Vaccination is a critical component of public health policy and has proven to be a cost-effective strategy in protecting human and animal populations. However, traditional classical vaccine technologies face inherent manufacturing challenges when developing vaccines against emerging pathogens with pandemic potential. Therefore, the development of platforms capable of rapid response to epidemic threats and effective adaptation to newly emerging escape variants is of paramount importance. Furthermore, such platforms should allow for plug-and-play modular designs or similar formulations to achieve rapid response capabilities, large-scale production, and facilitate smaller facility footprints, lower costs, and easier manufacturing deployment. Among these strategies, viral vector-based vaccines represent a viable approach.
Viral vector-based vaccines are derived from engineered viruses that encode the gene(s) of one or more antigens cloned into the vector backbone. Viral vectors can be designed as replication-defective (incapable of replication) while retaining the ability to infect cells and express encoded antigens. In contrast, replication-competent vectors are considered truly infectious, akin to live-attenuated vaccines. The production of viral vectors has been streamlined through multi-step processes, including plug-and-play genetic engineering methods, large-scale transfection, amplification in mammalian cell culture, harvest, purification, concentration, diafiltration, and formulation. The Vesicular Stomatitis Virus (VSV)-derived Ebola vaccine encoding the Ebola surface glycoprotein (Gp) serves as an example of a replication-competent vaccine. Approved by the FDA in December 2019, this vaccine was deployed as part of vaccination strategies to combat Ebola outbreaks. Conversely, non-replicating viral vectors do not produce productive infections and are generally considered safer and easier to manufacture. Replication-deficient human and chimpanzee adenoviruses (Ad and ChAd), adeno-associated viruses (AAV), modified VSV, modified Vaccinia Ankara (MVA), poxviruses, and Newcastle Disease Virus (NDV) are other examples extensively utilized in the development of safe, virus-based vaccines.
Typically, these platforms mimic natural infection to elicit robust humoral and cellular (CD4+ and CD8+) responses. The potent immune responses observed with these platforms are attributed to broad tropism, high transduction efficiency of vector entry into target cells, efficient antigen expression driven by strong promoters, long-term antigen expression, and the intrinsic immunogenicity of the vector viruses themselves, such as the presence of pathogen-associated molecular patterns (PAMPs).
Due to the versatility of production platforms and the capacity for rapid deployment during epidemics or pandemics, viral vectors are increasingly used for the production of prophylactic vaccines. Beyond their high immunogenicity, viral vector-based vaccines are easier to manufacture and, in some cases, safer compared to inactivated, attenuated, and recombinant protein technologies. As viral vector vaccines induce potent immune responses, they are often employed as single-dose regimens or as components of heterologous prime-boost vaccination schedules.
Key considerations in developing these platforms include pre-existing immunity against the viral vector and reduced efficacy upon subsequent administration due to anti-vector immunity. Strategies developed to circumvent these drawbacks include the use of chimeric vectors, vectors derived from different species (e.g., chimpanzee, bovine, and porcine), or vector serotypes known to have low seroprevalence in human populations. Since seropositivity rates may vary across regions, careful consideration is required during the development of such vaccines.
Adenoviral Vector Vaccines
Currently, several adenoviral vectors are employed as vaccine delivery systems, including Adenovirus serotype 5 (Ad5), Ad26, and chimpanzee adenoviruses. Adenoviruses (Ad) are a diverse family of DNA viruses causing respiratory, ocular, and gastrointestinal epithelial infections in hosts. Their genomes consist of linear double-stranded DNA ranging from 26–45 kb. Adenoviruses offer multiple advantages as viral vectors for vaccine development, including relatively low pathogenicity, genetic safety, and a lack of integration into the host genome. Other attractive features of Ad vectors for vaccine development include their robust immunogenicity, efficient infection of various cell types, and capacity for transgene incorporation. Adenoviral-based vectors can be replication-competent or replication-defective, depending on whether they contain the entire Early 1 (E1) region or parts thereof. Traditionally, Ad vectors are produced using adherent packaging cell lines, with virus vectors purified from lysates via cesium chloride density gradient centrifugation. However, for large-scale manufacturing, suspension culture bioreactors utilizing continuous cell lines are preferred for vector propagation. Purification processes typically involve several chromatography steps coupled with tangential flow filtration.
