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Biomedical subjects

E J Stott

Publications and source records attributed to E J Stott.

At least 19 recordsLinked to original sources
PubMed1994

Effects of natural sequence variation on recognition by monoclonal antibodies neutralize simian immunodeficiency virus infectivity.

The determinants of immune recognition by five monoclonal antibodies (KK5, KK9, KK17, Senv7.1, and Senv101.1) that neutralize simian immunodeficiency virus infectivity were analyzed. These five neutralizing monoclonal antibodies were generated to native SIVmac251 envelope glycoprotein expressed by a vaccinia virus recombinant vector. All five recognize conformational or discontinuous epitopes and require native antigen for optimal recognition. These monoclonal antibodies also recognize SIVmac239 gp120, but they do not recognize gp120 of two natural variants of SIVmac239, 1-12 and 8-22, which evolved during the course of persistent infection in vivo (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843-1854, 1991). Recombinant viruses which were constructed by exchanging variable regions between SIVmac239 and variant 1-12 were used to define domains important for recognition. Radioimmunoprecipitation analysis demonstrated that sequence changes in variable regions 4 and 5 (V4/V5) were primarily responsible for the loss of recognition of the 1-12 variant. Site-specific mutants were used to define precise changes that eliminate recognition by these neutralizing antibodies. Changing N-409 to D, deletion of KPKE, and deletion of KEQH in V4 each resulted in loss of recognition by all five monoclonal antibodies. SIVs with these natural sequence changes are still replication competent and viable. Changing A-417 to T or A/N-417/418 to TK in V4 or Q-477 to K in V5 did not alter recognition detectably. These results define specific, naturally occurring sequence changes in V4 of SIVmac that result in loss of recognition by one class of SIVmac neutralizing antibodies.

Amino Acid Sequence↗
PubMed1992

Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.

Amino Acid Sequence↗
PubMed1992

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.

The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.

Antigens, Viral↗
PubMed1992

Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies.

The regions of the fusion protein of respiratory syncytial virus (RSV) that react with neutralizing, fusion-inhibiting and highly protective bovine and murine monoclonal antibodies (MAbs) were mapped by two methods: (i) competitive binding assays and (ii) production and analysis of antibody-escape mutants. Competitive binding assays with 16 murine and 10 bovine MAbs identified 11 antigenic sites on the fusion (F) protein, many of which overlapped extensively, and indicated that cattle, a natural host for RSV, and mice recognize similar epitopes. Neutralizing MAbs identified four sites, two of which were also fusion-inhibiting and highly protective in mice. The pattern of reactivity of antibody-escape mutants with the MAbs confirmed the mapping of the protective epitopes deduced from competitive binding assays. A comparison of the biological properties of MAbs to the F protein indicated that protection against RSV infection correlated with fusion inhibition rather than neutralization titre or complement-dependent lysis of virus-infected cells.

Animals↗
PubMed1992

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus.

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Antibodies, Monoclonal↗
PubMed1992

Neutralizing antibodies modulate replication of simian immunodeficiency virus SIVmac in primary macaque macrophages.

Cultured macaque macrophages are permissive for the replication of SIVmac251, and inoculation with virus is followed by the production of viral p27. Neutralizing macaque polyclonal and murine monoclonal antibodies preincubated with the virus prevented infection but did not prevent cytopathic virus replication when added more than 3 days after inoculation with virus. However, application of the neutralizing antibodies to macrophages 24 h after inoculation with virus resulted in sustained, low-level production of viral antigen. Cell lysates and individual macrophages from treated cultures contained less viral protein by Western blot (immunoblot) and immunocytochemistry than untreated controls. In situ hybridization and polymerase chain reaction procedures for detecting and estimating relative amounts of viral RNA and DNA showed that both viral nucleic acids failed to increase beyond the levels obtained before the addition of neutralizing antibodies. The data suggest that macrophages may need to be infected with a minimum threshold of virus particles in order to reach their full potential for virus replication and that their exposure to neutralizing antibodies prior to reaching this threshold resulted in limited virus replication.

Animals↗
PubMed1992

Transmission studies with simian immunodeficiency virus of macaques; persistent infection of baboons.

