Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

Natural antibodies to CCR5 from breast milk block infection of macrophages and dendritic cells with primary R5-tropic HIV-1

In the present study, we demonstrate that breast milk of 66% and 83% of HIV-seronegative and seropositive women, respectively, contains natural Abs of the secretory IgA and IgG isotypes directed against the CCR5 coreceptor for R5-tropic strains of HIV-1. Abs to CCR5 were affinity purified on a matrix to which a synthetic peptide corresponding to the second extracellular loop of CCR5 had been coupled. The purified Abs bound to the CCR5 peptide in a dose-dependent fashion and to both native CCR5 expressed by Chinese hamster ovary cells transfected with CCR5 gene, macrophages, and immature dendritic cells. Although the avidity differed, the amount of anti-CCR5 Abs did not significantly differ between breast milk of HIV-seropositive and -seronegative women. Purified anti-CCR5 Abs inhibited up to 75% infection of macrophages and dendritic cells with HIV(BaL) and HIV(JR-CSF). Our observations provide evidence for a role of natural Abs to CCR5 in breast milk in controlling transmissibility of HIV through breastfeeding.     

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tag-deleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.     

Induction of autoantibodies to CCR5 in macaques and subsequent effects upon challenge with an R5-tropic simian/human immunodeficiency virus

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV(SF162P3) in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV(SF162P3). Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.     

Development of targeted gene transfer into human primary T lymphocytes and macrophages using high-titer recombinant HIV vectors

Primary human lymphocytes and macrophages are an important target cells for human immunodeficiency virus (HIV). For targeted gene transfer into CD4(+) lymphocytes and macrophages, we constructed HIV vectors with envelope glycoprotein (gp120) from the T-cell tropic BH10 strain and the macrophage tropic SF162, and developed an improved strategy for preparation of high-titer HIV vectors. Among several possible procedures, we found that ultrafiltration using CENTRIPREP columns was highly effective to concentrate HIV particles. The titer could be increased four orders of magnitudes. The total recovery was more than 80%. No replication-competent cytopathic HIV was detected in concentrated vector preparation. Using the high-titer HIV vector carrying the enhanced green fluorescent protein (EGFP) gene, we transduced human primary lymphocytes and macrophages. FACS analysis showed that the T-cell tropic vector could transduce 40-80% of CD4(+) T-cells stimulated with IL2 plus PHA and 20-50% of unstimulated cells. The macrophage tropic vector was shown to transduce approximately 20% of terminally differentiated macrophages. These results represent the initial report of targeted gene transfer into terminally differentiated macrophages. These results also indicate that these HIV vectors are useful for the manipulation of gene expression in HIV infectable cells and the development of gene therapy targeting lymphocytes and macrophages.     

Antibodies to C-C chemokine receptor 5 in normal human IgG block infection of macrophages and lymphocytes with primary R5-tropic strains of HIV-1

In the present study, we demonstrate that normal human IgG for therapeutic use (i.v. Ig) contains natural Abs directed against the CCR5 coreceptor for HIV-1. Abs to CCR5 were isolated from i.v. Ig using an affinity matrix consisting of a synthetic peptide corresponding to the N-terminus of CCR5 coupled to Sepharose. Natural anti-CCR5 Abs inhibited the binding of RANTES to macrophages, demonstrating their interaction with the coreceptor of R5-tropic HIV-1. Affinity-purified anti-CCR5 Ig further inhibited infection of lymphocytes and monocytes/macrophages with primary and laboratory-adapted strains of HIV-1, but did not inhibit infection with X4-tropic HIV. Our results suggest that anti-CCR5 Abs from healthy immunocompetent donors may be suitable for development of novel passive immunotherapy regimens in specific clinical settings in HIV infection.     

DNA-free recombinant SV40 capsids protect mice from acute renal failure by inducing stress response, survival pathway and apoptotic arrest

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses.     

Regulation of human immunodeficiency virus type 1 infection, beta-chemokine production, and CCR5 expression in CD40L-stimulated macrophages: immune control of viral entry

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+ peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and beta-chemokine production, and beta-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of beta-chemokines (macrophage inhibitory proteins 1alpha and 1beta and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-alpha led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the beta-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.     

Structural insights from binding poses of CCR2 and CCR5 with clinically important antagonists: a combined in silico study

Chemokine receptors are G protein-coupled receptors that contain seven transmembrane domains. In particular, CCR2 and CCR5 and their ligands have been implicated in the pathophysiology of a number of diseases, including rheumatoid arthritis and multiple sclerosis. Based on their roles in disease, they have been attractive targets for the pharmaceutical industry, and furthermore, targeting both CCR2 and CCR5 can be a useful strategy. Owing to the importance of these receptors, information regarding the binding site is of prime importance. Structural studies have been hampered due to the lack of X-ray crystal structures, and templates with close homologs for comparative modeling. Most of the previous models were based on the bovine rhodopsin and β2-adrenergic receptor. In this study, based on a closer homolog with higher resolution (CXCR4, PDB code: 3ODU 2.5 Å), we constructed three-dimensional models. The main aim of this study was to provide relevant information on binding sites of these receptors. Molecular dynamics simulation was done to refine the homology models and PROCHECK results indicated that the models were reasonable. Here, binding poses were checked with some established inhibitors of high pharmaceutical importance against the modeled receptors. Analysis of interaction modes gave an integrated interpretation with detailed structural information. The binding poses confirmed that the acidic residues Glu291 (CCR2) and Glu283 (CCR5) are important, and we also found some additional residues. Comparisons of binding sites of CCR2/CCR5 were done sequentially and also by docking a potent dual antagonist. Our results can be a starting point for further structure-based drug design.     

Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the delta ccr5 mutation.

The alpha-chemokine receptor fusin (CXCR-4) and beta-chemokine receptor CCR5 serve as entry cofactors for T-cell (T)-tropic and macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively, when expressed with CD4 in otherwise nonpermissive cells. Some M-tropic and dual-tropic strains can also utilize other beta-chemokine receptors, such as CCR2b and CCR3. A mutation of CCR5 (delta ccr5) was recently found to be common in certain populations and appears to confer protection against HIV-1 in vivo. Here, we show that this mutation results in a protein that is expressed intracellularly but not on the cell surface. Primary CD4 T cells from delta ccr5 homozygous individuals were highly resistant to infection with prototype M-tropic HIV-1 strains, including an isolate (YU-2) that uses CCR5 and CCR3, but were permissive for both a T-tropic strain (3B) and a dual-tropic variant (89.6) that uses CXCR-4, CCR5, CCR3, or CCR2b. These cells were also resistant to M-tropic patient isolates but were readily infected by T-tropic patient isolates. Primary macrophages from delta ccr5 homozygous individuals were also resistant to infection with M-tropic strains, including YU-2, but the dual-tropic strain 89.6 was able to replicate in them even though macrophages are highly resistant to CXCR-4-dependent T-tropic isolates. These data show that CCR5 is the essential cofactor for infection of both primary macrophages and T lymphocytes by most M-tropic strains of HIV-1. They also suggest that CCR3 does not function for HIV-1 entry in primary lymphocytes or macrophages, but that a molecule(s) other than CCR5 can support entry into macrophages by certain virus isolates. These studies further define the cellular basis for the resistance to HIV-1 infection of individuals lacking functional CCR5.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Depletion of CCR5-expressing cells with bispecific antibodies and chemokine toxins: a new strategy in the treatment of chronic inflammatory diseases and HIV

The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5(+) cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5(+) leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3(+) T cells against CCR5(+) target cells. The Ab specifically depletes CCR5(+) T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5(+) monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of PSEUDOMONAS: exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5(+) Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5(-) Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells.     

Charons 36 to 40: multi enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers.

New phage lambda based cloning vectors, Charons 36-40, have been constructed which allow cloning of large (up to 24 kb) DNA fragments with up to sixteen cloning enzymes. Several of these could not be used previously with lambda vectors. Clones produced with these vectors can be propagated under recombination deficient conditions. A novel polystuffer method has been developed that permits vector arms to be purified by simple precipitation and which allows reliable identification of clones that have reincorporated any part of the stuffer. Three of the vectors are available with amber mutations in essential genes.     

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.     

Structure prediction of GPCRs using piecewise homologs and application to the human CCR5 chemokine receptor: validation through agonist and antagonist docking

This article describes the construction and validation of a three-dimensional model of the human CC chemokine receptor 5 (CCR5) receptor using multiple homology modeling. A new methodology is presented where we built each secondary structural model of the protein separately from distantly related homologs of known structure. The reliability of our approach for G-protein coupled receptors was assessed through the building of the human C-X-C chemokine receptor type 4 (CXCR4) receptor of known crystal structure. The models are refined using molecular dynamics simulations and energy minimizations using CHARMM, a classical force field for proteins. Finally, docking models of both the natural agonists and the antagonists of the receptors CCR5 and CXCR4 are proposed. This study explores the possible binding process of ligands to the receptor cavity of chemokine receptors at molecular and atomic levels. We proposed few crucial residues in receptors binding to agonist/antagonist for further validation through experimental analysis. In particular, our study provides better understanding of the blockage mechanism of the chemokine receptors CCR5 and CXCR4, and may help the identification of new lead compounds for drug development in HIV infection, inflammatory diseases, and cancer metastasis.     

Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and down-regulation of C-C chemokine receptor-5 expression

Constitutive expression of IFN-beta by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release. Moreover, IFN-beta transduction totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-beta production also resulted in an increased production of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, and RANTES. RANTES was found to be involved in the HIV resistance observed, and this was correlated with a down-regulation of the CCR-5 HIV entry coreceptor. These results demonstrate the feasibility and the efficacy of such IFN-beta-mediated gene therapy. In addition to inhibiting HIV replication, IFN-beta transduction could have beneficial immune effects in HIV-infected patients by favoring cellular immune responses.