September 30, 2022

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Delivery system for non-viral coronavirus vaccine

Delivery system for non-viral coronavirus vaccine


Delivery system for non-viral coronavirus vaccine.   SARS-CoV-2 is a single-stranded RNA virus with a β-coronavirus composed of coronavirus (S) protein, envelope protein, membrane protein, nucleocapsid protein and accessory protein.

SARS-CoV-2 has a high degree of sequence similarity with SARS-CoV, and adopts a similar entry route to infect human cells. In other words, the receptor binding domain (RBD) of the S1 subunit of the S protein interacts with human angiotensin converting enzyme 2 (ACE2), and then membrane fusion mediated by the S2 subunit.

Therefore, S protein is an important part of SARS-CoV-2’s cell infection. Moreover, it was found that the functionally neutralizing antibody (NAb) produced in COVID-19 patients mainly targets the epitope in the S protein, which indicates that the S protein is a promising target for the anti-SARS-CoV-2 vaccine.

The process of developing a vaccine involves two key steps:

1) Identify the antigen;

2) Develop the delivery method of the antigen to achieve powerful cellular and humoral immunity.

In the case of SARS-CoV-2, previous experience with SARS-CoV and MERS-CoV has enabled rapid development of candidate vaccines. The whole virus is a traditional type of vaccine, historically used for major diseases such as smallpox, tuberculosis and yellow fever. This type of vaccine can be divided into two main types: attenuated live virus and inactivated virus.

The research team from the University of Michigan in the United States has launched research on “delivery” for “non-viral” vaccines. The research paper was published in “Advanced Drug Delivery Reviews” entitled “Non-viral COVID-19 vaccine delivery systems”

Delivery system for non-viral coronavirus vaccine

In this review, researchers introduce some of the leading non-viral vaccines in clinical development, and discuss delivery strategies to improve vaccine efficacy, protection period, safety, and mass vaccination.

They believe that in addition to these viral vector-based vaccines, the non-viral vaccine platform realized with the development of nanomedicine and vaccine delivery technology is also in the late stage of clinical trials (Table 1).

They include mRNA and DNA encoding SARS-CoV-2 protein antigens and vaccines based on protein antigens. The membrane-bound glycoprotein (called spike protein) on the corona virus is responsible for the virus to enter the host cell, so it is an ideal target for the anti-SARS-CoV-2 vaccine.

Vaccines based on mRNA and DNA focus on methods of delivering genetic material encoding antigen candidates into host cells, but due to limited cellular uptake and the instability of naked mRNA and DNA, they require the use of delivery vehicles or electrophoresis.

On the other hand, protein-based vaccines require the synthesis and characterization of protein antigens as part of vaccine production. Since protein antigens are synthesized by cells and secreted into the culture medium in a soluble form, it is often a challenge to ensure proper protein folding and maintain its antigenicity.

Therefore, when designing a gene vector to impart stability to the antigen protein before transfecting the antigen into the target cell, recombination technology is often involved. Compared with vaccines based on whole viruses or viral vectors, the subunit antigens used in vaccines based on mRNA, DNA, and proteins can cause a weaker immune response and therefore require co-administration of adjuvants.

Delivery system for non-viral coronavirus vaccine


Design Ideas of mRNA Vaccine

As shown in Table 1, all vaccines for COVID-19 mRNA that are being developed clinically are delivered through lipid nanoparticles (LNP). LNP encapsulates mRNA in a solid lipid structure and consists of four components (Figure 1): cationic or ionizable lipids for mRNA complexation, cholesterol for stabilizing nanoparticles, and for helping formation and intracellular The released auxiliary phospholipids, and pegylated lipids can reduce non-specific interactions. As a non-viral mRNA carrier, LNP has the following advantages:

1) LNP effectively encapsulates and concentrates mRNA;

2) LNP promotes the delivery of intracellular mRNA to the cytoplasm by increasing cellular uptake and triggering endosomal escape;

3) LNP increases the stability of mRNA by protecting it from degradation in the extracellular space;

4) LNP is composed of biocompatible materials suitable for human use;

5) Large-scale synthesis of GMP-grade LNP.

Delivery system for non-viral coronavirus vaccine

To develop effective LNP vectors to deliver COVID-19 mRNA vaccines, two key factors should be considered: The first factor is the selection of suitable cations or ionizable lipids. The second factor is to optimize the composition of cholesterol, auxiliary phospholipids and lipid-PEG and their relative proportions in LNP.

These factors may greatly affect the effectiveness and performance of mRNA vaccines. However, the optimization process of the recipe usually involves more variables and requires a lot of resources. In order to make the optimization process more effective, researchers have adopted a design of experiment (DOE) method, including fractional factorial design and deterministic screening.

Through these methods, multiple parameters such as lipid ratio and lipid structure can be adjusted at the same time, and it is found that incorporating DOPE and increasing the ionizable lipid:mRNA ratio can improve the efficiency of mRNA delivery.

Based on this, LNP containing C12-200 was optimized for erythropoietin mRNA delivery, and a 7-fold increase in efficacy was observed. Similar methods can also be used to optimize LNP for COVID-19 vaccine delivery.


Other DNA delivery strategies applicable to COVID-19

In the preclinical stage, there are other DNA delivery strategies applicable to COVID-19. One way to improve efficacy in vivo is by adding adjuvants or adjuvant-encoded plasmids. When co-delivered with antigen-encoded plasmids, adjuvants are secreted into the surrounding area, where they can stimulate local APCs and cells in draining lymph nodes (LN), resulting in a stronger and longer-lasting immune response. Another method is the transdermal application of microneedles (MN), which allows the DNA vaccine to be deposited on the epidermis and dermis rich in immune cells. Compared with the soluble DNA vaccine, the MN vaccine caused an antigen-specific IgG1 serum antibody 3 times more frequently, an excess of cytotoxic CD8 T cells 3 times, and inhibited the lung metastasis of melanoma.

