Adenoviruses are a group of common viruses that can cause a range of illnesses, often resembling the common cold or flu. These non-enveloped viruses contain a double-stranded DNA genome encased within an icosahedral protein shell called a capsid. Over 50 distinct types of adenoviruses can infect humans, leading to conditions like bronchitis, sore throat, or pink eye. While most adenovirus infections are mild, they can persist on surfaces and be shed from the body for days or weeks after symptoms resolve.
Why Adenoviruses Are Produced
Adenoviruses are engineered and produced for their utility in various therapeutic applications, leveraging their ability to efficiently deliver genetic material into a broad range of cell types. A primary application is in gene therapy, where modified adenoviruses serve as vectors to introduce therapeutic genes into cells to treat genetic disorders or cancer. For instance, they can deliver genes to correct deficiencies in conditions like cystic fibrosis or macular degeneration. These engineered viruses are replication-defective, meaning they cannot multiply on their own, ensuring their role is gene delivery.
Adenoviruses also function as platforms for vaccine development, delivering specific antigens to stimulate an immune response against various pathogens. This approach has been used in some COVID-19 vaccines, such as those employing the ChAdOx1 or Ad26.COV2.S platforms, which are designed to express the SARS-CoV-2 spike protein. The advantage of adenoviruses in vaccine development lies in their capacity to induce both strong cellular and humoral (antibody) immunity. Their natural ability to infect respiratory cells is beneficial for vaccines targeting respiratory infections.
Beyond gene therapy and vaccines, adenoviruses are engineered as oncolytic viruses for cancer treatment. These viruses are designed to infect and replicate within cancer cells, causing them to burst and die while minimizing harm to healthy cells. This process, known as oncolysis, also releases tumor antigens, which can stimulate the patient’s immune system to mount an anti-tumor response. Examples like Ad5-VirRX 007 are being investigated for their potential to selectively target and eliminate various cancer types.
The Production Journey
The production of adenoviruses for therapeutic purposes is a multi-stage process requiring specialized facilities and controlled environments. The journey begins with cell culture, where specific host cells are grown in large bioreactors. Human embryonic kidney 293 (HEK293) cells are used because they contain certain adenovirus genes that allow replication-defective adenoviruses to multiply. These cells are propagated and expanded to achieve high densities suitable for viral infection.
Once the host cell population reaches the desired density, the engineered adenovirus is introduced into the cell culture during the viral infection stage. The adenovirus enters the HEK293 cells, utilizing the cellular machinery and complementary viral genes provided by the host cells to replicate and produce new viral particles. This replication continues until a significant portion of the cells show signs of infection and lysis, indicating a high yield of new virus.
Following successful replication, the next step involves harvesting the virus. This includes collecting the cells and the surrounding culture medium, as viruses can be found in both locations. The harvested material then undergoes a series of purification steps to separate the adenovirus particles from cellular debris, host cell proteins, DNA, and other impurities. Common purification methods include chromatography, which separates components based on their chemical or physical properties, and ultracentrifugation, which separates particles by density or size.
The final stage of the production journey is formulation, where the purified adenovirus is prepared into a stable solution suitable for storage and administration. This involves suspending the virus in a buffer system containing specific excipients, such as sugars like sucrose or trehalose, and non-ionic surfactants like polysorbate-80. These components help maintain the virus’s integrity and activity, preventing aggregation or degradation during storage at refrigerated temperatures between 2°C and 8°C for up to 24 months.
Ensuring Quality and Safety
Ensuring the quality and safety of produced adenoviruses involves rigorous testing throughout and after the manufacturing process. Sterility testing is performed to confirm the absence of contamination by bacteria, fungi, or other adventitious agents that could pose risks to patients. This involves culturing samples from the viral preparation to detect any microbial growth.
Purity testing is conducted to verify that the final product is free from unwanted components, such as residual host cell DNA, proteins, or other viral contaminants. Techniques like quantitative PCR (qPCR) are used to measure host cell DNA levels, with international guidelines stipulating a maximum acceptable level, around 10 nanograms per dose. SDS-PAGE and chromatography methods are employed to assess protein purity and confirm the correct composition of viral capsid proteins.
Potency testing confirms that the viral preparation has the desired biological activity and concentration. This involves cell-based assays that measure the virus’s ability to infect target cells and express the therapeutic gene or antigen. Identity testing is also performed to confirm that the produced material is indeed the correct adenovirus vector, by verifying the viral genome and the expression of the intended transgene.
Regulatory bodies, such as the Food and Drug Administration (FDA) in the United States, play a role in overseeing the entire production process of these biological products. They establish guidelines and requirements for manufacturing practices and quality control, ensuring that gene therapy products and vaccines meet high standards for safety and effectiveness before they can be used in clinical trials or made available to the public.