Some highlights of virus research in 1991.
Explore the source record for details and available documents.
Research↗
Biomedical subjects
R E Randall
Publications and source records attributed to R E Randall.
Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen.
We have previously shown that immunization with solid matrix-antigen-antibody (SMAA) complexes induces both vigorous humoral and cell-mediated immune responses and have suggested that this method of vaccination may be developed for use in humans, and potentially as a vaccine against AIDS. Here we demonstrate that a small oligopeptide can act as a tag for the construction of SMAA complexes using a tag-specific monoclonal antibody and tag-linked antigens. We show that a 14-amino acid oligopeptide, present in the phospho (P) and V proteins of simian virus 5 (SV5), retains its antigenicity when attached to the C terminus of three 'foreign' proteins [p27 and gp110 of simian immunodeficiency virus (SIV) and glutathione S-transferase] such that these proteins can be incorporated into SMAA complexes using a monoclonal antibody (MAb) that was originally raised against the native SV5 P and V proteins. Mice were immunized with SMAA complexes containing recombinant p27-TAG and MAbs have been isolated that recognized native SIV p27. The significance of these results in terms of the development of SMAA complexes as human vaccines is discussed.
Amino Acid Sequence↗
Immunological relationships between parainfluenza virus type 4 and other paramyxoviruses studied by use of monoclonal antibodies.
These results demonstrated that cross-reactions between mumps virus, PIV-2, PIV-4, and SV5 involve both internal components and surface glycoproteins and that these viruses form a separate group within the paramyxovirus genus.
Animals↗
Sequence comparison between the haemagglutinin-neuraminidase genes of simian, canine and human isolates of simian virus 5.
The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene was determined for a simian (W3), human (LN) and two canine (CPI+/CPI-) isolates of simian virus 5 (SV5). A comparison of the predicted amino acid sequences revealed that the human and canine isolates varied from the simian isolate by 1.7% and 2.4% respectively. This lack of significant variation between the HN proteins of the four SV5 isolates suggests that insufficient differences have occurred between isolates to confine them to a specific host.
Amino Acid Sequence↗
Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics.
Two canine isolates of simian virus 5 (SV5), termed CPI+ and CPI-, were examined for their ability to react with a bank of monoclonal antibodies (MAbs) that had been previously raised against a human isolate of SV5. CPI- virus was originally isolated from the brain of a gnotobiotic dog infected with CPI+ virus and establishes persistent infections more readily than CPI+ in vitro. Of more than 50 MAbs tested, only one (P-k) reacted with CPI+ but not CPI-, enabling distinction between the two canine isolates. It had been shown previously that MAb P-k reacts with an epitope common to both the P and V proteins. In order to characterize further the epitope binding site of this MAb the P/V genes of CPI+ and CPI- were sequenced. There were four nucleotide differences between CPI+ and CPI-, three of which resulted in predicted amino acid substitutions. Synthetic peptides corresponding to regions encompassing these changes were made and radioimmune competition assays were used to identify the epitope binding site of MAb P-k. Sequence comparison of the P/V gene of CPI+ with the published sequence of a monkey isolate of SV5 (W3) revealed 14 nucleotide differences with five amino acid substitutions. The only amino acid substitution observed between CPI+, CPI- and W3 which altered the predicted secondary structures of the P and V proteins was a leucine to proline change that induced a predicted beta-turn and resulted in the loss of binding of MAb P-k.
Amino Acid Sequence↗
Solid matrix-antibody-antigen complexes induce antigen-specific CD8+ cells that clear a persistent paramyxovirus infection.