In addition, oral administration of DNA vaccines has been examined in preclinical studies. Unlike Symvivo’s bac-TRL-Spike vaccine, the M1 DNA plasmid encoding the H1N1 virus matrix protein is encapsulated in cationic liposomes and administered orally, resulting in a significant increase in IgG titers, T cell activity and immune memory. When challenged with homologous influenza A virus intranasally, the virus level was lower than the detectable level in the lungs of mice immunized with oral M1 pDNA liposomes, indicating a protective effect against respiratory tract attack. Considering the respiratory tract SARS-CoV-2 infection, it is particularly important to provide candidate vaccines that enhance respiratory protection.

In summary, DNA vaccine is a promising vaccine strategy against SARS-CoV-2. They have a simple design, convenient manufacturing process, and are cost-effective and stable with a long shelf life. Compared with other vaccine platforms, the main disadvantage of DNA vaccines is their relatively weak immunogenicity, but more research is being conducted to improve this aspect through various delivery strategies. Although there is no approved DNA-based vaccine on the market, the DNA vaccine may prove to be an effective platform for SARS-CoV-2 vaccination.




The adjuvant triggers the PRR on the adaptive immune cells and triggers different immune response pathways according to the type of PRR adjuvant to be activated. Therefore, the use of effective and well-matched adjuvants can greatly improve the effectiveness of the vaccine. Currently, many COVID-19 vaccine developers use adjuvants in their vaccines, including AS03 (GSK’s alpha-tocopherol and squalene in oil-in-water emulsions), CpG 1018 (Dynavax’s DNA-based TLR-9 agonist It has been shown in preclinical and clinical studies to greatly improve the efficacy of the vaccine.

For mRNA vaccines, mRNA itself can bind to certain pattern recognition receptors (PRR), such as TLR 3, 7, 8, RIG-1, PKR, OAS and MDA5, which in turn induce innate immune activation, type I interferon, And the production of pro-inflammatory cytokines, has a supporting role.

However, the activation of these receptors can trigger the natural antiviral mechanism, which inhibits the translation of foreign mRNAs through the phosphorylation of eiF2α and the overexpression of RNase L. Therefore, there is a balance between mRNA-induced innate immune activation and mRNA translation. Alternatively, the delivery system can be adjusted to an adjuvant or an additional adjuvant can be co-delivered.

For example, the lipid components of LNP have been screened against combinatorial libraries of lipids, which can induce effective antigen expression and appropriate innate stimulation of mRNA vaccines. After screening more than 1,000 lipid formulations, the authors identified a class of lipids with the best performance with unsaturated lipid tails, dihydroimidazole linkers and cyclic amine head groups. These lipids induce DC activation through stimulators of the interferon gene (STING) pathway, and have nothing to do with the TLR or RIG pathway, and trigger a strong antigen-specific T cell response in murine tumor models, and have therapeutic effects.


DNA vaccines also have the potential to induce innate immune activation, because many DNA plasmids produced in bacteria may contain unmethylated CpG motifs. In fact, the CpG motif has been intentionally added to the DNA plasmid backbone as an adjuvant to enhance vaccine efficacy.

Studies have shown that the addition of CpG greatly improves the immune response mediated by antigen-specific T cells, thereby protecting mice from mouse melanoma cells. On the other hand, similar to mRNA, there are DNA vaccine delivery systems with immunostimulatory properties. Vaxfectin adjuvant is one of them, which is a cationic lipid-based delivery platform.

According to reports, intramuscular injection of DNA vaccine by Vaxfectin can enhance antigen-specific IgG1 and IgG2a responses in mice. A mechanism study conducted in another study showed that after intramuscular injection of Vaxfectin DNA vaccine, genes in mice were regulated, resulting in transcripts related to antigen processing, presentation, and TLR pathways in muscle cells A large amount of enrichment can explain the adjuvant properties of mice. Vaxfectin. In addition, many adjuvants are being developed, which have great potential to improve protein-based vaccines.

The synthetic TLR-7/8 agonist 3M-052 has been used as an adjuvant with HIV-1 clade C 1086.C-derived gp140 envelope protein (Env) for anti-HIV-1 vaccines. Compared with conventional adjuvants such as alum, R848 (TLR-7/8 agonist), MPL and other vaccines, Env plus 3M-052 vaccination induces a higher level of antibody response and long-lived plasma in the bone marrow of rhesus monkeys. cell. And GLA (TLR-4 agonist). In fact, many studies are currently investigating methods to provide TLR7/8 agonists based on their potential to enhance the immune response of immune-susceptible populations (including children and the elderly).



Scientists believe that the accumulation of experience in similar diseases caused by SARS-CoV and MERS-CoV has laid the foundation for accelerating the development of SARS-CoV-2 vaccines. Our current global situation urgently requires rapid development of vaccines while complying with strict guidelines on the safety of SARS-CoV-2 vaccines.

At the same time, preparations should be made for the potential mutation of SARS-CoV-2 and its seasonal recurrence. In addition, we should develop effective countermeasures against other emerging pathogens. In particular, the structural fragility and suboptimal immunogenicity of many candidate vaccines should be solved.

In terms of stability, considering the global distribution of vaccines, cold chains and transportation, these may increase costs and restrict distribution. In the past two decades, nanoparticle-mediated vaccine delivery has achieved good results.

Future research should focus on simplifying the nanoparticle vaccine delivery system so that the final vaccine product can be more easily produced and formulated. The physical and chemical properties of vaccine preparations should be optimized, especially during long-term storage and transportation, for rapid deployment and large-scale vaccination on a global scale.



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