Viral Vector and DNA Vaccines

Viral vector and DNA vaccines are genetic vaccine architectures that deliver a double-stranded DNA or negative-sense RNA transgene encoding a target pathogen antigen into host cells, directing the host's own transcriptional and translational machinery to synthesize the immunogen in situ. Like mRNA vaccines, genetic delivery enables newly synthesized antigenic proteins to enter both the endogenous major histocompatibility complex (MHC) Class I pathway—priming cytotoxic CD8⁺ T lymphocytes (CTLs)—and the exogenous MHC Class II pathway, driving CD4⁺ follicular helper T cells and neutralizing antibodies.

Recombinant viral vector vaccines package the antigen transgene inside the protein capsid or envelope of an attenuated or replication-incompetent carrier virus—most notably adenoviruses (Ad26, ChAdOx1, Ad5), vesicular stomatitis virus (rVSV), or Modified Vaccinia Ankara (MVA)—exploiting the vector's natural receptor-mediated cell entry and intrinsic pathogen-associated molecular patterns (PAMPs) to eliminate the need for chemical adjuvants. Plasmid DNA vaccines dispense with a viral coat altogether, using in vivo electroporation or needle-free jet injection to drive supercoiled bacterial plasmid DNA across the plasma and nuclear membranes of host myocytes and dermal antigen-presenting cells.

Genomic Deletion of E1/E3 and Trans-Complementation in HEK293 or PER.C6 Cells

Adenoviruses (Adenoviridae) are non-enveloped icosahedral viruses (90–100 nm diameter) containing a linear, 36-kilobase double-stranded DNA genome flanked by inverted terminal repeats (ITRs). The capsid comprises 240 homotrimers of the major hexon protein, 12 vertex penton bases, and 12 projecting trimeric fiber proteins whose distal knob domain binds host cell-surface receptors (such as the Coxsackievirus and Adenovirus Receptor [CAR], CD46, or sialic acid/integrins).

To convert a wild-type adenovirus into a safe, replication-incompetent vaccine vector:

1. Deletion of the E1 Region (E1A and E1B): The immediate-early E1A and E1B genes encode master transcriptional transactivators required to initiate expression of all viral early (E2, E4) and late structural capsid genes and to drive host cells into S phase. Deleting the ~3.2-kb E1 locus renders the recombinant adenovirus incapable of replicating or producing progeny virions in normal human cells, and the target antigen expression cassette (driven by a strong human cytomegalovirus [CMV] immediate-early promoter) is inserted directly into the vacated E1 locus.
2. Deletion of the E3 Region: The E3 locus (~3.0–3.5 kb) encodes immune-evasion proteins that are completely dispensable for viral assembly; deleting E3 expands the vector's transgene packaging capacity to 7.5 to 8.0 kilobases.
3. Propagation in E1-Trans-Complementing Packaging Cell Lines: Because an ΔE1/ΔE3 adenovirus cannot replicate on its own, it is manufactured in specialized human packaging cell lines—specifically HEK293 (human embryonic kidney cells stably transformed with sheared Ad5 DNA bases 1–4,344) or PER.C6 (human embryonic retinal cells engineered with the exact Ad5 E1 region without overlapping flanking sequences, preventing homologous recombination from generating replication-competent adenovirus [RCA]). Inside HEK293 or PER.C6 bioreactors, the host cells supply E1A and E1B proteins in trans, allowing high-titer assembly of virions that undergo only a single abortive infection cycle when injected into a human vaccinee.

Overcoming Pre-Existing Anti-Vector Immunity: Ad26, ChAdOx1, and Heterologous Prime-Boost

First-generation adenoviral vectors utilized Human Adenovirus Serotype 5 (HAdV-5 / Ad5), a ubiquitous respiratory virus. However, 40% to >80% of the global adult population (and >80% in sub-Saharan Africa) harbors high-titer pre-existing neutralizing antibodies against the Ad5 hexon hypervariable loops from childhood colds. When an Ad5 vector is injected into a seropositive recipient, pre-existing anti-Ad5 antibodies neutralize the vector particles before they can transduce host cells, blunting transgene expression (and in the 2007 Merck STEP HIV-1 Ad5 gag/pol/nef trial, Ad5-seropositive uncircumcised men experienced transiently elevated HIV acquisition linked to activation of mucosal Ad5-specific CD4⁺ T cells).