We have previously shown that the adoptive transfer of splenocytes, isolated from mice immunized by infection with the paramyxovirus simian virus 5 (SV5), enhance the speed of clearance of SV5 from the lungs of immunodeficient mice; clearance is mediated primarily through CD8+ effector cells and not by serum neutralizing antibody (D.F. Young, R.E. Randall, J.A. Hoyle, and B.E. Souberbielle, J. Virol. 64:5403-5411, 1990). In this article we demonstrate that immunization of mice with solid matrix-antibody-antigen (SMAA) complexes also induces CD8+ effector cells that are responsible for clearing persistent SV5 infections in immunodeficient mice. The demonstration that immunization with SMAA complexes (an exogenous antigen) can induce class I-restricted cytotoxic T lymphocytes (CTLs) suggests that that these cells may be responsible for virus clearance in vivo. This premise is supported indirectly by the observation that immunization with SMAA complexes was less efficient in inducing class I-restricted CTLs (as measured in vitro) than was infectious virus and that splenocytes isolated from mice immunized with SMAA complexes were also less efficient in clearing virus from lungs of immunodeficient mice than were splenocytes isolated from mice immunized by infection with virus. This was not because the SMAA complexes were generally less immunogenic than infectious virus, since mice immunized with SMAA complexes (which contained the HN protein of SV5) produced higher levels of neutralizing antibody than mice immunized with infectious virus. In the majority of experiments, fixed and killed suspensions of Staphylococcus aureus Cowan strain A were used as the solid matrix in the construction of SMAA complexes. However, in this article we present evidence that alum-antibody-antigen complexes are as immunogenic as S. aureus A-antibody-antigen complexes. These results suggest that the immunological reactivity of the solid matrix itself does not influence the intensity of the immune response to the antigens of interest in the SMAA complexes. The significance of these results for vaccine design are discussed.
Animals↗
Two nontemplated nucleotide additions are required to generate the P mRNA of parainfluenza virus type 2 since the RNA genome encodes protein V.
The nucleotide sequence of the "P/V gene" of parainfluenza virus type 2 is presented. To determine the nature of any nontemplated additions of nucleotides that may arise during the synthesis of mRNA from this gene the polymerase chain reaction was used to amplify specific sequences of both genomic RNA and mRNA. These results demonstrated that the V protein is encoded by the genome while insertion of two nontemplated G residues are required to synthesize P mRNA. The predicted P protein has 44% identical amino acid homology with that of simian virus 5 and 37% with mumps virus; 25% of the amino acids is conserved between all three viruses.
Amino Acid Sequence↗
Differential expression of two immediate early genes of herpesvirus saimiri as detected by in situ hybridization.
Transcripts from two immediate early (IE) genes have been identified in cells infected with the gamma herpesvirus, herpesvirus saimiri. One is a 1.3 kb RNA transcribed from the HindIII-G fragment of virus DNA (IE-G), the other is a 1.6 kb RNA from the gene for the IE 52K phosphoprotein. Labelled oligonucleotide probes specific for each of these RNAs have been used in in situ hybridization experiments to compare their expression in individual cells in infected populations. In the presence of cycloheximide, the IE-G RNA accumulates synchronously throughout the population of infected cells and prior to the asynchronous accumulation of RNA from the gene for the IE 52K protein in the same population of cells. This heterogeneity in the timing of expression of RNA from the IE 52K gene is paralleled by the asynchronous accumulation of the protein product. We conclude that transcription of the IE-G RNA is independent of expression of the IE 52K gene and that expression of the 52K gene requires (or is prevented by) factors which do not affect accumulation of the RNA from the IE-G gene.
Animals↗
Sequence analysis of the HN gene of parainfluenza virus type 2.
A cDNA library was constructed in lambda gt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43% amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.
Amino Acid Sequence↗
Clearance of a persistent paramyxovirus infection is mediated by cellular immune responses but not by serum-neutralizing antibody.
Infection of the lungs of immunodeficient mice with the paramyxovirus simian virus 5 (SV5) was prolonged compared with the time course of infection in immunocompetent mice. Although there was a significant increase in both viral RNA and proteins, little infectious virus was produced. Adoptive transfer of immune lymphocytes (isolated from the spleens of mice previously infected with SV5) but not of nonimmune lymphocytes increased the speed of clearance of virus from the lungs of immunodeficient mice. In contrast, passive transfer of a pool of neutralizing monoclonal antibodies specific for the HN and F glycoproteins of SV5 did not have a significant effect on the speed of clearance of virus. Furthermore, no significant increase in the rate of virus clearance was observed upon adoptive transfer of purified immune B lymphocytes to SV5-infected immunodeficient mice despite production by the mice of high titers of neutralizing antibodies. Evidence is presented that CD8+ effector cells are primarily responsible for the clearance observed. The general significance of these results with respect to immune clearance of persistent virus infections is discussed.
Animals↗
Multiple sclerosis and paramyxovirus.
Explore the source record for details and available documents.
Antibodies, Viral↗
Solid matrix-antibody-antigen (SMAA) complexes for constructing multivalent subunit vaccines.
