Nano coronavirus recombinant vaccine taking graphene oxide as carrier

Patent no: CN112220919A


The invention belongs to the field of nano materials and biomedicine, and relates to a vaccine, in particular to development of 2019-nCoV coronavirus nuclear recombinant nano vaccine. The invention also comprises a preparation method of the vaccine and application of the vaccine in animal experiments. The new corona vaccine contains graphene oxide, carnosine, CpG and new corona virus RBD; binding carnosine, CpG and neocoronavirus RBD on the backbone of graphene oxide; the CpG coding sequence is shown as SEQ ID NO 1; the novel coronavirus RBD refers to a novel coronavirus protein receptor binding region which can generate a high-titer specific antibody aiming at the RBD in a mouse body, and provides a strong support for prevention and treatment of the novel coronavirus.

The invention belongs to the field of nano materials and biomedicine, and relates to development of a vaccine development platform. In particular to the development of 2019-nCoV coronavirus nuclear recombinant nano-vaccine. The invention also includes the use of the vaccine in animal testing.

The vaccine is an ultimate weapon for eliminating major infectious diseases, has the advantages of lowest cost and more advantages of prior enemy than other therapies, undoubtedly becomes hopeful to the public, the smallpox is eliminated by human beings through vaccination, the poliomyelitis cases are reduced by 99 percent, the infectious diseases such as diphtheria are rare, and the incidence rate of diseases such as measles, neonatal tetanus and the like is remarkably reduced. The effect of vaccines on human health is not excessive, and the birth of each new vaccine is a great victory for human beings to overcome an infectious disease! To date, no medical treatment has been able to have such an important, lasting and profound effect on human health as a vaccine; nor is any therapeutic available to eliminate a disease from the earth at the very least cost of a vaccine.After the occurrence of SARS-CoV-2 epidemic, different laboratories in China have completed the isolation of virus strains, and in order to make a big step forward in vaccine development, we believe that we will soon have a final weapon for the eradication of SARS-CoV-2, however, until now there is no approved vaccine or drug for the treatment of CoV infection, and there is a great need to develop an effective drug for the treatment or prevention of coronavirus infection and outbreak.According to the research of coronavirus vaccines such as SARS and MERS, the main target point of the existing coronavirus vaccine is the S protein of coronavirus. Vaccines need to induce not only humoral and cellular immune responses, but also mucosal immune responses, and with the aid of adjuvants to induce balanced Th1 and Th2 pathways to produce truly effective vaccines. At present, the research of more SARS and MERS vaccines mainly focuses on viral vector vaccines and subunit vaccines, and a large number of researches show that the difficulty of SARS and MERS lies in that long-term memory B cells cannot be stimulated to generate, the long-term memory cells in the healed SARS and MERS patients can only last for 2-3 years, immunological memory cannot be generated, and the vaccine development failure is caused, and only 6 potential coronavirus vaccines enter the clinical research stage at present, but no 1 effective vaccine is approved to be marketed.Disclosure of InventionThe invention aims to provide a coronavirus recombinant vaccine.Another purpose of the invention is to provide a preparation method of the virus recombinant vaccine.It is still another object of the present invention to provide the use of the recombinant vaccine of the virus.In view of various problems of the conventional vaccines at present, how to change the problems of the existing vaccines and enhance immune response is a problem which is always considered, in order to improve the immunocompetence of the immunogen and enhance the immune response capability of the body, the most basic method is to mix the immunogen with an adjuvant, and the immune adjuvant is a promoter capable of enhancing the immune response of the body to the immunogen. CpG Oligodeoxynucleotide (ODN) is a very promising adjuvant discovered in recent years. CpG ODN has been shown to have better adjuvant activity in vivo, in vitro and clinical studies in animals, and the best studies are CpG7909 and CpG 1018. 