Nucleic Acid Vaccine Booste: Vector

Nucleic Acid Vaccine Booste: Vector.  The launch of the COVID-19 mRNA vaccine opened a chapter in the use of nucleic acid vaccines in humans. Nucleic acid vaccines are favored by scientific researchers and pharmaceutical companies because of their rapid design, low production cost, easy transportation and storage, and ability to stimulate body fluids and cellular immunity. However, in clinical use, nucleic acid vaccines have problems of targeting, safety and low immunogenicity, which greatly restrict their development.

By using nano-carriers, such as polymers, lipids, inorganic materials, etc., some of the problems can be solved. Among them, biopolymer nanomaterials are worth looking forward to. The reasons are as follows:

1. Biocompatibility

2. Biodegradability

3. Sustainable availability

4. Adjustability of physical and chemical and biological characteristics (size, chemical composition, surface function).

In addition, the material’s solubility and stability, nucleic acid solidification ability, cell uptake, intracellular release and immunogenicity can be further improved.

The mRNA vaccine has higher safety because it does not integrate into the nucleus, but the DNA vaccine is more stable than the mRNA vaccine. With the launch of mRNA vaccines, it is believed that DNA vaccines will also be greatly developed. This article takes more DNA vaccines as an example to introduce the development of delivery vectors. Of course, this also applies to the application of mRNA.

1. Biopolymer carrier design

The biopolymer DNA vaccine system contains at least one core skeleton and plasmid DNA (pDNA). The biopolymer backbone can be modified with functional groups or covalent polymers to enhance its delivery function. pDNA can be located on the surface or inside of nanoparticles (NPs). On this basis, further modifications can be made, such as adding a polymer shell, a polymer matrix, and combining two different substances to form NPs. When designing DNA vaccine vectors, different biological barriers should be considered.

Such as in the extracellular environment, the inactivation of serum proteins and the degradation of DNase should be considered. After reaching the target cell, the DNA nanoparticle needs to be internalized through the process of membrane phagocytosis, and then escape, pass through the cytoplasm and enter the nucleus, where the DNA dissociates from the carrier and finally obtains the encoded antigen.

2. Methods to improve DNA polymerization

The effective combination of DNA and biopolymers is the key to delivery. Only in this way can nanoparticles provide protection for DNA and ensure effective uptake by cells. Unstable polymerization and improper release will make the transfection efficiency low, resulting in a lower immune effect.

At present, the most widely used method is to use positive charges. Because the DNA phosphate backbone carries negative charges, positively charged NPs provide a non-covalent mode of action. This phenomenon can also be seen under natural conditions. Chitosan, polylysine (PLL), protamine, polyarginine, etc. are all good DNA polymerizers. By introducing synthetic polymers, such as polyethyleneimine (PEI), polyurethane, glycerol methacrylate or positive resin, etc., positive charges can be introduced into the carrier core.

PEI is a synthetic polyamine frequently used in DNA delivery due to its high cation content, high buffering capacity, and easy availability. It is called the “gold standard” in polymer-mediated gene delivery. In addition, covalent polymers carrying cationic amino acids, polypeptides or small molecules can also be used for charge regulation. For example, arginine, histidine and lysine can be used to regulate chitosan, sodium alginate, human serum albumin (HSA), HA and chondroitin sulfate. Other small molecules used to modify biopolymers include quaternary amines, spermine, piperazine, cysteine, and succinyltetraethylenepentamine.

Moreover, the polymer backbone itself can be adjusted by chemical modification. For example, chitosan carries a positive charge under acidic pH conditions, and trimethylation at its primary amine can make it carry a positive charge under a wide range of pH conditions. The methyl esterification of the carboxyl group in albumin can reduce the negative charge and make the whole protein present a positive charge, thereby facilitating DNA polymerization.

In addition, the hydrophobic force can anchor pDNA to the vector. In order to achieve this goal, pDNA needs to be hydrophobic by cetyl bromide or dioleoyl-3-trimethylammonium propane. The advantage of this method is that the carrier material is not limited to positively charged polymers, and negatively charged or neutral polymers can also be used. Through this method, pDNA is effectively immobilized on the modified HA, and DNA can also be introduced into the hydrophobic core of PLGA.

