Biomedical subjects

E G Routledge

Publications and source records attributed to E G Routledge.

16 recordsLinked to original sources

PubMed2006

Protection of mice against Human respiratory syncytial virus by wild-type and aglycosyl mouse-human chimaeric IgG antibodies to subgroup-conserved epitopes on the G glycoprotein.

Monoclonal antibodies (mAbs) to conserved epitopes on the G glycoprotein of human respiratory syncytial virus (HRSV) subgroup A fail to neutralize the virus in cell culture in the absence of complement, but are protective in rodent models of infection. They may have potential as prophylactic agents in human infants. In order to investigate the role of Fc-dependent pathways in protection by one such antibody, 1C2, the V(H) and V(L) genes were isolated by RT-PCR and assembled with human kappa light-chain and human gamma1 heavy-chain constant-region genes to form two mouse-human chimaeras, which were expressed in NS0 cells. One of the chimaeras carried a wild-type gamma1 chain, whilst the other had an aglycosyl mutation in the C(H)2 domain rendering the antibody defective in complement activation and FcgammaR binding. Whilst both chimaeric antibodies exhibited similar avidity for HRSV in ELISA, only the fully glycosylated wild type was capable of neutralizing the virus in the presence of complement. In mice passively immunized with either murine or wild-type gamma1 chimaeric antibody, no virus could be recovered from the lungs 4 days after intranasal inoculation of HRSV. In mice immunized with the aglycosyl gamma1 chimaera, however, virus was present in the lungs following challenge, although virus titres were significantly reduced compared with controls (P < 0.005). These results indicate that the protective effect of this antibody is mediated by both Fc-dependent and Fc-independent pathways.

Animals↗

PubMed1999

Antigen-specific membrane fusion mediated by the haemagglutinin protein of influenza A virus: separation of attachment and fusion functions on different molecules.

Using genetic engineering techniques, two strategies for changing the receptor-binding specificity of the influenza A virus haemagglutinin (HA) protein whilst preserving its membrane fusion function, have been explored. The aim was to investigate whether the HA protein could be developed as an attachment/entry protein for targeting enveloped virus gene therapy vectors to specific cell populations. In the first strategy, a single chain antibody Fv region (scFv) specific for the hapten NIP was inserted between HA1 codons 139 and 145, to create a scFv-HA chimaeric protein. This protein was shown to possess anti-NIP binding activity, but membrane fusion activity could not be demonstrated. The possibility that linking the scFv domain directly to HA may have inhibited the HA fusion function led to the development of the second strategy. This involved separating the receptor-binding and membrane fusion functions of HA on to two different molecules. The feasibility of this strategy was tested by looking for fusion between NIP-conjugated red blood cells which lacked sialic acid (the HA protein's natural receptor) and Chinese hamster ovary cells that expressed both the above anti-NIP scFv-HA chimaeric protein (as a non-fusigenic, receptor-binding molecule) and wild-type HA protein (as a fusigenic, non-binding molecule) on their surface. Cell-to-cell fusion was observed in this system, indicating that the receptor-binding function of HA can be transferred to an adjacent molecule, and also changed in its specificity, without compromising its membrane fusion activity. This finding strongly suggests that the development of a two-molecule attachment and entry system for retargeting enveloped virus gene therapy vectors, based on HA, is a viable proposition.

Animals↗

PubMed1995

The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody.

