receptor for advanced glycation endproducts and dysfunctional high-density lipoprotein in persons infected with human immunodeficiency virus: ACTG NWCS332.

The role of high-density lipoprotein (HDL) function and advanced glycation end products (AGEs) in HIV-related atherosclerotic cardiovascular disease (CVD) is unclear. Both glycation and oxidation (HDLox) are major modifications of HDL that can alter its composition and function. Therefore, we explored the longitudinal association of HDLox with progression of glycation, as evaluated by measurement of circulating forms of receptor for AGE that predict morbidity (soluble Receptors for Advanced Glycation Endproducts [sRAGE], endogenous secretory Receptors for Advanced Glycation Endproducts [esRAGE]), in people with HIV-1 (PWH; HIV-1) and uninfected (HIV-1) individuals.We retrospectively assessed if levels of plasma sRAGE and esRAGE and HDL function (reduced antioxidant function is associated with increased HDL lipid hydroperoxide content; HDLox) in a subset of participants (n = 80) from a prospective 3-year study (AIDS Clinical Trials Group A5078). Primary outcomes were baseline and yearly rates of change over 96 of 144 weeks (Δ) in HDLox in HIV-1 versus uninfected HIV-1 controls (noted as HIV-1).Higher baseline levels of sRAGE in PWH on effective anti-retroviral therapy and with low CVD risk, but not in HIV-1 persons, were independently associated with higher HDLox. EsRAGE, but not sRAGE, had consistent inverse relationships with ΔHDLox in both HIV-1 and HIV-1 persons at baseline. In HIV-1 but not in HIV-1 persons, ΔHDLox had positive and inverse relationships with ΔRAGE and ΔesRAGE, respectively.Glycation and oxidation of HDL may contribute to impaired HDL function present in PWH

Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

BACKGROUND: Cellulose acetate phthalate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was shown to inhibit infection by the human immunodeficiency virus type 1 (HIV-1) and several herpesviruses. CAP formulations inactivated HIV-1, herpesvirus types 1 (HSV-1) and 2 (HSV-2) and the major nonviral sexually transmitted disease (STD) pathogens and were effective in animal models for vaginal infection by HSV-2 and simian immunodeficiency virus. METHODS: Enzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope. RESULTS: 1) CAP binds to HIV-1 virus particles and to the envelope glycoprotein gp120; 2) this leads to blockade of the gp120 V3 loop and other gp120 sites resulting in diminished reactivity with HIV-1 coreceptors CXCR4 and CCR5; 3) CAP binding to HIV-1 virions impairs their infectivity; 4) these findings apply to both HIV-1 IIIB, an X4 virus, and HIV-1 BaL, an R5 virus. CONCLUSIONS: These results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection

Cost-effectiveness of voluntary HIV-1 counseling and testing in reducing sexual transmission of HIV-1 in Kenya and Tanzania.

Background Access to HIV-1 voluntary counseling and testing (VCT) is severely limited in less-developed countries. We undertook a multisite trial of HIV-1 VCT to assess its impact, cost, and cost-effectiveness in less-developed country settings.\ud Methods\ud The cost-effectiveness of HIV-1 VCT was estimated for a hypothetical cohort of 10 000 people seeking VCT in urban east Africa. Outcomes were modelled based on results from a randomised controlled trial of HIV-1 VCT in Tanzania and Kenya. Our main outcome measures included programme cost, number of HIV-1 infections averted, cost per HIV-1 infection averted, and cost per disability-adjusted life-year (DALY) saved. We also modelled the impact of targeting VCT by HIV-1 prevalence of the client population, and the proportion of clients who receive VCT as a couple compared with as individuals. Sensitivity analysis was done on all model parameters.\ud Findings\ud HIV-1 VCT was estimated to avert 1104 HIV-1 infections in Kenya and 895 in Tanzania during the subsequent year. The cost per HIV-1 infection averted was US$249 and$346, respectively, and the cost per DALY saved was $12·77 and$17·78. The intervention was most cost-effective for HIV-1-infected people and those who received VCT as a couple. The cost-effectiveness of VCT was robust, with a range for the average cost per DALY saved of $5·16-27·36 in Kenya, and$6·58-45·03 in Tanzania. Analysis of targeting showed that increasing the proportion of couples to 70% reduces the cost per DALY saved to $10·71 in Kenya and$13·39 in Tanzania, and that targeting a population with HIV-1 prevalence of 45% decreased the cost per DALY saved to $8·36 in Kenya and$11·74 in Tanzania.\ud Interpretation\ud HIV-1 VCT is highly cost-effective in urban east African settings, but slightly less so than interventions such as improvement of sexually transmitted disease services and universal provision of nevirapine to pregnant women in high-prevalence settings. With the targeting of VCT to populations with high HIV-1 prevalence and couples the cost-effectiveness of VCT is improved significantly

Short communication: NKG2C+ NK cells contribute to increases in CD16+CD56- cells in HIV type 1+ individuals with high plasma viral load.

