Search

Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4 + T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect of in vitro HIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.

Summary A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of β1,2-xylosylation and core α1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core α1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core α1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and α1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

Anti-idiotypic antibody (Ab2) 3H6 is directed against the human monoclonal antibody 2F5, which is one of a few neutralising antibodies against HIV-1. Since the binding epitope of 2F5 is cryptic and no neutralising immune response could be elicited by several potential vaccines comprising this region, Ab2/3H6 represents a potent vaccine candidate for active immunisation. Here we describe the molecular features of Ab2/3H6 after changing the antigen binding specificity by single point mutations in the complementarity-determining region 3 of the Ab2/3H6 heavy chain. The resulting Ab2/3H6 mutants were compared in several experimental settings to the wild type Ab2/3H6 Fab fragment. Moreover, we report about an immunisation study with Ab2/3H6 Fab variants, which elicited a specific 2F5-like humoral immune response in BALB/c mice.

Antibody 2G12 is one of a small number of human immunoglobulin G (IgG) monoclonal antibodies exhibiting potent and broad human immunodeficiency virus-1 (HIV-1)-neutralizing activity in vitro, and the ability to prevent HIV-1 infection in animal models. It could be used to treat or prevent HIV-1 infection in humans, although to be effective it would need to be produced on a very large scale. We have therefore expressed this antibody in maize, which could facilitate inexpensive, large-scale production. The antibody was expressed in the endosperm, together with the fluorescent marker protein Discosoma red fluorescent protein (DsRed), which helps to identify antibody-expressing lines and trace transgenic offspring when bred into elite maize germplasm. To achieve accumulation in storage organelles derived from the endomembrane system, a KDEL signal was added to both antibody chains. Immunofluorescence and electron microscopy confirmed the accumulation of the antibody in zein bodies that bud from the endoplasmic reticulum. In agreement with this localization, N-glycans attached to the heavy chain were mostly devoid of Golgi-specific modifications, such as fucose and xylose. Surprisingly, most of the glycans were trimmed extensively, indicating that a significant endoglycanase activity was present in maize endosperm. The specific antigen-binding function of the purified antibody was verified by surface plasmon resonance analysis, and in vitro cell assays demonstrated that the HIV-neutralizing properties of the maize-produced antibody were equivalent to or better than those of its Chinese hamster ovary cell-derived counterpart.

A new scalable liposome production system is presented, which is based on the ethanol injection technique. The system permits liposome manufacture regardless of production scale, as scale is determined only by free disposable vessel volumes. Once the parameters are defined, an easy scale up can be performed by just changing the process vessels. These vessels are fully sterilizeable and all raw materials are transferred into the sanitized and sterilized system via 0.2 microm filters to guarantee an aseptic production. Liposome size can be controlled by the local lipid concentration at the injection point depending on process parameters like injection pressure, lipid concentration and injection rate. These defined process parameters are furthermore responsible for highly reproducible results with respect to vesicle diameters and encapsulation rates Compared to other technologies like the film method which is normally followed by size reduction through high pressure homogenization, ultrasonication or extrusion, no mechanical forces are needed to generate homogeneous and narrow distributed liposomes. Another important advantage of this method is the suitability for the entrapment of many different drug substances such as large hydrophilic proteins by passive encapsulation, small amphiphilic drugs by a one step remote loading technique or membrane association of antigens for vaccination approaches.

13 p. The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen. J.L.N. is supported by grants BIO2011-29792 and GIU-06/42 from The Ministerio de Ciencia e Innovacion (Spanish MICINN) and the Basque Government, respectively. J.M.V. is supported by The Ministerio de Ciencia e Innovacion (MICINN) grant BFU2010-15703/BMC and CAM (community of Madrid) grant S2009MAT-1507. A.A. received a pre-doctoral fellowship from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (VH) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the VHgenes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.

PLoS one 5(11), e13976 (2010). doi:10.1371/journal.pone.0013976, Published by PLoS [u.a.], Lawrence, Kan.

Recently we were able to show that the murine anti-idiotypic antibody (Ab2) 3H6 (Kunert et al., 2002; Antiidiotypic Antibody Inducing HIV-1 Neutralizing Antibodies Pending) significantly blocked the binding of the human mAb 2F5 to its synthetic epitope ELDKWA as well as to gp160. Furthermore, 3H6 was also capable of decreasing the in vitro neutralisation potency of 2F5 in a dose-related way. Finally, the murine 3H6 Fab fragments were capable to induce Ab1-like neutralising immune responses after applying the Ab2 to mice. Thus, Ab2/3H6 successfully mimics a neutralising epitope on gp41, which is either not or only poorly immunogenic on the native HIV-1 during natural infection. In the present study the murine Ab2/3H6 was partially humanized (the murine variable regions were fused with the corresponding human constant regions) and recombinantly expressed either as whole IgG1 (using two different expression strategies) or Fab fragment in CHO cells. Crude Ab2/3H6 IgG1 and Ab2/3H6 Fab expression supernatants were effectively purified using either protein A for Ab2/3H6 IgG1 (up to 90% recovery) or a combination of anion-exchange and size-exclusion chromatography in case of Ab2/3H6 Fab (up to 75% recovery). The purified Ab2/3H6 versions were further characterized by several specificity studies and competition experiments with neutralizing monoclonal antibody nMAb 2F5.

