Companies: Osivax

Nucleic acid based immunization exhibits a number of advantages. For example, the manufacture of nucleic acid based vaccines is straight forward, relatively inexpensive.

DNA Vaccines

Potential advantages: DNA based vaccines are stable for long-term storage. DNA vaccines offer advanatages overy many of vaccines. Since the antigen is produced with mammalin post translational modification, conformation and oligomerization, it is more likely to be similar or identical to the wild type protein produced by viral infection than recombinant or chemically modified proteins. However, this distinction may turn out to be a disadvantage for the application of bacterial antigens, since non-native post translational modification may result in reduced immunogenicity. In addition, viral surface proteins are not highly organized in the absence of matrix proteins.

DNA vaccine can induce humoral and cellular immunity and it also saves labour and time because of the siplicity of oepration (Zhang, J Gene Med 2007 9, 715-726).

Potential disadvantages: DNA based vaccines exhibit a variety of potential safety risks such as induction of anti-DNA antibodies, and potential integration of the transgene into the host genome. This may lead to the inactivation of cellular genes, an uncontrollable long term expression of the transgene, or oncogenesis, and thus, is generally not applicable for tumor-associated antigens with oncogenic potential such as erb-B2.

DNA vaccination in avian species:

Duan (US2005/0208628) dicloses a pcoress for producing antibodies to an antigen in an avian species by delivering a DNA dequence encoding the antigen operatively linke to a promoter capable of directing expression of the antigen in the avian species and recovering antibodies from the avian species. Preferably the avian species being vaccinated is a chicken or quail and the antibodies are recovered from egg yolk. The use of chicken offers a number of advantages such as cost.

RNA Vaccines

Two different strategies have been pursued for immunotherapy with in vitro transcribed RNA (IVT-RNA), which have both been successfully tested in various animal models. Either the RNA is directly injected into the patient by different immunization routes or dendritic cells are transfected with IVT-RNA using conventional transfection methods in vitro and then the transfected dendritic cells are administered to the patient. It has been shown that immunization with RNA transfected dendritic cells induces antigen-specific cytotoxic T-lymphocytes (CTL) in vitro and in vivo. Furthermore, it has been shown that direct injection of naked RNA into the lymph nodes of laboratory animals (intranodal injection) leads to uptake of said RNA primarily by immature dendritic cells, probably by a process called macropinocytosis. It is assumed that the RNA is translated and the expressed protein is presented on the MHC molecules on the surface of the antigen presenting cells to elicit an immune response.

Potential Advantages: Some of the advantages of RNA based immunization are the transient expression and the non-transforming character. Furthermore, RNA does not have to be transported into the nucleus for the transgene to be expressed, and moreover, cannot be integrated into the host genome. Similar to the injection of DNA, the injection of RNA may result in both a cellular as well as a humoral immune response in vivo.

RNA vaccines exhibit important safety features. RNA is more reactive than DNA and less stable but is resistant to ultra-violet radiation. mRNA does not integrate into the host chromosomes. The delivery of mRNA results in faster expression of the antigen of interest and requires fewer copies for expression. mRNA expression is transient, which adds to its safety. mRNA is more effective than DNA for protein production in post mitotic and non-dividing cells because DNA requires translocation through the nuclear member and plasma membrane, while mRNA requires translocation only through the plasma membrane. mRNA is not only a template for translation, but also acts as a ligand for toll-like receptors and is nuclease sensitive; thus it presents less concern for horizontal transmission. In addition, RNA vaccines elegantly integrate adjuvanticity and antigen expression, thereby mimicking relevant aspects of viral infections. This increases their efficacy compared to inactivated vaccines that require the use of adjuvants, simplifying handling and production. RNA can address a range of dedicated immunologic pattern recognition receptors, including TLRs 3, 7 and 8, RIG-1, MDA5, PKR and others that may act synergistically and serve to enhance the induction of antigen specific adaptive B and T cell responses. Importantly, by antigen synthesis in transfected host cells, mRNA vaccines directly introduce antigen into cellular antigen processing and presentation pathways, granting access to MHC molecules and triggering T cell responses, irrespective of the hosts MHC haplotype. This enables the induction of polyclonal T cell responses that may act synergistically with other immune responses, including B cells. (US 11,141,478).

For an infectious disease vacine, what one really wants to see is neutralizing antibodies.The mRNA vaccines are really good at geenrating those particular types of antibodies, adn they are showing that they can generate T cell responses too. (Grinstein, “Vaccine Developers Leverage mRNA and other powerful technologies” Genetic Engineerig & Biotechnology News, May 2024).

Potential Disadvantages: A major disadvantage of RNA based vaccination is the instability of the RNA in vivo, in particular in the cells of the immune system. Degradation of long-chain RNA from the 5′-end is induced in the cell by the so called “decapping” enzyme Dcp2 which cleaves m7GDP from the RNA chain. Thus, it is assumed that the cleavage occurs between the alpha- and beta-phosphate groups of the RNA-cap.