11/9.2017, the hepatitis B vaccine approved by Dynavax Technologies of the United states of America FDA and using CpG1018 as an adjuvant is on the market, is the first approved CpG ODN adjuvant vaccine in the world and is used for preventing HBV infection of adults 18 years old and older, and a plurality of different types of CpG ODN are used as adjuvants in a plurality of clinical trials. CpG is combined with TLR9 to activate immature pDC cells and induce natural and adaptive immune response, but a single CpG structure has limited activation effect on immune cells and is easy to be rapidly hydrolyzed by exonuclease, so that the stability of the CpG in vivo is insufficient, and side effects are also caused; CpG Oligodeoxynucleotide (ODN) synthesized in the sequence can also enhance the stimulation effect, and after the CpG is coupled with other proteins such as antigen and the like, the CpG oligodeoxynucleotide is combined, so that the CpG oligodeoxynucleotide has a very obvious immune activation effect.Graphene is a two-dimensional carbon nanomaterial consisting of carbon atoms in sp hybridized orbitals in a hexagonal honeycomb lattice. The basic structural unit of the material is the most stable benzene six-membered ring in the organic material, and the material is the most ideal two-dimensional material at present. Graphene Oxide (GO) is a Graphene oxide derivative, and is a exfoliated product. Due to the characteristics of unique SP2 hybridization, a perfect two-dimensional structure and high reactivity of the edge, the treatment platform based on the hybrid structure can be used as an ideal load and grafting carrier in design and development, and plays an important role in aspects of nano-drug delivery systems, biological detection, tumor treatment, cell imaging and the like.The present invention has been completed based on the above-mentioned studies.The invention discloses a brand-new vaccine development method based on a graphene oxide material serving as a framework for loading CpG molecules and recombinant proteins. Based on the technical platform, a new nano new crown vaccine is prepared by combining the recombinant protein of the RBD region of the Spike protein of the SAR-CoV-2. The prepared nano new corona vaccine has stronger immunogenicity in mouse experiments and can generate high-titer antibodies.In one aspect, the invention provides a coronavirus vaccine comprising graphene oxide, carnosine, CpG, and RBD. In a preferred embodiment of the invention, the vaccine is named GO-Car-carnosine-CpG-RBD vaccine.Graphene Oxide (GO) is an oxide of graphene, and after oxidation, oxygen-containing functional groups on the graphene oxide are increased, so that the graphene oxide is more active than graphene. For example, hydroxyl groups and epoxy groups are randomly distributed on a graphene oxide monolith, while carboxyl groups and carbonyl groups are introduced at the edge of the monolith. Common commercial products of graphene oxide are in the form of powder, flakes and solutions, and are brown-yellow in color.Carnosine, known by the scientific name β -alanyl-L-histidine, is a crystalline solid composed of a dipeptide consisting of two amino acids, β -alanine and L-histidine. Carnosine has strong antioxidant ability, and can scavenge Reactive Oxygen Species (ROS) and alpha-beta unsaturated aldehydes, which are formed by over-oxidation of fatty acids in cell membranes during oxidative stress.CpG motifs have the effect of activating the body’s immune system and can be used as adjuvants. Preferably, the CpG coding sequence is shown as SEQ ID NO 1.RBD (spike receptor binding domain), specifically a coronavirus protein (S protein) Receptor Binding Domain (RBD) in the present invention. For example, the RBD protein can be selected as follows:

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Methods for producing recombinant coronavirus

A helper cell for producing an infectious, replication defective, coronavirus (or more generally nidovirus) particle cell comprises (a) a nidovirus permissive cell; (b) a nidovirus replicon RNA comprising the nidovirus packaging signal and a heterologous RNA sequence, wherein the replicon RNA further lacks a sequence encoding at least one nidovirus structural protein; and (c) at least one separate helper RNA encoding the at least one structural protein absent from the replicon RNA, the helper RNA(s) lacking the nidovirus packaging signal. The combined expression of the replicon RNA and the helper RNA in the nidovirus permissive cell produces an assembled nidovirus particle which comprises the heterologous RNA sequence, is able to infect a cell, and is unable to complete viral replication in the absence of the helper RNA due to the absence of the structural protein coding sequence in the packaged replicon. Compositions for use in making such helper cells, along with viral particles produced from such cells, compositions of such viral particles, and methods of making and using such viral particles, are also disclosed.