The host range of SIVmac was investigated in three monkey species. Blood-borne and cell-adapted virus inocula obtained from a rhesus macaque infected with SIVmac251 were compared. African green monkeys were not susceptible to infection, whereas baboons and rhesus macaques became persistently infected and showed similar patterns of seroconversion. However, in contrast to the macaques, no clinical or histopathological evidence of disease was seen in the baboons 2 years after virus inoculation. Thus baboons could be used as an alternative to macaques in vaccine development studies with this particular isolate of SIVmac. Furthermore, this system may be useful for the investigation of factors responsible for disease progression.

Animals↗
PubMed1991

Vaccine-induced CD4+ T cells against the simian immunodeficiency virus gag protein. Epitope specificity and relevance to protective immunity.

We have examined the induction and epitope specificity of T cells for the simian immunodeficiency virus (SIV) gag p27 protein in macaques immunized with either a recombinant SIV gag protein or an inactivated SIV vaccine. CD4+ MHC class II-restricted T cell lines and clones derived from five immunized macaques recognized a total of seven peptides in three immunodominant regions of p27. Two T cell clones generated from one of the lines, recognized a single 20 amino acid peptide that overlapped with a region previously shown to include a CTL epitope from SIV-infected macaques. Although this epitope is in a conserved region of the gag protein of SIV, its recognition by a CD4+ T cell clone was abrogated by sequence variation in the equivalent HIV protein. The specificity of the T cell lines for synthetic peptides demonstrated considerable overlap between T cells generated by immunization with the recombinant gag protein and inactivated SIV. However, in contrast to the protective efficacy of the whole virus vaccine in the syntex adjuvant formulation, immunization with the p27 protein with alum failed to generate a protective immune response. Furthermore, despite the consistent gag-specific T cell responses induced by the recombinant protein, there was no evidence of an enhanced antibody response to envelope (env) after live SIV challenge.

Amino Acid Sequence↗
PubMed1991

Synthetic peptides corresponding to the F protein of RSV stimulate murine B and T cells but fail to confer protection.

We have previously located a major neutralization site of the fusion protein of respiratory syncytial virus (RSV) in the polypeptide region extending from amino acids Ile221 to Glu232. In this report, 8 peptides corresponding to the six major hydrophilic regions of the F1 subunit were selected to analyse their immunogenic and protective capacities as well as their ability to block the high neutralization activities of 4 monoclonal antibodies (MAbs). Only 5 of the 8 peptides tested induced specific antibodies while all induced an in vitro interleukin-2 response of splenocytes from immunized mice. Peptide 3 (Ile221-Phe237) was able to elicit neutralizing antibodies, confirming our previous hypothesis concerning the location of a neutralization site. However, immunization with the latter did not induce significant reduction of virus in lungs of BALB/c mice upon challenge, probably due to an inadequate level of circulating neutralizing antibodies. Interestingly, peptides 2 (Asn216-Glu232), 3 (Ile221-Phe237), and 5 (Ser275-Ile288) blocked in vitro neutralization by four different F1 specific MAbs. A hypothesis is proposed to explain these results.

Amino Acid Sequence↗
PubMed1991

Effects of recombinant human alpha A interferon in gnotobiotic calves challenged with respiratory syncytial virus.

The effects of recombinant human alpha A interferon were studied in gnotobiotic calves challenged with respiratory syncytial virus (RSV). Gnotobiotic calves given doses of interferon by intramuscular injection over five days showed a marked, dose-related, rise in rectal temperature and depression of circulating leucocytes. Differential counts showed decreases in both lymphocytes and neutrophils. No significant pathological differences were found between treated and untreated calves, nor could any difference be demonstrated in the pattern of RSV infection.

Animals↗
PubMed1991

Production and of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins.

Eighteen monoclonal antibodies (MAb) to simian immunodeficiency virus (SIV) envelope have been characterized. All MAb were shown to bind to viral antigens on the surface of unfixed SIV-infected cells and to precipitate surface glycoproteins of SIVmac251. In Western blot 11 MAb bound to gp160 and gp120, five bound to gp160 and the transmembrane protein gp41 and two MAb did not react with denatured antigen. Preliminary competition assays identified the existence of six competition groups; two groups were within gp41 and four were within gp120. Of the latter four groups, three contained MAb with neutralizing activity. Two of the neutralizing MAb (KK5 and KK9) did not react with denatured antigen in Western blot suggesting that they may recognize conformational epitopes. Enzyme-linked immunosorbent-assay titres of MAb against SIVmac251 ranged from 10(2.4) to 10(5.6) and although similar titres were obtained with some MAb against other SIV and HIV antigens, the presence of isolate specific and shared group epitopes was demonstrated.