Adeno-Associated Virus (AAV) Vector Vaccines
Adeno-associated viruses belong to the Parvoviridaefamily. They are non-enveloped viruses with a genome consisting of 4.8 kb of linear single-stranded DNA. AAV vectors are among the most popular choices in gene therapy applications due to their relatively low immunogenicity, high safety profile, broad tropism, and propensity for sustained long-term gene expression. AAV vectors are typically produced by transfecting Human Embryonic Kidney (HEK) 293T cells with plasmids containing the transgene, packaging, and helper functions. While AAV vectors are primarily used for treating ocular and muscular diseases, their utility as vaccine carriers has increased in recent years, including for infectious diseases such as HIV, HPV, and influenza. Several studies indicate that AAV vector vaccines can induce intense and durable antibody responses after a single dose without the need for adjuvants. In some cases, they yield higher or more persistent antibody responses compared to other vaccination strategies. However, AAV vectors are considered to possess lower immunogenicity relative to other viral vectors. The isolation and development of novel AAV serotypes and capsid variants provide opportunities to optimize prime/boost strategies, where switching AAV capsids can help evade neutralizing antibody responses against the capsid induced after priming. Major drawbacks of AAV vectors include limited transgenic capacity and widespread pre-existing human immunity. Current research is evaluating strategies to enhance AAV immunogenicity and bypass existing immunity.
Vesicular Stomatitis Virus (VSV) Vector Vaccines
Vesicular Stomatitis Virus (VSV), a member of the Rhabdoviridaefamily, is a single-stranded negative-sense RNA virus characterized by its bullet shape. VSV typically infects cattle, horses, pigs, and goats and may transmit to humans, though it most commonly causes asymptomatic infection. The viral genome is non-segmented and encodes the N, P, M, G, and L proteins. The G protein, or glycoprotein, mediates attachment of the virion to host cells. VSV represents a valuable vaccine platform due to its ability to replicate at high titers, low seroprevalence, and minimal pre-existing human immunity. VSV-based vaccines are produced by transfecting mammalian cells—including Baby Hamster Kidney cells stably expressing T7 polymerase (BHK-T7)—or HEK293T cells transfected with recombinant VSV plasmids expressing the target transgene in place of the viral G gene. For large-scale production of recombinant VSV (rVSV), ion exchange chromatography is typically used for purification, followed by further concentration via tangential flow ultrafiltration.
Poxvirus Vector Vaccines
Poxviruses are a complex family of enveloped viruses characterized by double-stranded DNA genomes. Unusually, they replicate and transcribe in the cytoplasm using viral polymerases. Poxvirus-based vaccines have been utilized against various infectious diseases, such as HIV-1, tuberculosis, and malaria. One advantage of using poxvirus vectors is their capacity to carry approximately 25 kb of heterologous genes, which is significantly larger than that of other vectors. This makes poxvirus vectors ideal candidates for multi-antigen vaccines targeting different pathogens. Poxvirus vectors are typically generated via homologous recombination in cells infected with vaccinia virus. Cells infected with vaccinia virus are transfected with recombinant transfer plasmids, and the resulting recombinant vectors can be further propagated in susceptible cells. Poxvirus vectors have proven highly immunogenic, eliciting robust immune responses. These viral vectors are either naturally replication-defective in humans due to host range restrictions or rendered replication-defective through continuous passage in avian cells, leading to the loss of genes necessary for infecting human cells. One poxvirus showing promise as a vaccine delivery platform is the Modified Vaccinia Ankara (MVA) vector. A study involving an MVA vector HIV-1 chimeric bivalent vaccine demonstrated that it elicited both humoral and cell-mediated immune responses in humans. The vaccine was safe, well-tolerated, and immunogenic.
Viral vectors have existed for over forty years, and many have been utilized and are currently employed as vaccines against infectious diseases. The SARS-CoV-2 pandemic accelerated the development of viral vector vaccines and highlighted the strengths and limitations of some of these platforms. Further modification of these vectors is required to optimize reactogenicity, efficacy, and vector dosing.
