Gene-Based Vaccines: How Do They Work?

An introduction to genetic modulation of immune cells by gene-based vaccines

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What Are Gene-Based Vaccines?

Gene-based vaccines (also known as nucleic acid-based vaccines) involve the delivery of a gene sequence encoding an antigen into host cells. The host can then mount an immune response against this antigen in order to prevent or treat the disease1-4

Therefore, gene-based vaccines can be:1,3,4

Preventive


Intended to generate neutralizing antibodies against the infectious pathogen

Therapeutic


Intended to stimulate a cellular immune response to kill diseased cells

Two main types of gene-based vaccines currently exist, based on whether the gene encoding the antigen is added as DNA or RNA:4,5

DNA Vaccines

RNA Vaccines

Both DNA and RNA vaccines work by allowing the expression of the foreign antigen in the patient\'s cells. This leads to an immune response against the infectious pathogen or diseased cells that express the antigen, with the ultimate goal of killing them4-8

General Mechanism of Gene-Based Vaccines7,9

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APC, antigen-presenting cell; DNA, deoxyribonucleic acid; MHC, major histocompatibility complex; RNA, ribonucleic acid.

DNA and RNA Vaccines: How Do They Work?

Following the delivery of the gene-based vaccine, the vaccine uses the host\'s transcriptional and translational machinery to produce the desired antigen that will be recognized by components of the immune system10

Antigen-presenting cells (APCs) play an essential role in the recognition of the encoded antigen and activation of the immune response10

DNA Vaccines

Antigen expression/uptake by APCs2,9,11,12

The antigen encoded by the DNA vaccine can be expressed or taken in by APCs via two distinct routes:

  • The direct route occurs when APCs are transfected directly with the DNA vaccine and subsequently express the antigen
  • The indirect route involves the uptake of the antigen by APCs from myocytes or keratinocytes:
    • Myocytes or keratinocytes are transfected with the DNA vaccine upon vaccine injection into muscle or skin
    • Myocytes or keratinocytes express the antigen and then release it via exosomes or apoptotic bodies
    • APCs take up the antigen through endocytosis

Antigen processing by APCs2,12

Inside APCs, the antigen is further processed and loaded onto the major histocompatibility complex (MHC) class I or II

Antigen presentation and priming of immune cells by APCs2,12

APCs subsequently travel to lymph nodes, where they present the processed antigen via MHC class II CD4+ T cells (helper T cells) or via MHC class I to CD8+ T cells (cytotoxic T cells), leading to the activation of CD4+ or CD8+ T cells, respectively

APC, antigen-presenting cell; BCR, B-cell receptor; CD, cluster of differentiation; DNA, deoxyribonucleic acid; MHC, major histocompatibility complex; TCR, T-cell receptor.
Figure adapted from Xu Y, et al. 2014.11

Co-stimulatory molecules and cytokines are released, which, together with the antigen presentation to T cells, provide the necessary signals to promote and amplify an immune cellular response13,14

In addition to this cellular immune response within the lymph nodes, a humoral immune response can be induced when B cells (naïve or memory B cells) are activated. This occurs when:2,9,13,15

  • Specific high-affinity B cell receptors expressed on the surface of B lymphocytes recognize the peptide antigen
  • B lymphocytes acquire help from preactivated antigen-specific CD4+ T cells
    • In response to peptide-bound MHC molecules and co-stimulatory secondary signals, activated CD4+ T cells secrete cytokines during cell-to-cell interaction with B cells

Subsequently, B cells proliferate and differentiate into antibody-secreting effector B cells15

RNA Vaccines

RNA vaccines work by introducing an mRNA sequence that encodes a disease-specific antigen4,16

Although the exact mechanism by which RNA vaccines instigate an immune response remains to be elucidated, their general mechanism has been described, similarly to DNA vaccines2,4,8,17

Once the vaccine's mRNA strand is inside the body's cells, the cells use the genetic information to produce the mRNA-encoded antigen8,18

  • The RNA vaccine is captured by APCs at the injection site, which then migrate to the lymph nodes. In the lymph nodes, APCs present the encoded antigen via MHC class I or MHC class II molecules to CD8+ T cells or CD4+ T cells, respectively, thus activating cellular and humoral responses

Delivery of Gene-Based Vaccines

Both DNA and RNA vaccines require a delivery vehicle to introduce the antigen gene into the host\'s cells16,19

The delivery vehicle can determine:16,19

  • The efficiency by which gene-based vaccines elicit an immune response against the desired target cells or pathogen
  • The efficient and safe delivery into the host\'s cells

DNA Vaccines

Depending on the delivery vehicle, DNA vaccines can be categorized as either viral vector- or plasmid-based vaccines2,3,20-24

Plasmid-based2,20,21

Despite the initial promising results in animal studies, DNA vaccines delivered via plasmids have shown poor immunogenicity in humans.

