A Method to Generate and Rescue Recombinant Adenovirus Devoid of Replication-Competent Particles in Animal-Origin-Free Culture Medium.

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA.

BCG administration promotes the long-term protection afforded by a single-dose intranasal adenovirus-based SARS-CoV-2 vaccine.

Recent publications have explored intranasal (i.n.) adenovirus-based (Ad) vaccines as an effective strategy for SARS-CoV-2 in pre-clinical models. However, the effects of prior immunizations and infections have yet to be considered. Here, we investigate the immunomodulatory effects of Mycobacterium bovis BCG pre-immunization followed by vaccination with an S-protein-expressing i.n. Ad, termed Ad(Spike). While i.n. Ad(Spike) retains some protective effect after 6 months, a single administration of BCG-Danish prior to Ad(Spike) potentiates its ability to control viral replication of the B.1.351 SARS-CoV-2 variant within the respiratory tract. Though BCG-Danish did not affect Ad(Spike)-generated humoral immunity, it promoted the generation of cytotoxic/Th1 responses over suppressive FoxP3+ TREG cells in the lungs of infected mice. Thus, this vaccination strategy may prove useful in limiting future pandemics by potentiating the long-term efficacy of mucosal vaccines within the context of the widely distributed BCG vaccine.

Preclinical Development and Characterization of Novel Adeno-Associated Viral Vectors for the Treatment of Lipoprotein Lipase Deficiency.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Inducible HEK293 AAV packaging cell lines expressing Rep proteins.

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7 weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1 L in a wave bioreactor using serum-free suspension culture.

Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning.

Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements. To accelerate clone isolation and ensure monoclonality, the packaging genes were transfected simultaneously into human embryonic kidney cells (293SF-3F6) previously engineered with the induction system, and clones were isolated after limiting dilution into nanowell arrays using a robotic cell picking instrument with scanning capability. The method's effectiveness to isolate colonies derived from single cells was demonstrated using mixed populations of cells labeled with two different fluorescent markers. Because the recipient cell line grew in suspension culture, and all the procedures were performed without serum, the resulting clones were readily adaptable to serum-free suspension culture. The best producer clone produced LVs expressing GFP at a titer of 2.3 × 108 transduction units (TU)/mL in the culture medium under batch mode without concentration.

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

A low dose adenovirus vectored vaccine expressing Schistosoma mansoni Cathepsin B protects from intestinal schistosomiasis in mice.

BACKGROUND: Schistosomiasis is an underestimated neglected tropical disease which affects over 236.6 million people worldwide. According to the CDC, the impact of this disease is second to only malaria as the most devastating parasitic infection. Affected individuals manifest chronic pathology due to egg granuloma formation, destroying the liver over time. The only FDA approved drug, praziquantel, does not protect individuals from reinfection, highlighting the need for a prophylactic vaccine. Schistosoma mansoni Cathepsin B (SmCB) is a parasitic gut peptidase necessary for helminth growth and maturation and confers protection as a vaccine target for intestinal schistosomiasis. METHODS: An SmCB expressing human adenovirus serotype 5 (AdSmCB) was constructed and delivered intramuscularly to female C57BL/6 mice in a heterologous prime and boost vaccine with recombinant protein. Vaccine induced immunity was described and subsequent protection from parasite infection was assessed by analysing parasite burden and liver pathology. FINDINGS: Substantially higher humoral and cell-mediated immune responses, consisting of IgG2c, Th1 effectors, and polyfunctional CD4+ T cells, were induced by the heterologous administration of AdSmCB when compared to the other regimens. Though immune responses favoured Th1 immunity, Th2 responses provided by SmCB protein boosts were maintained. This mixed Th1/Th2 immune response resulted in significant protection from S. mansoni infection comparable to other vaccine formulations which are in clinical trials. Schistosomiasis associated liver pathology was also prevented in a murine model. INTERPRETATION: Our study provides missing preclinical data supporting the use of adenoviral vectoring in vaccines for S. mansoni infection. Our vaccination method significantly reduces parasite burden and its associated liver pathology - both of which are critical considerations for this helminth vaccine. FUNDING: This work was supported by the Canadian Institutes of Health Research, R. Howard Webster Foundation, and the Foundation of the McGill University Health Centre.

A rapid Focus-Forming Assay for quantification of infectious adenoviral vectors.

Currently available methods to titrate adenoviral vectors (AdV) in the absence of a gene reporter such as GFP, are either time-consuming or not very reproducible. A Focus-Forming Assay (FFA) for quantification of infectious AdV particles followed by automated focus counting was developed using new monoclonal antibodies (mAbs) against the human adenovirus type 5. Briefly, in this method, 96-well plates of HEK293A cells were infected with 2-fold dilutions of AdV at seeding time. Forty eight hours post-infection, the cells were fixed with methanol. The cells were then incubated with each mAb followed by a FITC conjugated anti-mouse antibody. The plates were scanned and positive cells counted using an automated fluorescence microscopy system. The results of the FFA were compared with the plaque assay and the TCID50 assay. The titer of six different recombinant AdV were compared using the FFA along with a commercial kit. The results were similar, but in contrast to the commercial kit for which the stained cells are counted manually, the software automatically counts the positives cells in the FFA. The automatic counting of positive cells makes the FFA a more precise and reliable assay compared to the commercial kit for titration of AdV.

