CHO cells for virus-like particle and subunit vaccine manufacturing.

Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development. We also discuss the challenges associated with vaccine production in CHO cells, with a focus on ensuring viral clearance for eVLP products.

MicroRNAs and long non-coding RNAs during transcriptional regulation and latency of HIV and HTLV.

Human immunodeficiency virus (HIV) and human T cell leukemia virus (HTLV) have replicative and latent stages of infection. The status of the viruses is dependent on the cells that harbour them and on different events that change the transcriptional and post-transcriptional events. Non-coding (nc)RNAs are key factors in the regulation of retrovirus replication cycles. Notably, micro (mi)RNAs and long non-coding (lnc)RNAs are important regulators that can induce switches between active transcription-replication and latency of retroviruses and have important impacts on their pathogenesis. Here, we review the functions of miRNAs and lncRNAs in the context of HIV and HTLV. We describe how specific miRNAs and lncRNAs are involved in the regulation of the viruses' transcription, post-transcriptional regulation and latency. We further discuss treatment strategies using ncRNAs for HIV and HTLV long remission, reactivation or possible cure.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.

Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters.

The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5' end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5' transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.

Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain.

Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.