Co-receptor signaling in the pathogenesis of neuroHIV.

The HIV co-receptors, CCR5 and CXCR4, are necessary for HIV entry into target cells, interacting with the HIV envelope protein, gp120, to initiate several signaling cascades thought to be important to the entry process. Co-receptor signaling may also promote the development of neuroHIV by contributing to both persistent neuroinflammation and indirect neurotoxicity. But despite the critical importance of CXCR4 and CCR5 signaling to HIV pathogenesis, there is only one therapeutic (the CCR5 inhibitor Maraviroc) that targets these receptors. Moreover, our understanding of co-receptor signaling in the specific context of neuroHIV is relatively poor. Research into co-receptor signaling has largely stalled in the past decade, possibly owing to the complexity of the signaling cascades and functions mediated by these receptors. Examining the many signaling pathways triggered by co-receptor activation has been challenging due to the lack of specific molecular tools targeting many of the proteins involved in these pathways and the wide array of model systems used across these experiments. Studies examining the impact of co-receptor signaling on HIV neuropathogenesis often show activation of multiple overlapping pathways by similar stimuli, leading to contradictory data on the effects of co-receptor activation. To address this, we will broadly review HIV infection and neuropathogenesis, examine different co-receptor mediated signaling pathways and functions, then discuss the HIV mediated signaling and the differences between activation induced by HIV and cognate ligands. We will assess the specific effects of co-receptor activation on neuropathogenesis, focusing on neuroinflammation. We will also explore how the use of substances of abuse, which are highly prevalent in people living with HIV, can exacerbate the neuropathogenic effects of co-receptor signaling. Finally, we will discuss the current state of therapeutics targeting co-receptors, highlighting challenges the field has faced and areas in which research into co-receptor signaling would yield the most therapeutic benefit in the context of HIV infection. This discussion will provide a comprehensive overview of what is known and what remains to be explored in regard to co-receptor signaling and HIV infection, and will emphasize the potential value of HIV co-receptors as a target for future therapeutic development.

Existence of Replication-Competent Minor Variants with Different Coreceptor Usage in Plasma from HIV-1-Infected Individuals.

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations.IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.

Modelling coupled within host and population dynamics of [Formula: see text] and [Formula: see text] HIV infection.

Most existing models have considered the immunological processes occurring within the host and the epidemiological processes occurring at population level as decoupled systems. We present a new model using continuous systems of non linear ordinary differential equations by directly linking the within host dynamics capturing the interactions between Langerhans cells, CD4[Formula: see text] T-cells, R5 HIV and X4 HIV and the without host dynamics of a basic compartmental HIV/AIDS model. The model captures the biological theories of the cells that take part in HIV transmission. The study incorporates in its analysis the differences in time scales of the fast within host dynamics and the slow without host dynamics. In the mathematical analysis, important thresholds, the reproduction numbers, were computed which are useful in predicting the progression of the infection both within the host and without the host. The study results showed that the model exhibits four within host equilibrium points inclusive of three endemic equilibria whose effects translate into different scenarios at the population level. All the endemic equilibria were shown to be globally stable using Lyapunov functions and this is an important result in linking the within host dynamics to the population dynamics, because the disease free equilibrium point ceases to exist. The effects of linking were observed on the endemic equilibrium points of both the within host and population dynamics. Linking the two dynamics was shown to increase in the viral load within the host and increase in the epidemic levels in the population dynamics.

HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies.

UNLABELLED: Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. IMPORTANCE: While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

The two human CXCR4 isoforms display different HIV receptor activities: consequences for the emergence of X4 strains.

CXCR4 is a chemokine receptor that plays key roles with its specific ligand, CXCL12, in stem cell homing and immune trafficking. It is also used as a coreceptor by some HIV-1 strains (X4 strains), whereas other strains (R5 strains) use an alternative coreceptor, CCR5. X4 strains mainly emerge at late stages of the infection and are linked to disease progression. Two isoforms of this coreceptor have been described in humans: CXCR4-A and CXCR4-B, corresponding to an unspliced and a spliced mRNA, respectively. In this study, we show that CXCR4-B, but not CXCR4-A, mediates an efficient HIV-1 X4 entry and productive infection. Yet, the chemotactic activity of CXCL12 on both isoforms was similar. Furthermore, HIV-R5 infection favored CXCR4-B expression over that of CXCR4-A. In vitro infection with an R5 strain increased CXCR4-B/CXCR4-A mRNA ratio in PBMCs, and this ratio correlated with HIV RNA plasma level in R5-infected individuals. In addition, the presence of the CXCR4-B isoform favored R5 to X4 switch more efficiently than did CXCR4-A in vitro. Hence, the predominance of CXCR4-B over CXCR4-A expression in PBMCs was linked to the ability of circulating HIV-1 strains to use CXCR4, as determined by genotyping. These data suggest that R5 to X4 switch could be favored by R5 infection-induced overexpression of CXCR4-B. Finally, we achieved a specific small interfering RNA-mediated knockdown of CXCR4-B. This represents a proof of concept for a possible gene-therapeutic approach aimed at blocking the HIV coreceptor activity of CXCR4 without knocking down its chemotactic activity.