In addition to positive charges and hydrophobic interactions, hydrogen bonds can also be used to polymerize DNA. For example, the guanidine groups in arginine can form hydrogen bonds with the DNA phosphate backbone, and these groups can interact with the cell surface to significantly increase cell uptake.

The use of multivalent metal cations can also achieve DNA polymerization. Zinc ions can promote the polymerization of DNA phosphate backbone and two different biopolymers (histidine linked to PLL and dipyrylamine modified HA), but the use of other ions has a clear range (safety range), especially those that are possible Effective adjuvants include nickel, beryllium, cobalt and palladium. In addition to being an excellent coordination ion, zinc can also modify histidine to increase endosome release and increase transfection rate.

Picture Figure 1 DNA polymerization mode

3. Improve the stability and solubility of DNA biopolymer carrier system

The stability and solubility of DNA vaccine preparations are not only related to the safety and effectiveness of the vaccine after immunization, but also related to the long-term effective storage of the product. The stability of DNA vaccines needs to be considered when deciding the method of immunization (oral or parenteral immunization) and the type of preparation (dry powder or suspension).

One problem that affects the stability of nanoformulations is agglutination, especially in suspensions. NPs have high surface potential energy due to their high surface area and volume ratio, and they have the characteristics of achieving low surface potential energy through agglomeration, which is especially reflected in biological materials. In order to prevent agglutination, the surface of NPs is coated with a hydrophilic polymer, such as polyethylene glycol (PEG), which can prevent the interaction between particles and increase the blood circulation time, thereby increasing the possibility of reaching target cells. PEG surface modification has been realized in PLGA, PDA, colloid, and polyspermine.

Agglutination of the cationic delivery system can also be eliminated by the anionic polymer chondroitin sulfate. PLL, polyarginine and protamine NPs are treated with chondroitin sulfate to reduce the agglutination of red blood cells, and the cytotoxicity associated with high cations is also reduced. Hashimoto and colleagues found that functional chitosan containing hydrophilic lactose residues not only reduced self-aggregation, but also reduced its aggregation with serum proteins.

The blood is rich in a large number of proteins, such as HSA and BSA, which play an important role in the transportation of various biologically important molecules from hormones to fatty acids. They are also used as carriers in the process of gene delivery. Because it naturally exists in the blood circulation and has the least interference with other serum proteins, the level of agglutination is relatively low and the pharmacokinetics is relatively good.

From the perspective of chemical stability, pDNA should be prevented from being degraded by DNase. Encapsulation of pDNA inside NP can block enzymatic hydrolysis. However, experiments have shown that pDNA on the surface of the carrier can also reduce enzymatic degradation. This may be due to the conformational change of pDNA and the nano surface layer or the steric hindrance caused by the carrier backbone. .

Oral administration is superior to intradermal or intramuscular injection in terms of painlessness, convenience and low sterility requirements. For local gastrointestinal diseases, such as dental caries, colorectal cancer, inflammatory bowel inflammation and ulcerative colitis, oral administration is undoubtedly a good way, but it faces many chemical stability challenges. Under oral administration, nano-formulations will be treated in a strong acid environment, leading to degradation of the carrier material and affecting DNA delivery.

This can be eliminated by the appropriate design of the carrier. For example, experiments have shown that the surface layer of sodium alginate can effectively protect DNA from passing through the gastrointestinal tract, and at the same time, it facilitates the ingestion of the epidermis of the gastrointestinal tract with its superior mucosal adsorption and penetration. In addition, the oral administration effect of the three-component system consisting of sodium alginate, chitosan and pDNA is significantly better than that of the chitosan and pDNA group.

This may be due to the dissociation of the sodium alginate-chitosan crosslink at pH 1.5, resulting in the formation of an insoluble sodium alginate protective layer on the surface of the carrier, which provides protection. The introduction of polycaprolactone and 2-hydroxyethyl methacrylate into gelatin and chitosan NPs, respectively, can protect the DNA load against the harsh conditions of the gastrointestinal tract.

The protein zein can be used as an oral DNA vaccine carrier. Because of its hydrophobic amino acid content of more than 50% and its high glutamine content, it has amphiphilic characteristics, which is beneficial to the formation of NPs with a hydrophobic core and a hydrophilic outer layer. In an acidic environment, such as the stomach environment, Zein nanocarriers are insoluble, can resist low pH, and protect the internal load from degradation by enzymes. The test surface showed that mice immunized with chitosan carrier outsourcing zein protein had a higher level of immunity compared with the control group lacking zein protein.

It is important to develop a DNA carrier system that does not require refrigeration to ensure that the DNA vaccine remains stable during storage and transportation, not only for energy needs, but also suitable for low-income countries. NPs coated with HA and chitosan can be stored in freeze-dried or liquid form at room temperature for more than 12 months.

The solubility of carrier materials is related to stability, which is the key to the effectiveness of DNA preparations. Many biopolymers used in the design of DNA vaccines are soluble in water-soluble media. However, commonly used biopolymers, such as chitosan, have limited solubility at physiological pH. The hydrophilicity of the polymer backbone can be improved by chemical modification, such as the deacetylation of acetylated amine residues, and the introduction of carboxymethyl or larger hydrophilic groups such as N-(2- Hydroxy-3-trimethylammonium) propyl chloride and methacrylic acid polymer ester, etc.

4. Improve the carrier’s cell uptake capacity

In order to increase the immune response and the therapeutic activity of DNA vaccines, effective uptake and release of genetic material in target cells are critical. Cell uptake is the most important step to ensure the biological activity of the vaccine. It depends on the interaction between the cell membrane and the carrier and involves multiple modes of endocytosis. The internalization process depends on the cell type (different cells have different types and amounts of membrane proteins and lipids) and the physical and chemical characteristics of the carrier.

1) Non-specific cell uptake

In the case of non-specific, inhaled endocytosis, cationic polymers are easier to achieve than anionic and neutral molecules. Endocytosis is achieved by adsorbing positively charged DNA-polymers on the outer cell membrane of negatively charged proteoglycans. Non-specific cell uptake can be enhanced by CPPs. CPPs are short positively charged polypeptides that can pass through cell membranes without destroying their integrity, and were first found in viral proteins.

The commonly used CPP is the HIV-1 transduction sequence transactivator (Tat), which can increase the efficiency of gene transfection. The introduction of Tat and arginine-glycine-aspartate (RGD) polypeptides can significantly enhance the uptake efficiency of HSA nanocarriers. The degree of crosslinking of the polymer backbone significantly affects the characteristics of the carrier. In addition to positively charged polymers and CPPs, Ca2+ is also used to assist cell uptake and endosomal release. Ca2+ mediates several cellular pathways, including endocytosis. The difference in the natural concentration of these ions inside and outside the cell plays an important role in gene delivery (the Ca2+ concentration is 104 times higher in the cell than outside the cell). Ca2+ modified sodium alginate-sulfate NPs as DNA delivery carriers can achieve clathrin-mediated endocytosis.

When there is a large number of positive charges, especially when the density is high, it will cause cytotoxicity, which will cross-link with serum proteins that carry negative charges, and then be cleared by the reticuloendothelial tissue. Therefore, it is important to control the positive charge content and density. One way to reduce the positive charge content without affecting the uptake of non-specific cells is to introduce hydrophobic groups such as phenylalanine or enoyl. The interaction between the hydrophobic group and the cell membrane reduces toxicity and enhances cell uptake. Care must be taken when introducing these modifications to prevent drastically reducing solubility and system stability.

Figure 2 Different cell endocytosis modes and DNA vaccine mechanism of action

Figure 3 Non-specific and specific cell uptake

2) Specific cell uptake

Target ligands can enhance the accumulation of carrier systems in specific tissues or cells, reduce non-specific uptake, and promote internalization. Small molecules, such as folic acid, sulfondronate, lactose, mannose, and TLR7 agonists, as well as polypeptides, oligonucleotides, etc., have been introduced into biopolymer nanocarriers to improve specific receptor targeting. In addition to ligand selection, controlling its surface density is also necessary to improve target efficiency and internalization.

Specific delivery and enhancement of DNA vaccine intake in APC are related to the immune effect. Lectin-binding receptors, such as mannose receptors CD206 and DEC-205, are abundantly expressed on the surface of APCs, including macrophages and DCs. The introduction of mannose can promote APC uptake.

DEC-205 receptor can trigger MHC I and MHC II responses, thereby activating CD8+ and CD4+ T cells. In order to ensure the targeting of DEC-205, Suresh et al. fused an anti-DEC-205 antibody to pDNA chitosan and designed a DNA vaccine for SARS-CoV. Experiments have shown that the vector targets nasal DCs and triggers stronger immunity in the case of low-dose DNA vaccines.

In addition to lectin-binding receptors, TLRs can also be used for macrophage targeting. Chitosan NPs were modified by TLR (TLR-7 and TLR-2), and compared with chitosan NPs alone, it significantly enhanced IL-8 levels in THP-1 macrophages.

DNA vaccines can induce long-term immune responses. It is very useful for inducing memory immunity and system immunity in cancer treatment. In addition to targeting APC receptors, different cancer cells can also be targeted by introducing CTLs epitopes. In addition, folic acid and CD44 receptors are overexpressed on the surface of many tumor cells. Therefore, the introduction of folic acid into chitosan, polyspermine, and chondroitin sulfate can enhance the immunity of liver, lung, and ovarian cancer cells. Biopolymer backbones, such as endogenous polysaccharides, HA and chondroitin sulfate, specifically bind to the CD44 receptor.

5. Improve the carrier’s endosome release capacity

The endosome is the organelle responsible for intracellular processing and contains various enzymes. If the genetic material is released in the endosome, it will be degraded, resulting in a low transfection rate. The endosomal maturation process experienced a pH drop from physiological 7.4 to early 6.5, late 6.0, and lysosome 5.0 (proton pumping, that is, ATP hydrolysis pumps protons into endosomes and lysosomes). Cationic polymers such as PEI, polyamide, succinyl tetraethylene pentamine, spermine and imidazole molecules and histidine can rupture the endosome.

Presumably, the reason is that these molecules interact with protons, causing protons to be pumped out in large numbers. Subsequently, it causes the accumulation of chloride ions and water inside and outside the body, osmosis and swelling, and finally causes the inner body to rupture. Although this is a general explanation, it still needs to be considered. However, molecules with high buffering capacity play a major role in endosomal escape and the release of genetic material. For example, Cheng et al. designed a histidine-rich polypeptide dextran vector to deliver genes.

In this experiment, dextran (less) combined with arginine-histidine polypeptide (more), showing low toxicity and high gene expression . The presence and proportion of histidine are important for DNA enrichment and endosome release. Arginine was originally used for DNA enrichment and enhanced cellular uptake.

In addition to strong buffering groups, endosomal escape can also be enhanced by molecules that can penetrate the lysosome. Such as chloroquine, cationic lipids and membrane disrupting peptides. The latter are highly regarded because they are derived from viral and bacterial vectors because they can escape the endosome. Membrane destruction peptides such as HA2 (a fusion peptide derived from influenza virus), and LLO (a Listeria monocytogenes cholesterol-dependent toxin) are used to modify the pDNA delivery system to enhance the cytoplasmic delivery of target cells. Despite its mode of different action, can realize the inner body escape. HA2 undergoes a conformational change after acidification and can fuse with endosomal membranes. LLO is active at low pH and degrades in the cytoplasm. The endosome escapes by perforating the lipid bilayer.

6. Improve the dissociation of vector and pDNA and guide pDNA into the nucleus

Dissociation of pDNA from the vector is the key to high transfection efficiency. Effective release can be achieved by shielding the strong electrostatic interaction between pDNA and the carrier or introducing a stimulus reaction degradation system. The shielding of positive charges can generally be achieved by introducing a second polymer.

For example, the introduction of anionic polymers such as sodium alginate and poly (γ-glutamic acid) or negatively charged proteins such as α-casein on chitosan nanocarriers can reduce the strength of interaction between DNA and particles, which is easy to release and increase Transfection. It is worth noting that this will also lead to a decrease in the DNA transported to the target cell.

The net positive charge can also be reduced by hydrophobic molecules such as phenylalanine or elastic large groups such as pullulan. Compared with traditional cationic polymers, the PEI25k carrier system is based on a reversible polymer, which will achieve low toxicity, and the transfection efficiency can be as high as 8 times.

An easier way to control the release of DNA is to use a stimulus response linker. This is generally achieved by introducing disulfide bonds and pH-sensitive bonds. Disulfide bonds can be cleaved into SH groups under the action of intracellular glutathione. The concentration of intracellular glutathione is generally 100-1000 times that of extracellular (2-10mM vs. 2-20uM), which is beneficial to disulfide The bond modifies the efficient cleavage of polymers. This method is suitable for the dissociation of pDNA from disulfide bond modified cationic chitosan, HA, polyarginine and LLO polymers.

In addition to disulfide bonds, acid-degradable ketone esters, diacrylate crosslinkers, and bisamide bonds are also used to improve the release of DNA from the polyspermine gene delivery system in an acidic environment. Liu et al. reported that pDNA was released from the chitosan carrier through an acidic environment pH<6.5, and the c=n bond in the schiff base was broken. In another study, Wang and his colleagues designed a pda-pei nanocarrier that is sensitive to pH. The complex is stable at physiological pH and breaks after being internalized by the endosome. In addition, near-infrared (nir) light irradiation and photothermal sensitive carrier systems can achieve rapid release of the inner body. In the acidic environment of cancer cells and nir light irradiation treatment, nanocarriers can overcome multiple obstacles to achieve gene delivery.

Once released, pdna needs to enter the nucleus and be transcribed into a DNA vaccine. In order to assist the nuclear transcription of genetic material, the carrier needs to additionally modify the nuclear localization signal (nls), a short peptide rich in positive charges of lysine and arginine, derived from eukaryotic nuclear proteins and viral proteins, which can effectively mediate the nucleus Within the transfer. Guan et al. introduced nls, a source of sv40 large t antigen, and added it to the hsa-dna cation system to effectively increase gene expression in vivo and in vitro. Similarly, protamine, an arginine-rich protein commonly used for dna enrichment and membrane transport, also shows the function of assisting nuclear uptake. When protamine is combined with biodegradable anionic polymers such as chondroitin sulfate or hydrophobic groups such as cholesterol, the level of nuclear transport is increased, which is due to the existence of nls-like regions, that is, repeats containing 4-6 arginines sequence.

7. The immune stimulating effect of DNA vaccine delivery vector

Immune stimulants, such as IFN-γ, IL-2 or IL-4, play an important role in the immune response, that is, stimulating T cell differentiation. This is why adjuvants are used in vaccines. Adjuvants are generally organic or inorganic additives, such as aluminum salts in hepatitis vaccines or single phospholipids in shingles vaccines that can induce a strong immune response, which is achieved by stimulating the secretion of immune stimulants.

Nanocarriers also have an adjuvant effect. For example, Jiang et al. reported that modification of chitosan NPs with methacrylate-based polymers not only significantly stabilized DNA, but also resulted in increased antibody levels and IFN levels. In addition, Yue et al. found that the introduction of CpG motifs on chitosan-NP vectors increased the proliferation of T cells, the production and release of IFN-γ, IL-2, and IL-4. The CpG motif is an extensively studied gene delivery adjuvant, consisting of a short synthetic single-stranded DNA sequence, which mainly contains cytosine and guanine elements.

In nature, unmethylated CpG motifs can be found in bacterial genomes, but they are very rare in vertebrate genomes. Therefore, when being brought into vertebrate organisms, the foreign CpG DNA is recognized as an invasive species, which can lead to a strong immune response. In short, CpG represents a pathogen-associated molecular pattern (PAMP), which is recognized by a specific pattern recognition system, APC’s TLR-9.

Interestingly, the size of the carrier complex also plays a role in the initiation of immune responses, a phenomenon that occurs in various inorganic molecular and biopolymer delivery systems. However, no universal, linear size-immune response relationship has been derived, indicating that other factors, including shape, chemical composition, surface modification, and carrier charge also contribute to it.

However, Yue et al. found that small CpG modified chitosan NPs induced a stronger immune response than large particles. Kim et al. studied the ability of monodisperse polypyrrole NPs and found that medium-sized NPs (60 nm in diameter) have a stronger immune response than smaller or larger NPs.

Therefore, when designing a nucleic acid vaccine vector, the above factors that affect the stability, solubility, cell uptake, endosome release, pDNA dissociation, nuclear targeting and immunostimulatory activity of the vector must be combined to achieve the best effect.

(source:internet, reference only)

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