Animals↗
PubMed1991

Comparison of the virulence of wild-type thymidine kinase (tk)-deficient and tk+ phenotypes of vaccinia virus recombinants after intranasal inoculation of mice.

A recombinant vaccinia virus vector was constructed which expressed the major surface glycoprotein G of human respiratory syncytial virus (RSV) and the thymidine kinase (tk) gene of vaccinia virus. The virulence of this tk+ recombinant virus was compared with that of a tk- recombinant and the wild-type (wt) virus after intranasal inoculation of mice. Respiratory infection with wt virus resulted in a lethal infection with widespread dissemination of virus. In contrast, infection with the tk- recombinant was not lethal and the virus had a reduced ability to disseminate to extrapulmonary tissue compared with wt virus. Insertion of the tk gene restored the virulence of the recombinant virus to the level of that of the wt virus. Despite a dramatic reduction in virulence of the tk- recombinant, virus could occasionally be recovered from the brains of mice. The expression of the attachment glycoprotein of RSV appeared to enhance the ability of the tk- recombinant virus to replicate in the lungs when compared with recombinants expressing fusion or nucleoprotein genes. The results confirm that inactivation of the tk gene results in a dramatic reduction of virulence for mice but suggest that there is still a potential danger of infection of the brain following intranasal administration of virus.

Animals↗
PubMed1990

Histopathological changes in simian immunodeficiency virus infection.

The histological lesions were studied in seven rhesus and three cynomolgus monkeys infected with simian immunodeficiency virus for periods ranging from nine weeks to 18 months. Lymphoreticular changes included hyperplasia, follicular involution and depletion, and one animal had amyloidosis of the spleen. Hyperplastic changes also took place in mucosa-associated lymphoid tissue and infiltrations occurred in the vaginal mucosa of one animal, which could be significant in sexual transmission of the infection. The range of opportunistic infections was small compared with that in human AIDS patients, although two monkeys had Pneumocystis carinii pneumonia. Enterocolitis was a common finding and brown adipose tissue was transformed into a large vacuolated type. Lesions of the central nervous system were found in five of nine monkeys, and consisted of foci of glial activity and perivascular and meningeal lymphocytic infiltration. A lymphoma involving the lumbar spinal cord developed in one animal.

Adipose Tissue, Brown↗
PubMed1990

The use of the polymerase chain reaction for the detection of Simian immunodeficiency virus in experimentally infected macaques.

A rapid, non-radioactive assay for the detection of proviral Simian immunodeficiency virus (SIV) in tissue-culture cells is described. The assay is based on the co-amplification of the SIV env and gag genes by the polymerase chain reaction (PCR). When the gag PCR product is blotted onto a nylon membrane and hybridised to a radioactive oligonucleotide probe, the assay can also be used to detect the SIV gag gene in DNA isolated directly from experimentally infected cynomolgus macaque lymphocytes. This provides a valuable assay for the presence of proviral SIV during animal trials of AIDS vaccines and chemotherapeutics.

Animals↗
PubMed1989

Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs.

We have characterized the proteins and mRNAs of bovine respiratory syncytial (BRS) virus strain 391-2 and constructed cDNA clones corresponding to 9 of the 10 BRS virus mRNAs. The proteins of BRS virus-infected cells were compared with the proteins from human respiratory syncytial (HRS) virus-infected cells. Nine proteins specific to BRS virus-infected cells, corresponding to nine HRS virus proteins, were identified. Only a BRS virus polymerase protein remains to be identified. The BRS virus G glycoprotein showed major antigenic differences from the HRS virus G glycoprotein by immunoprecipitation and Western (immuno-) blot analysis, whereas the BRS virus F, N, M, and P proteins showed antigenic cross-reactivity with their HRS virus counterparts. Analysis of RNAs from BRS virus-infected cells showed virus-specific RNAs which had electrophoretic mobilities similar to those of mRNAs of HRS virus but which hybridized poorly or not at all with HRS virus-specific probes in Northern (RNA) blot analysis. To analyze the BRS virus RNAs further, cDNA clones to the BRS virus mRNAs were generated. Nine separate groups of clones were identified and shown to correspond to nine BRS virus mRNAs by Northern blot analysis. A 10th BRS virus large mRNA was identified by analogy with the HRS virus polymerase mRNA. These data show that like HRS virus, BRS virus has 10 genes coding for 10 mRNAs.

Antibodies, Monoclonal↗