Induction of HIV-blocking anti-CCR5 IgA in Peyers's patches without histopathological alterations.

UNLABELLED: The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. IMPORTANCE: The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.

Regulation of adaptive immunity by the fractalkine receptor during autoimmune inflammation.

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells, and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the CNS during experimental autoimmune encephalomyelitis (EAE). Our results show that, in models of active EAE, Cx3cr1(-/-) mice exhibited more severe neurologic deficiencies. Bone marrow chimeric mice confirmed that CX3CR1 deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115(+)Ly6C(-)CD11c(+) dendritic cells into EAE-affected brains that correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ- and IL-17-producing T cells in lymphoid tissues of CX3CR1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient dendritic cells. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein35-55-reactive wild-type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared with wild-type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.

HIV-1 neutralizing antibodies display dual recognition of the primary and coreceptor binding sites and preferential binding to fully cleaved envelope glycoproteins.

The gp120 CD4 binding site (CD4bs) and coreceptor binding site (CoRbs) are two functionally conserved elements of the HIV-1 envelope glycoproteins (Env). We previously defined the presence of CD4bs-neutralizing antibodies in the serum of an HIV-1-infected individual and subsequently isolated the CD4bs-specific monoclonal antibodies (MAbs) VRC01 and VRC03 from the memory B cell population. Since this donor's serum also appeared to contain neutralizing antibodies to the CoRbs, we employed a differential fluorescence-activated cell sorter (FACS)-based sorting strategy using an Env trimer possessing a CoRbs knockout mutation (I420R) to isolate specific B cells. The MAb VRC06 was recovered from these cells, and its genetic sequence allowed us to identify a clonal relative termed VRC06b, which was isolated from a prior cell sort using a resurfaced core gp120 probe and its cognate CD4bs knockout mutant. VRC06 and VRC06b neutralized 22% and 44% of viruses tested, respectively. Epitope mapping studies revealed that the two MAbs were sensitive to mutations in both the gp120 CoRbs and the CD4bs and could cross-block binding of both CD4bs and CoRbs MAbs to gp120. Fine mapping indicated contacts within the gp120 bridging sheet and the base of the third major variable region (V3), which are elements of the CoRbs. Cell surface binding assays demonstrated preferential recognition of fully cleaved Env trimers over uncleaved trimers. Thus, VRC06 and VRC06b are Env trimer precursor cleavage-sensitive neutralizing MAbs that bind to a region of gp120 that overlaps both the primary and the secondary HIV-1 receptor binding sites.

CX3CR1+ CD8alpha+ dendritic cells are a steady-state population related to plasmacytoid dendritic cells.

Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8alpha. Here we describe a subset of CD8alpha(+) DC in lymphoid organs of naïve mice characterized by expression of the CX(3)CR1 chemokine receptor. CX(3)CR1(+) CD8alpha(+) DC lack hallmarks of classical CD8alpha(+) DC, including IL-12 secretion, the capacity to cross-present antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX(3)CR1(+) CD8alpha(+) DC resemble CD8alpha(-) cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX(3)CR1(+) CD8alpha(+) DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D-J Ig gene rearrangements and that development of CX(3)CR1(+) CD8alpha(+) DC requires E2-2, the critical transcriptional regulator of PDC. Thus, CX(3)CR1(+) CD8alpha(+) DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8alpha(+) and CD8alpha(-) DC, and should assist the identification of human counterparts of murine DC subsets.

Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy.

