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DNA Replicons and Infectious Diseases: History
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Contributor: Kenneth Lundstrom

DNA-based gene therapy and vaccine development have received plenty of attention lately. DNA replicons based on self-replicating RNA viruses such as alphaviruses and flaviviruses have been of particular interest due to the amplification of RNA transcripts leading to enhanced transgene expression in transfected host cells. Moreover, significantly reduced doses of DNA replicons compared to conventional DNA plasmids can elicit equivalent immune responses. DNA replicons have been evaluated in preclinical animal models for cancer immunotherapy and for vaccines against infectious diseases and various cancers. Strong immune responses and tumor regression have been obtained in rodent tumor models. Immunization with DNA replicons has provided robust immune responses and protection against challenges with pathogens and tumor cells. DNA replicon-based COVID-19 vaccines have shown positive results in preclinical animal models.

  • DNA replicons
  • self-replication RNA viruses
  • RNA amplification

1. Introduction

Plasmid DNA-based delivery of transgenes has been used for gene therapy [1], which to a large extent has involved cancer therapy [2]. Both prophylactic and therapeutic efficacy have been achieved in the areas of cancer therapy [3], cancer immunotherapy [4], and cancer vaccines [5]. The aim in cancer therapy is to overexpress toxic, antitumor, or suicide genes to kill tumor cells, leading to tumor eradication and, in the best-case scenario, cure [3]. This approach has often been aided by the utilization of tumor-specific promoters [6]. In cancer immunotherapy, immunostimulatory factors such as cytokines and chemokines are overexpressed to strengthen the immune system, which has been weakened by the presence of tumors [7]. In the case of cancer vaccines, immunogenic proteins or their epitopes targeting cancer cells are introduced into plasmid DNA vectors for immunization studies in animal models and cancer patients [8]. Moreover, DNA vaccines against infectious diseases have also been engineered [9]. Similarly, surface proteins or their epitopes have been used as antigens for induction of antibody responses and protection against challenges with pathogenic viruses, bacteria, protozoans, and parasites.
Plasmid DNA has largely been administered as intramuscular, intradermal, and subcutaneous injections [10][11]. The easy handling and rapid and inexpensive manufacturing of plasmid DNA have made DNA-based approaches attractive alternatives. However, in contrast to mRNA-based gene delivery, plasmid DNA needs to translocate to the nucleus for mRNA transcription and transport to the cytoplasm before translation can occur [12], which has hampered the efficacy of delivery and transgene expression. To address delivery issues, various technologies, such as electroporation [13], gene gun [14], and liposome [15] and polymer-based [16] nanoparticle formulations, have been applied. Especially lipid nanoparticles (LNPs) have demonstrated enhanced delivery capacity, improved transgene expression, and superior therapeutic efficacy compared to naked plasmid DNA. An alternative approach to generate improved transgene expression has been to apply DNA vectors based on self-replicating RNA viruses, which have been named DNA replicon vectors [17].

