Attenuated flavivirus virus and uses thereof

Abstract

The present invention provides an attenuated virus of the genus Flavivirus containing a polyadenylic acid (poly(A)) sequence and use thereof, in which a part of the nucleotide sequence of the 3' untranslated region (3'UTR) of the Flavivirus virus is replaced with the polyadenylic acid (poly(A)) sequence, and in the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus obtained by replacing the part of the nucleotide sequence of the Flavivirus virus, at least the 3' terminal stem loop region (3'SL) is retained, and use thereof. The attenuated virus is used for preparing a safe and effective attenuated vaccine strain.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, and in particular to an attenuated virus of the Flavivirus genus and its use. [Background technology]

[0002] The genus Flavivirus belongs to the family Flaviviridae and includes more than 70 species of viruses, many of which are spread by mosquitoes and ticks, among which Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), dengue virus (DENV), and zika virus (ZIKV) are important human pathogens causing high morbidity and mortality worldwide. Severe flavivirus infections can result in death, including hemorrhagic fever (YFV and DENV), encephalitis and neurological sequelae (JEV, TBEV, WNV, SLEV and MVEV); mortality rates for JEV, YFV, TBEV and DENV can range from 5% to 30%.

[0003] According to data from the World Health Organization, DENV is currently endemic in more than 100 countries, causing 20,000 deaths per year, JEV is endemic in Southeast Asia and the Western Pacific, with 68,000 cases reported per year, WNV broke out in New York in 1999, causing 9,862 infections and 264 deaths (CDC statistics), and spread rapidly from North America to Central America and finally South America, with the epidemic areas still expanding, ZIKV had a small outbreak in Yap Islands, Micronesia, and Gabon in 2007, and then an unexpected large-scale explosion in Latin America in 2016, and this ZIKV infection was associated with severe neurological disease (Guillain-Barré syndrome) and neonatal head malformations, and was declared a "public health emergency of international concern." In 2016, approximately 4 million cases of ZIKV infection were recorded in the United States alone. ZIKV then spread rapidly to regions such as French Polynesia, South America, Africa, and Asia. Flavivirus infections have therefore become one of the major public health problems facing the world.

[0004] At present, there are no effective antiviral drugs or specific treatments for flavivirus infection, and there are only three types of flavivirus vaccines for human prevention, so the development of a new, more effective flavivirus vaccine is of important strategic significance for preventing flavivirus infection in China and around the world. Among the many types of vaccines, attenuated live vaccines have attracted attention in vaccine development because they have strong immunogenicity, can induce humoral and cellular immune responses with a single immunization, and can induce more complete and long-term immune protection.

[0005] The flavivirus genome is a single-stranded positive-stranded RNA of approximately 11,000 bases in length, consisting of a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and a long open reading frame in the center. The 5' end of the genome contains a 7-methylguanosine cap (mCd) similar to that of mRNA. 7Although the genome has a polyadenylic acid tail (poly(A)), the 3' end of the genome does not have a polyadenylic acid tail (poly(A)) but a single conservative 5'-CU OH -3' sequence. The untranslated regions (UTRs) of flavivirus genomes contain conserved RNA sequences that form highly complex secondary and tertiary structures and play multiple roles in viral replication, translation, transmission, and pathogenesis (Viruses. 2014 Jan 27;6(2):404-27).

[0006] The 5'UTR is about 100 bases in length and contains i) a conserved stem-loop structure (SL) that allows SL-A to bind to NS5-RdRp (Genes Dev. 2006 Aug 15; 20(16): 2238-49) and initiate the synthesis of negative-strand RNA, and ii) a cyclization sequence located upstream of the AUG start codon (5'upstream of AUG region (5'UAR), 5'cyclization sequences (5'CS) and 5'downstream of AUG region (5'DAR)) that mediates long-range RNA-RNA interactions (Virus Res. 2009 Feb; 139(2): 230-9). The 3'UTR is relatively long, about 400 to 700 bases, forms multiple conserved RNA secondary structures, and plays an important role in viral replication, pathogenicity, and propagation. Structurally, the 3'UTR is divided into i) stem-loop region (SL), ii) dumbbell region (DB), and iii) short hairpin 3' stem-loop region (sHP-3'SL). Among them, sHP-3'SL is the most conserved RNA secondary structure among flaviviruses, and the complementary circularization sequences 3'CS, 3'UAR, and 3'DAR contained therein complementarily pair with the corresponding 5'CS, 5'UAR, and 5'DAR to cause genome circularization, which is an essential cis action for initiating replication in RNA replication. The stem-loop region (SL) and dumbbell region (DB) are not well conserved in the Flavivirus genus as enhancers during the genome replication process, and their size and structure vary depending on the virus. Although the stem-loop region (SL) and dumbbell region (DB) play non-essential roles in genome replication, multiple studies based on the attenuation or enhancement of virulence by different degrees of deletion or point mutation in the SL and DB regions have shown that the stem-loop region (SL) and dumbbell region (DB) play important roles in aspects such as regulating viral virulence, host immune response and host fitness, making the stem-loop region (SL) and dumbbell region (DB) two important targets for designing attenuated vaccines.