Impact of dextran sulfate in culture media on titration of vesicular stomatitis virus.

Viral vectors derived from vesicular stomatitis virus (VSV) are important vectors for the development of vaccines and for the treatment of cancer. The efficiency of therapy based on VSV is dependent on the dose of virus used. Therefore it is essential to measure accurately and reproducibly the amount of functional vectors in the samples to be tested. Two common methods used to measure the titer of VSV are TCID50% and plaque assay. In the current study, we compared these two titration methods by using a recombinant VSV expressing the green fluorescent protein (VSV-GFP) as a model virus. Some culture media developed for suspension mammalian cells contain dextran sulfate. We observed that plaque assay, but not TCID50%, can underestimate the virus titer up to 10 fold when VSV-GFP was produced in culture media containing dextran sulfate. Dextran sulfate is commonly used in serum-free culture media to reduce cell aggregation in suspension culture. The inhibitory effect of dextran sulfate on the titration of VSV-GFP was confirmed by supplementing the culture medium with this compound during virus production. Our results also demonstrated that extending the incubation time during plaque assay and TCID50% increases virus titer.

Optimization of production of vesicular stomatitis virus (VSV) in suspension serum-free culture medium at high cell density.

During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium.

The IGF-Trap: Novel Inhibitor of Carcinoma Growth and Metastasis.

The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1. The IGF-Trap has a high affinity for hIGFI and hIGFII but low affinity for insulin, as revealed by surface plasmon resonance. It efficiently blocked IGFIR signaling in several carcinoma cell types and inhibited tumor cell proliferation, migration, and invasion in vitro. In vivo, the IGF-Trap showed favorable pharmacokinetic properties and could suppress the growth of established breast carcinoma tumors when administered therapeutically into tumor-bearing mice, improving disease-free survival. Moreover, IGF-Trap treatment markedly reduced experimental liver metastasis of colon and lung carcinoma cells, increasing tumor cell apoptosis and reducing angiogenesis. Finally, when compared with an anti-IGFIR antibody or IGF-binding protein-1 that were used at similar or higher concentrations, the IGF-Trap showed superior therapeutic efficacy to both inhibitors. Taken together, we have developed a targeted therapeutic molecule with highly potent anticancer effects that could address limitations of current IGFIR-targeting agents.

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications.

Wild bird influenza survey, Canada, 2005.

Of 4,268 wild ducks sampled in Canada in 2005, real-time reverse transcriptase-PCR detected influenza A matrix protein (M1) gene sequence in 37% and H5 gene sequence in 5%. Mallards accounted for 61% of samples, 73% of M1-positive ducks, and 90% of H5-positive ducks. Ducks hatched in 2005 accounted for 80% of the sample.

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada.

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.

The ribonucleotide reductase domain of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is essential for R1 antiapoptotic function.

The R1 subunit (ICP10) of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR), which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 aa, protects cells against apoptosis. As the NH(2) domain on its own is not antiapoptotic, it has been postulated that both domains of R1 or part(s) of them could be necessary for this function. Here, N- and C-terminal deletions were introduced in HSV-2 R1 to map the domain(s) involved in its antiapoptotic potential. The results showed that, whereas most of the NH(2) domain including part of the recently described putative alpha-crystallin domain is dispensable for antiapoptotic activity, it is the integrity of the structured RR domain that is required for protection. As the alpha-crystallin domain appears to play an important role in protein folding and oligomerization, the N-terminal boundary of the antiapoptotic domain could not be defined precisely. In addition, this study provided evidence that overexpression of HSV-2 R2 at levels up to 30-fold more than HSV-2 R1 did not decrease protection from tumour necrosis factor alpha, indicating that the R1 surface where R2 binds is not involved in antiapoptotic activity. Importantly, this result suggests that the co-expression of both RR subunits during the lytic cycle should not affect protection from this cytokine.

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor.

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.

DNA delivery to cells in culture: generation of adenoviral libraries for high-throughput functional screening.

In functional genomics, the use of expression libraries of DNA variants in combination with potent screening techniques is a powerful tool for gene discovery. They allow study of gene and protein function, generation of peptide variants with novel properties, as well as identification of functional short DNA and RNA motifs. In proteomics, generation of large expression libraries of protein variants with random substitutions ("directed evolution") and further screening for novel or improved functions has been commonly used for isolation of proteins with novel characteristics, for improving enzymes, for rapid isolation of antibodies, and for functional protein studies. Most commonly, peptide libraries are expressed and screened in prokaryotic systems. Such systems have the advantage of rapid and simple generation of clones expressing single variants, allow high diversity (up to 10(11)), and can be combined with phage- or cell-surface display technique (2). The main disadvantage of bacterial systems is the absence of posttranslational modifications and native folding of many mammalian proteins, leading to limited applications, particularly when enzyme-substrate-, protein-protein, or protein-RNA interactions are to be studied.