The human monoclonal antibody 2G12 is a member of a small group of broadly neutralizing antibodies against human immunodeficiency virus type 1. 2G12 adopts a unique variable heavy domain-exchanged dimeric configuration that results in an extensive multivalent binding surface and the ability to bind with high affinity to densely clustered high mannose oligosaccharides on the "silent" face of the gp120 envelope glycoprotein. Here, we further define the amino acids responsible for this extraordinary domain-swapping event in 2G12.

P>The use of vaginal microbicides containing human immunodeficiency virus (HIV)-neutralizing antibodies (nAbs) is a promising strategy to prevent HIV-1 infection. Although antibodies are predominantly manufactured using mammalian cells, elastin-like peptide (ELP) fusion technology improves the stability of recombinant, plant-produced proteins and facilitates their purification, making plants an alternative platform for antibody production. We generated transgenic tobacco plants accumulating four different formats of the anti-HIV-1 antibody 2G12 in the endoplasmic reticulum (ER), i.e. with ELP on either the light or heavy chain, on both, or on neither. Detailed analysis of affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters of all formats were identical to 2G12 lacking ELP produced in Chinese hamster ovary (CHO) cells. Importantly, protein purification from seeds by inverse transition cycling (ITC) did not affect the binding kinetics. Analysis of heavy chain N-glycans from leaf-derived antibodies showed that retrieval to the ER was efficient for all formats. In seeds, however, N-glycans on the naked antibody were extensively trimmed compared with those on the ELP fusion formats, and were localized to a different subcellular compartment. The in vitro HIV-neutralization properties of the tobacco-derived 2G12 were equivalent to or better than those of the CHO counterpart.

Artificial Toll-like receptor 7/8 (TLR7/8) ligands can endow plasmacytoid dendritic cells (pDCs) with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–dependent lytic properties. Keeping in mind that ssRNA serves as natural TLR7/8 ligand, we searched for TRAIL-expressing cells in persons infected with HIV and identified TRAIL+ pDCs in HIV-1 viremic persons, but not in nonviremic and healthy persons. TRAIL expression on pDCs was directly correlated with individual viral loads. Conversely, HIV-1 viremia was found to be associated with the up-regulation of the apoptosis-transmitting receptor TRAIL R1 on activated CD4+ T cells. As a consequence, the latter became susceptible to TRAIL-dependent pDC-mediated killing. In contrast, initiation of antiretroviral therapy led to the up-regulation of apoptosis-inhibiting TRAIL R4 on CD4+ T cells, which subsequently became resistant against pDC-mediated cellular injury. Definition of pDCs as killers of CD4+ T cells implies a new mechanism of disease progression in HIV infection.

A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.

To date only a few neutralizing antibodies against HIV-1 exist. Since these neutralizing antibodies are only rarely found in sera of HIV-1 infected individuals an active vaccine is required. We recently developed murine anti-idiotypic antibody Ab2/3H6 against monoclonal antibody (mAb) 2F5, which is one of the most prominent neutralizing antibodies. Anti-idiotypic antibody Ab2/3H6 has been partially humanized and expressed as whole immunoglobulin G in Chinese hamster ovary cells in order to minimize the human anti-mouse antibody response. Here we describe the expression, purification, and immunohistochemical characterization of the chimeric Ab2/3H6 Fab fragment, which was finally used beside the whole IgG1 as an antigen for immunization of guinea pigs. The crude sera were screened for specific antibodies against the epitope of mAb 2F5 ELDKWA as well as for reactivity against HIV-1 gp41.

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5. The results of these studies support the high specificity of the recombinantly expressed Ab2s to the idiotype. Apparent affinities were designated by end point measurement and were similar compared to the murine Ab2/3H6. Moreover, the inhibition potency of chimeric Ab2/3H6 analyzed by in vitro studies could be shown to be the same as that detected for the hybridoma-derived murine Ab2/3H6.

Objective: The human monoclonal antibodies (mAb) 2F5, 2G12, and 4E10 are three of the most broadly neutralizing antibodies against HIV-1. Although they have been shown to prevent de novo infection in vivo, their potential for treatment of chronic infection is less clear. One major obstacle may be the emergence of resistant viruses during mAb treatment. Design: To assess whether escape mutants can be generated in vitro which are resistant to all three mAbs, two neutralization-sensitive T-cell line-adapted viruses and two primary isolates were passaged in the presence of increasing concentrations of 2F5, 2G12, 4E10, and a 1: 1: 1 mixture. To get insight into viral escape in vivo, viruses were isolated from eight patients treated with repeated infusions of 2F5/2G12/4E10. Results: In vitro, viruses resistant to a single mAb emerged after 3–22 weeks. Generation of viruses resistant to the triple-combination was a slower process characterized by recurrent loss of virus replication. Some generated triple-resistant viruses seemed to be impaired in their replicative fitness. Neutralization resistance to 2F5 and partly 4E10 could be attributed to amino acid mutations in the mAb epitopes, but not for 2G12. In vivo, none of the patients developed detectable viruses that escaped neutralization by all three mAbs within the 77-day observation period. Virus escape occurred only to 2G12 in three patients. Conclusions: In summary, the findings of the in vivo study and the difficulty in generating multi-resistance in vitro together with the fact that some generated viruses seemed to have impaired replication fitness indicate that 2F5, 2G12, and 4E10 may be useful for therapy in HIV-1 infection.