Chronic HIV-1 infection results in the expansion of both NKG2C+ and CD16+CD56- human natural killer cells. NKG2C+ cells proliferate in response to human cytomegalovirus (HCMV) and expansion of the dysfunctional CD56-CD16+ natural killer (NK) cells is associated with HIV-1 viremia. Here we report an association between increased proportions of CD56-CD16+ NK cells in viremic HIV-1+ individuals and an increased contribution of NKG2C+ cells to this subset. These data, in addition to anti-HCMV IgG serology, indicate a potential contribution of both HCMV and HIV-1 to NK cell dysfunction in HIV-1-infected individuals

Breast Milk from Tanzanian Women has Divergent Effects on Cell-Free and Cell-Associated HIV-1 Infection in Vitro.

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-Lewis(X) from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding

A Physical Theory of the Competition that Allows HIV to Escape from the Immune System

Competition within the immune system may degrade immune control of viral infections. We formalize the evolution that occurs in both HIV-1 and the immune system quasispecies. Inclusion of competition in the immune system leads to a novel balance between the immune response and HIV-1, in which the eventual outcome is HIV-1 escape rather than control. The analytical model reproduces the three stages of HIV-1 infection. We propose a vaccine regimen that may be able to reduce competition between T cells, potentially eliminating the third stage of HIV-1.Comment: 5 pages, 2 figures, to appear in Phys. Rev. Let

Characterization of the longitudinal HIV-1 quasispecies evolution in HIV-1 infected individuals co-infected with Mycobacterium tuberculosis

