As the COVID-19 pandemic continues, nucleic acid vaccines are among the many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidates under investigation. These vaccines offer several benefits compared to peptide or protein vaccines and live-attenuated or inactivated virus vaccines and viral vector vaccines.

A recent review paper in the journal Current Opinion in Virology in September 2020 shows how a new generation of RNA vaccines called self-amplifying RNA vaccines are more potent than conventional mRNA vaccines and could hold the key to the global production of cost-effective potent COVID-19 vaccines.

Study: A new generation of vaccines based on alphavirus self-amplifying RNA. Image Credit: nobeastsofierce / Shutterstock

DNA-Based Vaccines

The first plasmid DNA-based nucleic acid vaccines showed their effectiveness in eliciting both antibodies and cellular immunity against a range of antigens. These were optimized to promote adequate levels of immunity by several techniques, including complexing the nucleic acid with lipids or polymers, in order to decrease the size so it could enter the cell through the cell membrane. Inducing pores in the membrane via electroporation, or using a gene gun to insert the nucleic acid, were later physical methods.

DNA vaccines can function only within the cell nucleus, which presents some technical issues. Besides this, such vaccines caused DNA to be present in the nucleus, with the possibility of its being inserted into the genome, with future oncogenic potential. Human immune responses have generally been weaker than in the preclinical animal models.

Conventional mRNA Vaccines

With no approved DNA vaccines on the scene as yet, mRNA vaccines have also been investigated. This molecule can express itself in the cytoplasm, making transfection easier, while its inability to integrate into the genome makes it safer. However, ribonucleases in skin and blood degrade it rapidly. To prevent this, it can be complexed with other compounds to increase its cellular uptake. Newer synthetic technologies have made it possible to produce stable mRNA and to insert it into the cell more efficiently in vivo.

Self-Amplifying RNA Vaccines Express More Antigen

Self-amplifying RNA (saRNA) is a newer type of RNA vaccine with high immunogenicity. Derived from alphaviruses or flaviviruses, it contains the viral replicase enzyme that allows it to amplify itself. Meanwhile, it expresses the substituted genes rather than those that encode the viral structural proteins.

A brief description of the mechanism of this saRNA is as follows: it enters the cytoplasm of the host cell, translates the replicase, which makes a complementary negative copy of the mRNA. This long mRNA strand becomes the template used by Rep to synthesize more saRNA. At the same time, the replicase also binds to a subgenomic promoter in the negative strand to synthesize subgenomic mRNA at ten times greater concentration than genomic RNA, encoding the viral antigen.

Since saRNA is already more immunogenic than other RNA molecules, activating several Toll-like receptors, for instance, this elevated expression of viral antigen in vivo causes extremely robust immune responses. The saRNA packaged in lipid nanoparticles expresses the antigen for much longer than mRNA.

Direct delivery of saRNA

The current study ignores the potential to package saRNA into viral particles or deliver it via plasmids, instead targeting direct delivery of an saRNA vector. This comprises a positive-strand RNA that contains genes encoding the replicase and the viral antigen. Also called naked saRNA, this is the easiest way to deliver RNA into cells. Many earlier papers have reported the successful use of naked saRNA for vaccination of mice against the influenza virus, tumor cells, rabies virus, and HIV. Though there is an initial delay, its immunogenicity can be improved by electroporation or by forming complexes between naked saRNA and lipids/polymers, as for DNA vaccines.

Electroporation in saRNA Vaccines

Electroporation (EP) enhances vaccine efficacy by increasing the efficiency with which the transgene is transferred to the host cell. Intradermal EP presents a non-invasive means of immunization. The use of saRNA with EP produces an immune reaction in mice when conventional mRNA does not, probably because the replication of RNA is vital to the immune response. The innate immune response with saRNA delivered by EP may be lower than with LNPs.

Large animal models like the pig have shown that saRNA vaccines produced a longer period of expression, over at least 12 days. Since pig skin is very similar to human skin, this finding is relevant to human research.

Placental RNAase inhibitor may also be added to saRNA before intradermal injection, in order to enhance the efficacy of vaccination, since the skin contains high concentrations of RNAases to protect itself against pathogens.

LNP-Based Vaccines

Lipid nanoparticles (LNPs) are the basis on which several vaccines have been manufactured. Such formulations have been refined to be more stable and less immunogenic, even while eliciting high levels of humoral and cellular immunity against the specific antigen. There have been several types of LNP-mounted saRNAs, some of which can be separately stored and mixed with saRNA before administration. The RNA on these particles, though exposed, is preserved from RNAases in these LNPs.

Some, like the cationic nanoemulsion-based system, produce robust immune responses, similar to a live attenuated vaccine, but without the risk of reversion. Certain researchers propose that intramuscular injection of the vaccine causes the antigen to be expressed in muscle cells, after which it is transferred to antigen-presenting cells, which could indicate it had a cross-priming mechanism for CD8 T cells.

Researchers are further refining saRNA vaccines using various types of LNPs or including sugar-lipid conjugates in the LNPs to engage the mannose receptors on the antigen-producing cells.

Much work is going on to increase the stability of the vaccine and to optimize the immune responses to obtain broadly neutralizing antibodies by incorporating self-assembling bacterial proteins or other lipids.

Conjugates of saRNA with PEI

Another avenue of research is the compaction of mRNAs into small particles using cationic polymers such as polyethyleneimine (PEI) since the primary amines in them enhance the condensation of RNA, protect it from degradation and promote its uptake into the cell. It has been found that the molecular weight of the PEI, the w/w ratio of saRNA to PEI, and the inclusion of peptides that penetrate the cell are determinants of immune efficacy. Many promising formulations are under development with higher efficacy than naked saRNA.

Optimizing the saRNA Molecule

Again, a trans-amplifying RNA vaccine has been developed where the antigen-encoding gene is expressed from an saRNA that lacks replicase and supplying the replicase through a non-replicating mRNA. This does not produce more significant immune responses but might be more adaptable and easier to manufacture because the mRNA replicase can be stored separately.

Implications and Promising Candidates

The strong innate immune response in the host is one potential limitation of saRNA vaccines, which could reduce the intensity and duration of transgene expression. This is counteracted by also administering interferon inhibitors. Another different approach is to include mutations that increase subgenomic RNA expression, thus increasing its immune-stimulatory activity.

The researchers point to three prototype saRNA vaccines, of which one is based on the Venezuela equine encephalitis virus (VEEV) saRNA expressing the SARS-CoV-2 spike protein in the pre-fusion stabilized form, packaged in an LNP capsule. The result of the administration of this vaccine to mice was observed in the form of highly specific neutralizing antibodies and immune cells. It is now being evaluated in a phase I clinical trial in the UK. Another similar vaccine has been found to elicit neutralizing antibodies in mice and in nonhuman primates, which were still detectable after 70 days.

The use of highly efficient delivery methods to insert saRNA into the cell has led to the production of strong innate and adaptive immune responses, both humoral and cellular, in mice and large animals. This vaccine formulation appears to have several advantages over mRNA vaccines because of its similarity to a live viral vaccine. In this way, it elicits multiple immune recognition signals which induce a robust and broad immune response, but without apparent adverse effects.

The new format is quickly and easily adapted to different antigens by merely switching around the sequence that encodes for the subgenomic RNA since this does not require a shut-down of the production run. Thus, this vaccine is amenable to GMP with the same workflow and processes used for mRNA. The speed and ease of manufacture of saRNA vaccines are key advantages when it comes to producing vaccine doses to supply a global-scale demand for universal immunization against SARS-CoV-2, as well as to contain the outbreaks of other dangerous viruses like Zika or Ebola.

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