[0007] Considering these factors together, there is a need for alternative attenuated flavivirus vaccine candidates with improved efficacy and safety profiles. Summary of the Invention

[0008] The present disclosure is based on an infectious clone of a Flavivirus virus, and replaces all or part of the nucleotide sequence of the 5'-end stem-loop region (SL) of the 3' untranslated region (3' UTR), all or part of the nucleotide sequence of the circularization sequence region (CS), and / or all or part of the nucleotide sequence of the dumbbell region (DB) with a poly(A) sequence to construct an attenuated poly(A) virus strain of a Flavivirus virus, and extensive in vivo studies have fully demonstrated that the attenuated poly(A) virus strain can be used as an attenuated vaccine candidate strain of a wild-type virus. Such a virus provides the basis for a safe vaccine.

[0009] An object of the present disclosure is to provide an attenuated Flavivirus virus having attenuated characteristics and still having good immunogenicity, the attenuated Flavivirus virus comprising a polyadenylic acid (poly(A)) sequence, wherein the polyadenylic acid (poly(A)) sequence replaces a part of the nucleotide sequence of the 3' untranslated region (3' UTR) of the Flavivirus virus, and after the part of the nucleotide sequence of the Flavivirus virus is replaced, at least the 3' stem-loop region (3' SL) is retained in the 3' untranslated region (3' UTR) of the attenuated Flavivirus virus obtained.

[0010] Another object of the present disclosure is to provide a DNA that can be transcribed to produce the attenuated Flavivirus RNA genome.

[0011] Another object of the present disclosure is to provide a cell comprising the attenuated Flavivirus virus or DNA.

[0012] Another object of the present disclosure is to provide a vaccine comprising the attenuated Flavivirus virus, DNA and / or cells.

[0013] Another object of the present disclosure is to provide a pharmaceutical composition comprising the attenuated Flavivirus, DNA, cells and / or vaccine, and a pharma- ceutically acceptable carrier.

[0014] Another object of the present disclosure is to provide a method for preparing the attenuated Flavivirus virus, which comprises replacing a wild-type virus 3' untranslated region (3' UTR) nucleotide sequence or a part of the nucleotide sequence with a polyadenylic acid (poly(A)) sequence.

[0015] Another object of the present disclosure is to provide a use of said attenuated Flavivirus virus, DNA and / or cells in the preparation of a live-attenuated Flavivirus virus vaccine.

[0016] Another object of the present disclosure is to provide a use of the attenuated Flavivirus virus, DNA, cells, vaccine and / or pharmaceutical composition in the preparation of a drug for treating or preventing a disease caused by a Flavivirus virus.