This application claims priority under 35 U.S.C. § 371 from PCT Application No. PCT/US02/12453, filed in English on Apr. 19, 2002, which claims the benefit of U.S. Application Ser. No. 60/285,320 and U.S. Application Ser. No. 60/285,318, both filed on Apr. 20, 2001, the disclosures and contents of which are incorporated by reference herein in their entireties.STATEMENT OF FEDERAL SUPPORT

This invention was made possible with government support under grant numbers AI23946 and GM63228 from the National Institutes of Health. The United States government has certain rights to this invention.FIELD OF THE INVENTION

The present invention relates to methods of producing recombinant nidovirus vectors, particularly coronavirus vectors, and expressing heterologous genes from said vectors.BACKGROUND OF THE INVENTION

Transmissible gastroenteritis (TGE) is an economically important, acute enteric disease of swine, which is often 100% fatal in newborn piglets (Enjuanes, et al. (1995) Dev. Biol. Stand. 84:145-152; Enjuanes, et al. (1995) Adv. Exp. Med. Biol. 380:197-211; Laude, et al. (1990) Vet. Microbiol. 23:147-154). TGE virus (TGEV), the causative agent of TGE, is a member of the Coronaviridae family and the order Nidovirales. In addition to the Coronaviridae, the order Nidovirales also includes the Arteriviridae family, of which the swine pathogen porcine reproductive and respiratory syndrome virus (PRRSV) is a member (Cavanagh and Horzinek (1993) Arch. Virol. 128:395-396; de Vries, et al. (1997) Semin. Virol. 8:33-47; Siddell, et al. (1983) J. Gen. Virol. 64:761-776). Despite significant size differences (˜13 to 32 kb), the polycistronic genome organization and regulation of gene expression from a nested set of subgenomic mRNAs are similar for all members of the order (de Vries, et al. (1997) Semin. Virol. 8:33-47; Snijder and Horzinek (1993) J. Gen. Virol. 74:2305-2316).

