Adenoviral Vector: Insights, Mechanisms, and Applications

Adenoviral vectors are essential tools in gene therapy and vaccine development due to their efficiency in delivering genetic material into cells. Their ability to induce strong immune responses has made them particularly valuable for vaccine platforms, including those used during the COVID-19 pandemic.

Their widespread use stems from advantages such as high transduction efficiency, transient gene expression, and adaptability for various therapeutic applications. However, challenges like preexisting immunity and potential inflammatory responses must be managed.

Structure And Composition

Adenoviral vectors are derived from non-enveloped, double-stranded DNA viruses with an icosahedral capsid approximately 90–100 nm in diameter. This capsid consists of three primary structural proteins: hexon, penton base, and fiber. Hexon proteins form most of the capsid surface, providing structural integrity. Penton base proteins, located at the vertices, mediate cell entry, while fiber proteins extend outward and facilitate receptor binding, a crucial step in host cell recognition.

The adenoviral genome is a linear, double-stranded DNA molecule ranging from 26 to 45 kilobases. It is divided into early (E) and late (L) transcription units, which regulate viral replication and protein synthesis. In gene therapy applications, vectors are engineered to remove specific early genes, such as E1 and E3, to prevent replication and create space for therapeutic transgenes. The deletion of E1 renders the virus replication-deficient, ensuring genetic material is delivered without producing infectious progeny. The E3 region is often removed to accommodate larger transgenes.

Capsid stability is reinforced by minor proteins such as protein IX, which enhances rigidity, and protein VI, which facilitates endosomal escape. These components work together to protect the viral genome and ensure efficient gene delivery. Additionally, terminal proteins at the ends of the viral DNA play a role in genome replication during natural infections, though this function is largely redundant in replication-deficient vectors used for therapy.

Mechanism Of Gene Delivery

Adenoviral vectors transfer genes through a series of interactions between viral components and host cellular machinery. The process begins when fiber proteins bind to specific cell surface receptors, typically the coxsackievirus and adenovirus receptor (CAR) for many serotypes, while others use alternative receptors such as desmoglein-2. Secondary interactions between penton base proteins and integrins, particularly αvβ3 and αvβ5, trigger receptor-mediated endocytosis. The virus is internalized via clathrin-coated pits, forming an early endosome.

Inside the endosome, acidic conditions induce conformational shifts in the capsid proteins, particularly protein VI, which disrupts the endosomal membrane, allowing the virion to escape into the cytoplasm. This step prevents lysosomal degradation and ensures the viral genome reaches the nucleus intact. The virus is transported along microtubules toward the nuclear envelope, utilizing dynein motor proteins.

At the nuclear pore complex, the remaining capsid components dissociate, enabling the viral genome to enter the nucleus. Unlike retroviruses and lentiviruses, which integrate into the host genome, adenoviral DNA remains episomal, reducing the risk of insertional mutagenesis. Once inside the nucleus, the transgene is transcribed by the host’s transcriptional machinery, leading to the production of the desired therapeutic or immunogenic protein.

Types Of Adenoviruses

Adenoviruses are classified into seven species, labeled A through G, with over 100 distinct serotypes identified in humans and animals. These serotypes exhibit varying degrees of tropism, influencing their suitability for gene therapy and vaccines.

Species C, which includes serotypes Ad2 and Ad5, is widely used due to its high transduction efficiency in epithelial and liver cells. Its well-characterized genetic structure has enabled the development of replication-deficient variants that maximize safety while maintaining strong transgene expression. The episomal nature of these vectors ensures transient gene expression, which is beneficial for controlled protein production.

Species B adenoviruses, including serotypes Ad3, Ad7, Ad11, and Ad35, target CD46, a complement regulatory protein. This receptor preference allows efficient transduction of hematopoietic cells, making them attractive for cancer gene therapy and hematologic disorders. Their reduced liver tropism compared to Ad5 also lowers off-target effects in hepatic tissues. Ad35-based vectors have been explored for vaccine development, particularly for populations with preexisting immunity to Ad5.

Species D encompasses the largest number of human adenovirus serotypes, many of which have strong ocular and gastrointestinal tropism. Ad26 and Ad48 have gained attention in vaccine development due to their ability to elicit robust immune responses while maintaining distinct serological profiles from more commonly used vectors like Ad5. Ad26-based vectors were used in COVID-19 vaccines, demonstrating their potential for large-scale immunization. Their moderate liver targeting makes them viable candidates for applications requiring controlled transgene expression.