One of the earliest and most striking observations made about HIV is the extensive genetic variation that the virus has within individual hosts, particularly in the hypervariable regions of the env gene which is divided into 5 variable regions (V1-V5) and 5 more constant (C1-C5) regions. HIV evolves at any time over the course of an individual’s infection and infected individuals harbours a population of genetically related but non-identical viruses that are under constant change and ready to adapt to changes in their environment. These genetically heterogeneous populations of closely related genomes are called quasispecies [65]. Tuberculosis or tubercle forming disease is an acute and/or chronic bacterial infection that primarily attacks the lungs, but which may also affect the kidneys, bones, lymph nodes, and brain. The disease is caused by Mycobacterium tuberculosis (MTB), a slow growing rod-shaped, acid fast bacterium. It is transmitted from person to person through inhalation of bacteria-carrying air droplets. Worldwide, one person out of three is infected with Mycobacterium tuberculosis – two billion people in total. TB currently holds the seventh place in the global ranking of causes of death [73]. In 2008, there were an estimated 9.4 (range, 8.9–9.9 million) million incident cases (equivalent to 139 cases per 100 000 population) of TB globally [75]. A complex biological interplay occurs between M. tuberculosis and HIV in coinfected host that results in the worsening of both pathologies. HIV promotes progression of M. tuberculosis either by endogenous reactivation or exogenous reinfection [77, 78] and, the course of HIV-1 infection is accelerated subsequent to the development of TB [80]. Active TB is associated with an increase in intra-patient HIV-1 diversity both systemically and at the infected lung sites [64,122]. The sustainability or reversal of the HIV-1 quasispecies heterogeneity after TB treatment is not known. Tetanus toxoid vaccinated HIV-1 infected patients developed a transient increase in HIV-1 heterogeneity which was reversed after few weeks [121]. Emergence of a heterogeneous HIV-1 population within a patient may be one of the mechanisms to escape strong immune or drug pressure [65,128]. The existence of better fitting and/or immune escape HIV-variants can lead to an increase in HIV-1 replication [129,130]. It might be that TB favourably selected HIV-1 variants which are sources for consistent HIV-1 replication. Understanding the mechanisms underlying the impacts of TB on HIV-1 is essential for the development of effective measures to reduce TB related morbidity and mortality in HIV-1 infected individuals. In the present study we studied whether the increase in HIV-1 quasispecies diversity during active TB is reversed or preserved throughout the course of antituberculous chemotherapy. For this purpose Two time point HIV-1 quasispecies were evaluated by comparing HIV-1 infected patients with active tuberculosis (HIV-1/TB) and HIV-1 infected patients without tuberculosis (HIV-1/non TB). Plasma samples were obtained from the Frankfurt HIV cohort and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty five clones were sequenced from each patient. Various phylogenetic analyses were performed including tree inferences, intra-patient viral diversity and divergence, selective pressure, co-receptor usage prediction and two time point identity of quasispecies comparison using Mantel’s test. We found out from this study that: 1) Active TB sustains HIV-1 quasispecies diversity for longer period 2. Active TB increases the rate of HIV-1 divergence 3) TB might slow down evolution of X4 variants And we concluded that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV- 1 may be predominant for R5 viruses. The use of CCR5-coreceptor inhibitors for HIV-1/TB patients as therapeutic approach needs further investigation.Eine der ersten und überraschenden Beobachtungen, welche bei der Analyse des HI-Virus gemacht wurden ist seine ausgeprägte Genetische Variabilität besonders die hypervariable Region des env Genes betreffen. Dieses wird in 5 variable Regionen (V1-V5) sowie 5 stärker konservierte Regionen (C1-C5) unterteilt. HIV wandelt sich zu jedem Zeitpunkt im Verlauf der Infektion und jedes infizierte Individuum ist Träger einer Population von genetisch verwandten jedoch nicht identischen Viren, welche sich kontinuierlich verändern und an die Erfordernisse innerhalb der Umgebung anpassen. Diese genetisch heterogenen, jedoch eng verwandten Populationen werden Quasispecies genannt. Tuberkulose ist eine mykobakterielle Infektion, welche sowohl akute als auch chronische Verläufe zeigt. Neben den Lungen als primärem Manifestationsort können auch die Nieren, Knochen und andere Organe befallen sein. Eine von drei Personen weltweit ist mit Mycobacterium tuberculosis infiziert, insgesamt 2 Milliarden Menschen. In HIV/TB Co-Inifzierten Menschen entsteht ein komplexes Zusammenspiel zwischen HIV und M. tuberculosis, welches zu einer Verschlechterung beider Krankheitsbilder führt. HIV führt durch endogene Rekativierung oder exogene Re-Infektion zu einer Progression der Tuberkulose, welche im weiteren Verlauf die Krankheitsprogression von HIV beschleunigt. Sowohl Morbidität als auch Mortalität sind in HIV-1/TB Co-Infizierten Menschen erhöht. Aktive Lungentuberkulose und Miliartuberkulose gehen mit dem Anstieg der Diversifität der HIV Viren innerhalb eines Wirtes einher. Wie lange diese erhöhte Heterogenität der HIV Quasispecies nach der erfolgreichen Behandlung einer Tuberkulose bestehen bleibt ist bisher noch unklar. Das Verständnis des dem Zusammenspiel von HIV und TB zugrundeliegenden Mechanismus ist essentiell für die Entwicklung von effektiven Massnahmen zur Senkung der Morbidität und Mortalität in HIV/TB Co-infizierten Menschen. Die gegenwärtige Forschungsarbeit folgte daher der Frage, ob wärend einer aktiven TB Infektion eine Zunahme der Diversität der HIV-1 Quasispecies zu beobachten ist und ob diese Diversität während einer TB Therapie erhalten bleibt oder sich zurück bildet. Hierfür wurden die HIV-1 Quasispecies zu zwei Zeitpunkten untersucht, wobei Proben von HIV-1 infizierten Patienten mit aktiver Tuberkulose (HIV-1/TB) und HIV infizierte Patienten ohne Tuberkulose (HIV-1/non TB) verglichen wurden. Aus Plasmaproben der Frankfurter HIV Cohorte wurde HIV-1 RNA isoliert. C2V5 env wurde durch PCR amplifiziert und molekular cloniert. Acht bis fünfundzwanzig Clone wurden für jeden Patienten sequenziert. Mehrere phylogenetische Analysen wurden durchgeführt, welche tree inferences, Intra-Patienten- und virale Diversität und Divergenz, Selektionsdruckanalysen, Vorhersage der Co-Rezeptornutzung sowie Zweipunktanalysen der Identität von Quasispecies mit Hilfe des Mantel’s Test miteinschlossen. Die Analysen ergaben die folgenden Ergebnisse: 1) Eine aktive TB erhält die Diversität von HIV-1 Quasispecies über einen längeren Zeitraum. 2. Eine aktive TB verstärkt die HIV -1 Divergenz 3) TB könnte zu einer langsameren Evolution von X4 Varianten führen. Schlussfolgerung: eine aktive TB beeinflusst die Entwicklung der viralen Diversität und Divergenz von HIV-1 im Verlauf der Krankheit. Der Einfluss der aktiven TB auf die longitudinale Evolution von HIV-1 könnte insbesondere R5 Viren betreffen. Der Einsatz von CCR5-Corezeptor Inhibitoren in HIV-1/TB coinifizerten Patienten sollte daher in Langzeitstudien untersucht werden

Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

BACKGROUND: Cellulose acetate phthalate (CAP), a promising candidate microbicide for prevention of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted disease (STD) pathogens, was shown to inactivate HIV-1 and to block the coreceptor binding site on the virus envelope glycoprotein gp120. It did not interfere with virus binding to CD4. Since CD4 is the primary cellular receptor for HIV-1, it was of interest to study CAP binding to HIV-1 complexes with soluble CD4 (sCD4) and its consequences, including changes in the conformation of the envelope glycoprotein gp41 within virus particles. METHODS: Enzyme-linked immunosorbent assays (ELISA) were used to study CAP binding to HIV-1-sCD4 complexes and to detect gp41 six-helix bundles accessible on virus particles using antibodies specific for the α-helical core domain of gp41. RESULTS: 1) Pretreatment of HIV-1 with sCD4 augments subsequent binding of CAP; 2) there is synergism between CAP and sCD4 for inhibition of HIV-1 infection; 3) treatment of HIV-1 with CAP induced the formation of gp41 six-helix bundles. CONCLUSIONS: CAP and sCD4 bind to distinct sites on HIV-1 IIIB and BaL virions and their simultaneous binding has profound effects on virus structure and infectivity. The formation of gp41 six-helical bundles, induced by CAP, is known to render the virus incompetent for fusion with target cells thus preventing infection