TGEV possesses a single-stranded, positive-sense ˜28.5-kb RNA genome enclosed in a helical nucleocapsid structure that is surrounded by an envelope containing three viral proteins, including the S glycoprotein, the membrane (M) glycoprotein and a small envelope (E) protein (Eleouet, et al. (1995) Virology 206:817-822; Enjuanes and van der Zeijst (1995) In: S. G. Siddell (ed.), The Coronaviridae. Plenum Press, New York, N.Y., p. 337-376; Rasschaert and Laude (1987) J. Gen. Virol. 68:1883-1890; Risco, et al. (1996) J. Virol. 70:4773-4777). Remarkably, only the E and M proteins are absolutely required for particle formation, defining a novel model for virion budding (Fischer, et al. (1998) J. Virol. 72:7885-7894; Vennema, et al. (1996) EMBO J. 15:2020-2028). The TGEV genome contains eight large open reading frames (ORFs), which are expressed from full-length or subgenomic-length mRNAs during infection (Eleouet, et al. (1995) Virology 206:817-822; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). The 5′-most ˜20 kb contains the replicase genes in two ORFs, 1A and 1B, the latter of which is expressed by ribosomal frameshifting (Almazan, et al. (2000) Proc. Natl. Acad. Sci. USA 97:5516-5521; Eleouet, et al. (1995) Virology 206:817-822). The 3′-most ˜9 kb of the TGEV genome contains the structural genes, each preceded by a highly conserved transcription regulatory element (TSE) [ACTAAAC; SEQ ID NO:1]. The size of the functional TSE is subject to debate, but ranges from ˜7-15+ nucleotides in length when analyzed in recombinant defective interfering RNAs (Enjuanes, et al. (2001) J. Biotechnology 88:183-204; Jeong, et al. (1996) Virology 217:311-322; Krishnan, et al. (1996) Virology 218:400-405; Joo and Makino (1995) J. Virol. 69:3339-3346). In general, TSE length affects the function of individual mutations because longer elements are generally more resistant to “debilitating” mutations (Enjuanes, et al. (2001) J. Biotechnology 88:183-204). As the leader RNA sequence is also defined by a TSE at its 3′ end, some degree of base-pairing between the leader RNA and body TSE likely mediate virus transcription of subgenomic RNAs (Baker and Lai (1990) EMBO J. 9:4173-4179; Baric, et al. (1983) J. Virol. 48:633-640; Makino, et al. (1986) Proc. Natl. Acad. Sci. USA 83:4204-4208; Makino, et al. (1991) J. Virol. 65:6031-6041; Siddell, S. G. 1995. The coronaviridae, An introduction. In: The coronaviridae, eds. S. G. Siddell, Plenum Press, New York. pp 1-10). The subgenomic mRNAs are arranged in a co-terminal nested set structure from the 3′ end of the genome, and each contains a leader RNA sequence derived from the 5′ end of the genome. Although each mRNA is polycistronic, the 5′-most ORF is preferentially translated, necessitating the synthesis of a distinct mRNA species for each ORF (Lai and Cavanagh (1997) Adv. Virus Res. 48:1-100; McGoldrick, et al. (1999) Arch. Virol. 144:763-770; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). Both full-length and subgenomic-length negative-strand RNAs are also produced and have been implicated in mRNA synthesis (Baric and Yount (2000) J. Virol. 74:4039-4046; Sawicki and Sawicki (1990) J. Virol. 64:1050-1056; Schaad and Baric (1994) J. Virol. 68:8169-8179; Sethna, et al. (1991) J. Virol. 65:320-325; Sethna, et al. (1989) Proc. Natl. Acad. Sci. USA 86:5626-5630). Subgenomic RNA synthesis occurs by a method of discontinuous transcription, most likely by transcription attenuation during negative-strand synthesis (Baric and Yount (2000) J. Virol. 74:4039-4046; Sawicki and Sawicki (1990) J. Virol. 64:1050-1056).

The coronavirus E and M proteins function in virion assembly and release, which involve the constitutive secretory pathway of infected cells. Coexpression of the E and M proteins results in virus-like particle formation in cells, defining a novel, nucleocapsid-independent mechanism of enveloped-virus assembly (Vennema, et al. (1996) EMBO J. 15:2020-2028). The role of the E protein in virus assembly was further confirmed by reverse genetic analysis using targeted recombination (Fischer, et al. (1998) J. Virol. 72:7885-7894) and the development of TGEV replicon viruses (Curtis, et al. (2002) J. Virol. 76(3):1422-34). The TGEV M protein may serve to initiate the viral particle assembly process through interactions with genomic RNA and nucleoprotein in pre-Golgi compartments (Narayanan, et al. (2000) J. Virol. 74:8127-8134). The precise role of E in the assembly and release of coronavirus particles is not clear. Although an interaction between the E and M proteins has not yet been demonstrated, such an interaction likely occurs and would serve to facilitate the budding of viral particles. Additionally, E protein has been suggested to “pinch off the neck” of the assembled viral particles during the final stages of budding (Vennema, et al. (1996) EMBO J. 15:2020-2028).