The factors affecting the immunogenicity of a humanized gamma 1 CD3 monoclonal antibody (mAb) were investigated in transgenic mice that express the human CD3 antigen epsilon polypeptide (the mAb target antigen). Two derivatives of the mAb were employed, one with a normal, glycosylated Fc region (gamma 1 CD3 mAb), and the other with an aglycosylated Fc region (aglycosyl gamma 1 CD3 mAb). Comparisons of the antiglobulin responses elicited by the two derivatives in transgenic and nontransgenic mice demonstrated that Fab-mediated cell binding activity, dependent on target antigen expression, was a major positive determinant of CD3 mAb immunogenicity. A second positive factor was mAb Fc region glycosylation. At low dose levels the gamma 1 CD3 mAb consistently produced a higher antiglobulin response than the aglycosyl gamma 1 CD3 mAb. This was probably a result of the nonspecific, in vivo T cell activating property of the gamma 1 CD3 mAb, a consequence of its ability to cross-link T cells to Fc gamma receptor-bearing cells. (The aglycosyl gamma 1 CD3 mAb has a reduced Fc binding affinity for Fc gamma receptors and so does not activate T cells in vivo.) In support of this hypothesis, the gamma 1 CD3 mAb was able to nonspecifically enhance humoral immunity to an unrelated, coadministered antigen, whereas the aglycosyl gamma 1 CD3 mAb was not. The lower immunogenicity of the aglycosyl gamma 1 CD3 mAb correlated with a longer in vivo half-life and an improved capacity to block the target CD3 antigen. These results suggest that, as well as reducing the cytokine-induced side effects normally associated with CD3 mAb therapy, the nonactivating aglycosyl gamma 1 CD3 mAb will be less likely than the activating gamma 1 CD3 mAb to stimulate a neutralizing antiglobulin response.

Animals↗

PubMed1994

Engineering multiple-domain forms of the therapeutic antibody CAMPATH-1H: effects on complement lysis.

Antibody-mediated lysis of cells involves a complex interaction between the cell, the target antigen, the antibody and host effector mechanisms. One such mechanism, complement-mediated cell lysis, requires the interaction of C1q with the antibody heavy chain constant regions, and in particular the CH2 domain. Here we investigate the potential benefit of multiple-domain forms of the therapeutic monoclonal antibody CAMPATH-1H. This antibody is directed against the CDw52 antigen expressed by human lymphocytes and has proven lytic abilities both in vitro and in vivo. Using target cells with either high or low antigen density, engineered antibodies that contained additional domains in tandem (CH2, hinge-CH2 or Fc intramolecular repeats) showed no improvement in complement-mediated lysis when compared with controls. However, a homodimeric form of the antibody that was engineered by mutation of a serine residue to cysteine near the carboxy-terminal of the CH3 domain, exhibited markedly improved lysis using target cells expressing antigen at low density. Interestingly, no improvement was seen using cells expressing antigen at high density. These results suggest that dimeric forms of antibodies could be useful for converting cells with low density antigens into useful targets for therapy.

Alemtuzumab↗

PubMed1991

Reshaping a therapeutic CD4 antibody.

An immunosuppressive rat antibody (Campath-9) against human CD4 has been reshaped for use in the management of autoimmunity and the prevention of graft rejection. Two different forms of the reshaped antibody were produced that derive their heavy chain variable region framework sequences from the human myeloma proteins KOL or NEW. When compared to a chimeric form of the CD4 antibody, the avidity of the KOL-based reshaped antibody was only slightly reduced, whereas that of the NEW-based reshaped antibody was very poor. The successful reshaping to the KOL-based framework was by a procedure involving the grafting of human framework sequences onto the cloned rodent variable region by in vitro mutagenesis.

Amino Acid Sequence↗

PubMed1991

A humanized monovalent CD3 antibody which can activate homologous complement.

The rat monoclonal antibody (mAb) YTH12.5, specific for the CD3 antigen complex on human T cells has been modified in order to improve its efficacy in human therapy. With the aim of rendering it less immunogenic, it has been humanized using the method of framework grafting. During this process sequence analysis of the YTH12.5 VL gene indicated that it was of the lambda subclass, however, it was markedly dissimilar from previously published rat and mouse V lambda gene sequences and may represent a new V lambda gene family. The humanization of this light chain represents the first successful reshaping of a lambda light chain V region. To improve the effector function of the antibody we have created a monovalent form (1 Fab, 1 Fc) using a novel method involving the introduction of an N-terminally truncated human IgG1 heavy chain gene into cells producing the humanized CD3 mAb. Comparison of the mono- and bivalent humanized mAb in a complement-mediated cell lysis assay revealed that the monovalent antibody mediated lysis of human T cell blasts whereas the bivalent form did not. The availability of a humanized, complement-fixing CD3 mAb may improve opportunities for human therapy, in the management of organ rejection, autoimmunity and the treatment of T cell lymphoma.

Amino Acid Sequence↗

PubMed1989

Monoclonal antibodies to feline calicivirus.