Viral vector-based3,20,22-24

Viral vectors have emerged as a promising platform for DNA vaccines. Many clinical trials are evaluating the use of viral vector-based vaccines to treat or prevent human diseases.

RNA Vaccines

Because mRNA is degraded by normal cellular processes, RNA vaccines need to be delivered via methods that protect the mRNA from degradation while facilitating efficient delivery into the target cells at the same time16

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Some of these methods include:16

  • Ex vivo transfection of dendritic cells with naked mRNA
  • Direct injection of naked mRNA in vivo
  • Physical delivery methods in vivo (e.g. gene gun, electroporation)
  • Cationic peptide protamine
  • Cationic lipid and polymer-based nanoparticles (e.g. lipid nanoparticles)

Two main types of RNA vaccines currently exist:8,16,17

Nonreplicating mRNA vaccines

    • Carry only the coding sequence of the antigen surrounded by regulatory regions
    • The mRNA strand is packaged and administered directly to the patient, where it is taken up by the cells to produce the antigen
  • Alternatively, dendritic cells can be extracted from the patient\'s blood, subsequently transfected with the RNA vaccine, and then delivered back into the patient to stimulate an immune reaction

Self-replicating mRNA vaccines

  • In addition to viral elements, they encode the antigen that allows for intracellular RNA amplification and protein expression

Advantages and Limitations of DNA Vaccines

Plasmid-based DNA vaccines offer some advantages, which include:4,9,19,25,26

  • Stimulation of humoral and cellular immune responses

    Potential for prophylactic and therapeutic applications

  • Noninfectious

    Low risk of reverting into a disease-causing form because they are nonlive, nonreplicating, and nonspreading

  • Potential for easy commercialization

    Easy design and subsequent alteration and manufacturing

    Relatively low production cost in comparison with protein vaccines

    Stable at room temperature (facilitates transportation and storage)

Plasmid-based DNA vaccines have important disadvantages that have limited their use, which include:4,21,25,30

  • Limited transfection efficacy

    Due to lack of chemical stability, vulnerability to nuclease degradation, rapid removal from the body, and inefficient transport to the lymph nodes

  • Low immunogenicity

    Challenges in improving the immunogenicity in humans still remain


    May require multiple booster doses


  • Risk of toxicities

    Potential for induction of anti-DNA antibodies


    Potential integration into the host\'s genome


Advantages of viral vector-based DNA vaccines include:4,12,19,27-32

  • Stimulation of humoral and cellular immune responses

    Potential for prophylactic and therapeutic applications

  • High gene transfer efficacy

    Ability to transfer the genetic material to the target cell with high efficiency

  • Potent immunogenicity

    Stimulate humoral and cellular immune responses
    Confer a robust and long-lasting T-cell response

  • Specificity

    Can be engineered to target specific cell types

  • Potential safety profile

    Low risk to revert into virulent forms (unlike live-attenuated virus vaccines)

  • Potential for easy commercialization

    Allows for scalability

Limitations of viral vector-based DNA vaccines include:4,30

  • Risk of immune rejection

    Potential of an immune response against the viral vector

  • Unknown efficacy against neoantigens in tumors

    The efficacy against neoantigens in humans remains to be elucidated

Advantages and Limitations of RNA Vaccines

Advantages of RNA vaccines include:4,16,17,30

  • Stimulation of humoral and cellular immune responses

    Potential for prophylactic and therapeutic applications

  • Flexible design

    The mRNA can be formulated with carrier molecules to allow rapid intake by cells and prolonged expression of the antigen

  • Lower barrier for antigen expression compared with DNA vaccines

    mRNA-mediated antigen expression does not require nuclear delivery

  • Potential safety profile

    Noninfectious and nonintegrating, with no risk of infection or insertional mutagenesis

    Naturally degraded

    The inherent immunogenicity can be downmodulated

  • Potential for easy commercialization

    Allows for a rapid, inexpensive, and scalable production

Some challenges remain, which limit the clinical application of RNA vaccines:4,16-18,30

  • Mechanism of immune stimulation remains to be elucidated

    Further insights into the mechanism of action need to be elucidated to fully understand the impact on theimmune response

