Maureen M. Goodenow

Microbial translocation induces persistent macrophage activation unrelated to HIV-1 levels or T cell activation following therapy

Objective:HIV-1 replication and microbial translocation occur concomitant with systemic immune activation. This study delineates mechanisms of immune activation and CD4 T-cell decline in pediatric HIV-1 infection. Design:Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric study volunteers prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral therapy (ART). All HIV-1-infected patients reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus. Methods:Systemic immune activation was determined by polychromatic flow cytometry of blood lymphocytes and ELISA for plasma soluble CD27, soluble CD14, and tumor necrosis factor. Microbial translocation was evaluated by limulus amebocyte lysate assay to detect bacterial lipopolysaccharide (LPS) and ELISA for antiendotoxin core antigen immunoglobulin M (IgM) antibodies. Immune activation markers were compared with viral load, CD4 cell percentage, and LPS by regression models. Comparisons between healthy and HIV-1-infected or between different viral outcome groups were performed by nonparametric rank sum. Results:Microbial translocation was detected in healthy infants but resolved with age (P < 0.05). LPS and soluble CD14 levels were elevated in all HIV-1-infected patients (P < 0.05 and P < 0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T-cell reconstitution but viral rebound following ART continued to display high levels of soluble CD27. Conclusion:Microbial translocation in pediatric HIV-1 infection is associated with persistent monocyte/macrophage activation independent of viral replication or T-cell activation.

HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes

HIV‐1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4+‐transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell‐ and viral strain‐specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV‐1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis.

Persistence of multiple maternal genotypes of human immunodeficiency virus type I in infants infected by vertical transmission.

The extent of nucleotide variation within the HIV-1 env hypervariable domains serves as a marker of virus genotypes within infected individuals and as a means to track transmission of the virus between individuals. We analyzed env V1 and V2 sequences in longitudinal samples from two HIV-1-infected mothers, each with three children infected by maternal transmission of the virus. Sequences in samples that were obtained from two infants at 2 d and 4 wk after birth displayed more variation in V1 and V2 than maternal samples obtained at the same times. Multiple HIV-1 genotypes were identified in each mother. In each family, multiple maternal HIV-1 genotypes were transmitted to the infants. Specific amino acid residues in the hypervariable domains were conserved within sequences from each family producing a family-specific amino acid signature pattern in V1 and V2. Viruses that were highly related to maternal viruses in signature pattern persisted for as long as 4 yr in the older children. Results support a model of transmission involving multiple HIV-1 genotypes with development of genetic variation from differential outgrowth and accumulation of genetic changes within each individual.

CD4+ memory T cells are the predominant population of HIV-1-infected lymphocytes in neonates and children

Background: CD4+ memory T cells express CD45RO and are the principal viral reservoir in HIV‐infected adults. In infants and children, CD45RO T cells comprise the minority of the CD4+ T‐cell population. The majority of blood CD4+ T cells are naive, expressing CD45RA. Objective: To determine the developmental stage at which pediatric CD4+ T cells become susceptible to HIV‐1 infection in vivo by determining which T‐cell population harbors HIV‐1 proviral DNA. Design: A prospective, cross‐sectional analysis of peripheral blood CD8+ T cells, CD45RA, or CD45RO CD4+ T cells obtained from 10 HIV‐infected neonates and children were analysed for provirus. Methods: Semi‐quantitative polymerase chain reaction methods were used to detect HIV‐1 proviral DNA within purified lymphocyte populations selected using immunoaffinity magnetic microspheres. Results: CD8+ T cells harbored no detectable HIV‐1, indicating that infection of common thymocytes does not contribute to the population of infected blood T cells. Infive children and two of the five neonates, the CD4+ CD45RO memory T lymphocytes contained 10–100‐fold greater numbers of infected cells than the CD4+ CD45RA naive T‐cell population. Three neonates, who exhibited rapid disease progression, demonstrated high proviral levels in their CD4+ CD45RA T cells. The normal age‐related predominance of CD4+ CD45RA T cells was preserved independent of CD4+ T‐cell attribution. Conclusions: The majority of HIV‐1‐infected blood CD4+ T cells in infants and children are restricted to the small population of terminally differentiated CD4+ CD45RO memory T cells. Neonates with rapid CD4+ T‐cell attrition display high levels of provirus in their CD4+ CD45RA T‐cell population.

Immunoreconstitution after ritonavir therapy in children with human immunodeficiency virus infection involves multiple lymphocyte lineages.

OBJECTIVE To evaluate lymphocyte reconstitution after protease inhibitor therapy in children with human immunodeficiency virus (HIV) infection. STUDY DESIGN Forty-four HIV-infected children receiving ritonavir monotherapy followed by the addition of zidovudine and didanosine were evaluated during a phase I/II clinical trial. The cohort had a median age of 6.8 years and advanced disease (57% Centers for Disease Control and Prevention stage C, 73% immune stage 3) and was naive to protease inhibitor therapy. RESULTS After 4 weeks of therapy, there was a significant increase in CD4(+) and CD8(+) T cells. CD4(+) T cells continued to increase, whereas CD8(+) T cells returned to baseline by 24 weeks. Unexpectedly, there was a significant increase in B cells. Changes in CD4(+) T-cell subsets revealed an initial increase in CD4(+) CD45RO T cells followed by a sustained increase in CD4(+) CD45RA T cells. Children <6 years of age had the highest increase in all lymphocyte populations. Significant improvement in CD4(+) T-cell counts was observed even in those children whose viral burden returned to pre-therapy levels. CONCLUSIONS Early increases in lymphocytes after ritonavir therapy are a result of recirculation, as shown by increases in B cells and CD4(+) CD45RO and CD8(+) T cells. Children exhibited a high potential to reconstitute CD4(+) CD45RA T cells even with advanced disease and incomplete viral suppression.