Immune Response

Adenoviral vectors interact with the immune system through both innate and adaptive mechanisms, influencing their efficacy in gene therapy and vaccination. Upon administration, host cells detect viral components through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. These sensors recognize adenoviral DNA and capsid proteins, triggering signaling cascades that activate pro-inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and type I interferons. This immune activation recruits dendritic cells and macrophages, which process viral antigens.

As antigen-presenting cells present adenoviral proteins via major histocompatibility complex (MHC) molecules, the adaptive immune system responds. CD8+ cytotoxic T lymphocytes (CTLs) recognize infected cells and eliminate them, while CD4+ helper T cells facilitate antibody production by B cells. Neutralizing antibodies against adenoviral capsid proteins, particularly hexon and fiber proteins, can limit vector efficacy upon repeated administration by blocking cellular entry. This humoral response is a key consideration in therapeutic applications requiring multiple doses, as preexisting immunity to common serotypes like Ad5 can reduce effectiveness.

Steps In Manufacturing

The production of adenoviral vectors involves tightly controlled steps to ensure purity, potency, and safety. Each phase optimizes vector yield while minimizing contaminants, such as replication-competent adenoviruses (RCAs), which can arise from recombination events. The process begins with generating a seed stock using a cell line, such as HEK293, that provides essential viral proteins for replication-deficient vectors. These cells are transfected with a plasmid containing the engineered adenoviral genome, allowing controlled viral amplification under bioreactor conditions. Once sufficient virus is produced, cells are lysed to release viral particles, which undergo purification steps, including ultracentrifugation and chromatography, to remove host cell debris.

Following purification, vectors undergo rigorous quality control testing to assess genetic stability, potency, and absence of contaminants. Analytical methods such as PCR and next-generation sequencing confirm the absence of unwanted genetic recombination, while potency assays evaluate transduction efficiency. Stability studies determine appropriate storage conditions, as adenoviral vectors require careful handling to maintain functionality. Once these parameters meet regulatory standards, the final product is formulated and vialed for clinical or research use. Production facilities must adhere to Good Manufacturing Practices (GMP) guidelines set by regulatory agencies such as the FDA and EMA to ensure consistency and safety.

Species C

Adenoviral vectors from Species C, particularly Ad2 and Ad5, are widely used in gene therapy and vaccine development. Their broad tropism and high transduction efficiency make them effective for delivering genetic material to various cell types, including epithelial and liver cells. Ad5-based vectors have been employed in vaccine platforms, such as those developed for HIV and COVID-19, due to their ability to induce strong antigen expression and immune responses. However, widespread natural exposure to Ad5 has led to preexisting immunity, which can reduce vector effectiveness upon repeated administration.

To address this challenge, modifications such as chimeric fiber proteins or alternate serotype backbones have been explored to evade neutralizing antibodies. Researchers have also investigated helper-dependent adenoviral vectors, which lack all viral coding sequences, to minimize immune recognition and prolong transgene expression.

Species B

Adenoviruses from Species B, including Ad3, Ad7, Ad11, and Ad35, exhibit distinct receptor interactions. Unlike Ad5, which primarily binds to CAR, many Species B adenoviruses utilize CD46, a complement regulatory protein expressed on various human cells. This receptor preference allows efficient transduction of hematopoietic cells, making them valuable for hematologic diseases and oncology.

Their reduced liver tropism compared to Ad5 presents advantages in systemic gene therapy by lowering off-target effects. Ad35-based vectors have been investigated for tuberculosis and Ebola vaccines, where their distinct serological profile helps circumvent preexisting immunity to Ad5.

Species D

Species D, the largest adenovirus group, includes Ad26 and Ad48, which have been prominent in vaccine development. Their ability to elicit strong immune responses while maintaining low seroprevalence in human populations has made them key candidates for vaccine platforms. The Ad26-based vector used in the Johnson & Johnson COVID-19 vaccine demonstrated its effectiveness in inducing protective immunity with a single dose.

Beyond vaccines, Species D adenoviruses are being explored for gene therapy applications requiring efficient transduction without excessive liver targeting. Their moderate ability to transduce epithelial and immune cells while avoiding strong preexisting immunity makes them well-suited for controlled gene expression.