To inhibit the decapping process and thus increase the stability of RNA in vivo, the effect of phosphorothioate-cap-analogs on the stability of said RNA has been studied. It has been shown that the substitution of an oxygen atom for a sulphur atom at the beta-phosphate group of the 5′-cap results in stabilization against Dcp2. The phosphorothioate modification of the RNA 5′-cap has been combined with an “anti-reverse cap analog” (ARCA) modification that inhibits the reverse integration of the cap into an RNA chain. (Sahin, 9,295,717).

Due to its negative charge, mRNA does not easily enter cells, and it can be rapidly degraded by nucleases such as the enzyme RNase. Lipid nanoparticle (LNP) encapsulation, which is used in the current mRNA based COVID vaccines, helps mitigate this problem, as do other methods involving substitution of modified bases or design of the mRNA. Alternatively, physical methods such as electroportion can be employed. This approach has gained traciton in ex vivo adminsitered therapeutic cancer vaccines but is not highly efficient. (Cytiva, “mRNA vaccines and therapeutics: current trends and perspectives” Fe 17, 2025.)

RNA vaccine – cationic lipid nanoparticle:

Huang (US 10,022,435) discloses Aspects nucleic acid vaccines (NAVs) comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide, formulated within a cationic lipid nanoparticle.

Against Specific Microbies:

During mRNA vaccine development scientists must identify a target protein form the pathogen of itnerest such as the spike (S) protein in the case of SAR-CoV-2. The target protein must be sufficiently different from porteins in human  cells so the resulting immune response detects and supresses only the pathogen. After target identification, scientists insert the DNA sequence coding for the gene of itnerest(e.g., gene encoding for the S prtoein) into a plasmid, which is amplified in host bacteria (e.g., E. coli), which then udnergoes purification and linerization. Becasue mRNA is highly susceptible to degradation , a lipid nanoparticle (LNP) or other suitable divery system must be developed to package the mRN and deliver it to the cells.

––Coronaviruses are enveloped, positive-sense single-stranded RNA viruses. They have the largest genomes (26-32 kb) among known RNA viruses, and are phylogenetically divided into four genera (α, β, γ, δ), with betacoronaviruses further subdivided into four lineages (A, B, C, D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Of the six known human coronaviruses, four of them (HCoV-OC43, HCoV-229E, HCoV-HKU1 and HCoV-NL63) circulate annually in humans and generally cause mild respiratory diseases, although severity can be greater in infants, elderly, and the immunocompromised. In contrast, the Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged into the human population from animal reservoirs within the last 15 years and caused outbreaks with high case-fatality rates.

Corbett (10,960,070) discloses recombinant coronavirus S ectodomain trimers comprising protomers comprising one or more proline substitution(s) that stabilize the S protein trimer in the prefusion conformation. One class of mutation, comprising one or more (such as two) proline substitutions at or near the boundary between a Heptad Repeat 1 (HR1) and a central helix of the protomers of the coronavirus S ectodomain trimer was found to be surprisingly effective for stabilization of coronavirus S protein trimers in the prefusion conformation. Embodiments of such prefusion-stabilized coronavirus S ectodomain trimers are demonstrated to produce a superior immune response in an animal model compared to corresponding coronavirus S ectodomain trimers that are not stabilized in the prefusion conformation.

–Herpes Simplex Virus (HSV): (US 11,141,478) discloses compositions that include one or more nucleoside modified mRNAs, wherein each of said nucleoside modified mRNAs encodes a herpes Simplex Virus (HSV) glycoprotein or immunogenic fragment thereof, and wherein said nucleoside modified mRNA includes one or more pseudouridine residues. The HSV glycoprotein can include glycoprotein D, C, E, B, H, L or I.

–Zika virus: (US 11,241,490) discloses a composition for inducing an immune response against Zika virus in a subject that includes at least one isolated nucleoside modified RNA encoding at least one Zika virus antigen. In one embodiment, the nucleoside modified RNA includes pseudouridine.

Miles (US 2010/0003285) discloses a vaccine for human hookworm that includes at least one L3 larval stage antigen, at least one adult stage human hookworm antigen and adjuvants.

–SARS: Severe acute respiratory syndrome (SARS) emerged in Guangdong Province in South China in 2002, ultimately spreading to five continents where it caused 8,000 respiratory infections and 800 deaths. The SARS coronavirus (SARS-CoV) was identified as the etiologic agent of SARS in 2003. (US 2016/0376321) discloses methods and compositions related to the SARS-CoV spike protein useful for treatment or prevention of SARS.

–Chagas disease: also known as American Trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma cruzi. It is a leading cause of heart disease in Latin America, with up to 10 million infected people in the Western Hemisphere. The disease burden of Chagas based on disability-adjsuted life years is five times greater than malaria and about one-fifth that of HIV/AIDS in the LAC region. Additionally, the annual economic toll for treatment exceeds 7 billion globablly. Most of the deaths and disability attributed to Chagas diase result form chronic Chagas cardiomyopathy which develops in about 30% of infected individuals years to decades after the initial infection due to cascading effects of parasite induced pathologic changes including inflammation, cardiomyocyte hypertrophy, and fibrosis. (US 2021/0268080) discloses methods for preparing T cruzi antigen presenting dendritic cells which includes transducing the DCs with a vector for the expression of T. cruzi antigens.