[0017] Another object of the present disclosure is to provide a method for stimulating an immune response against a Flavivirus virus, comprising administering the attenuated Flavivirus virus, DNA, cells, vaccine and / or drug composition by immunization. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 shows a schematic diagram of the construction of clones of attenuated flavivirus strains in which the 3'UTR is replaced with a poly(A) sequence and a diagram showing viral rescue, in which A shows IFA verification of WNV-poly(A) clone construction and viral rescue, B shows IFA verification of JEV-poly(A) clone construction and viral rescue, C shows IFA verification of DENV-2(NGC)-poly(A) clone construction and viral rescue, and D shows IFA verification of YFV-poly(A) clone construction and viral rescue. [Diagram 2] FIG. 1 shows a schematic diagram of clone construction and virus rescue of another attenuated strain of West Nile virus. [Diagram 3] FIG. 1 shows a comparison of the growth curves of attenuated flavivirus strains in which the 3'UTR is replaced with a poly(A) sequence and the wild-type virus, in which A shows a comparison of the growth curves of the WNV-poly(A) attenuated strain and the corresponding wild-type virus strain, B shows a comparison of the growth curves of the JEV-poly(A) virulent strain and the corresponding wild-type virus strain, C shows a comparison of the growth curves of the DENV-2(NGC)-poly(A) virulent strain and the corresponding wild-type virus strain, and D shows a comparison of the growth curves of the YFV-poly(A) virulent strain and the corresponding wild-type virus strain. [Figure 4] FIG. 1 shows genetic stability verification of attenuated flavivirus strains in which the 3'UTR is replaced with a poly(A) sequence, where A shows genetic stability verification of a WNV-poly(A) attenuated strain, B shows genetic stability verification of a JEV-poly(A) attenuated strain, C shows genetic stability verification of a DENV-2(NGC)-poly(A) attenuated strain, and D shows genetic stability verification of a YFV-poly(A) attenuated strain. [Diagram 5] FIG. 1 shows the detection of pathogenicity of WNV-poly(A) in C57BL / 6 mice, where A shows the survival rate of mice immunized with an attenuated WNV-poly(A) strain, B shows the virus titer of mice immunized with an attenuated WNV-poly(A) strain, and C shows the body weight of mice immunized with an attenuated WNV-poly(A) strain. [Figure 6]FIG. 1 shows an immune protection test of WNV-poly(A) in a C57BL / 6 mouse model, in which A shows the IgG antibody titers produced in mice immunized once with different doses of an attenuated WNV-poly(A) strain, B shows the neutralizing antibody titers produced in mice immunized once with different doses of an attenuated WNV-poly(A) strain, C shows the survival status after challenge of mice immunized once with different doses of an attenuated WNV-poly(A) strain, D shows the body weight status after challenge of mice immunized once with different doses of an attenuated WNV-poly(A) strain, and E shows the virus titers after challenge of mice immunized once with different doses of an attenuated WNV-poly(A) strain. [Figure 7] FIG. 1 shows cellular immune evaluation in a C57BL / 6 mouse model of attenuated WNV-poly(A) strains, in which A shows ELISpot detection of 7d CD8+IFN-γ+ T cells from mice immunized once with the attenuated WNV-poly(A) strain, and B shows the percentage of 7d CD8+IFN-γ+ T cells from mice immunized once with the attenuated WNV-poly(A) strain. [Figure 8] This is a long-term immune protection study of the attenuated WNV-poly(A) strain in a C57BL / 6 mouse model. A shows the long-term maintenance of IgG antibody titers in mice immunized once with the attenuated WNV-poly(A) strain, B shows the long-term maintenance of neutralizing antibody production in mice immunized once with the attenuated WNV-poly(A) strain, C shows the long-term survival of mice immunized once with the attenuated WNV-poly(A) strain after challenge, and D shows the long-term virus titers in mice immunized once with the attenuated WNV-poly(A) strain after challenge. [Figure 9] This figure shows the detection of pathogenicity of DENV-poly(A) in AG129, where A shows the survival rate of mice immunized with the attenuated DENV-poly(A) strain, B shows the virus titer of mice immunized with the attenuated DENV-poly(A) strain, and C shows the body weight of mice immunized with the attenuated DENV-poly(A) strain. [Figure 10]FIG. 1 shows an immune protection test of DENV-poly(A) in the AG129 mouse model, in which A shows the IgG antibody titers produced in mice immunized once with different doses of the attenuated DENV-poly(A) strain, B shows the neutralizing antibody titers produced in mice immunized once with different doses of the attenuated DENV-poly(A) strain, C shows the survival status after challenge of mice immunized once with different doses of the attenuated DENV-poly(A) strain, D shows the body weight status after challenge of mice immunized once with different doses of the attenuated DENV-poly(A) strain, and E shows the virus titers after challenge of mice immunized once with different doses of the attenuated DENV-poly(A) strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] I. Definition In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology used herein are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0020] As used herein, the term "about" or "approximately" means, unless otherwise specified, within ±10% of a given value or range. When required to be an integer, the term shall be rounded up or down to the nearest integer within ±10% of the given value or range.

[0021] As used herein, Flavivirus includes, but is not limited to, Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), dengue virus (DENV), zika virus (ZIKV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), Omsk hemorrhagic fever virus, and Kyasanur Forest Disease Virus, or flaviviruses related to any of them.

[0022] As used herein, the terms 5'-terminal stem-loop region (SL) of the flavivirus 3' untranslated region, dumbbell region (DB), repeat circularization sequence region 3 (RCS3), circularization sequence region 3 (CS3), repeat circularization sequence region 2 (RCS2), circularization sequence region 2 (CS2), circularization sequence region 1 (CS1), and short hairpin-3' terminal stem-loop region following circularization sequence region 1 (sHP-3'SL) are all terms of the corresponding field. In other studies, the stem-loop region (SL) is sometimes referred to as the variable region (VR).

[0023] The term "live attenuated vaccine" or "attenuated vaccine" as used herein refers to a virus that has been altered from the original parent or wild-type virus such that its ability to infect a host, replicate in a host, package, reinfect a host, or a combination thereof is attenuated. Generally, such attenuation may occur in some or all of the virus's hosts, or may occur in only one or some of the virus's hosts. Thus, the attenuation of a live attenuated virus (i.e., the reduction in its ability to infect a host, replicate in a host, package, reinfect a host, or a combination thereof) is usually associated with one or more of the virus's hosts, and is not significantly or measurably attenuated in one or more of the virus's other hosts. The live attenuated viruses disclosed herein are usually genetically modified or may be referred to as mutants, variants, genetically engineered modifications, recombinants, or combinations.

[0024] The term "live attenuated vaccine" or "attenuated vaccine" as used herein refers to a pharmaceutical composition comprising a live attenuated pathogen, e.g., a virus. The pharmaceutical composition comprises at least one immunoactive component that induces an immune response in a subject against the virus, thereby protecting the subject from death and / or potential death due to the virus, and optionally other components that enhance the immunoactivity of the active component or components. The vaccine may further comprise other components typical of pharmaceutical compositions. The at least one immunoactive component is one or more live attenuated viruses as described herein.

