Viral vector

CDC poster explaining viral vector vaccines

A 2021 U.S. Centers for Disease Control and Prevention poster on the COVID-19 viral vector vaccines

A viral vector is a modified virus designed to deliver genetic material into cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, agriculture, and medicine.

Viruses have evolved specialized molecular mechanisms to transport their genomes into infected hosts, a process termed transduction. This capability has been exploited for use as viral vectors, which may integrate their genetic cargo—the transgene—into the host genome, although non-integrative vectors are also commonly used. In addition to agriculture and laboratory research, viral vectors are widely applied in gene therapy: as of 2022, all approved gene therapies were viral vector-based. Further, compared to traditional vaccines, the intracellular antigen expression enabled by viral vector vaccines offers more robust immune activation.

Many types of viruses have been developed into viral vector platforms, ranging from retroviruses to cytomegaloviruses. Different viral vector classes vary widely in strengths and limitations, suiting some to specific applications. For instance, relatively non-immunogenic and integrative vectors like lentiviral vectors are commonly employed for gene therapy. Chimeric viral vectors—such as hybrid vectors with qualities of both bacteriophages and eukaryotic viruses—have also been developed.

Viral vectors were first created in 1972 by Paul Berg. Further development was temporarily halted by a recombinant DNA research moratorium following the Asilomar Conference and stringent National Institutes of Health regulations. Once lifted, the 1980s saw both the first recombinant viral vector gene therapy and the first viral vector vaccine. Although the 1990s saw significant advances in viral vectors, clinical trials had a number of setbacks, culminating in Jesse Gelsinger's death. However, in the 21st century, viral vectors experienced a resurgence and have been globally approved for the treatment of various diseases. They have been administered to billions of patients, notably during the COVID-19 pandemic.

Characteristics

Structure of a virus

Structure of a virus, specifically the hepatitis C virus

Viruses, infectious agents composed of a protein coat that encloses a genome, are the most numerous biological entities on Earth.Template:SfnTemplate:Sfn As they cannot replicate independently, they must infect cells and hijack the host's replication machinery in order to produce copies of themselves.Template:Sfn Viruses do this by inserting their genome—which can be DNA or RNA, either single-stranded or double-stranded—into the host.Template:Sfn Some viruses may integrate their genome directly into that of the host in the form of a provirus.Template:Sfn

This ability to transfer foreign genetic material has been exploited by genetic engineers to create viral vectors, which can transduce the desired transgene into a target cell.Template:Sfn Viral vectors consists of three components:Template:SfnTemplate:Sfn

1. A protein capsid and sometimes an envelope that encapsidates the genetic payload. This determines the range of cell types that the vector infects, termed its tropism.
2. A genetic payload: the transgene that results in the desired effect when expressed.
3. A "regulatory cassette" that controls transgene expression, whether integrated into a host chromosome or as an episome. The cassette comprises an enhancer, a promoter, and auxiliary elements.

Applications

Genetically engineered mice glowing green

Mice transduced by a lentiviral vector fluoresce under UV-illuminationTemplate:Sfn

Basic research

Viral vectors are routinely used in a basic research setting and can introduce genes encoding, for instance, complementary DNA, short hairpin RNA, or CRISPR/Cas9 systems for gene editing.Template:Sfn Viral vectors are employed for cellular reprogramming, like inducing pluripotent stem cells or differentiating adult somatic cells into different cell types.Template:Sfn Researchers also use viral vectors to create transgenic mice and rats for experiments.Template:Sfn Viral vectors can be used for in vivo imaging via the introduction of a reporter gene. Further, transduction of stem cells can permit the tracing of cell lineage during development.Template:Sfn

Gene therapy

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Viral vector-based gene therapy

Gene therapy seeks to modulate or otherwise affect gene expression via the introduction of a therapeutic transgene. Gene therapy by viral vectors can be performed by in vivo delivery by directly administering the vector to the patient, or ex vivo by extracting cells from the patient, transducing them, and then reintroducing the modified cells into the patient.Template:Sfn Viral vector gene therapies may also be used for plants, tentatively enhancing crop performance or promoting sustainable production.Template:Sfn

There are four broad categories of gene therapy: gene replacement, gene silencing, gene addition, or gene editing.Template:SfnTemplate:Sfn Relative to other non-integrative gene therapy approaches, transgenes introduced by viral vectors offer multi-year long expression.Template:Sfn

Vaccines

Shipments of the Russian-made Sputnik V COVID-19 vaccine, an adenoviral vector, are lined up in a storage facility in Guatemala, 2021

Shipments of the Russian-made Sputnik V COVID-19 vaccine, an adenoviral vector, are seen in Guatemala in 2021.