Three bioengineering strategies solved pre-existing anti-Ad5 immunity:

• Rare Human Serotype 26 (Ad26, Dan Barouch & Janssen): Human Adenovirus 26 exhibits low global seroprevalence (typically <10%–20% neutralizing titers in Western cohorts and low baseline titers globally) and utilizes CD46 and sialic acid rather than CAR for cell entry. Ad26 underpins Ad26.COV2.S (encoding the SARS-CoV-2 Spike stabilized with both the 2P K986P/V987P mutations and a mutated furin cleavage site R682S/R685G) and the licensed Ebola prime vaccine Ad26.ZEBOV (Zabdeno).
• Simian Chimpanzee Adenoviruses (ChAdOx1, Sarah Gilbert & Adrian Hill at Oxford): Derived from chimpanzee adenovirus isolate Y25, ChAdOx1 (used in ChAdOx1 nCoV-19 / AZD1222 [Vaxzevria]) has <1% pre-existing neutralizing seroprevalence in human populations while maintaining full transduction efficiency of human cells and potent TLR9/cGAS–STING innate activation.
• Heterologous Prime–Boost Regimens: Because the first dose of any viral vector induces de novo neutralizing antibodies against its own capsid that can blunt a homologous second dose, heterologous prime–boost pairs two antigenically distinct vectors encoding the same insert—such as Ad26.ZEBOV prime followed 8 weeks later by MVA-BN-Filo (Mvabea) boost for Ebola, or rAd26 prime followed by rAd5 boost in Sputnik V (Gam-COVID-Vac).

Mechanistic Pathogenesis of Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

During the global rollout of hundreds of millions of doses of ChAdOx1 nCoV-19 and Ad26.COV2.S in early 2021, pharmacovigilance systems identified a rare, idiosyncratic prothrombotic syndrome—Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT), also termed Thrombosis with Thrombocytopenia Syndrome (TTS)—occurring at an incidence of ~1 to 2 cases per 100,000 first doses (higher in adults under 50 years) and presenting 5 to 30 days post-vaccination with unusual venous or arterial thromboses (notably cerebral venous sinus thrombosis [CVST] and splanchnic vein thrombosis), severe thrombocytopenia (<150,000 platelets/μL), and elevated D-dimer.

As elucidated by Andreas Greinacher, Marie Scully, and colleagues in April 2021, VITT closely mirrors autoimmune heparin-induced thrombocytopenia (HIT), except that patients have had no exposure to heparin:

1. Electrostatic Hexon–PF4 Complex Formation: Cryo-EM and Brownian dynamics simulations demonstrated that the outer surface of the adenoviral hexon capsid protein (specifically its negatively charged hypervariable loops) binds via strong electrostatic attraction to Platelet Factor 4 (PF4 / CXCL4)—a strongly cationic tetrameric chemokine released from α-granules of activated platelets.
2. Neoantigen Presentation and Anti-PF4 Autoantibodies: In the inflammatory milieu triggered by microvascular leakage of adenoviral particles and EDTA/host-cell protein excipients, macromolecular hexon–PF4 complexes cross-link B-cell receptors on splenic marginal-zone B cells, breaking self-tolerance and stimulating rapid clonal expansion of pathogenic IgG1 autoantibodies targeting the heparin-binding site on PF4.
3. FcγRIIa Cross-Linking and NETosis: Ultra-large immune complexes of PF4 + anti-PF4 IgG bind and cross-link low-affinity IgG receptors (FcγRIIa / CD32a) on the surface of platelets, monocytes, and neutrophils. This triggers massive intravascular platelet activation, microparticle shedding, and neutrophil extracellular trap release (NETosis)—forming extruded chromatin/histone scaffolds that propagate catastrophic venous thrombosis while consuming circulating platelets. Recognition of this mechanism enabled rapid diagnostic ELISA screening for anti-PF4 antibodies and life-saving treatment with high-dose intravenous immunoglobulin (IVIG) (which competitively blocks FcγRIIa) and non-heparin anticoagulants (direct oral factor Xa/thrombin inhibitors or argatroban).

Replication-Competent rVSV-ZEBOV (Ervebo) and Attenuated Poxvirus (MVA-BN) Vectors

Beyond adenoviruses, two distinct viral vector families play critical roles in outbreak response:

• Recombinant Vesicular Stomatitis Virus (rVSV-ZEBOV / Ervebo): Engineered by Heinz Feldmann, Thomas Geisbert, and Gary Kobinger from Vesicular Stomatitis Virus (VSV), a bullet-shaped negative-sense single-stranded RNA rhabdovirus of livestock that rarely infects humans. By deleting the gene encoding the native neurotropic VSV surface glycoprotein (G) and replacing it with the transmembrane surface glycoprotein (GP) of Zaire Ebolavirus (rVSVΔG-ZEBOV-GP), the chimeric virus becomes attenuated while its cellular tropism is entirely redirected by the Ebola GP protein. Because rVSV-ZEBOV is replication-competent, a single intramuscular dose undergoes rapid transient replication, activating RIG-I/TLR7 and eliciting protective neutralizing IgM and IgG within 7 to 10 days. Deployed via ring vaccination in the 2015 Guinea Ebola ça Suffit! trial and the 2018–2020 DRC outbreak, a single dose of Ervebo achieved 97.5% to 100% clinical vaccine efficacy from day 10 onward.
• Modified Vaccinia Ankara (MVA-BN / Jynneos / Imvanex): Derived by Anton Mayr through >570 serial passages of chorioallantois vaccinia virus Ankara in primary chick embryo fibroblasts, resulting in six major genomic deletions totaling ~30 kilobases (~15% of the viral genome), including host-range and immune-evasion genes. Unlike classic Vaccinia, MVA cannot replicate productively in human or mammalian cells (assembly is blocked at the late immature virion stage), yet because poxviruses transcribe their genes entirely in the host cytoplasm using their own virion-packaged RNA polymerase, MVA drives high-level antigen synthesis and safe cross-protective immunity against smallpox and mpox (monkeypox), even in immunocompromised patients.

