Recombinant envelope glycoproteins prepared from a subtype B (MN) strain and a subtype E (CM244) strain of HIV-1 were combined to create a bivalent vaccine (B/E) effective against viruses circulating in the United States and Asia. Combining the two antigens resulted in formulations that increased the breadth and potency of the inter-subtype neutralizing response. Antibodies to the bivalent vaccine formulation neutralized viruses possessing diverse phenotypes, including syncytia-inducing and non-syncytia-inducing primary isolates, viruses using either the CCR5 or the CXCR4 chemokine receptors, and viruses differing in their sensitivity to soluble CD4. These studies demonstrate for the first time that the magnitude and quality of the immune response to HIV-1 can be improved by combining recombinant envelope glycoproteins from different genetic subtypes.

Strategies that prevent the attachment of N-linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N-linked glycosylation sites. Site-directed mutagenesis was used to delete N-linked glycosylation sites from a chimeric protein, TNFR-IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR-IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N-terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport.

Annals of the New York Academy of SciencesVolume 782, Issue 1 p. 70-78 Gene Transfer and Amplification in CHO Cells Efficient Methods for Maximizing Specific Productivity and Assessment of Genetic Consequences FLORIAN M. WURM, FLORIAN M. WURM Genentech Inc. Process Sciences South San Francisco, California 94080 Current address (corresponding author): Florian M. Wurm, Professor of Biotechnology, Swiss Federal Institute of Technology Lausanne (EPFL), Department of Chemistry, CH-1015 Lausanne, Switzerland.Search for more papers by this authorADRIANA JOHNSON, ADRIANA JOHNSON Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorTHOMAS RYLL, THOMAS RYLL Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorCHRISTIANE KÖHNE, CHRISTIANE KÖHNE Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorHARRY SCHERTHAN, HARRY SCHERTHAN Department of Human Genetics University of Kaiserslautern Kaiserslautern, GermanySearch for more papers by this authorFRANK GLAAB, FRANK GLAAB Department of Human Genetics University of Kaiserslautern Kaiserslautern, GermanySearch for more papers by this authorYOLANDA S. LIE, YOLANDA S. LIE Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorCHRISTOS J. PETROPOULOS, CHRISTOS J. PETROPOULOS Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorW. ROBERT ARATHOON, W. ROBERT ARATHOON Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this author FLORIAN M. WURM, FLORIAN M. WURM Genentech Inc. Process Sciences South San Francisco, California 94080 Current address (corresponding author): Florian M. Wurm, Professor of Biotechnology, Swiss Federal Institute of Technology Lausanne (EPFL), Department of Chemistry, CH-1015 Lausanne, Switzerland.Search for more papers by this authorADRIANA JOHNSON, ADRIANA JOHNSON Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorTHOMAS RYLL, THOMAS RYLL Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorCHRISTIANE KÖHNE, CHRISTIANE KÖHNE Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorHARRY SCHERTHAN, HARRY SCHERTHAN Department of Human Genetics University of Kaiserslautern Kaiserslautern, GermanySearch for more papers by this authorFRANK GLAAB, FRANK GLAAB Department of Human Genetics University of Kaiserslautern Kaiserslautern, GermanySearch for more papers by this authorYOLANDA S. LIE, YOLANDA S. LIE Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorCHRISTOS J. PETROPOULOS, CHRISTOS J. PETROPOULOS Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this authorW. ROBERT ARATHOON, W. ROBERT ARATHOON Genentech Inc. Process Sciences South San Francisco, California 94080Search for more papers by this author First published: May 1996 https://doi.org/10.1111/j.1749-6632.1996.tb40548.xCitations: 23 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume782, Issue1Recombinant DNA Biotechnology III: The Integration of Biological and Engineering ScienceMay 1996Pages 70-78 RelatedInformation

To express recombinant proteins, we routinely co-transfect DHFR-deficient CHO cells with DNA that encodes a recombinant protein together with a plasmid that encodes the enzyme DHFR. Subsequently, cells producing DHFR are selected and screened for recombinant protein expression. We and others have shown that Chinese hamster ovary (CHO) cells contain several hundred copies of endogenous retrovirus-like sequences that are closely related to mammalian A-type or C-type retroviruses (1,2,3,4). Since both A-type and C-type sequences are actively transcribed in many CHO cell lines(2,3), some elements must reside in transcriptionally active regions of chromatin. Using fluorescence in situ hybridization we have demonstrated that CHO retroviral sequences are distributed randomly among all chromosomes. By using DHFR plasmids containing defective A-type and C-type CHO sequences, we can improve our transfection efficiencies and increase recombinant protein production(5). This paper extends our studies on the use of endogenous retroviral sequences and begins to address questions concerning the molecular basis for the observed effects. Our findings have lead us to speculate that A-type and C-type retrovirus sequences may mediate the integration of transfected DNA into transcriptionally active regions of the CHO genome (referred to as "retrotargeting" RTT), thereby increasing the level of protein production.

CHO cells, like many other rodent cells, contain hundreds and perhaps thousands of copies of sequences reminiscent of A-type and C-type retrovirus genomes1,2,3,4. RNA molecules derived from these pseudo virus genomes are abundantly represented in CHO cells indicating that these sequences are in locations of active gene transcription. We hoped that the inclusion of such CHO-derived retroviral sequences in the DNA preparations for transfection would mediate integration into transcriptionally active chromosomal regions. Two phenomena have been studied: the effect of CHO derived retroviral sequences on (1) the transfection efficiency and (2) their effects on the expression levels of clonal cell lines derived from these transfections.

Using fluorescence in situ hybridization (FISH)(1) we studied distribution, structures and statistical trends of amplified sequences in recombinant CHO cell lines. We found a high proportion of cells (40–60%) with multiple and/or unusually structured and extended chromosomal regions containing amplified sequences in the presence but not in the absence of methotrexate (MTX). Removal of MTX from culture media resulted in rapid disappearance of cells containing those amplified sequences represented by multiple and heterogeneous integrations. In cloned lines, a single, defined "master integration" became the dominant representative of amplified sequences in these cells. We studied long term genetic stability and specific productivity in the absence of MTX in uncloned, heterogeneous populations of cells representing multiple independent integration and amplification events. In order to allow for maximal growth rates and enhanced chances for selection of low producer subpopulations we used continuous high density perfusion cultures. We found no evidence for selection of subpopulations without or with significantly reduced amounts of amplified sequences. Also, specific productivity of these cell lines did not decrease over a time of about 100 days. Conclusions were: MTX is responsible for continuing rearrangements of amplified (and nonamplified) DNA sequences in recombinant CHO cells. In the absence of MTX clonal cell lines are characterized by genetically stable, single, unique, identifiable integrations of amplified sequences which can serve as identifying genetic markers.