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease.

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6(54/84) variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6(54/84) variant bound to ritonavir and indinavir shows structural changes in the 80s loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10s and 30s loops, which suggest possible changes in the dynamics of flap opening and closing.

Increased Replication of Non-Syncytium-Inducing HIV Type 1 Isolates in Monocyte-Derived Macrophages Is Linked to Advanced Disease in Infected Children

Non-syncytium-inducing (NSI) strains of HIV-1 prevail among most infected children, including pediatric patients who develop advanced disease, severe immune suppression, and die. A study was designed to address the hypothesis that genotypic and/or phenotypic markers can distinguish NSI viruses isolated during early infection from NSI viruses found in advanced disease. Primary HIV-1 isolates, which were obtained from 43 children, adolescents, and adults who displayed a cross-section of clinical disease and immune suppression but were untreated by protease inhibitor antiretroviral therapy, were characterized for replication phenotype in different cell types. Most individuals (81%) harbored NSI viruses and almost half had progressed to advanced disease or severe immune deficiency. About 51% of NSI isolates produced low levels of p24 antigen (median, 142 pg/ml) in monocyte-derived macrophages (MDMs), 31% produced medium levels (median, 1584 pg/ml), and 17% produced high levels (median, 81,548 pg/ml) (p < 0.001). Seven of eight syncytium-inducing isolates also replicated in MDMs and displayed a dual-tropic phenotype that was associated with advanced disease. Replication of NSI viruses in MDMs varied as much as 100- to 1000-fold and was independent of replication in peripheral blood mononuclear cells. Replication in MDMs provided a clear biological feature to distinguish among viruses that were otherwise identical by NSI phenotype, V3 genotype, and CCR5 coreceptor usage. Low-level MDM replication was characteristic of viruses isolated from asymptomatic individuals, including long-term survivors. Enhanced MDM replication was related to morbidity and mortality among patients. Replication levels in MDMs provide a novel prognostic indicator of pathogenic potential by NSI viruses.

HIV-1 Activates Macrophages Independent of Toll-Like Receptors

Background Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1). Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection. Methodology/Principal Findings To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK), and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR) pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1β, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication. Conclusions/Significance HIV-1 induced a primed, proinflammatory state, M1HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute to immune pathogenesis, and provide important targets for therapeutic approaches.

Transforming growth factor-β1 increases CXCR4 expression, stromal-derived factor-1α-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages

Stromal‐derived factor‐1 (SDF‐1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus‐1 (HIV‐1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV‐1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV‐1, blood and tissue levels of transforming growth factor‐β1 (TGF‐β1) are increased. The purpose of this study was to evaluate the effects of TGF‐β1 on CXCR4 expression and function in primary human monocyte‐derived macrophages (MDMs) and rat microglia. TGF‐β1 up‐regulated CXCR4 and enhanced SDF‐1α‐stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual‐tropic CXCR4‐using HIV‐1 (D‐X4). In contrast, TGF‐β1 failed to increase CCR5 expression or infection by a CCR5‐using virus in MDMs. Our data demonstrate that TGF‐β1 enhances macrophage responsiveness to SDF‐1α stimulation and susceptibility to HIV‐1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual‐tropic X4 viral variants at later stages of HIV‐1 infection.

High-Resolution Molecular Epidemiology and Evolutionary History of HIV-1 Subtypes in Albania

Background HIV-1 epidemic in Western Europe is largely due to subtype B. Little is known about the HIV-1 in Eastern Europe, but a few studies have shown that non-B subtypes are quite common. In Albania, where a recent study estimated a ten-fold increase of AIDS incidence during the last six years, subtype A and B account for 90% of the know infections. Methodology/Principal Findings We investigated the demographic history of HIV-1 subtype A and B in Albania by using a statistical framework based on coalescent theory and phylogeography. High-resolution phylogenetic and molecular clock analysis showed a limited introduction to the Balkan country of subtype A during the late 1980s followed by an epidemic outburst in the early 1990s. In contrast, subtype B was apparently introduced multiple times between the mid-1970s and mid-1980s. Both subtypes are growing exponentially, although the HIV-1A epidemic displays a faster growth rate, and a significantly higher basic reproductive number R0. HIV-1A gene flow occurs primarily from the capital Tirane, in the center of the country, to the periphery, while HIV-1B flow is characterized by a balanced exchange between center and periphery. Finally, we calculated that the actual number of infections in Albania is at least two orders of magnitude higher than previously thought. Conclusions/Significance Our analysis demonstrates the power of recently developed computational tools to investigate molecular epidemiology of pathogens, and emphasize the complex factors involved in the establishment of HIV-1 epidemics. We suggest that a significant correlation exists between HIV-1 exponential spread and the socio-political changes occurred during the Balkan wars. The fast growth of a relatively new non-B epidemic in the Balkans may have significant consequences for the evolution of HIV-1 epidemiology in neighboring countries in Eastern and Western Europe.