2. DNA Replicons and Infectious Diseases

In the context of DNA replicon vectors mainly alphaviruses have been utilized for vaccine development against infectious diseases (Table 1). Among alphaviruses, Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus (VEE) DNA replicons have been used. For example, SIN DNA replicons expressing the herpes simplex virus-1 glycoprotein B (HSV-1-gB) elicited virus-specific antibodies and cytotoxic T-cells in immunized mice and provided protection against lethal challenges with HSV-1 [18]. Furthermore, a single intramuscular immunization with SIN-HSV-1-gB protected mice from lethal challenges with HSV-1. In another application of SIN DNA replicons, the measles virus (MV) hemagglutinin (pMSIN-H) and the fusion of hemagglutinin and the MV F fusion protein (pMSINH-FdU) have been administered to cotton rats [19]. Injection of pMSIN-H provided 100% protection against pulmonary measles, whereas protection with pMSINH-Fdu administration was achieved only after booster vaccination with a live MV vaccine [19]. Moreover, SFV DNA replicons have been subjected to several studies in preclinical animal models. For example, SFV DNA replicons expressing the bovine viral diarrhea virus (BVDV) p80 (NS3) were administered to the quadricep muscles of BALB/c mice, which elicited statistically significant cytotoxic T-cell (CTL) responses and cell-mediated immune (CMI) responses against BVDV [20]. In a comparative study, both SFV DNA replicons expressing the classical swine fever virus (CSFV) E2 glycoprotein and an adenovirus expressing CSFV E2 (rAdV-E2) showed strong immune responses in a pig model [21]. A heterologous vaccination regimen of prime immunization with pSFV1CS-E2 followed by rAdV-E2 booster vaccination induced significantly higher CSFV-specific neutralizing antibody titers compared to two immunizations with rAdV-E2. Furthermore, the pSFV1CS-E2/rAdV-E2 approach prevented viremia and clinical symptoms in pigs, which was not the case for the homologous immunization strategy [21]. In another prime-boost approach, efficient priming of a low dose of only 0.2 μg of SFV DNA replicons expressing the HIV Env protein and a Gag-Pol-Nef fusion protein was achieved in combination with the poxvirus Ankara (MVA) strain expressing the same HIV proteins in mice, resulting in greatly enhanced immune responses [22]. Moreover, prime-boost vaccination with different SFV DNA replicons expressing the Core, E1, E2, or non-structural p7-NS2-NS3 of the hepatitis C virus (HCV) followed by administration of the MVA strain expressing nearly the full-length HCV genome elicited high levels of HCV-specific CTL responses and humoral immune responses in mice [23]. Alphavirus DNA replicons have also been employed for the expression of the Ebola virus (EBOV) glycoprotein (GP) gene for co-expression of the GP and EBOV VP40 genes of Sudan or Zaire EBOV strains, which elicited both binding and neutralizing antibodies in mice [24]. The antibodies also showed cross-reactivity against another EBOV strain. Moreover, SFV DNA replicon vaccines showed superior immunogenicity compared to a recombinant MVA vaccine. SFV DNA replicon-based co-expression of EBOV GP and VP40 induced significantly stronger EBOV-specific humoral and cellular responses than either EBOV GP or VP40 alone in mice [25]. In another approach, a DNA replicon-based vaccine pMG4020 expressing the full-length rearranged genome of the V4020 virus provided protection against challenges with VEE in mice [26]. Moreover, immunization with the pMG4020 DNA replicon resulted in the protection of rhesus macaques against VEE challenges [27]. More recently, SFV DNA replicons have been subjected to the expression of the full-length SARS-CoV-2 Spike (S) protein (DREP-S) or the S protein ecto-domain stabilized in a prefusion conformation (DREP-Secto) [28]. Both DREP-S and DREP-Secto induced binding and neutralizing antibodies. Superior vaccine potency was obtained for DREP-S, eliciting high titers of SARS-CoV-2-specific IgG antibodies in mice receiving a single injection [28].
Table 1. Examples of DNA replicon-based vaccines against infectious agents and toxins.
Disease DNA Vector/Target μg DNA Findings
Viral      
HSV-1 SIN/HSV-1 gB 0.01–3 Protection in mice against HSV-1 after single injection [18]
MV SIN/MV-H 100 100% protection against MV in cotton rats [19]
MV SIN/MV-H-Fdu 100 Protection only after booster with live MV vaccine in rats [19]
BVDV SFV/BVDV p80 100 CTL and CMI responses against BVDV in mice [20]
CSFV SFV/CSFV-E2 + rAdV-E2 100 Heterologous prime-boost strategy superior in a pig model [21]
HIV SFV/HIV Env, GagPolNef 0.2 Greatly enhanced immune responses after MVA booster [22]