For use as vaccine platforms, viral vectors can be engineered to carry a specific antigen associated with an infectious disease or a tumor antigen.Template:SfnTemplate:Sfn Conventional vaccines are not suitable for protection against some pathogens due to unique immune evasion strategies and differences in pathogenesis.Template:Sfn Viral vector-based vaccines, for instance, could eventually offer immunity against HIV-1 and malaria.Template:Sfn

While traditional subunit vaccines elicit a humoral response,Template:Sfn viral vectors allow for intracellular antigen expression that activates MHC pathways via both direct and crosspresentation pathways. This induces a robust adaptive immune response.Template:SfnTemplate:Sfn Viral vector vaccines also have intrinsic adjuvant properties via innate immune system activation and the expression of pathogen-associated molecular patterns, negating the need for any additional adjuvant.Template:SfnTemplate:Sfn In addition to a more robust immune response in comparison to other vaccine types, viral vectors offer efficient gene transduction and can target specific cell types.Template:Sfn Pre-existing immunity to the virus used as the vector, however, can be a significant issue.Template:Sfn

Prior to 2020, viral vector vaccines were widely administered but confined to veterinary medicine.Template:Sfn In the global response to the COVID-19 pandemic, viral vector vaccines played a fundamental role and were administered to billions of people, particularly in low and middle-income nations.Template:Sfn

Types

Retroviruses

Retroviruses—enveloped RNA viruses—are popular viral vector platforms due to their ability to integrate genetic material into the host genome.Template:Sfn Retroviral vectors comprise two general classes: gamma retroviral and lentiviral vectors. The fundamental difference between the two are that gamma retroviral vectors can only infect dividing cells, while lentiviral vectors can infect both dividing and resting cells.Template:Sfn Notably, retroviral genomes are composed of single-stranded RNA and must be converted to proviral double-stranded DNA, a process known as reverse transcription—before it is integrated into the host genome via viral proteins like integrase.Template:Sfn

The most commonly used gammaretroviral vector is a modified Moloney murine leukemia virus (MMLV), able to transduce various mammalian cell types. MMLV vectors have been associated with some cases of carcinogenesis.Template:Sfn Gammaretroviral vectors have been successfully applied to ex vivo hematopoietic stem cell to treat multiple genetic diseases.Template:Sfn

Lentiviral vectors

File:Lentiviral vector.png

Packaging and transduction by a lentiviral vector.

Most lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV-1), although modified simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV), and the equine infectious anaemia virus (EIAV) have also been utilized.Template:Sfn As all functional genes are removed or otherwise mutated, the vectors are not cytopathic and can be engineered to be non-integrative.Template:Sfn

Lentiviral vectors are able to carry up to 10 kb of foreign genetic material, although 3-4 kb was reported as optimal as of 2023.Template:SfnTemplate:Sfn Relative to other viral vectors, lentiviral vectors possess the greatest transduction capacity, due to the formation of a three-stranded "DNA flap" during retro-transcription of the single-strand lentiviral RNA to DNA within the host.Template:Sfn

Although largely non-inflammatory,Template:Sfn lentiviral vectors can induce robust adaptive immune responses by memory-type cytotoxic T cells and T helper cells.Template:Sfn This is largely due to lentiviral vectors' high tropism for dendritic cells, which activate T cells.Template:Sfn However, they can infect all types of antigen-presenting cells.Template:Sfn Moreover, as they are the only retroviral vectors able to efficiently transduce both dividing and non-dividing cells, make them the most promising vaccine platforms.Template:Sfn They have also been trialed as vaccines against cancer.Template:Sfn

Lentiviral vectors have been used as in vivo therapies, such as directly treating genetic diseases like haemophilia B and for ex vivo treatments like immune cell modification in CAR T cell therapy.Template:Sfn In 2017, the US Food and Drug Administration (FDA) approved tisagenlecleucel, a lentiviral vector, for acute lymphoblastic leukaemia.Template:Sfn

Adenoviruses

File:Icosahedral Adenoviruses.jpg

Adenoviruses (visualized via electron micrograph at left and right and depicted graphically at center) are commonly used as viral vector platforms. Note the icosahedron capsid structure.