[0025] As used herein, the term "virus rescue" refers to the process of introducing an in vitro construct containing a viral sequence into a suitable cell to produce a transmissible or infectious virus. The term "recombinant virus" refers to a genetically engineered virus produced by recombinant DNA technology, and can be distinguished from naturally occurring viruses in that the viral sequence can be artificially engineered with any genetic manipulation, such as deletion, insertion, inversion, and substitution.

[0026] As used herein, the term "recombinant RNA virus" refers to a virus according to the present application that contains heterologous RNA.

[0027] As used herein, in the case of a virus, the term "wild type" refers to the type of virus that is prevalent, spreads naturally, and causes typical disease outbreaks. In other embodiments, in the case of a virus, the term "wild type" refers to the parent virus.

[0028] As used herein, the term "multiplicity of infection" or "MOI" refers to the average number of infectious viral particles per cell infected. The MOI is calculated by dividing the number of infectious viral particles added (ml added x PFU / ml) by the number of cells added (ml added x cells / ml).

[0029] II. MODE FOR CARRYING OUT THE DISCLOSURE In one aspect, the present disclosure provides an attenuated Flavivirus virus comprising a polyadenylic acid (poly(A)) sequence, wherein a portion of the nucleotide sequence of a 3' untranslated region (3'UTR) of the Flavivirus virus is replaced with the polyadenylic acid (poly(A)) sequence, and after the portion of the nucleotide sequence of the Flavivirus virus has been replaced, at least a 3'-terminal stem-loop region (3'SL) is retained in the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus obtained.

[0030] In the attenuated Flavivirus virus of the above embodiment, the portion of the nucleotide sequence includes all or part of the 5'-terminal stem-loop region (5'SL) in the 3' non-coding region (3'UTR), all or part of the circularization sequence region (CS), and / or all or part of the nucleotide sequence of the dumbbell region (DB).

[0031] In some preferred embodiments of the present disclosure, a portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, and then at least the circularization sequence region 1 (CS1) of the 3' untranslated region and the short hairpin-3' terminal stem-loop region (sHP-3'SL) following the circularization sequence region 1 are retained in the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus.

[0032] In some preferred embodiments of the present disclosure, the 5'-terminal stem-loop region (5'SL) in the 3' non-coding region (3'UTR) is selected from stem-loop region I (SLI), stem-loop region II (SLII), stem-loop region III (SLIII) and stem-loop region IV (SLIV).

[0033] In some preferred embodiments of the present disclosure, the cyclization sequence region (CS) is selected from cyclization sequence region 1 (CS1), cyclization sequence region 2 (CS2), cyclization sequence region 3 (CS3), repeat cyclization sequence region 2 (RCS2), and repeat cyclization sequence region 3 (RCS3).

[0034] In some preferred embodiments of the present disclosure, the dumbbell region (DB) is selected from dumbbell region 1 (DB1) and dumbbell region 2 (DB2).

[0035] In the attenuated Flavivirus virus according to any one of the above embodiments, the polyadenylic acid (poly(A)) sequence contains 10 to 200 adenylic acids.

[0036] In some preferred embodiments of the present disclosure, the polyadenylic acid (poly(A)) sequence contains 50 to 180, 100 to 160 adenylic acids, more preferably 130 to 150 adenylic acids, preferably 130 or 150 adenylic acids.

[0037] In some preferred embodiments of the present disclosure, the Flavivirus is selected from Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), dengue virus (DENV), zika virus (ZIKV), St. Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVEV), Omsk hemorrhagic fever virus, and Kyasanur Forest Disease Virus.

[0038] In some preferred embodiments of the present disclosure, the attenuated Flavivirus has a plaque titer of 5×10 4 ~5×10 6 PFU / ml.

[0039] In some preferred embodiments of the present disclosure, the attenuated Flavivirus virus has a virulence reduced by 10 to 100 times compared to the parent wild-type virus.

[0040] In some preferred embodiments of the present disclosure, the attenuated Flavivirus virus is rescued in baby hamster kidney cells BHK-21 and / or amplified in African green monkey kidney cells Vero cells.

[0041] In some preferred embodiments of the present disclosure, the dengue virus is selected from dengue virus types 1, 2, 3, and 4.

[0042] In some preferred embodiments of the present disclosure, the attenuated Flavivirus is an attenuated West Nile virus. In some preferred embodiments of the present disclosure, the attenuated West Nile virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence set forth in SEQ ID NO.1, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, and more preferably a nucleotide sequence having 98% or more or more 99% identity. In one specific embodiment of the present disclosure, the attenuated West Nile virus has the nucleotide sequence of SEQ ID NO. 1.