For successful vaccination to many diseases, it is necessary to induce both humoral and cell-mediated immune responses to the infectious agent: this may require the incorporation of multiple antigens from the same microbe into the vaccine. In this article, Richard Randall proposes that one of the most practical and effective ways of producing multivalent vaccines may be through the construction of solid matrix-antibody-antigen (SMAA) complexes. The advantages of such vaccines and their future potential is discussed.
Animals↗
Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface.
The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is a type II integral membrane protein that is expressed at the infected cell surface. The intracellular assembly and transport of HN in CV1 cells was examined using conformation-specific HN mAbs and sucrose density sedimentation analysis. HN was found to oligomerize with a t1/2 of 25-30 min and these data suggest the oligomer is a tetramer consisting primarily of two noncovalently associated disulfide-linked dimers. As HN oligomers could be found that were sensitive to endoglycosidase H digestion and oligomers formed in the presence of the ER to the Golgi complex transport inhibitor, carbonylcyanide m-chlorophenylhydrazone (CCCP), these data are consistent with HN oligomerization occurring in the ER. Unfolded or immature HN molecules that could not be recognized by conformation-specific antibodies were found to specifically associate with the resident ER protein GRP78-BiP. Immunoprecipitation of BiP-HN complexes with an immunoglobulin heavy-chain binding protein (BiP) antibody indicated that newly synthesized HN associated and dissociated from GRP78-BiP (t1/2 20-25 min) in an inverse correlation with the gain in reactivity with a HN conformation-specific antibody, suggesting that the transient association of GRP78-BiP with immature HN is part of the normal HN maturation pathway. After pulse-labeling of HN in infected cells, it was found that HN is rapidly turned over in cells (t1/2 2-2.5 h). This led to the finding that the vast majority of HN expressed at the cell surface, rather than being incorporated into budding virions, is internalized and degraded after localization to endocytic vesicles and lysosomes.
Animals↗
Immunization against multiple viruses by using solid-matrix-antibody-antigen complexes.
We have previously shown that mice immunized with solid-matrix-antibody-antigen (SMAA) complexes in the absence of adjuvants show vigorous humoral and cell-mediated immune responses to the immunizing antigen. Here we report that various proteins involved in inducing protective immune responses to different viruses can easily and simply be incorporated into SMAA complexes and that such complexes act as powerful multivalent immunogens. Construction of such SMAA complexes may be one of the most practical and effective ways of producing multivalent subunit vaccines for use in humans and animals.
Animals↗
Humoral and cytotoxic T cell immune responses to internal and external structural proteins of simian virus 5 induced by immunization with solid matrix-antibody-antigen complexes.
Monoclonal antibodies (MAbs) were complexed to solid matrices and used to purify virus proteins from simian virus 5 (SV5)-infected tissue culture cells, ideally in such a way as to bind equimolar amounts of antigen to antibody. The resulting solid matrix-antibody-antigen (SMAA) complexes were then used as immunogens and successfully induced specific humoral and cytotoxic T cell responses. By attaching more than one MAb to the solid matrix and using such complexes to purify the respective proteins multivalent immunization was achieved. Analysis of the cytotoxic T cell response of immunized animals indicated that both surface and internal SV5 structural proteins can act as target antigens. Immunization with SMAA complexes, in the absence of adjuvant, induced higher levels of antibody than the antigen alone precipitated on alum. However, immunization with SMAA complexes resulted in relatively less antibody being produced to the antigenic determinant through which the protein is coupled, via antibody, to the SMAA complex compared with the amount of antibody produced against other antigenic determinants on that protein. The particulate solid matrix used to form the SMAA complexes in most of the experiments was a 'fixed' and killed suspension of the Cowan A strain of Staphylococcus aureus, although preliminary results indicated that Protein A-Sepharose could also be used as a solid matrix. Prior immunization with S. aureus alone did not reduce the level of the immune response to the appropriate antigen on subsequent immunization with S. aureus-antibody-antigen complexes. In fact on immunization of mice with these complexes the level of antibody produced to the S. aureus matrix itself was less when S. aureus-antibody-antigen complexes were used as immunogens than when S. aureus or S. aureus-antibody complexes were used. Furthermore, rabbits immunized with S. aureus-mouse MAb-antigen complexes showed a vigorous immune response to the antigen.