We have prepared monoclonal antibodies to the capsid protein of feline calicivirus (FCV). These antibodies are directed against two close but distinct epitopes, only one of which is involved in virus neutralization. We have used these antibodies and immune cat serum in immunoprecipitation and Western blotting experiments and have identified novel proteins in FCV-infected cells which we term P78, P41, P35 and P29. The number and sizes of FCV proteins now known resemble those made by other caliciviruses.

Animals↗

PubMed1989

IgG and IgM antibodies to viral glycoproteins in respiratory syncytial virus infections of graded severity.

Serum antibodies to the fusion (F) and large glycoprotein (G) of respiratory syncytial virus in the serum of 57 infected infants were measured by enzyme linked immunosorbent assay (ELISA). Most serum samples taken at the time of admission to hospital contained antibodies to both glycoproteins, and overall there was no significant evidence of a selective deficiency of antibody to either viral antigen. Less than a quarter of the infants showed rising IgG antibody titres to either glycoprotein after infection, whereas over threequarters produced an IgM response. There was a significant correlation between IgG response to viral glycoproteins and the age of the infant. The correlation of age with the IgM response was less pronounced, and there was no correlation between serum IgG antibody derived transplancentally in the acute phase of infection and IgM response to either glycoprotein. Neither IgG or IgM responses correlated with a clinical assessment of the severity of infection in the infants. IgM responses, however, were weakly correlated with reduced secretion of infectious virus in the upper respiratory tract.

Antibodies, Viral↗

PubMed1988

The purification of four respiratory syncytial virus proteins and their evaluation as protective agents against experimental infection in BALB/c mice.

The fusion (F) glycoprotein, large glyco- (G) protein, phospho- (P) protein and 22K protein of respiratory syncytial (RS) virus A2 strain were purified by a combination of immunoaffinity adsorption and preparative SDS-PAGE. All four proteins elicited serum antibody in mice after repeated inoculation in adjuvant, although the magnitude of the response as measured by ELISA varied from mouse to mouse. The F protein generated neutralizing antibodies in only 50% of the mice determined to be seropositive by ELISA. The G protein also induced neutralizing antibodies but in this instance neutralization tests and ELISA titres were more closely correlated. No neutralizing activity was detected in mice immunized with the P or 22K proteins although all produced antibody detectable by ELISA. Mice immunized with either the F or the G protein were found to be protected against subsequent RS virus challenge, whether they had developed neutralizing antibody or not. Mice inoculated with the P or 22K proteins were not protected.

Animals↗

PubMed1987

NCL-5D3: a new monoclonal antibody recognizing low molecular weight cytokeratins effective for immunohistochemistry using fixed paraffin-embedded tissue.

Production of a new monoclonal antibody designated NCL-5D3 is described. The antibody recognizes several low molecular weight cytokeratins, in particular cytokeratin Moll number 8 as determined by immunoblotting studies, and is highly effective for immunocytochemistry using routinely processed paraffin-embedded material. Staining is enhanced by prior treatment of the sections with trypsin. Assessment using a wide variety of normal and neoplastic tissue indicates reactivity with all tissues of simple or glandular epithelial origin, and in addition with many squamous carcinomas. Thus the antibody should prove of value in diagnostic histopathology.

Antibodies, Monoclonal↗

PubMed1987

Strain variation of respiratory syncytial virus.

Western blotting and immunoperoxidase staining with monoclonal antibodies were employed to analyse epitopic and polypeptide molecular weight variation among isolates of respiratory syncytial (RS) virus collected in Newcastle between 1965 and 1986. One group of isolates resembled the A2 and Long prototype subgroup A strains of RS virus in possessing a P protein of Mr 34,000. Isolates in this subgroup showed two patterns of reactivity with subgroup A-specific monoclonal antibodies to the G glycoprotein and 22K protein. Isolates with both reactivity patterns were isolated throughout the period studied. Isolates in the second group resembled the 8/60 subgroup B prototype strain in their lack of reactivity to subgroup A-specific monoclonal antibodies but were heterogeneous in P protein molecular weight. The earliest isolate only, made in 1965, possessed a P protein of Mr 31,000 resembling the prototype strain. All subsequent subgroup B isolates possessed a higher Mr, 33,000, P protein. Overall, subgroup A viruses were isolated most frequently although subgroup B strains may have predominated in some epidemics.