[0043] In some preferred embodiments of the present disclosure, the attenuated Flavivirus virus is an attenuated Japanese encephalitis virus. In some preferred embodiments of the present disclosure, the attenuated Japanese encephalitis virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence shown in SEQ ID NO.2, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, more preferably a nucleotide sequence having 98% or more or 99% or more identity. In one specific embodiment of the present disclosure, the attenuated Japanese encephalitis virus has a nucleotide sequence of SEQ ID NO. 2.

[0044] In some preferred embodiments of the present disclosure, the attenuated Flavivirus is an attenuated dengue virus. In some preferred embodiments of the present disclosure, the attenuated dengue virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence set forth in SEQ ID NO.3, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, and more preferably a nucleotide sequence having 98% or more or more 99% identity. In one specific embodiment of the present disclosure, the attenuated dengue virus has a nucleotide sequence of SEQ ID NO.3.

[0045] In some preferred embodiments of the present disclosure, the attenuated Flavivirus is an attenuated yellow fever virus. In some preferred embodiments of the present disclosure, the attenuated yellow fever virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence set forth in SEQ ID NO.4, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, and more preferably a nucleotide sequence having 98% or more or more 99% identity. In one specific embodiment of the present disclosure, the attenuated yellow fever virus has the nucleotide sequence of SEQ ID NO.4.

[0046] In some preferred embodiments of the present disclosure, the attenuated Flavivirus is an attenuated Zika virus. In some preferred embodiments of the present disclosure, the attenuated Zika virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence set forth in SEQ ID NO.5, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, and more preferably a nucleotide sequence having 98% or more or more 99% identity. In one specific embodiment of the present disclosure, the attenuated Zika virus has the nucleotide sequence of SEQ ID NO.5.

[0047] In some preferred embodiments of the present disclosure, the attenuated flavivirus is an attenuated tick-borne encephalitis virus. In some preferred embodiments of the present disclosure, the attenuated tick-borne encephalitis virus comprises a nucleotide sequence having 80% or more than 80% identity to the nucleotide sequence shown in SEQ ID NO.6, preferably a nucleotide sequence having 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity, more preferably a nucleotide sequence having 98% or more or 99% or more identity. In one specific embodiment of the present disclosure, the attenuated tick-borne encephalitis virus has a nucleotide sequence of SEQ ID NO.6.

[0048] In a preferred embodiment of the present disclosure, a portion of the nucleotide sequence includes 5'-terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), and circularization sequence region 3 (CS3), and after the portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus retains dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region 2 (DB2), circularization sequence region 2 (CS2), circularization sequence region 1 (CS1), and a short hairpin-3' terminal stem-loop region (sHP-3'SL) following circularization sequence region 1 in the 3' untranslated region.

[0049] In a preferred embodiment of the present disclosure, a portion of the nucleotide sequence includes dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region 2 (DB2), circularization sequence region 2 (CS2), and circularization sequence region 1 (CS1), and after a portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus retains the 5'-terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), and short hairpin-3'-terminal stem-loop region (sHP-3'SL) in the 3' untranslated region.

[0050] In a preferred embodiment of the present disclosure, a portion of the target nucleotide sequence includes 5'-terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region (DB2), and circularization sequence region 2 (CS2), and after a portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus retains the circularization sequence region 1 (CS1) of the 3' untranslated region and the short hairpin-3' terminal stem-loop region (sHP-3'SL) following the circularization sequence region 1.

[0051] In a preferred embodiment of the present disclosure, a portion of the nucleotide sequence includes a 5'-terminal stem-loop region II (SLII), a repeat circularization sequence region 3 (RCS3), a stem-loop region IV (SLIV), a circularization sequence region 3 (CS3), a dumbbell region 1 (DB1), a repeat circularization sequence region 2 (RCS2), a dumbbell region (DB2), and a circularization sequence region 2 (CS2), and after a portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, the 3' untranslated region (3'UTR) of the attenuated Flavivirus virus retains the circularization sequence region 1 (CS1) of the 3' untranslated region and the short hairpin-3' terminal stem-loop region (sHP-3'SL) following the circularization sequence region 1.

[0052] In a preferred embodiment of the present disclosure, a portion of the nucleotide sequence includes a 5'-terminal stem-loop region II (SLII), a repeat circularization sequence region 3 (RCS3), a dumbbell region (DB), a repeat circularization sequence region 2 (RCS2), a dumbbell region (DB2), and a circularization sequence region 2 (CS2), and after a portion of the nucleotide sequence is replaced with a polyadenylic acid (poly(A)) sequence, the 3' untranslated region (3'UTR) of the attenuated Flavivirus genus virus retains the 5'-terminal stem-loop region (SL), the circularization sequence region 1 (CS1), and a short hairpin-3'-terminal stem-loop region (sHP-3'SL) following the circularization sequence region 1 of the 3' untranslated region.

[0053] In one aspect, the disclosure provides DNA that can be transcribed to produce the RNA genome of the attenuated Flavivirus virus.

[0054] In some preferred embodiments of the present disclosure, the DNA is an infectious clone of an attenuated strain of a Flavivirus virus.

