Alternative for the neurovirulence test.

The neurovirulence test established by Albert B. Sabin is required according to different pharmaceutical monographies (WHO Technical Report Series, US-Pharmacopoeia, European Pharmacopoeia, etc) and is used for evaluation of sufficient and consistent attenuation of life monovalent poliomyelitis virus bulk lots (Sabin) which are used for the manufacture of oral poliomyelitis vaccines (OPV). Since this animal experiment is based on the use of relatively high numbers of primates (at least 22 animals per test vaccine for the virus serotypes 1 and 2 and at least 36 animals for serotype 3) it is searched for alternatives since several years.

Alternative for the neurovirulence test.

For safety testing and release for attenuated oral poliomyelitis virus (OPV) vaccine lots, national and international authorities require the in vivo neurovirulence test (NVT). This test is extremely expensive, time consuming and is carried out in socially sensitive monkeys. In this test the test vaccine is injected into the spinal chord of monkeys and the mean lesion score of neurovirulence is evaluated and compared to that of a reference vaccine.

Increased safety level of serotype 3 Sabin oral poliomyelitis vaccine lots by improved seed virus, and tissue culture and virus infection conditions.

The content of 472U to 472C revertant virus in serotype 3 oral poliomyelitis monovalent bulk vaccines can be quantified by MAPREC (Mutant Analysis by PCR and Restriction Enzyme Cleavage). Besides other wildtype reversions identified in propagated type 3 Sabin strain populations, the 472U to 472C reversion correlates most prominently with neurovirulence in the monkey neurovirulence test. Therefore, the results can be used for the discrimination of 'good' and 'bad' vaccines on the molecular level.

Mutations in Sabin 2 strain of poliovirus and stability of attenuation phenotype.

In this study, we attempted to identify the molecular determinants in the genome of the attenuated Sabin 2 vaccine strain of poliovirus that may change during vaccine production and result in an increase in monkey neurovirulence. An extensive search for suitable vaccine lots identified six batches that had failed the monkey neurovirulence test (MNVT). On repeated tests, these batches were found to have acceptable levels of monkey neurovirulence.

Status and transcriptional activity of a bovine-papillomavirus-I-based expression vector in a recombinant production cell line.

We have analysed the status and transcriptional activity of the bovine papillomavirus-I (complete BPV-I genome)-based expression vector pCES in CI27i-cell-line-derived 3TI cells used for the industrial production of recombinant human erythropoietin (rhuEpo). Complete tandem head-to-tail integration of about 600 vector copies at a single site of the cellular genome was observed. Deletions, insertions or rearrangements of pCES-specific sequences or extrachromosomal copies of vector sequences were not detected.

N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells.

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt.

Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization.

Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs.

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale.

Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced.

Evidence for the location of the receptor-binding site of human erythropoietin at the carboxyl-terminal domain.

Five different peptides (P1: 84-95; P2: 152-166; P3: 52-63; P4: 7-23; P5: 110-123) homologous to relatively hydrophilic regions of human erythropoietin (huEpo) have been synthesized to identify biologically active domains of the hormone. All peptides were able to induce high titers of peptide-specific antibodies in rabbits. Antisera from rabbits induced by recombinant huEpo (rhuEpo) contained a relatively high amount of antibodies preferentially directed against three peptides (P2, P4, and P5), of which P4 comprised the amino-terminal region, P2 the carboxyl-terminus, and P5 an interior region previously described as the receptor-binding site.

Coding sequence and expression of the homeobox gene Hox 1.3.

We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6.

Primary structure and nuclear localization of a murine homeodomain protein.

The murine homeobox Hox 1.1 (m6) is the first of a cluster of six boxes on chromosome 6. Using probes and synthetic peptides derived from the Hox 1.

Late-phase expression of a murine cytomegalovirus immediate-early antigen recognized by cytolytic T lymphocytes.

The cloned murine cytolytic T-lymphocyte line IE1-IL and several sublines detect a murine cytomegalovirus immediate-early (IE) membrane determinant in conjunction with Ld class I major histocompatibility glycoprotein. The lines retained cytolytic activity, strict antigen specificity, and self-restriction even when adapted to long-term, antigen-independent growth in the presence of interleukin-2 only (M. J.

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription.

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters.

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated.

Anti-idiotypes against anti-H-2 antibodies VI. Detection of shared idiotypes among monoclonal anti-H-2 antibodies.

Anti-idiotypes produced against monoclonal anti-H-2 antibodies have been used to examine idiotope sharing among a panel of anti-major histocompatibility complex (MHC) antibodies detecting the same and different specificities. Both xenogeneic anti-100-30 and anti-3-83, which did not react with predominant idiotypes in conventional alloantisera, detected idiotopes on 3-83 and 100-30 as well as on 2 other monoclonal anti-H-2Kk antibodies. All 4 monoclonal antibodies recognized the same epitope cluster on the Kk molecule and detected the same serological specificity.