Adenoviruses are double-stranded DNA viruses belonging to the family Adenoviridae.Template:SfnTemplate:Sfn Their relatively large genomes, of approximately 30-45 kb, make them ideal candidates for genetic delivery;Template:Sfn newer adenoviral vectors can carry up to 37 kb of foreign genetic material.Template:Sfn Adenoviral vectors display high transduction efficiency and transgene expression, and can infect both dividing and non-dividing cells.Template:Sfn

The adenoviral capsid, an icosahedron, features a fibre "knob" at each of its 12 vertices. These fibre proteins mediate cell entry—greatly affecting efficacy and contribute to its broad tropism—notably via coxsackie–adenovirus receptors (CARs).Template:SfnTemplate:Sfn Adenoviral vectors can induce robust innate and adaptive immune responses.Template:Sfn Its strong immunogenicity is particularly due to the transduction of dendritic cells (DC), upregulating the expression of both MHC I and II molecules and activating the DCs.Template:Sfn They have a strong adjuvant effect, as they display several pathogen-associated molecular patterns.Template:Sfn One disadvantage is that pre-existing immunity to adenovirus serotypes is common, reducing efficacy.Template:SfnTemplate:Sfn The use of chimpanzee adenoviruses may circumvent this issue.Template:Sfn

While the activation of both innate and adaptive immune responses is an obstacle for many therapeutic applications, it makes adenenoviral vectors an ideal vaccine platform.Template:Sfn The global response to the COVID-19 pandemic saw the development and use of multiple adenoviral vector vaccines, including Sputnik V, the Oxford–AstraZeneca vaccine, and the Janssen vaccine.Template:Sfn

Adeno-associated viruses

File:Viral mediated delivery of genes to neurons 2.jpg

Lentivirus (upper panel) – To produce lentiviruses with the gene of interest as the lentiviral DNA construct, first transfect cells with a packaging plasmid and the envelope vector (VSVG). Adeno Associated Virus (AAV) (lower panel) – To produce AAV, package a gene of interest into the AAV-ITR vector and transfect cells with a Helper vector and the Rep/Cap DNA integration vector.

Adeno-associated viruses (AAVs) are relatively small single-stranded DNA viruses belonging to Parvoviridae and, like lentiviral vectors, AAVs can infect both dividing and non-dividing cells.Template:Sfn AAVs, however, require the presence of a "helper virus" such as an adenovirus or herpes simplex virus to replicate within the host, although it can do so independently if cellular stress is induced or the helper virus genes are carried by the vector.Template:Sfn

AAVs insert themselves into a specific site in the host genome, particularly AAVS1 on chromosome 19 in humans. However, recombinant AAVs have been designed that do not integrate. These are instead stored as episomes that, in non-dividing cells, can last for years.Template:Sfn One disadvantage is that they are not able to carry large amounts of foreign genetic materials. Furthermore, the need to express the complementary strand for its single-stranded genome may delay transgene expression.Template:Sfn

As of 2020, 11 different AAV serotypes—differing by capsid structure and consequently by tropism—had been identified.Template:Sfn The tropism of adeno-associated viral vectors can be tailored by creating recombinant versions from multiple serotypes, termed pseudotyping.Template:Sfn Due to their ability to infect and induce longlasting effects within nondividing cells, AAVs are commonly used in basic neuroscience research.Template:Sfn Following the approval of the AAV Alipogene tiparvovec in Europe in 2012,Template:Sfn in 2017, the FDA approved the first AAV-based in vivo gene therapy—voretigene neparvovec—which treated RPE65-associated Leber congenital amaurosis.Template:Sfn As of 2020, 230 clinical trials using AAV-based treatments were either underway or had been completed.Template:Sfn

Vaccinia

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An electron micrograph of vaccinia

Vaccinia virus, a poxvirus, is another promising candidate for viral vector development.Template:Sfn Its use as the smallpox vaccine—first reported by Edward Jenner in 1798—led to the eradication of smallpox and demonstrated vaccinia as safe and effective in humans.Template:SfnTemplate:Sfn Moreover, manufacturing procedures developed to mass-produce smallpox vaccine stockpiles may expedite vaccinia viral vector production.Template:Sfn

Vaccinia possesses a large DNA genome and can consequently carry up to 40 kb of foreign DNA.Template:SfnTemplate:SfnTemplate:SfnTemplate:Sfn Further, vaccinia are unlikely to integrate into the host genome, decreasing the chance of carcinogenesis.Template:Sfn Attenuated strains—replicating and non-replicating—have been developed.Template:Sfn Although widely characterized due to its use against smallpox, as of 2019 the function of 50 percent of the vaccinia genome was unknown. This may lead to unpredictable effects.Template:Sfn