[0055] In some preferred embodiments of the present disclosure, the infectious clone is a plasmid.

[0056] In one aspect, the present disclosure provides a cell comprising the attenuated Flavivirus virus or the DNA.

[0057] In one aspect, the present disclosure provides a vaccine comprising the attenuated Flavivirus virus, DNA and / or cells.

[0058] In one aspect, the disclosure provides a pharmaceutical composition comprising the attenuated Flavivirus, DNA, cells and / or vaccine, and a pharma- ceutically acceptable carrier.

[0059] In one aspect, the present disclosure provides a method for preparing the attenuated Flavivirus virus, comprising replacing a nucleotide sequence of a wild-type virus 3' untranslated region (3'UTR) or a portion of the nucleotide sequence with a polyadenylic acid (poly(A)) sequence.

[0060] In some preferred embodiments of the present disclosure, the attenuated Flavivirus virus can be rescued in baby hamster kidney cells BHK-21 and / or amplified in African green monkey kidney cells Vero cells.

[0061] In one aspect, the disclosure provides the use of the attenuated Flavivirus virus, DNA, or cells in the preparation of a live-attenuated Flavivirus virus vaccine.

[0062] In one preferred embodiment of the present disclosure, the use is of the attenuated West Nile virus in the preparation of a live attenuated West Nile virus vaccine.

[0063] In one preferred embodiment of the present disclosure, the use is the use of the attenuated Japanese encephalitis virus in the preparation of a live attenuated Japanese encephalitis virus vaccine.

[0064] In one preferred embodiment of the present disclosure, said use is the use of said attenuated dengue virus in a live attenuated dengue virus vaccine.

[0065] In one preferred embodiment of the present disclosure, said use is of said attenuated yellow fever virus in the preparation of a live attenuated yellow fever virus vaccine.

[0066] In one preferred embodiment of the present disclosure is the use of said attenuated Zika virus in the preparation of a live attenuated Zika virus vaccine.

[0067] In one aspect, the disclosure provides the use of the attenuated Flavivirus, DNA, cells, vaccines and / or pharmaceutical compositions in the preparation of a medicament for treating or preventing a Flavivirus disease.

[0068] In one preferred embodiment of the present disclosure, the use provides for the use of the attenuated West Nile virus in the preparation of a medicament for treating or preventing a disease caused by West Nile virus.

[0069] In one preferred embodiment of the present disclosure, the use provides the use of the attenuated Japanese encephalitis virus in the preparation of a medicament for treating or preventing a disease caused by the Japanese encephalitis virus.

[0070] In one preferred embodiment of the present disclosure, the use provides for the use of the attenuated dengue virus in the preparation of a medicament for treating or preventing a disease caused by dengue virus.

[0071] In one preferred embodiment of the present disclosure, the use provides for the use of the attenuated yellow fever virus in the preparation of a medicament for treating or preventing a disease caused by the yellow fever virus.

[0072] In one preferred embodiment of the present disclosure, the use provides for the use of the attenuated Zika virus in the preparation of a medicament for treating or preventing disease caused by Zika virus.

[0073] In one aspect, the disclosure provides a method for stimulating an immune response against a Flavivirus comprising administering the attenuated Flavivirus, DNA, cell, vaccine and / or drug composition in an immunization regimen.

[0074] In an embodiment herein, a composition is provided for administration to a subject in a biocompatible form suitable for in vivo administration of the drug. "Biocompatible form suitable for in vivo administration" refers to a form of an active ingredient (e.g., a drug of the embodiment) that is administered such that the therapeutic effect of the active agent exceeds any toxic effects. Administration of a therapeutically effective amount of a therapeutic composition is defined as an amount effective at a dosage and for a period of time necessary to achieve a desired result. For example, a therapeutically active amount of a compound may vary depending on factors such as, for example, the disease state, age, sex, and weight of an individual, as well as the ability of an antibody to elicit a desired response in an individual. Dosage regimens may be adjusted to provide an optimal therapeutic response.

[0075] In one embodiment, the compound (e.g., a live attenuated virus of the embodiments) may be administered in a convenient manner, for example, subcutaneously, intravenously, orally, by inhalation, intradermal, transdermal application, intravaginal application, topical administration, intranasally, or rectally. Depending on the route of administration, the active compound may be contained in a protective buffer (e.g., albumin and trehalose, poloxamer 407 / trehalose / albumin (FTA)). In one embodiment, the composition may be administered orally. In another embodiment, the composition may be administered intravenously. In one embodiment, the composition may be administered intranasally, for example, by inhalation. In another embodiment, the composition may be administered intradermally by a needle-free system (e.g., Pharmajet®) or other intradermal administration system.