Animals↗
Immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, simian virus 5.
A mouse model system has been developed to examine the ability of purified virus proteins to protect mice from infection with the paramyxovirus simian virus 5. The system is based on the infection of mouse lungs by intranasal administration of infectious virus. The relative amounts of virus proteins and nucleic acid present within infected lungs were estimated either by Western blot analysis of disrupted lung tissues or by in situ hybridization studies using cryostat sections of infected lungs. During a normal time course of infection in non-immunized mice increasing amounts of virus protein and nucleic acid were detected in the lungs until 3 days post-infection (p.i.). Thereafter the amount of virus present within the lungs remained relatively constant until 7 days p.i. when there was a rapid decrease. Cytotoxic T cells, but not neutralizing antibody, could be detected at the time when the amount of virus within the lungs was decreasing. Prior immunization of mice with solid matrix-antibody-antigen (SMAA) complexes containing either surface or internal virus structural proteins reduced the amount of virus replication within infected lungs, the greatest degree of protection being observed when nucleoprotein or matrix protein was used to immunize the mice. There was no correlation between the degree of protection observed and the level of neutralizing antibody present in immunized animals; no neutralizing antibody was detected in mice immunized with internal virus proteins even at the time of sacrifice 5 days p.i. We have previously shown that immunization of mice with SMAA complexes containing either surface or internal virus structural proteins can induce cytotoxic T cells and thus conclude that the most likely explanation for the protection observed in immunized mice is through the induction of cytotoxic T cells.
Animals↗
Comparison between parainfluenza virus type 2 and simian virus 5: monoclonal antibodies reveal major antigenic differences.
Marked differences in the apparent Mrs of the HN, NP and F proteins of simian virus 5 (SV5) and parainfluenza virus type 2 (PF-2) were revealed by SDS-PAGE. To examine the antigenic relationships between SV5, PF-2 and other paramyxoviruses, monoclonal antibodies (MAbs) specific to PF-2 were isolated. These antibodies had specificities for the HN, NP and P proteins and together with 54 MAbs to SV5 were tested for their ability to react with SV5, PF-2, PF-3, mumps and measles virus proteins. Most of these MAbs (55 out of 60) reacted with homologous virus only. However, five reacted with both SV5 and PF-2. These antibodies had reactivities to the NP, M and P proteins. Furthermore, one of the antibodies with reactivity to the P protein also reacted with mumps virus. Although none of the 21 MAbs with specificities for the HN protein of either SV5 or PF-2 cross-reacted with heterologous virus, some antigenic similarities between the HN protein of SV5 and PF-2 could be detected. This was demonstrated by raising a series of polyclonal antisera to purified preparations of SV5 or PF-2 HN proteins in BALB/c mice, and testing for their ability to neutralize both SV5 and PF-2 and also to immunoprecipitate the HN proteins of these viruses. Surprisingly, while low levels of cross-neutralizing antibody could be detected in some sera (e.g. neutralization of SV5 1:1600 and of PF-2 1:80), other sera with similar neutralization titres against homologous virus failed to neutralize heterologous virus. Furthermore, only a minority of the anti-HN antisera showed any immune-precipitating activity against the heterologous HN protein.
Animals↗
Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates.
Hybridomas secreting monoclonal antibodies to simian virus 5 (SV5) were obtained following immunization of mice with purified preparations of a human isolate (LN) of SV5. Immune precipitation studies showed that these monoclonal antibodies had specificities for the haemagglutinin-neuraminidase (HN), fusion (F), nucleo-, matrix and phospho- (P) proteins of SV5. By use of a radioimmune competition assay the monoclonal antibodies to the HN protein were assigned to four groups, members of which recognized different antigenic sites on the protein. All the anti-HN antibodies and the anti-F antibody neutralized virus infectivity. The 54 monoclonal antibodies obtained were used to determine whether there were antigenic differences between five human, two canine and one simian isolate of SV5. Although most of the monoclonal antibodies reacted with all isolates, a few did reveal antigenic differences in the HN, F and P proteins. Furthermore, analysis by SDS-PAGE showed that while the electrophoretic mobilities of most of the virus polypeptides of these isolates were similar some differences could be detected. In particular the P protein showed the most marked mobility differences between the human, canine and simian isolates. Slight differences in the mobility of the F1 glycoprotein could also be visualized.
Animals↗