CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Liu

et al. 2019

Vaccine

a b s t r a c tThe novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.

Section: Discussioncontrasting

confidence: 72%

Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development

Liu

et al. 2019

Vaccine

“…6A) between the binding affinity of rgp120 A224-N332 produced in MGAT1 - CHO cells as measured by EC50 (1.33 nM) for PG9, when compared to the RV144 antigen, rgp120 A224, produced in CHO S cells (no binding plateau). This result is in concordance with data from previous transient transfection studies (57, 58). Binding of bN-mAbs to protein produced in the 5F MGAT1 - CHO cell line was indistinguishable to protein produced by transient transfection in MGAT1 - CHO cells.…”

Section: Resultssupporting

confidence: 93%

“…6F). This difference was also previously reported (57). VRC01 is an anti-CD4 binding site antibody with low affinity for glycan in glycan-array assay (65).…”

Section: Resultssupporting

confidence: 90%

“…Estimating that the frequency of cells expressing high levels of rgp120 might be in the range of one in 10 −4 (59-61), we calculated that we needed to screen 10- 100 thousand transfectants. To optimize transfection efficiency, we replaced cationic lipid transfection that we had previously used to transiently produce rgp120’s (36, 57, 58, 62) with electroporation. Use of the MaxCyte STX system resulted in reproducible transfections with efficiencies typically greater than 80% in CHO-S or MGAT1 - CHO cells when GFP expression was quantified by flow cytometry, and viabilities greater than 95% measured by trypan-blue exclusion (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine for production

O’Rourke

Byrne

Tatsuno

et al. 2018

Preprint

Self Cite

2 The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging.3 The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is 4 typically 10-100 fold lower than other glycoproteins of pharmaceutical interest.5 Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing 6 glycans compared to virus associated Envs that possess mainly high-mannose 7 carbohydrates. This difference alters the net charge and biophysical properties of Envs 8 and impacts their antigenic structure. Here we employ a novel gene-edited CHO cell 9 line (MGAT1 -CHO) to address the problems of low expression, high sialic acid content, 10 and poor antigenic structure. We demonstrate that stable cell lines expressing high 11 levels of gp120, potentially suitable for biopharmaceutical production can be created 12 using the MGAT1 -CHO cell line. We also show that the efficiency of this process can be 13 greatly improved with robotic selection. Finally, we describe a MGAT1 -CHO cell line 14 expressing A244-rgp120 that exhibits improved binding of three major families of bN-15 mAbs compared to Envs produced in normal CHO cells. The new strategy described 16 has the potential to eliminate the bottleneck in HIV vaccine development that has limited 17 the field for more than 25 years. 18 3 19 1 Introduction 20The development of a safe, effective, and affordable HIV vaccine is a global 21 public health priority. After more than 30 years of HIV research, a vaccine with these 22properties has yet to be described. To date, the only clinical study to show that 23 vaccination can prevent HIV infection is the 16,000-person RV144 trial carried out in 24 Thailand between 2003 and 2009 (1). This study involved immunization with a 25 recombinant canarypox virus vector to induce cellular immunity (2-4) and a bivalent 26 recombinant gp120 vaccine designed to elicit protective antibody responses (5-7).27 Although statistically significant, the protective efficacy of this vaccination regimen was 28 low (31.2%, P=0.04). Several correlates of protection studies suggested that the 29 protection observed was primarily due to antibodies to rgp120 (8-10). Thus, there is 30 considerable interest in finding ways to improve the level of protection that can be 31 achieved with rgp120 vaccine regimens. Improving an existing vaccine such as RV144, 32 with an established record of safety, would be faster and more cost-effective than 33 developing a new vaccine concept from scratch. A roadmap to improve the rgp120 34 vaccine used in the RV144 trial has been provided by the recent studies of broadly 35 neutralizing monoclonal antibodies (bN-mAbs) to gp120 as well as studies of the 36 carbohydrate content of virion associated Env proteins. Beginning in 2009, studies of 37 bN-mAbs isolated from HIV infected subjects revealed that many recognized unusual 38 glycan dependent epitopes requiring high-mannose glycans that are early intermediates 39 in the N-linked glycosylation pathway (11)(12)(13)(14)(15)(16)(17)(18)(...

Biotech & Bioengineering

“…ERV‐edited clones were found to proliferate at comparable rates as polyclonal populations, empty vector‐treated and untreated cell controls, with a density reaching approximately 12.5×10 6 cells/ml after 5 days in culture (Figure S8A). Such a cell density concords with the expected CHO‐K1 doubling time of roughly 20 hr (Byrne et al, ). However, two Myr2‐edited clones (C02, D12) and one PPYP6‐mutated clone (K14) showed slightly modified cell cycle durations, and the maximal viable cell density of C02 was reduced, although this effect was not statistically significant.…”

Section: Resultssupporting

confidence: 64%

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

Duroy

Bosshard

Schmid‐Siegert

et al. 2019

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.