[0076] In some embodiments, the attenuated live virus vaccine (e.g., West Nile virus vaccine) may be administered to children or adolescents as a stabilized formulation (e.g., albumin and trehalose, FTA, or other formulations for stabilizing the attenuated live virus). As used herein, the term "pharmaceutical acceptable carrier" may further include diluents, e.g., saline and aqueous buffers. It may be necessary to combine the attenuated live virus formulation with a material that prevents its inactivation, or to administer the compound simultaneously with a material that prevents its inactivation. In some embodiments, one or more formulations of the attenuated live West Nile virus may be administered to a subject at an initial dose, followed by a booster subcutaneously or intradermally. They may also be prepared as dispersions in glycerol, liquid polyethylene glycols and mixtures thereof, and oils. Under normal conditions of storage and use, these formulations may include preservatives or other stabilizing compounds (e.g., those for stabilizing the attenuated live flavivirus or other stabilizing compounds) to prevent the growth of microorganisms.

[0077] Pharmaceutical compositions suitable for injectable use can be administered by means known in the art. For example, sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of injectable sterile solutions or dispersions may be used. In some embodiments, the compositions may be sterile, easily injectable fluids. The pharma-ceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. In the case of dispersions, the required particle size may be maintained by coating materials such as lecithin, and the proper fluidity may be maintained by surfactants. Various antibacterial or antifungal agents or other preparations may be added to prevent microorganisms.

[0078] In some embodiments, when prepared, solutions may be administered in a form compatible with the dosage form formulation and in a therapeutically effective amount. In accordance with these embodiments, the formulations can be readily administered in a variety of dosage forms, such as, for example, the injectable solution types described above.

[0079] In some embodiments, a subject may be administered a single or multiple doses of an attenuated flavivirus (e.g., West Nile virus) formulation. In some embodiments, a subject may be treated with a single dose of the formulation. In other embodiments, a subject may be treated with at least two doses of a live attenuated West Nile virus formulation. In some embodiments, a subject is administered an attenuated West Nile virus composition on day 0 and a booster dose is administered within about three months after the first dose. In some embodiments, a subject is a subject who has never been exposed to West Nile virus and has no immunity (seronegative). In other embodiments, a subject has previously been exposed to and / or infected with West Nile virus (seropositive). According to these embodiments, a seronegative subject is treated on day 0 and may receive a booster dose within six months, five months, four months, three months, or less of the first dose to provide an enhanced immune response to West Nile virus. In some embodiments, a child may be a child between about 2 years of age and about 17 years of age. In other embodiments, the child is between 2 and 17 years old.

[0080] In other embodiments, a nasal solution or spray, aerosol or inhalant can deliver a virus formulation of a live attenuated flavivirus (e.g., West Nile virus) to a subject. Some formulations may contain excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0081] The drug composition can be prepared using a carrier, which protects the active ingredient from rapid elimination from the body, for example, as a sustained release formulation or a coating material. Such carriers include controlled release formulations, for example, microencapsulated delivery systems, and biodegradable biocompatible polymers, including, but not limited to, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other known materials.

[0082] In some embodiments, the dose range for the live attenuated flavivirus (e.g., West Nile virus) is initially about 10 2 ~about 10 6 A PFU may be administered, followed optionally by at least a second dose within 30 days or within the following 12 months, as needed.

[0083] In some embodiments, the drug compositions disclosed herein may be administered to a subject in one dose or multiple doses. In some embodiments, the immunogenic compositions disclosed herein may be administered to a subject in one dose, two doses, or more doses within a predetermined time period, such as within about 6 months, within about 120 days, within about 90 days, within about 80 days, within about 70 days, within about 60 days, within about 50 days, within about 40 days, within about 30 days, within about 20 days, within about 10 days, within about 5 days, or less, or within a few hours or minutes on the same day, or simultaneously at the same or different anatomical sites. In some embodiments, the drug compositions disclosed herein may be administered within about 90 days of each other, within about 60 days of each other, within about 30 days of each other, and less than about 30 days of each other. In some embodiments, the compositions disclosed herein may be administered to a subject subcutaneously or intradermally. Administration to two or more anatomical sites may include any combination of administrations, including administration to two or more anatomical sites in the same manner, or administration to two separate anatomical sites in two different manners. According to these embodiments, the two or more anatomical sites may include different limbs or different regions of the body. In some embodiments, two doses of the vaccine composition may be introduced into a subject sequentially at the same or multiple anatomical sites on day 0, for example, to provide protection against all flavivirus (e.g., West Nile virus) serotypes (e.g., cross-protection). In other embodiments, the drug composition may include a combination of live attenuated West Nile virus and other immunogenic agents against other flaviviruses (e.g., Zika virus, Japanese encephalitis, West Nile virus, St. Louis encephalitis virus, yellow fever, or other viruses). In some embodiments, the vaccines against West Nile virus disclosed herein are used to reduce other related viral infections, e.g., Zika virus infection.

[0084] In some embodiments, the drug compositions disclosed herein may be used to increase a subject's immune response to a targeted formulation, such as by administering an immunogenic composition against a flavivirus (e.g., West Nile virus) virus and CD8 to a child or adolescent who receives such a drug composition against a flavivirus (e.g., West Nile virus) virus. + This is done by combining agents to enhance T cell responses or other immune responses to West Nile Virus.