As a vaccine platform, vaccinia vectors display highly effective transgene expression and create a robust immune response.Template:Sfn The virus fast-acting: its life cycle produces mature progeny vaccinia within 6 hours, and has three viral spread mechanisms.Template:Sfn Vaccinia also has an adjuvant effect, activating a strong innate response via toll-like receptors.Template:Sfn A significant disadvantage that can reduce its efficacy, however, is pre-existing immunity against vaccinia in those who received the smallpox vaccine.Template:Sfn

Herpesviruses

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Herpes simplex virus I

Of the nine herpesviruses that infect humans, herpes simplex virus 1 (HSV-1) is the most well characterized and most commonly used as a viral vector.Template:Sfn HSV-1 offers several advantages: it has broad tropism and can deliver therapeutics via specialized expression systems.Template:Sfn Moreover, HSV-1 can cross the blood brain barrier if medically-disrupted, enabling it to target neurological diseases. Also, HSV-1 does not integrate into the host genome and can carry large amounts of foreign DNA. The former feature prevents harmful mutagenesis, as can occur with retroviral and adeno-associated vectors. Replication-deficient strains have been developed.Template:Sfn

In 2015, talimogene laherparepvec—an HSV-1 vector that triggers an anti-tumor immune response—was approved by the FDA to treat melanoma.Template:Sfn As of 2020, HSV-1 vectors have been experimentally applied against sarcomas and cancers of the brain, colon, prostate, and skin.Template:Sfn

Cytomegalovirus (CMV), a herpesvirus, has also been developed for use as a viral vector.Template:Sfn CMV can infect most cell types and can thus proliferate throughout the body. Although a CMV-based vaccine provided significant immunity against SIV—closely related to HIV—in macaques, development of CMV as a reliable vector was reported to still be in early stages as of 2020.Template:SfnTemplate:Sfn

Plant viruses

Plant viruses are also engineered viral vectors for use in agriculture, horticulture, and biologic production.Template:Sfn These vectors have been employed for a range of applications, from increasing the aesthetic quality of ornamental plants to pest biocontrol, rapid expression of recombinant proteins and peptides, and to accelerate crop breeding.Template:Sfn The use of engineered plant viruses has been proposed to enhance crop performance and promote sustainable production.Template:Sfn

Replicating virus-based vectors are typically used.Template:Sfn RNA viruses used for monocots include wheat streak mosaic virus and barley stripe mosaic virus and, for dicots, tobacco rattle virus. Single-stranded DNA viruses like geminiviruses have also been utilized.Template:Sfn Viral vectors can be administered to plants via several pathways termed "agro-inoculation", including via rubbing, a biolistic delivery system, agrospray, agroinjection, and even via insect vectors.Template:SfnTemplate:Sfn However, Agrobacterium-mediated delivery of viral vectors—in which bacteria are transformed with plasmid DNA encoding the viral vector construct—is the most common approach.Template:Sfn

Bacteriophages

Chimeric vectors combining both bacteriophages and eukaryotic viruses have been developed and are capable of infecting eukaryotic cells.Template:SfnTemplate:Sfn Unlike eukaryotic virus-based vectors, such bacteriophage vectors have no innate tropism for eukaryotic cells, allowing them to be engineered to be highly specific for cancer cells.Template:Sfn

Bacteriophage vectors are also commonly used in molecular biology.Template:Sfn For instance, bacteriophage vectors are used in phage-assisted continuous evolution, promoting rapid mutagenesis of bacteria.Template:Sfn Although limited to mycobacteriophages and some phages of gram-negative bacteria, bacteriophages can be used for direct cloning.Template:Sfn

Manufacture

File:Bioreactor principle.svg

A typical suspension culture bioreactor

Viral vector manufacturing methods often vary by vector, although most utilize an adherent or suspension-based system with mammalian cells.Template:Sfn For viral vector production on a smaller, laboratory setting, static cell culture systems like Petri dishes are typically used.Template:Sfn

Those techniques used in the laboratory are difficult to scale, requiring different approaches on an industrial scale.Template:Sfn Large single-use disposable culture systems and bioreactors are commonly used by manufacturers.Template:Sfn Vessels such as those with gas permeable surfaces are used to maximize cell culture density and solution transducing units.Template:Sfn Depending on the vessel, viruses can be directly isolated from the supernatant or isolated via chemical lysis of the cultured cells or microfluidization.Template:Sfn In 2017, The New York Times reported a manufacturing backlog of inactivated viruses, delaying some gene therapy trials by years.Template:Sfn