PRRSV is endemic in most swine producing countries. Virions are enveloped 45-70 nm particles that contain 5 envelope proteins and an icosahedral nucleocapsid (N), which surrounds a single-stranded positive polarity RNA genome of about 15 kb (Pancholi, et al. (2000) J. Infect. Dis. 182:18-27; Pirzadeh and Dea (1998) J. Gen. Virol. 79:989-99). The 15 kDa N protein is most abundant and contains common conformational antigenic sites that are conserved of European and North American strains (Pirzadeh and Dea (1998) J. Gen. Virol. 79:989-99). N is likely multimerized to form icosahedral core structures (20-30 nm), which can be observed by EM. The major envelope proteins include a 25 kDa glycoprotein (GP5) and an 18-19 kDa unglycosylated M protein (Eleouet, et al. (1995) Virology 206:817-22; Meulenberg, et al. (1997) Vet. Microbiol. 55:197-202). GP5 (ORF 5) heterogeneity ranges from 50-90% amino acid identity among isolates, contains at least two neutralizing sites, and expression causes apoptosis (Eleouet, et al. (1995) Virology 206:817-22; Pirzadeh, et al. (1998) Can. J. Vet. Res. 62:170-7; Saif (1999) Transmissible gastroenteritis and porcine respiratory coronavirus, p. 295-325. In B. Straw, D’Allaire, S, Mengeling, W L and Taylor, D J (ed.), Diseases of Swine 8th edition. Iowa State University Press, Ames, Iowa; Tresnan, et al. (1996) J. Virol. 70:8669-74). The M protein (ORF6) contains 3 hydrophobic domains and accumulates in the ER of infected cells, where it forms disulfide-linked heterodimers with GP5 and may function in virus assembly (Meng (2000) Vet. Microbiol. 74:309-29). As with equine arterivirus, it is likely that coexpression of M and GP5 are needed for appropriate post-translational modification, folding and function, and for inducing high neutralizing antibody titers (Balasuriya, et al. (2000) J. Virol. 74:10623-30; Eleouet, et al. (1995) Virology 206:817-220).

Live, attenuated PRRSV vaccines causes viremia and may spread to other pigs. DNA immunization with a plasmid encoding GP5 of PRRSV induces specific neutralizing antibodies and reduces viremia and lung pathology in swine following challenge (Risco, et al. (1996) J. Virol. 70:4773-7). Recombinant adenovirus and vaccinia viruses encoding various PRRSV antigens are also being developed with encouraging results (Budzilowicz, et al. (1985) J. Virol. 53:834-40; Tresnan, et al. (1996) J. Virol. 70:8669-74). Several groups have concluded that effective PRRSV recombinant vaccines must induce high neutralizing titers, induce cellular immunity, induce heterotypic immunity and provide protection at mucosal surfaces (Eleouet, et al. (1995) Virology 206:817-22; Meulenberg, et al. (1997) Vet. Microbiol. 55:197-202). Achieving these goals is complicated by the generally low immunogenicity of the PRRSV envelope proteins and high genomic heterogeneity present in field isolates (Meulenberg, et al. (1997) Vet. Microbiol. 55:197-2020). Hence, improved vaccines are needed.

Recently, a simple and rapid approach for systematically assembling a full-length cDNA copy of the TGEV genomic RNA from which infectious transcripts can be produced has been described (Yount, et al. (2000) J. Virol. 74:10600-10611). This approach, as well as that of Almazan et al. ((2000) Proc. Natl. Acad. Sci. USA 97:5516-5521), will facilitate reverse genetic methods that impact all aspects of coronavirology, however, the production of infectious TGEV replicon particles is still limited.

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Recent progress of graphene oxide as a potential vaccine carrier and adjuvant


Vaccine is one of the most effective strategies for preventing and controlling infectious diseases and some noninfectious diseases, especially cancers. Adjuvants and carriers have been appropriately added to the vaccine formulation to improve the immunogenicity of the antigen and induce long-lasting immunity. However, there is an urgent need to develop new all-purpose adjuvants because some adjuvants approved for human use have limited functionality. Graphene oxide (GO), widely employed for the delivery of biomolecules, excels in loading and delivering antigen and shows the potentiality of activating the immune system. However, GO aggregates in biological liquid and induces cell death, and it also exhibits poor biosolubility and biocompatibility. To address these limitations, various surface modification protocols have been employed to integrate aqueous compatible substances with GO to effectively improve its biocompatibility. More importantly, these modifications render functionalized-GO with superior properties as both carriers and adjuvants. Herein, the recent progress of physicochemical properties and surface modification strategies of GO for its application as both carriers and adjuvants is reviewed.

