Chimeric Glycoprotein Research Articles

Increased Risk of Non-Q Wave Myocardial Infarction After Directional Atherectomy Is Platelet Dependent: Evidence From the EPIC Trial

Objectives. We sought to determine the effects of platelet glycoprotein IIb/IIIa receptor blockade on adverse outcomes, especially non-Q wave myocardial infarction, in patients undergoing directional atherectomy in the Evaluation of c7E3 for the Prevention of Ischemic Complications (EPIC) trial.Background. Randomized trials comparing directional atherectomy with percutaneous transluminal coronary angioplasty (PTCA) have demonstrated modest benefits favoring atherectomy but at a cost of increased acute ischemic complications, notably non-Q wave myocardial infarction. The mechanism for this excess risk is unknown.Methods. Of 2,038 high risk patients undergoing coronary intervention in the EPIC trial, directional atherectomy was performed in 197 (10%). Patients randomly received the chimeric glycoprotein IIb/IIIa antibody 7E3 (c7E3), as a bolus or a bolus and 12-h infusion or placebo. Study end points included death, myocardial infarction, repeat intervention or bypass surgery.Results. Patients undergoing directional atherectomy had a lower baseline risk for acute complications but had a higher incidence of any myocardial infarction (10.7% vs. 6.3%, p = 0.021) and non-Q wave myocardial infarction (9.6% vs. 4.9%, p = 0.006). Bolus and infusion of c7E3 reduced non-Q wave myocardial infarctions by 71% after atherectomy (15.4% for placebo vs. 4.5% for bolus and infusion, p = 0.046). Non-Q wave myocardial infarction rates after PTCA were not affected by c7E3, although Q wave myocardial infarctions were reduced from 2.6% to 0.8% (p = 0.017).Conclusions. The EPIC trial confirmed the increased risk of non-Q wave myocardial infarction with directional atherectomy use compared with PTCA. A bolus and 12-h infusion of the glycoprotein IIb/IIIa receptor inhibitor c7E3 abolished this excess risk. Directional atherectomy-related non-Q wave myocardial infarction appears to be platelet aggregation dependent.

Chimeric envelope glycoproteins constructed between amphotropic and xenotropic murine leukemia retroviruses.

A set of chimeric envelope proteins between amphotropic and xenotropic murine leukemia retroviruses (MuLV), two closely related members in the MuLV family, were constructed. The purpose was to examine the regions that could be successfully exchanged between these two similar viral envelope proteins. The data indicate that fully active chimeras can be built when the junction is either at the EcoRI site (amino acid 169) 42 amino acids N-terminal to the polyproline hinge of gp70 (named CH4) or at the ScaI site (aa 593) in the membrane spanning portion of p15E (CH1). However, a chimera at the AflII site (aa 125, CH5) and two in the C-terminal end of gp70 (aa 418, CH2; aa 326, CH3) were inactive. These results, taken together with other data from our laboratory and others, suggest that the entire gp70/p15E structure is sensitive to alterations and that even envelope proteins that are very similar have only a limited ability to exchange sequences.

Vpu-Mediated Proteolysis of gp160/CD4 Chimeric Envelope Glycoproteins in the Endoplasmic Reticulum: Requirement of Both the Anchor and Cytoplasmic Domains of CD4

The Vpu protein of HIV-1 induces degradation of CD4 in the endoplasmic reticulum. Previous studies have elucidated the role of the CD4 cytoplasmic domain in the Vpu-induced degradation process, and the minimal Vpu responsive element mapped to a small region in the CD4 tail. In the present study, we have carried out both biochemical and biological experiments to analyze the role of the CD4 anchor domain in the Vpu-induced degradation process. We generated chimeric proteins that possessed the ecto-anchor domains of gp160 and the cytoplasmic domain of CD4. The chimeric envelope glycoproteins were functionally active in the fusion of HeLa CD4+ cells, with the exception of those having the arginine to isoleucine (R to I) substitution in the gp160 anchor domain. Coexpression studies revealed that these chimeric glycoproteins were stable and functionally active in the presence of Vpu, as opposed to those having the anchor-cytoplasmic domains of CD4. The half-life of Vpu-sensitive chimeric glycoproteins was calculated to be approximately 60-90 min, whereas Vpu-resistant envelope glycoproteins exhibited relatively longer half-lives in the presence of Vpu. Taken together, these studies strongly suggest that the CD4 anchor domain appears to provide critical sequence or structural elements through which the Vpu protein could access CD4 or glycoproteins hearing the Vpu responsive element for degradation in the endoplasmic reticulum.

Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin antiplatelet antibody Fab 7E3 in high-risk coronary angioplasty.

Thrombosis has been implicated as central to the clinical complications of coronary angioplasty (PTCA). Chimeric monoclonal 7E3 Fab (c7E3 Fab) is the first of a new class of antiplatelet drugs directed at the platelet glycoprotein IIb/IIIa integrin. This study was performed to determine the pharmacodynamics of c7E3 Fab administration during PTCA and to gain an initial clinical experience with this novel agent. The study was a multicenter, open-label, dose-escalation study conducted in two stages. Enrollment included 56 patients scheduled for elective PTCA who were estimated to be at moderate to high risk of sustaining ischemic complications. All patients were given aspirin and heparin. The study drug was given at least 10 minutes before PTCA. In stage 1, increasing bolus doses of c7E3 Fab were given to 15 patients; a bolus dose of 0.25 mg/kg was found to result in blockade of > 80% of the receptors and reduce platelet aggregation to < 20% compared with baseline, establishing this dose as that necessary to sufficiently suppress platelet activity. In stage 2, additional c7E3 Fab was administered by continuous infusion to 32 patients for progressively longer periods of time (up to 24 hours) to confirm that platelet inhibition could be maintained with prolonged drug infusion. Also, 9 patients otherwise meeting entry criteria were given placebo. There were no thrombotic events among patients receiving c7E3 Fab. Overall procedural and clinical success and complication rates as well as rates of bleeding were statistically similar among groups. However, minor bleeding was more frequent with administration of the active drug. The novel antiplatelet agent c7E3 Fab can be administered during PTCA in combination with aspirin and heparin. Suppression of platelet activity is dose dependent and can be maintained for up to 24 hours. Further evaluation will be required to determine the extent of improvement in ischemic complication and restenosis rates and to provide additional insight into the safety profile of this potent monoclonal platelet antibody.

Purification of a Recombinant Human Respiratory Syncytial Virus Chimeric Glycoprotein Using Reversed-Phase Chromatography and Protein Refolding in Guanidine Hydrochloride

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu2+-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Evidence for a functional interaction between the V1/V2 and C4 domains of human immunodeficiency virus type 1 envelope glycoprotein gp120

The domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein that are required for envelope function have been partially characterized. Little is known, however, about the nature of the interactions between these domains. To identify regions of the HIV-1 envelope glycoprotein that are involved in interactions necessary for proper envelope function, we constructed a series of 14 envelope recombinants between the env genes of two HIV-1 isolates. The envelope chimeras were examined for their ability to induce syncytia, to be proteolytically processed, and to function during a spreading viral infection. Our results demonstrate that the exchange between the two isolates of the first and second hypervariable regions (V1/V2) of gp120 results in defects in envelope glycoprotein processing, syncytium formation, and infectivity. Long-term passage of cultures infected with virus bearing a V1/V2 chimeric envelope glycoprotein leads to the emergence of a revertant virus with replication characteristics comparable to those of the wild type. Analysis of the revertant indicated that an Ile-->Met change in the C4 region of gp120 (between hypervariable regions V4 and V5) is responsible for the revertant phenotype. This single amino acid change restores infectivity without significantly affecting gp160 processing, CD4 binding, or the levels of virion-associated gp120. While the Ile-->Met change in C4 greatly enhances the fusogenic potential of the V1/V2 chimeric envelope glycoprotein, it has a detrimental effect on syncytium formation when analyzed in the context of the wild-type envelope. These results suggest that an interaction required for proper envelope glycoprotein function occurs between the V1/V2 and C4 regions of gp120.

Modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive alkylation.

CD4 immunoadhesin (CD4-IgG) is a chimeric glycoprotein molecule comprised of the gp120-binding portion of human CD4 fused to the hinge and Fc portions of human IgG. As a candidate for human therapeutic use, CD4-IgG represents an important advance over soluble CD4, insofar as the systemic clearance in humans of CD4-IgG is significantly slower. In an effort to prolong its in vivo residence time even further, we have modified CD4-IgG chemically by attaching monomethoxypoly(ethylene glycol) (MePEG) moieties to lysine residues via reductive alkylation. We synthesized MePEG aldehyde and investigated reaction conditions for adding a range of MePEG moieties per protein molecule. At neutral pH in the presence of sodium cyanoborohydride, the reaction was sufficiently slow to allow for significant control over the extent of MePEGylation. Addition of 7.7 or 14.4 MePEG moieties to CD4-IgG resulted in an approximately 4- or 5-fold increase, respectively, in the persistence of the protein in rats, as compared with unmodified CD4-IgG. These results suggest that the therapeutic utility of a human receptor IgG chimera can be improved by MePEGylation technology, provided that the modified immunoadhesin retains its biological activity in vivo. Such modification can lead to a significant additional increase in the in vivo residence time of the protein.