Antibodies, Monoclonal↗

PubMed1987

Heterogeneity of the respiratory syncytial virus 22K protein revealed by Western blotting with monoclonal antibodies.

Respiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23,000 in our gel system). Three novel polypeptides with mol. wt. of 24,000, 21,000 and 17,000 were stained in addition to the 23,000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under non-reducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24,000, 21,000 and 17,000 polypeptides were not visible, the number of mobility variants of the 23,000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23,000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.

Antibodies, Monoclonal↗

PubMed1987

Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells.

Immunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h post-infection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.

Capsid↗

PubMed1986

A neutralizing monoclonal antibody to respiratory syncytial virus which binds to both F1 and F2 components of the fusion protein.

A virus-neutralizing monoclonal antibody (1E3) specifically immunoprecipitated the 70000 mol. wt. (70K) fusion (F) protein from respiratory syncytial (RS) virus-infected HeLa cells. Western blotting analysis of polypeptides from such cells separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that 1E3 was peculiar in that it bound to both F1 (50K) and F2 (20K) components of the F protein. Antibody subsequently eluted from either the F1 or the F2 regions of immunoblots re-bound to both F1 and F2 regions of the SDS-PAGE blot. These results show that monoclonal antibody 1E3 reacts with an epitope which is found on both F1 and F2 subunits of RS virus fusion protein.

Antibodies, Monoclonal↗

PubMed1986

Respiratory syncytial virus glycoprotein expression in human and simian cell lines.

Glycoproteins synthesized in both human (HeLa and HEp-2) and simian (Vero and BS-C-1) cell lines following infection with two different strains of respiratory syncytial virus (A2 and Long) were analysed by SDS-PAGE following immunoprecipitation with monoclonal antibodies. Minor virus strain-dependent differences in the large glycoprotein, G, and the fusion protein polypeptides F1 and F2 were observed together with minor cell line-dependent differences in the size of the F2 polypeptide. Major quantities of two glycoproteins, termed Ga (50K) and Gb (45K), were detected in A2 strain-, and to a lesser extent in Long strain-, infected simian cells. These proteins were also present in infected human cells, but in much reduced amounts. Immunoprecipitation with anti-G monoclonal antibodies demonstrated that Ga and Gb shared different epitopes with G.

Animals↗

PubMed1985

The development of monoclonal antibodies to respiratory syncytial virus and their use in diagnosis by indirect immunofluorescence.

Twelve clones of murine hybridoma cells secreting antibody specific for respiratory syncytial (RS) virus were classified into four groups on the basis of their pattern of staining of unfixed RS virus-infected HEp-2 cells in an indirect immunofluorescence test. Three of the groups reacted with virus antigens present on the membrane of the cells, whilst the fourth group failed to stain most live cells, suggesting specificity for an antigen expressed internally. Representative monoclonals from the membrane antigen staining groups immunoprecipitated the 86K glycoprotein (G), 50K plus 19K glycoprotein (F1,2) and a 23K non-glycosylated protein (VP23). A representative monoclonal from the fourth group that appeared to stain an internally expressed protein immunoprecipitated the virion 34K phospho-protein (P). All four monoclonals stained acetone-fixed tissue culture cells infected with either the Long strain of RS virus or with strains isolated in Newcastle during the 1965, 1972, and 1983 winter epidemics. The anti-fusion protein antibody stained acetone-fixed cells from all of 26 nasopharyngeal secretions from infants with RS virus infection. The anti-G glycoprotein antibody and the anti-VP23 antibody stained cells from secretions poorly or not at all, whilst the anti-P protein antibody stained cells in half the secretions tested but reacted with only a small proportion of cells in comparison with the anti-F or polyclonal antibodies. A pool of all four monoclonals produced more intense staining than the anti-F monoclonal alone and gave a more clearly defined staining reaction than the polyclonal antiserum used for routine diagnosis in over half the secretions. These results indicate that monoclonal antibodies will be of value in the diagnosis of RS virus by indirect immunofluorescence if care is taken in the selection of a suitable pool.

Antibodies, Monoclonal↗