Phylogenetic characterization of canine distemper virus from stray dogs in Kathmandu Valley.

Canine distemper is a highly contagious, often fatal disease caused by canine distemper virus (CDV) in domestic dogs and wild carnivores. The virus has caused mass epidemics in both wild and captive carnivores of high conservation value such as tigers, lions and leopards. Hence, understanding and managing CDV outbreaks is particularly important in Nepal, which is home to many species of threatened wild carnivores including tigers, leopards, snow leopards, dholes and wolves, and also contains a large population of stray dogs. Previous studies have suggested that CDV may pose a threat to wild carnivores, but there have not been any studies characterizing the genetic strains of the virus circulating in Nepal's carnivores. We collected invasive and non-invasive biological samples from stray dogs in Kathmandu Valley and genetically characterized the strains of CDV in the dogs to belong to the Asia-5 lineage by using phylogenetic analysis. The same lineage also contained CDV strains sequenced from dogs, civets, red panda and lions in India. Based on our phylogenetic analysis, we think it is likely that CDV is maintained through sylvatic cycle among sympatric carnivores allowing the recurring spillovers and outbreaks. It is crucial to prevent the virus transmission from reservoir hosts to other species, especially threatened populations of large carnivores in Nepal. Hence, we recommend for regular surveillance of CDV targeting wild carnivores in addition to the domestic dogs.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Development and Validation of an Anion Exchange High-Performance Liquid Chromatography Method for Analysis of Empty Capsids and Capsids Encapsidating Genetic Material in a Purified Preparation of Recombinant Adeno-Associated Virus Serotype 5.

The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the laboratory to the clinic. The active deployment of these analytical technologies for process and product characterization has helped define critical quality attributes and improve the quality of the clinical grade material. In this article, we report an anion exchange high-performance liquid chromatography (AEX-HPLC) method for relative and as well as absolute quantification of empty capsids (EC) and capsids encapsidating genetic material (CG) in purified preparations of adeno-associated virus (AAV) using serotype 5 as a model. The selection of optimal chromatographic buffer composition and step-gradient elution protocol offered baseline separation of EC and CG in the form of two peaks, as validated with the respective reference standards. The native amino acid fluorescence-based detection offered excellent linearity with a correlation coefficient of 0.9983 over two-log dilutions of the sample. The limit of detection and limit of quantification values associated with the total AAV5 capsid assay are 3.1E + 09 and 9.5E + 09, respectively. AEX-HPLC showed method comparability with the analytical ultracentrifugation (AUC) method for determination of relative proportions of EC and CG, supporting the reported HPLC method as an easy-to-access alternative to AUC with operational simplicity. Moreover, rapid and easy adaptation of this method to AAV8 material also demonstrated the robustness of the proposed approach.

Advancements in molecular design and bioprocessing of recombinant adeno-associated virus gene delivery vectors using the insect-cell baculovirus expression platform.

Despite rapid progress in the field, scalable high-yield production of adeno-associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC-BEVS for rAAV production. Since the first report of baculovirus-induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac-vector expression and a reduced number of baculovirus-coinfections. The latter streamlining strategy led to the eventual development of the Two-Bac, One-Bac, and Mono-Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large-scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell-specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges.

Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9.

Removal of empty capsids from adeno-associated virus (AAV) manufacturing lots remains a critical step in the downstream processing of AAV clinical-grade batches. Because of similar physico-chemical characteristics, the AAV capsid populations totally lacking or containing partial viral DNA are difficult to separate from the desired vector capsid populations. Based on minute differences in density, ultracentrifugation remains the most effective separation method and has been extensively used at small scale but has limitations associated with availabilities and operational complexities in large-scale processing. In this paper, we report a scalable, robust, and versatile anion-exchange chromatography (AEX) method for removing empty capsids and subsequent enrichment of vectors of AAV serotypes 5, 6, 8, and 9. On average, AEX resulted in about 9-fold enrichment of AAV5 in a single step containing 80% ± 5% genome-containing vector capsids, as verified and quantified by analytical ultracentrifugation. The optimized process was further validated using AAV6, AAV8, and AAV9, resulting in over 90% vector enrichment. The AEX process showed comparable results not only for vectors with different transgenes of different sizes but also for AEX runs under different geometries of chromatographic media. The herein-reported sulfate-salt-based AEX process can be adapted to different AAV serotypes by appropriately adjusting elution conditions to achieve enriched vector preparations.

Achieving High-Yield Production of Functional AAV5 Gene Delivery Vectors via Fedbatch in an Insect Cell-One Baculovirus System.

Despite numerous advancements in production protocols, manufacturing AAV to meet exceptionally high demand (1016-1017 viral genomes [VGs]) in late clinical stages and for eventual systemic delivery poses significant challenges. Here, we report an efficient, simple, scalable, robust AAV5 production process utilizing the most recent modification of the OneBac platform. An increase in volumetric yield of genomic particles by ∼6-fold and functional particles by ∼20-fold was achieved by operating a high-cell-density process in shake flasks and bioreactors that involves an Sf9-based rep/cap stable cell line grown at a density of about 10 million cells/mL infected with a single baculovirus. The overall volumetric yields of genomic (VG) and bioactive particles (enhanced transducing units [ETUs]) in representative fedbatch bioreactor runs ranged from 2.5 to 3.5 × 1014 VG/L and from 1 to 2 × 1011 ETU/L. Analytical ultracentrifugation analyses of affinity-purified AAV vector samples from side-by-side batch and fedbatch production runs showed vector preparations with a full and empty particle distribution of 20%-30% genomic and 70%-80% empty particles. Moreover, the stoichiometric analysis of capsid proteins from fedbatch production in shake flask and bioreactor run samples demonstrated the incorporation of higher VP1 subunits, resulting in better functionality.

Synthesis and antibacterial activity of nisin-containing block copolymers.

Nisin, an antibacterial peptide proven to be an effective inhibitor of Gram-positive bacteria, was incorporated into novel block copolymer constructs and tested for retained antibacterial activity. Covalent coupling was achieved by chemical modification of the N-terminal isoleucine to introduce a thiol group. Thiolated-nisin derivatives were then linked to poly[ethylene oxide]-poly[propylene oxide]-poly[ethylene oxide] (PEO-PPO-PEO) triblocks that had been end-activated such that terminal hydroxyl groups of the PEO chains were replaced with pyridyl disulfide moieties. The nisin-containing block copolymers were separated from free nisin by dialysis and showed antimicrobial activity against the Gram-positive indicator strain Pediococcus pentosaceus. The contribution to antimicrobial activity from nisin that was covalently linked was not distinguished from the contribution of nisin that had associated with the PEO-PPO-PEO triblocks through noncovalent interactions. However, nisin that was covalently linked showed activity upon reduction of the disulfide bond and release from the end-activated PEO.