Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 2 de 2
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
PLoS One ; 15(5): e0233866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470085

RESUMO

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.


Assuntos
Vacinas contra a AIDS/imunologia , Edição de Genes , Proteólise , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , Cricetulus , Fator VIII/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Proteases/química , Serina Proteases/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , Trombina/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
2.
Biotechnol Bioeng ; 116(9): 2130-2145, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31087560

RESUMO

Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s-/- MGAT1- CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.


Assuntos
Sistemas CRISPR-Cas , Complemento C1s/genética , Complemento C1s/metabolismo , Técnicas de Inativação de Genes , Proteína gp120 do Envelope de HIV/biossíntese , HIV-1 , Proteólise , Animais , Células CHO , Cricetulus , Proteína gp120 do Envelope de HIV/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA