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Ferritin: a new platform for vaccine design

Ferritin

  The structure of ferritin is conserved. Ferritin or recombinant ferritin of higher eukaryotes can be assembled by two subunits of light chain and heavy chain in different proportions. Usually, 24 subunits self-assemble into a hollow spherical nanostructure. . The spherical structure of human ferritin has an outer diameter of 12 nanometers and an inner diameter of 8 nanometers, and has 8 triplet symmetry axes and 6 quadruple symmetry axes on the surface. Divalent iron ions can enter the hollow cavity through surface channels, and are oxidized to ferric iron, which is finally stored inside ferritin in the form of ferrihydrite (5Fe2O3 9H2O). This biomineralization process mediated by ferritin can prevent excessive intracellular ferrous ions from causing damage to cells. After removing the iron inner core of ferritin, apoferritin can be obtained, and the hollow inner cavity can be used to encapsulate various types of small molecule drugs. The outside of ferritin can display different ligands through genetic engineering modification, chemical modification and other means to achieve further multi-functionalization of the ferritin platform.

  In recent years, with the development of technology, researchers have found that the display function of ferritin can make it a carrier platform for vaccines. The antigen is modified on the ferritin subunit by genetic engineering or chemical means, and then through the self-assembly of ferritin, multiple antigens can be displayed on the outer surface of ferritin. In addition, the triple axis of symmetry of ferritin allows multimeric antigens to be displayed in a trimer form, which better mimics the state of multimeric antigens on the virus surface under natural conditions than monomeric forms of antigens. At the same time, the light chain and heavy chain of ferritin can also carry different antigens, so that two antigens can be displayed on the surface at the same time. The ferritin subunits modified with different antigens are mixed and assembled into recombinant ferritin, which can realize the simultaneous display of different types of antigens on the surface of ferritin, and it is expected to directly regulate a single iron by changing the ratio of different ferritin subunits in the assembly system. Proportion of different antigens on protein assemblies. At present, in view of the unique advantages of ferritin, researchers have designed a variety of ferritin vaccines and are striving to achieve the goal of clinical application.
Genetically engineered ferritin vaccine

  Since the 21st century, with the rapid development of the biomedical field, there have been many breakthroughs in vaccine research, and various types of vaccines have been extended from the earliest attenuated or inactivated vaccines. According to the current use, human vaccines can be mainly divided into vaccines with or without live microorganisms [1]. Vaccines containing live microorganisms mainly refer to live attenuated vaccines and live microbial carrier vaccines, which have the characteristics of high transportation cost and low safety. Vaccines that do not contain live microorganisms can be divided into inactivated vaccines, nucleic acid vaccines, genetically engineered subunit vaccines, etc. These vaccines are more abundant and safer than live microorganism vaccines.
  Inactivated vaccines are the most widely used vaccines currently in clinical use. Such vaccines have a very mature preparation process and can be quickly produced and prepared. However, such vaccines also have disadvantages such as unstable virulence and easy to cause strong toxic side effects. In order to improve the protective efficacy of the vaccine and reduce adverse reactions, the researchers isolated the antigenic subunit of the virus to make a genetically engineered subunit vaccine. Epitope peptides are conserved peptides on the surface of viruses that can elicit an immune response to this type of virus. Compared with intact inactivated virus, epitope peptides do not have the risk of causing cell infection and therefore have a higher safety profile. However, the free antigenic peptide has poor stability and weak immune response, so it needs to be fixed on the carrier platform.
  The ferritin vaccine prepared by using ferritin as a carrier and immobilizing antigenic subunits on ferritin is a recombinant vaccine of genetic engineering subunits. Ferritin has good safety, stability and biocompatibility, and can reduce side effects. At the same time, the ferritin vaccine can stabilize antigenic subunits, display multivalent antigens, and improve the effect of immune response. A variety of peptides can be displayed on the surface of ferritin by means of genetic engineering modification, chemical cross-linking, and enzymatic linkage [2]. Among them, the method of genetic engineering modification has attracted much attention because of its simple and controllable characteristics.
  Ferritin itself has the characteristics of easy genetic engineering modification. The gene sequence of the antigenic subunit can be directly connected to the end of the ferritin gene sequence, and the recombinant plasmid can be transferred into expression systems such as bacteria or cells to achieve mass production of the target protein. Express. During the expression process, the ferritin subunits can self-assemble into spherical shapes, and the linked antigenic subunits will be expressed and assembled together with the ferritin subunits. After assembly, the antigenic subunits can be directly displayed on the outer surface of ferritin and bound to the target antibody. This method is easy to operate, and through rational design, the modified antigenic subunit will not affect the self-assembly and physiological function of ferritin. Compared with other methods of expressing and purifying and then modifying and linking, the method of genetic engineering modification can directly display the target peptide on the outer surface of ferritin. Therefore, genetic engineering technology has developed rapidly in recent years and has been widely used in the field of ferritin display.
Genetic engineering modified ferritin vaccine carrier platform

  In the display of ferritin vaccine antigens, the genetic engineering modification method has many unique advantages in addition to its simple operation. First, through the assembly of 24 subunits of ferritin vaccine, multivalent antigens can be displayed, the antigen presentation effect and affinity can be improved, and the half-life of antigens can be prolonged. Secondly, the symmetry axis structure of ferritin enables the assembly of the multimeric antigen at the symmetry axis, which can better restore the true morphological structure of the multimeric antigen. Thirdly, part of ferritin can be composed of two different subunits of light chain and heavy chain, and different antigenic peptides are connected to the two subunits respectively, so that two different types of antigens can be displayed on the surface of ferritin.

  Easy-to-use short peptide antigen
  display Ferritin display short peptide antigen is the simplest ferritin vaccine. Some short peptide antigens with sequences less than 200 amino acids can be directly linked to ferritin subunits by genetic engineering modification, and displayed on the outer surface of ferritin along with the self-assembly of ferritin to achieve multivalent display of antigens. Since the peptide segment formed by the short peptide antigen is smaller, the steric hindrance during the self-assembly process of ferritin is also smaller. In peptide design, a reasonable sequence can be found after relatively simple attempts. A research team has truncated the epitope of the hand-foot-mouth disease virus EV71 into short peptides of different lengths (less than 20 amino acids), and used genetic engineering modification methods to connect them to the N-terminus, C-terminus and loop of ferritin respectively. The effect of recombination position and truncation length on immune response was systematically studied [3]. The results show that the antigenic peptides linked to the N-terminal and loop regions can display 24 short peptide antigens on the outer surface of ferritin, while the peptides linked to the C-terminal are displayed in the lumen of ferritin. It is worth noting that the ferritin vaccine displayed on the outer surface of the antigen elicited a significantly stronger immune response than the internal display, and the ferritin vaccine with the antigen peptide linked to the loop region showed the best protective effect in mice.

  Trimeric long peptide antigen display
  Although the method of direct modification of short peptides on ferritin subunits can easily obtain a large number of recombinant ferritin vaccines. However, studies have shown that the shorter the peptide, the lower the antibody titer and the worse the immune response. This is due to the limited information that short peptide antigens can display, and cannot completely and truly restore the true state of the antigen on the surface of the virus. In addition, many antigens exist in multimeric structures on the surface of viruses. Therefore, a powerful vaccine must realize the simulation of the structure of antigenic multimers, which not only needs to meet the length requirements of antigenic peptides, but also meet the requirements of the spatial structure of antigenic peptides that can be polymerized. In this regard, the triple or quadruple symmetry axis of ferritin can satisfy the spatial display of antigenic peptides in the form of trimers or tetramers, respectively. Combined with the current situation that most antigens are trimers, the researchers have continuously improved the ferritin vaccine, and have successfully realized the display of long peptide antigens on the outer surface of ferritin in the form of trimers.
  In the design of ferritin vaccines with long peptide antigen display, it is very challenging to ensure the complete display and assembly of antigens without destroying the self-assembly of ferritin. Taking the long peptide antigen on the surface of the new coronavirus as an example, each antigenic peptide segment contains more than 1,000 amino acid sequences, and the complete surface antigen is assembled from three peptide segments to form a trimer structure. The steric hindrance of long peptide antigens is much greater than that of short peptides. If they are directly linked to the ferritin subunit by genetic engineering modification, the expression and assembly of ferritin will usually be affected, and a complete recombinant ferritin vaccine cannot be obtained.
  In order to prepare a potent ferritin recombinant vaccine carrying long peptide antigens, researchers have carried out a lot of exploration and improvement work on the connection or expression and purification of long peptide antigens and ferritin. In 2013, American scientists successfully developed a method for expressing and purifying ferritin in animal cells, effectively overcoming the effect of long peptides on ferritin assembly [4]. They directly linked the hemagglutinin spike of influenza virus to the end of ferritin by genetic engineering, and transferred the recombinant plasmid into mammalian cells for expression. In mammalian cells, the long peptide antigen-modified ferritin successfully completed the normal expression and self-assembly. Electron microscopy results showed that hemagglutinin spikes were displayed on the surface of ferritin in the form of trimers. The correct spatial display structure makes the antibody titer elicited by the ferritin vaccine more than 10 times higher than that of the common influenza virus inactivated vaccine.
  Ferritin linked to long peptides cannot be expressed in E. coli, but can be expressed and assembled normally in mammalian cells, which may benefit from the abundant endomembrane system in mammalian cells. Mammalian cells contain the endoplasmic reticulum and the Golgi apparatus, and these organelles carry heat shock proteins that assist peptides that cannot fold normally to complete the folding. The reconstituted ferritin probably assembles normally with the help of heat shock proteins. Theoretically, the ferritin vaccine expressed in mammalian cells has better immune response effect. Because the protein expressed in mammalian cells can undergo glycosylation modification of the inner membrane system, the obtained antigen is also closer to the real state after the virus infects the human body.
  Since then, vaccines that display long peptide antigens on ferritin have become the focus of attention, and researchers have developed vaccines against HIV, Epstein-Barr virus, respiratory syncytial virus and other viruses [5]. In 2021, scientists from Stanford University in the United States also used this method to express the ferritin linked to the new coronavirus spike in mammalian cells to prepare an efficient new coronavirus vaccine [5].

  Although the ferritin vaccine expressed and purified by mammalian cells in vitro has the advantage of strong immune response, it also has the disadvantages of higher cost and complicated operation because mammalian cells have higher culture requirements. In this regard, Chinese scientists bypassed the process of in vitro expression, encapsulated ferritin mRNA linked to the new coronavirus antigen sequence in liposomes, prepared an mRNA vaccine and injected it into mice, realizing the new coronavirus antigen display iron Direct expression of proteins in mammals [6]. More importantly, the ferritin vaccine expressed in vivo showed strong immune activation ability, which could effectively trigger the body’s immune system response.
  Two types of antigen display
  In prokaryotes, ferritin is usually composed of a single subunit, but in higher eukaryotes ferritin can be composed of two subunits, a light chain and a heavy chain. Different antigens are connected to the ends of the light chain and the heavy chain respectively, the light chain and the heavy chain can be assembled into a complete spherical shape with each other, and the light chain and the heavy chain can respectively form a three-fold symmetry axis structure, and display two trimers at the same time. antigen. Scientists at the American Vaccine Research Center used insect-derived ferritin to produce a vaccine that simultaneously displays two influenza hemagglutinin trimerization antigens [7]. This ferritin is composed of 12 light chains and 12 heavy chains, and H1N1 and B influenza antigens can be attached to the ends of the light and heavy chains, respectively. Through the self-assembly process of ferritin, the final complete ferritin has 12 two different antigens, and the two antigens can respectively form four trimeric antigen clusters at the triple axis of symmetry of ferritin. In addition, the simultaneous display of two HIV antigens and HIV antigens combined with influenza antigens was successfully achieved. This method utilizes specific ratios of heavy and light chains to assemble, providing a combinatorial display platform for multiple trimeric antigens.

The application prospect of ferritin vaccine

  The stable, safe and high biocompatibility of ferritin makes it a flexible and reliable vaccine carrier platform. In addition, the easy genetic modification of ferritin provides technical support for the design of ferritin vaccines. The trimeric spatial structure display of ferritin and the simultaneous display of dual antigens make ferritin stand out among many vaccine carriers. However, in the future clinical transformation of ferritin vaccines, in order to further optimize the application of ferritin vaccines, the following difficulties still need to be overcome:
  some ferritin derived from bacteria (such as Pyrococcus furiosus, Helicobacter pylori), their own May be immunogenic and pose a risk for in vivo applications. And ferritin extracted from bacteria usually carries endotoxin-like impurities with strong immunogenicity. Such impurities may not only cause a strong immune response in the body and cause damage to experimental animals, but also easily lead to false positives in immune detection. Therefore, purification and extraction of ferritin vaccines from bacteria need to ensure that there are steps to remove endotoxins.
  Although the display of long peptide antigens can greatly improve the efficacy of the immune response, the preparation of such ferritin vaccines is more complicated. Compared with the E. coli expression system, the expression conditions for obtaining ferritin vaccines based on mammalian cells are severe and the production cost is high. Subsequent studies also need to improve the preparation method of ferritin vaccines displaying long peptide antigens to reduce production costs.

  Correctly understand and use ferritin targeting. Human ferritin can recognize and bind to transferrin receptor 1 (TfR1), and some literatures believe that TfR1 is highly expressed on the surface of dendritic cells, so researchers try to use this feature to target dendritic cells for presentation antigens to enhance the stimulation of the immune system by vaccines [8]. But in human and mouse cells, ferritin may bind to different receptors. Without a clear understanding of this, it is easy to make mistakes in experiments. For example, researchers have tried to design ferritin vaccines that target dendritic cells to present tumor-specific antigens [9]. However, the receptor for ferritin in mice is not TfR1. Meanwhile, there is currently no evidence that ferritin can bind TfR1 in mice [10, 11]. Therefore, before designing and conducting experiments with ferritin vaccines, special attention should be paid to the differences in their targeting in mice and humans.
  Strengthen innovation in vaccine design. At present, the design of ferritin vaccines mostly stays at the stage of antigen display, and the improvement effect of antigen display is also minimal. Future research should create more new possibilities and broaden the development of ferritin vaccines. For example, it is possible to make full use of the encapsulation function of the internal cavity of ferritin, to enhance the immune effect by loading adjuvants, and to use computer 3D simulation methods to design and evaluate the rationality of ferritin vaccine sequences in high-throughput, before the actual expression. Preliminary screening of feasible designs. This can effectively reduce experimental operations, further reduce production costs, and improve research efficiency [12].
  All in all, ferritin is a potential vaccine carrier platform. While researchers are working hard to explore the advantages of ferritin, they also need to focus on solving the problems faced in the application of ferritin vaccines. With the in-depth research, it is believed that more unique properties of ferritin will be discovered, which will bring new impetus to the development of ferritin vaccines. It is hoped that the ferritin vaccine can really go to the clinic in the near future and realize the protection of human beings.

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