Plasmid DNA Vaccines and Physical Delivery (Electroporation and Jet Injection)

Plasmid DNA vaccines consist of a closed circular, supercoiled bacterial plasmid propagating in E. coli that contains a prokaryotic origin of replication, an antibiotic-resistance or RNA-OUT selection marker, and a mammalian expression cassette (a viral CMV promoter, an intron, the codon-optimized antigen ORF, and a bovine growth hormone polyadenylation signal). Although DNA plasmids are exceptionally thermostable (stable for months at 25 °C and years at 2–8 °C) and elicit zero anti-vector immunity, naked DNA injected via a standard syringe needle shows poor immunogenicity in humans because the large, polyanionic macromolecule (~3–6 MDa) must cross two physical lipid membranes: the plasma membrane and the nuclear envelope (via the nuclear pore complex) to reach host RNA polymerase II.

To overcome this delivery bottleneck, clinical DNA vaccines utilize physical intracellular delivery devices:

• In Vivo Electroporation (e.g., Inovio CELLECTRA): Immediately following intramuscular or intradermal DNA injection, brief millisecond electrical pulses generate transient hydrophilic nanopores in the cell membrane and create an electrophoretic field that drives the negatively charged plasmid DNA into the cell and nucleus, increasing antigen expression 100- to 1,000-fold (demonstrating regression of HPV-16/18 cervical intraepithelial neoplasia with VGX-3100).
• Needle-Free Spring-Powered Intradermal Jet Injection (ZyCoV-D): Developed by Zydus Cadila and authorized in India in 2021 as the world's first licensed human DNA vaccine, ZyCoV-D uses a needle-free high-pressure laminar fluid jet (** Tropis injector) to deliver a plasmid encoding the SARS-CoV-2 Spike protein directly into the epidermis and dermis—a tissue layer densely populated with Langerhans cells and dermal dendritic cells—achieving 66.6% Phase III efficacy** against symptomatic COVID-19 during the Delta variant wave.

How can a replication-deficient adenoviral vector be manufactured in billions of doses if its E1 replication genes are deleted?

Deleting the immediate-early E1A and E1B genes prevents the recombinant adenovirus from initiating viral DNA replication or late capsid synthesis inside normal human somatic cells. To manufacture the vaccine in industrial bioreactors, the E1-deleted vector is propagated inside engineered trans-complementing human packaging cell lines—such as HEK293 or PER.C6—which have the adenoviral E1A and E1B genes permanently integrated into their own cellular chromosomes. Inside the bioreactor, the host cells supply the missing E1 proteins in trans to package infectious virions; once purified and injected into a human arm, the virions infect host cells once to express the Spike transgene but cannot produce progeny virus.

Why must patients with Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) avoid heparin anticoagulation?

VITT is caused by pathogenic IgG1 autoantibodies triggered by electrostatic complexes between the negatively charged adenoviral hexon capsid and positively charged Platelet Factor 4 (PF4). Unlike classic heparin-induced thrombocytopenia (HIT) antibodies that bind a heparin-dependent site on PF4, VITT autoantibodies bind directly within the heparin-binding groove of PF4 without requiring heparin. However, administering unfractionated or low-molecular-weight heparin can still cross-link PF4 tetramers or aggravate prothrombotic immune complexes in a subset of patients; therefore, clinical hematology guidelines mandate non-heparin anticoagulants (such as apixaban, rivaroxaban, argatroban, or fondaparinux) combined with high-dose intravenous immunoglobulin (IVIG) to competitively block platelet FcγRIIa receptors.

Why do plasmid DNA vaccines require nuclear entry whereas mRNA and poxvirus vectors do not?

Plasmid DNA vaccines contain a double-stranded DNA gene driven by a mammalian promoter (such as the CMV promoter) that can only be transcribed into mRNA by host RNA Polymerase II, an enzyme that resides strictly inside the cell nucleus. Consequently, a plasmid DNA vaccine must cross both the outer plasma membrane and the inner nuclear envelope (typically aided by electroporation or jet injection) before any mRNA or protein can be made. In contrast, mRNA vaccines are already mature transcripts ready for immediate translation by cytoplasmic ribosomes, and poxvirus vectors (MVA) package their own DNA-dependent RNA polymerase inside the virion core to transcribe genes directly within the cytoplasm.

1. Greinacher, A., Thiele, T., Warkentin, T. E., Weisser, K., Kyrle, P. A., & Eichinger, S. (2021). Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination. New England Journal of Medicine, 384(22), 2092–2101. https://doi.org/10.1056/NEJMoa2104840
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