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.

Induction of local and systemic immunity against human respiratory syncytial virus using a chimeric FG glycoprotein and cholera toxin B subunit

Local IgA and IgG antibodies against respiratory syncytial virus (RSV) were induced in the respiratory tract of mice following intranasal vaccination with an RSV chimeric FG glycoprotein and cholera toxin B (CTB) as a mucosal adjuvant. Local antibody production was not induced following parenteral immunization with FG administered in alum adjuvant. While both vaccination protocols induced serum antibodies against RSV and protected the lower respiratory tract from RSV infection, only intranasal FG/CTB afforded protection of the upper respiratory tract. These data suggest that vaccination via the mucosal route may be superior to vaccination by a parental route in providing complete protection against RSV.

5284764 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3

5284764 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

5284764 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3

5284764 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

5194595 Chimeric glycoproteins containing immunogenic segments of the glycoproteins of human respiratory syncytial virus: Michael W Wathen assigned to The Upjohn Company

Sequences determining the pH dependence of viral entry are distinct from the host range-determining region of the murine ecotropic and amphotropic retrovirus envelope proteins.

The entry of ecotropic and amphotropic murine leukemia retroviruses (MuLV) into cells was investigated by using viral vector particles carrying chimeric amphotropic-ecotropic envelope glycoproteins on their surface. Chimeras were made by joining, at or near the polyproline hinge, the N-terminal portion of the amphotropic (4070A) gp70 onto the C-terminal portion of the ecotropic (Moloney) gp70 and p15E (constructs AE2 and AE4) or vice versa (AE12). Transduction efficiency of the constructs was tested on target cells that either have only ecotropic receptors (CHO-2 and CHO-11 cells), only amphotropic receptors (mink lung fibroblasts and Cos 1 cells), or both types of receptors (NIH 3T3 cells). The assay made use of the fact that the mechanism for viral entry of ecotropic viruses is pH dependent while that of amphotropic viruses is pH independent. Treatment of target cells with NH4Cl, which prevents the reduction of pH within endosomes, reduced the titers of viral particles bearing the C-terminal moiety from the ecotropic envelope but did not reduce the titers of particles which had a C-terminal moiety from the amphotropic envelope. In addition, in contrast to other low-pH-dependent enveloped viruses, brief acid treatment did not allow surface-bound viruses to bypass the NH4Cl block. The results indicate that the pH dependence of viral entry is a property of the sequences C terminal to the polyproline hinge.

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.

Development of a novel subunit vaccine that protects cotton rats against both human respiratory syncytial virus and human parainfluenza virus type 3.

A cotton rat model of experimental human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FRHNP, a novel chimeric glycoprotein which contains the extracellular regions of the fusion glycoprotein of RSV and the attachment glycoprotein of PIV-3, as a single subunit vaccine against these two viruses. This work was prompted by previous cotton rat studies that demonstrated that the major protective antigens of the two viruses were these glycoproteins. FRHNP was expressed in insect cells using a recombinant baculovirus. Vaccination with FRHNP resulted in induction of both RSV and PIV-3 neutralizing antibody and doses of 200 ng completely protected rats from either RSV or PIV-3 challenge. These results demonstrate that in the cotton rat animal model a single chimeric glycoprotein can be an effective vaccine against both RSV and PIV-3.

Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins

Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome.

5169628 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3 : Michael Wathen assigned to The Upjohn Company

5169628 Chimeric glycoproteins containing immunogenic segments of human parainfluenza virus type 3 : Michael Wathen assigned to The Upjohn Company

Chimeric glycoprotein comprising extracellular domains of PIV-3 F and HN proteins; parainfluenza virus-3F, HN chimeric gene construction and chimeric glycoprotein expression for use in recombinant vaccine

Chimeric glycoprotein comprising extracellular domains of PIV-3 F and HN proteins; parainfluenza virus-3F, HN chimeric gene construction and chimeric glycoprotein expression for use in recombinant vaccine