History

Template:Multiple image In 1972, Stanford University biochemist Paul Berg developed the first viral vector, incorporating DNA from the lambda phage into the polyomavirus SV40 to infect kidney cells maintained in culture.Template:SfnTemplate:SfnTemplate:Sfn The implications of this achievement troubled scientists like Robert Pollack, who convinced Berg not to transduce DNA from SV40 into E. coli via a bacteriophage vector. They feared that introducing the purportedly cancer-causing genes of SV40 would create carcinogenic bacterial strains.Template:SfnTemplate:Sfn These concerns and others in the emerging field of recombinant DNA led to the Asilomar Conference of 1975, where attendees agreed to a voluntary moratorium on cloning DNA.Template:Sfn

In 1977, the National Institutes of Health (NIH) issued formal guidelines confining viral DNA cloning to rigid BSL-4 conditions, practically preventing such research. However, the NIH loosened these rules in 1979, permitting Bernard Moss to develop a viral vector utilizing vaccinia.Template:Sfn In 1982, Moss reported the first use of a viral vector for transient gene expression.Template:Sfn The following year, Moss used the vaccinia vector to express a hepatitis B antigen, creating the first viral vector vaccine.Template:Sfn

Every realm of medicine has its defining moment, often with a human face attached. Polio had Jonas Salk. In vitro fertilization had Louise Brown, the world's first test-tube baby. Transplant surgery had Barney Clark, the Seattle dentist with the artificial heart. AIDS had Magic Johnson. Now gene therapy has Jesse Gelsinger.

— Sheryl Gay Stolberg, The New York Times MagazineTemplate:Sfn

Although a failed gene therapy attempt utilizing wild-type Shope papilloma virus had been made as early as 1972, Martin Cline attempted the first gene therapy utilizing recombinant DNA in 1980. It proved unsuccessful.Template:SfnTemplate:Sfn In the 1990s, as genetic diseases were further characterized and viral vector technology improved, there was overoptimism about the capabilities the technology. Many clinical trials proved failures.Template:Sfn There were some successes, such as the first effective gene therapy for severe combined immunodeficiency (SCID); it employed a retroviral vector.Template:Sfn

However, during a 1999 clinical trial at the University of Pennsylvania, Jesse Gelsinger died from a fatal reaction to an adenoviral vector-based gene therapy.Template:SfnTemplate:Sfn It was the first death related to any form of gene therapy.Template:Sfn Consequently, the FDA suspended all gene therapy trials at the University of Pennsylvania and investigated 60 others across the US.Template:Sfn An anonymous editorial in Nature Medicine noted that it represented a "loss of innocence" for viral vectors.Template:Sfn Shortly thereafter, the field's reputation was further damaged when 5 children treated with a SCID gene therapy developed leukemia due to an issue with the retroviral vector.Template:SfnTemplate:Efn

Viral vectors experienced a resurgence when they were successfully employed for ex vivo hematopoietic gene delivery in clinical settings.Template:Sfn In 2003, China approved the first gene therapy for clinical use: Gendicine, an adenoviral vector encoding p53.Template:SfnTemplate:Sfn In 2012, the European Union issued its first approval of a gene therapy, an adeno-associated viral vector.Template:Sfn During the COVID-19 pandemic, viral vector vaccines were used to an unprecedented extent: administered to billions of people.Template:SfnTemplate:Sfn As of 2022, all approved gene therapies were viral vector-based and over 1000 viral vector clinical trials targeting cancer were underway.Template:Sfn

In popular culture

Burnt and abandoned car props are situated near post-apocalyptic Washington Square Park in New York City as part of a set for the film I am Legend.

The film I Am Legend (set pictured) depicts a viral vector-created apocalypse.

In film, viral vectors are often portrayed as unintentionally causing a pandemic and civilizational catastrophe.Template:Sfn The 2007 film I Am Legend depicts a cancer-targeting viral vector as unleashing a zombie apocalypse.Template:SfnTemplate:Sfn Similarly, a viral vector therapy for Alzheimer's disease in Rise of the Planet of the Apes (2011) becomes a deadly pathogen and causes an ape uprising. Other films featuring viral vectors include The Bourne Legacy (2012) and Resident Evil: The Final Chapter (2016).Template:Sfn An advanced form of viral vector vaccine is a critical story element in Jurassic World Dominion (2022), in which it is used to cure a character's genetic disorder and later to stop a man-made ecological crisis.