CXC-chemokine receptor (CXCR)-4/fusin, a newly discovered co-receptor for T-cell line (T)-tropic HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. The occurrence of T-tropic HIV viruses is associated with CD4-positive cell decline and progression to AIDS, suggesting that the T-tropic HIV-1 contributes to AIDS pathogenesis. In this study, we used a novel strategy to inactivate CXCR-4 by targeting a modified CXC-chemokine to the endoplasmic reticulum (ER) to block the surface expression of newly synthesized CXCR-4. The genetically modified lymphocytes expressing this intracellular chemokine, termed "intrakine", are immune to T-tropic virus infection and appear to retain normal biological features. Thus, this genetic intrakine strategy is uniquely targeted at the conserved cellular receptor for the prevention of HIV-1 entry and may be developed into an effective treatment for HIV-1 infection and AIDS.

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells.

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.

Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier.

Contact between various epithelial cell lines and HIV chronically infected mononuclear cell lines results in a massive and rapid budding of HIV virions toward the epithelium followed by their internalization into epithelial endosome-like structures. Here it is shown that as early as 30 minutes after apical contact, primary virus isolates generated from primary peripheral blood leukocytes from HIV-infected patients can cross an epithelial cell line barrier using transcytosis, the characteristic epithelial transcellular vesicular pathway. As the next step in the spread of infection, transcytosed HIV particles can productively infect mononuclear cells located at the basolateral side of the epithelial barrier. These observations suggest an alternative, rapid and efficient mechanism for transmission of HIV across an intact epithelial barrier.

Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis.

Leukocyte migration in response to cell attractant gradients or chemotaxis is a key phenomenon both in cell movement and in the inflammatory response. Chemokines are quite likely to be the key molecules directing migration of leukocytes that involve cell polarization with generation of specialized cell compartments. The precise mechanism of leukocyte chemoattraction is not known, however. In this study, we demonstrate that the CC chemokine receptors CCR2 and CCR5, but not cytokine receptors such as interleukin (IL)-2Ralpha, IL-2Rbeta, tumor necrosis factor receptor 1, or transforming growth factor betaR, are redistributed to a pole in T cells that are migrating in response to chemokines. Immunofluorescence and confocal microscopy studies show that the chemokine receptors concentrate at the leading edge of the cell on the flattened cell-substratum contact area, induced specifically by the signals that trigger cell polarization. The redistribution of chemokine receptors is blocked by pertussis toxin and is dependent on cell adhesion through integrin receptors, which mediate cell migration. Chemokine receptor expression on the leading edge of migrating polarized lymphocytes appears to act as a sensor mechanism for the directed migration of leukocytes through a chemoattractant gradient.

Heterosexual transmission of human immunodeficiency virus type 1 subtype C: Macrophage tropism, alternative coreceptor use, and the molecular anatomy of CCR5 utilization.

Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Simian immunodeficiency virus SIVrcm, a unique CCR2-tropic virus, selectively depletes memory CD4+ T cells in pigtailed macaques through expanded coreceptor usage in vivo.

Simian immunodeficiency virus SIVrcm, which naturally infects red-capped mangabeys (RCMs), is the only SIV that uses CCR2 as its main coreceptor due to the high frequency of a CCR5 deletion in RCMs. We investigated the dynamics of SIVrcm infection to identify specific pathogenic mechanisms associated with this major difference in SIV biology. Four pigtailed macaques (PTMs) were infected with SIVrcm, and infection was monitored for over 2 years. The dynamics of in vivo SIVrcm replication in PTMs was similar to that of other pathogenic and nonpathogenic lymphotropic SIVs. Plasma viral loads (VLs) peaked at 10(7) to 10(9) SIVrcm RNA copies/ml by day 10 postinoculation (p.i.). A viral set point was established by day 42 p.i. at 10(3) to 10(5) SIVrcm RNA copies/ml and lasted up to day 180 p.i., when plasma VLs decreased below the threshold of detection, with blips of viral replication during the follow-up. Intestinal SIVrcm replication paralleled that of plasma VLs. Up to 80% of the CD4(+) T cells were depleted by day 28 p.i. in the gut. The most significant depletion (>90%) involved memory CD4(+) T cells. Partial CD4(+) T-cell restoration was observed in the intestine at later time points. Effector memory CD4(+) T cells were the least restored. SIVrcm strains isolated from acutely infected PTMs used CCR2 coreceptor, as reported, but expansion of coreceptor usage to CCR4 was also observed. Selective depletion of effector memory CD4(+) T cells is in contrast with predicted in vitro tropism of SIVrcm for macrophages and is probably due to expansion of coreceptor usage. Taken together, these findings emphasize the importance of understanding the selective forces driving viral adaptation to a new host.

The CX(3)C-chemokine fractalkine in kidney diseases.