HCV SFV/HCV C, E1, E2, NS2/3 0.5–50 CTL and humoral responses after MVA booster in mice [23]
EBOV SFV/EBOV GP, VP40 5 Binding and neutralizing Abs in mice [24]
EBOV SFV/EBOV GP, VP40 10 Superior humoral, cellular responses after co-injection [25]
VEE VEE/V4020 genome 100 Protection against VEE in mice [26]
VEE VEE/V4020 genome 100 Protection against VEE in rhesus macaques [27]
COVID-19 SFV/SARS-CoV-2 S 10 Neutralizing Abs, superior IgG Abs in mice after 1 injection [28]
COVID-19 SFV/SARS-CoV-2 Secto 10 Neutralizing Abs in mice [28]
Bacterial      
TB SIN/M. tuberculosis p85 0.5–50 Specific Ab responses, protection against challenges in mice [29]
TB VEE/Acr-Ag85B fusion 20 Inhibition of bacterial growth in lungs and spleen of mice [30]
Botulism SFV/BoNT/A Hc, GM-CSF 100 Prolonged survival after BoNT/A challenges in mice [31]
Botulism SFV/BoNT/E, BoNT/F 100 Protection against challenge with BoNT/E-BoNT/F mixture [32]
Botulism SFV/BoNT/A, B, E, F 100 Protection against 4 BoNT serotypes in mice [33]
Tetanus SFV/TeNT 100 Protection against TeNT in mice [33]
Anthrax SFV/anthrax PA 100 Protection against B. anthracis A16R strain in mice [34]
Protozoan      
TP SFV/Tg-NPase II 100 Protection against acute infection, toxoplasmosis in mice [35]
Parasites      
LD SFV/PpSP15-LmST11 0.5–2 Superior expression from RNA than DNA replicons [36]
Abs, antibodies; BoNT, botulinum neurotoxin; BVDV, bovine viral diarrhea virus; CMI, cell-mediated immune; CSFV, classical swine fever virus; CTL, cytotoxic T-cell; EBOV, Ebola virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HSV-1 gB, herpes simplex virus-1 glycoprotein B; LD, leishmaniasis disease; MV, measles virus; MVA, modified poxvirus Ankara strain; MV-H, MV hemagglutinin, MV-H-Fdu, MV hemagglutinin-fusion protein; rAdV, recombinant adenovirus; SFV, Semliki Forest virus; SIN, Sindbis virus; TB, tuberculosis; TP, toxoplasmosis.
Bacterial infections and toxins have also been targeted with DNA replicons. For example, SIN DNA expressing the Mycobacterium tuberculosis p85 antigen elicited strong p85-specific immune responses in mice [29]. Moreover, immunized mice showed long-term protection against challenges with lethal doses of M. tuberculosis. Application of VEE DNA replicons for the expression of a fusion protein of the M. tuberculosis α-crystallin (Acr) and Ag85B antigens induced strong CD4+ and CD8+ T-cell responses in mice and prevented bacterial growth in lungs and spleen of mice after challenge with aerosol-based M. tuberculosis [30]. In the context of Clostridium botulinum neurotoxin, the serotype A (BoNT/A) Hc gene was co-expressed with the granulocyte-macrophage colony-stimulating factor (GM-CSF) from SFV DNA replicons [31]. Mice immunized with SFV-BoNT/A Hc and SFV-GM-CSF DNA replicons showed significantly prolonged survival of mice after challenges with BoNT/A. In another approach, the BoNT/E and BoNT/F serotype Hc genes were expressed from a dual SFV DNA replicon showing resistance against challenges with a BoNT/E and BoNT/F mixture in mice [32]. Moreover, SFV DNA replicons have been engineered for the expression of the tetravalent BoNT Hc genes for the A, B, E, and F serotypes and the tetanus neurotoxin (TeNT) [33]. Initially, the SFV-TeNT DNA replicon provided strong antibody responses and protection in BALB/c mice [33]. Furthermore, the SFV-TeNT DNA replicon was combined with the BoNT serotypes in a pentavalent DNA replicon vaccine, which induced specific antibody responses and provided protection against the five targets in mice [33]. Related to anthrax, SFV DNA replicons have been utilized for the expression of the anthrax protective antigen (PA) [34]. Both SFV-PA replicons and viral particles elicited stronger PA-specific immune responses than conventional plasmid DNA vectors, resulting in potent protection against challenges with the Bacillus anthracis A16R strain in mice. In the context of protozoans, SFV DNA replicon-based expression of the nucleoside triphosphate hydrolase-II of Toxoplasma gondii (TgNTPase-II) provided partial protection against acute infection and toxoplasmosis in mice [35].
In the context of parasites, the fusion protein of the SP15 protein from the insect Phlebotomus papatasi and the Leishmania major stress inducible protein 1 (PpSP15-LmSTI1) was expressed from SFV RNA replicons, SFV DNA replicons, and conventional plasmid DNA vectors [36]. In this case, superior expression was observed from SFV RNA replicons in comparison to DNA replicons or conventional DNA plasmids, suggesting that SFV RNA replicons are preferred for further vaccine development against leishmaniasis.

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics15030947

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