Statement of Significance

Due to its unique physicochemical properties, graphene oxide is widely employed in medicine for purposes of photothermal treatment of cancer, drug delivery, antibacterial therapy, and medical imaging. Our work describes the surface modification of graphene oxide and for the first time summarizes that functionalized graphene oxide serves as a vaccine carrier and shows significant adjuvant activity in activating cellular and humoral immunity. In the future, it is expected to be introduced into vaccine research to improve the efficacy of vaccines.

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Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms


Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.


Graphene, which is isolated from crystalline graphite, is a flat monolayer composed of single-atom-thick, two-dimensional sheets of a hexagonally arranged honeycomb lattice [1]. Because of its unique structural, specific surface area and mechanical characteristics, the functions and applications of graphene have gained considerable attention since the discovery of the material in 2004 [23]. Graphene and its derivatives include monolayer graphene, few-layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO), graphene nanosheets (GNS), and graphene nanoribbons, etc. [47]. GO is one of the most vital chemical graphene derivatives of the graphene-family nanomaterials (GFNs), which attracts increasing attention for its potential biomedical applications. Graphene-based materials usually have sizes ranging from several to hundreds of nanometer and are 1-10 nm thick [89], which is also the definition of ‘nanoparticles’ or ‘nanomaterials’. Due to their exceptional physical and chemical properties, graphene materials have been widely used in various fields, including energy storage; nanoelectronic devices; batteries [1012]; and biomedical applications, such as antibacterials [1314], biosensors [1518], cell imaging [1920], drug delivery [82122], and tissue engineering [2325].

Along with the application and production of GFNs increasing, the risk of unintentional occupational or environmental exposure to GFNs is increasing [26]. And recently, there are some investigation on GFNs exposure in occupational settings and published data showed that the occupational exposure of GFNs had potential toxicity to the workers and researchers [2729]. GFNs can be delivered into bodies by intratracheal instillation [30], oral administration [31], intravenous injection [32], intraperitoneal injection [33] and subcutaneous injection [34]. GFNs can induce acute and chronic injuries in tissues by penetrating through the blood-air barrier, blood-testis barrier, blood-brain barrier, and blood-placenta barrier etc. and accumulating in the lung, liver, and spleen etc. For example, some graphene nanomaterials aerosols can be inhaled and substantial deposition in the respiratory tract, and they can easily penetrate through the tracheobronchial airways and then transit down to the lower lung airways, resulting in the subsequent formation of granulomas, lung fibrosis and adverse health effects to exposed persons [229]. Several reviews have outlined the unique properties [3536] and summarized the latest potential biological applications of GFNs for drug delivery, gene delivery, biosensors, tissue engineering, and neurosurgery [3739]; assessed the biocompatibility of GFNs in cells (bacterial, mammalian and plant) [74041] and animals (mice and zebrafish) [42]; collected information on the influence of GFNs in the soil and water environments [43]. Although these reviews discussed the related safety profiles and nanotoxicology of GFNs, the specific conclusions and detailed mechanisms of toxicity were insufficient, and the mechanisms of toxicity were not summarized completely. The toxicological mechanisms of GFNs demonstrated in recent studies mainly contain inflammatory response, DNA damage, apoptosis, autophagy and necrosis etc., and those mechanisms can be collected to further explore the complex signalling pathways network regulating the toxicity of GFNs. It needs to point out that there are several factors which largely influence the toxicity of GFNs, such as the concentration, lateral dimension, surface structure and functionalization etc. Herein, this review presents a comprehensive summary of the available information on the mechanisms and regulating factors of GFNs toxicity in vitro and in vivo via different experimental methods, with the goals of providing suggestions for further studies of GFNs and completing the toxicology mechanisms to improve the biological safety of GFNs and facilitate their wide application.

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