  • Need for intracellular delivery

    Due to the presence of extracellular ribonucleases that can degrade the mRNA


    Require encapsulation methods for efficient intracellular delivery


  • Concerns of instability and low transfection

    Extracellular ribonucleases can degrade the mRNA


    Sequences in the mRNA can affect their stability and translation into protein


  • Potential toxicity

    Due to the effect of free extracellular mRNA (e.g. coagulation, thrombus formation)


    Inflammation due to enhanced interferon response


  • Storage limitations

    Due to the need to be stored at cold temperatures

Clinical Applications of Gene-Based Vaccines

The most active areas of research for immune regulation with DNA- and RNA-based vaccines are infectious diseases and cancer2,4

In the US, there are hundreds of clinical trials that focus on gene-based vaccination, mostly targeting viral infections, such as influenza, human immunodeficiency virus, rabies, Zika, and cancer, such as acute myeloid leukemia, colorectal cancer, glioblastoma, melanoma, ovarian cancer, and prostate cancer2,4,16,33,34

In July 2020, Ab26.ZEBOV was approved in the EU35-38 and became the first gene-based vaccine approved for use in humans

  • Ab26.ZEBOV is composed of a single recombinant, replication-incompetent human adenovirus type 26 vector-based vaccine that encodes the full-length glycoprotein of the Zaire ebolavirus
  • Ab26.ZEBOV is used in combination with the multivalent vaccine MVA-BN-Filo as a two-dose regimen for the prevention of Ebola virus disease in subjects of at least 1 year of age

Coronavirus Disease 2019 (COVID-19)

Most recently, there has been a big surge in prophylactic RNA and DNA vaccine initiatives for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-1939

While over 100 vaccines of differing strategies are in clinical or preclinical development, following the declaration of the COVID-19 pandemic in 2020, RNA and DNA vaccines were among the first to reach Phase 340

The RNA and DNA vaccines in the most advanced stages of development were designed to lead to an immune response against the spike protein of SARS-CoV-241-43

SARS-CoV-2

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No RNA vaccines have previously obtained regulatory approval. However, Phase 3 data from RNA vaccines have shown high efficacy and safety in the target populations, and two RNA vaccines have received regulatory authorization for emergency use40,41,44-47

The following are examples of gene-based vaccines that target the spike protein of
SARS-CoV-241,43,45,48-54

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RNA vaccine

mRNA-127344,48,49,55-58

  • A lipid nanoparticle-encapsulated mRNA vaccine encoding the full-length, prefusion-stabilized spike protein of SARS-CoV-2
  • It is currently under investigation in Phase 2 (NCT04405076) and Phase 3 (NCT04470427) studies in the US
  • An interim analysis of the Phase 3 study indicated that the vaccine was 94.1% effective
    • These results are based on 196 cases of COVID-19; of these, 11 cases occurred in the vaccine group vs 185 cases in the placebo group
    • The study has enrolled more than 30,000 participants in the US
    • The results of this study have led to the authorization of this vaccine for emergency or temporary/conditional use in the US, UK, and the EU for individuals aged 18 years or over

BNT162b242,46,47,53,59,60

  • A lipid nanoparticle-formulated, nucleoside-modified RNA vaccine encoding the full-length, prefusion-stabilized spike protein of SARS-CoV-2
  • It is currently under investigation in a Phase 2/3 (NCT04368728) study across six countries (Argentina, Brazil, Germany, South Africa, Turkey, and the US)
  • An interim analysis of the Phase 3 study has indicated that the vaccine is more than 90% effective in participants without prior SARS-CoV-2 infection (first primary objective) measured from 7 days after the second dose
    • These results are based on 94 cases of COVID-19
    • The study has enrolled 43,538 participants
    • The results of this study have led to the authorization of this vaccine for emergency use or temporary/conditional use in the US, UK, and in the EU for individuals aged 16 years or over
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Viral vector-based vaccine

ChAdOx1-S43,51,61,62

  • A DNA vaccine delivered via a replication-defective chimpanzee adenovirus vector that expresses the full-length spike protein of SARS-CoV-2
  • Currently under investigation in a Phase 2/3 study in the UK (NCT04400838) and in expansion to other countries, which include Brazil, South Africa, and the US
  • A pooled interim analysis of patients who received the two-dose regimens and investigated in the Phase 1-3 studies (in the UK, Brazil, and South Africa) indicates that the vaccine is well tolerated, with an overall efficacy of 70.4%
  • These results are based on data from 11,636 participants between the ages of 18 and 55 years
  • The results of this study have led to the authorization of this vaccine for temporary/conditional use in the UK and EU for individuals aged 18 years or over

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