The chemokine CX(3)C-L/FKN is expressed in both soluble and transmembrane/mucin hybrid forms, thus combining chemoattractant functions together with receptor/adhesion molecule properties. In contrast to other chemokine receptors, CX(3)C-R is expressed not only on lymphoid cell populations, but also on several intrinsic cells including tubular epithelial cells and renal fibroblasts where it regulates various aspects of cell viability, matrix synthesis and degradation, migration, inflammation as well as oxidative stress. In the kidney, the chemokines/receptor pair has been shown to play a role in nephrogenesis as well as in the pathogenesis primary and secondary nephropathies. In several animal models and human specimens with acute and chronic renal failure including allograft nephropathy, CX(3)C-L/CX(3)C-R has been shown to exert immune and non-immune mediated renal damages. A blockade of this chemokine system ameliorated acute and chronic renal damages, though the latter to a more robust extent. There seems to a role of the CX(3)C-L/CX(3)C-R pair in mediating acute renal inflammation as well as in progressive chronic renal failure. However, functional studies are lacking for many aspects and further studies are necessary to better define the functional properties of CX(3)C-L/FKN and its receptor.

Evidence for cell-surface association between fusin and the CD4-gp120 complex in human cell lines.

Accessory cell-surface molecules involved in the entry of human immunodeficiency virus-type 1 into cells have recently been identified and shown to belong to the family of chemokine receptors. Treatment of human cell lines with soluble monomeric gp120 at 37 degrees C induced an association between the surface CD4-gp120 complex and a 45-kilodalton protein, which can be down-modulated by the phorbol ester phorbol 12-myristate 13-acetate. The three proteins were coprecipitated from the cell membranes with antibodies to CD4 or to gp120. The 45-kilodalton protein comigrated with fusin on sodium dodecyl sulfate gels and reacted with rabbit antisera to fusin in protein immunoblots. No 45-kilodalton protein could be coprecipitated from similarly treated nonhuman cells. However, infection of 3T3.CD4.401 cells with vaccinia-fusin recombinant virus (vCBYF1), followed by gp120 treatment, resulted in coprecipitation of fusin and CD4.401 molecules from their membranes. Together these data provide evidence for physical association between fusin and the CD4-gp120 complex on cell membranes.

LPS reduces HIV-1 replication in primary human macrophages partly through an endogenous production of type I interferons.

It has been proposed that the systemic immune activation state seen in HIV-1-infected patients is caused by circulating microbial products such as lipopolysaccharide (LPS). Given that macrophages play a key role in HIV-1 pathogenesis, we investigated the LPS-mediated effect on HIV-1 replication in cells of the myeloid lineage. We demonstrate that LPS promotes virus gene expression in a monocytic cell line while it diminishes virus production in primary human monocyte-derived macrophages (MDM). The incapacity of LPS to drive HIV-1 production in MDM was not due to its inability to activate the ubiquitous transcription factor NF-kappaB even in virus-infected cells. Neutralization of type I interferons (IFN) with B18R, a soluble vaccinia virus-coded type I IFN receptor, significantly but not totally diminished the antiviral activity of LPS. Therefore, inhibition of HIV-1 replication in MDM treated with microbial-derived LPS resulted from the induction of type I interferons and a yet to be defined soluble factor.

Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120.

During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation.

HIV-1 neutralization by chimeric CD4-CG10 polypeptides fused to human IgG1.

The envelope glycoprotein of HIV-1 is the principal target for entry inhibitors. The use of soluble CD4 has been found to be impractical as most clinical isolates are resistant to neutralization at feasible concentrations. CG10 is one of a small group of monoclonal antibodies specific to CD4-induced epitopes, which are structurally associated with the chemokine receptor-binding site and are capable of blocking the interaction of gp120 with its obligatory co-receptor. We have reasoned that fusing the single chain Fv of CG10 with CD4 can lead to increased HIV-1 neutralization activity and that this effect could be further enhanced by engrafting this chimeric construct onto an IgG Fc. Here we report the cloning of the genes encoding the variable regions of CG10 heavy and light chains and demonstrate that when attached to human IgG1 Fc, the single chain Fv of CG10 retains the binding properties of the original mouse antibody. Fusing CG10 single chain Fv with the gp120-binding portion of CD4 on a human IgG1 Fc backbone results in stronger binding of gp120 of different tropisms and in enhanced neutralization of laboratory-adapted strains and most, but not all, clade B and clade C isolates tested. Our findings underscore the potential use of CD4-based fusion proteins in the design of HIV immuno-therapeutics.