[0085] Topical administration is accomplished by topical application of a cream, jelly, rinse, etc., containing a therapeutically effective amount of the serine protease inhibitor. Transdermal administration is accomplished by administration of a cream, jelly, rinse, etc., that penetrates the skin and enters the bloodstream. Osmotic pumps may also be used for administration. The dosage required will vary depending on the particular condition being treated, the method of administration, and the rate at which the molecule is cleared from the body.

[0086] Other embodiments relate to methods for reducing inactivation of live attenuated viruses, including, but not limited to, combining one or more live attenuated viruses with compositions capable of reducing inactivation of a live attenuated virus (e.g., a flavivirus), including, but not limited to, one or more protein reagents, one or more sugar or polyol reagents, and any one or more EO-PO block copolymers, that are capable of reducing inactivation of or stabilizing a live attenuated virus.

[0087] In some embodiments, the compositions herein may be partially or completely dehydrated or hydrated. In other embodiments, the stable protein reagents used in the drug or non-drug compositions herein include, but are not limited to, whey proteins, human serum albumin, recombinant human serum albumin (rHSA), bovine serum albumin (BSA), other serum albumins or members of the albumin gene family. The sugar or polyol reagents include, but are not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fructose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylase, cyclodextrin, chitosan, or cellulose. In some embodiments, the surfactant includes, but is not limited to, nonionic surfactants such as alkyl poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (EO-PO block copolymers), poly(vinylpyrrolidone), alkyl polyglucosides (e.g., sucrose monostearate, lauryl diglucoside or sorbitan monolaurate, octyl glucoside and decyl maltoside), fatty alcohols (cetyl alcohol or oleyl alcohol) or cocamides (cocamide MEA, cocamide DEA, and cocamide TEA).

[0088] In other embodiments, the surfactant includes, but is not limited to, poloxamer 407 (e.g., Pluronic F127®), substituted poloxamer 407 or poloxamer 335 other than poloxamer 407, poloxamer 338 or poloxamer 238, or other EO-PO block copolymers with properties similar to F127®.

[0089] In some embodiments, the vaccine composition includes one or more protein reagents that are serum albumin, one or more trehalose saccharides, and one or more surfactant polymer reagents, such as, but not limited to, EO-PO block copolymers, poloxamer 407, or, more specifically, Pluronic F127®.

[0090] In other embodiments, the formulation for stabilizing a live virus may include one or more live flaviviruses, one or more carbohydrate reagents, and one or more amino acids or salts, esters, or amide derivatives thereof. In other embodiments, the attenuated live flaviviruses stabilized with the formulations used herein are used in commercial applications. In some embodiments, the composition further includes a buffering agent. According to these embodiments, the buffering agent includes, but is not limited to, phosphate buffered saline (PBS). According to these embodiments, the buffering agent may include at least one of sodium chloride (NaCl), sodium dihydrogen phosphate and / or disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). In some embodiments, the buffering agent of the composition may include sodium chloride at a concentration of 25 mM to 200 mM. In other embodiments, the compositions disclosed herein may include other suitable reagents, such as urea and / or MSG.

[0091] In some embodiments, a live attenuated flavivirus, such as West Nile virus, can be stabilized in a formulation that includes, but is not limited to, recombinant HSA at a concentration of 0.1% to 0.2% (w / v), and / or sucrose at a concentration of about 4.0% to about 6.0% (w / v), and / or mannitol at a concentration of about 2% to 4% (w / v), and / or alanine at a concentration of about 8.0 mM to about 22.0 mM, and / or methionine at a concentration of about 1.0 mM to about 5.0 mM, and / or MSG at a concentration of about 8.0 mM to 12.0 mM, and / or urea at a concentration of about 0.1% to about 0.3% (w / v). In some embodiments, the composition may include recombinant HSA, trehalose, mannitol, alanine, methionine, MSG, and urea. In other embodiments, the stabilizing composition may include HSA at a concentration of about 0.1% to about 0.2% (w / v), trehalose at a concentration of about 4% to about 6% (w / v), mannitol at a concentration of about 2% to about 4% (w / v), alanine at a concentration of 8 mM to 22 mM, methionine at a concentration of 1 mM to 5 mM, MSG at a concentration of 8 mM to 12 mM, and urea at a concentration of 0.1% to 0.3% (w / v). Some formulations for stabilizing live attenuated viruses include, but are not limited to, recombinant HSA, sucrose, alanine, and urea. According to these embodiments, the HSA concentration may be about 0.1% to about 0.2% (w / v), the sucrose concentration may be about 4% to about 6% (w / v), the alanine concentration may be about 8.0 mM to about 22 mM, and the urea concentration may be about 0.1% to about 0.3% (w / v). Other stabilized formulations may include recombinant HSA, sucrose, methionine, and urea. The recombinant HSA concentration may be about 0.1% to about 0.2% (w / v), the sucrose concentration may be about 4.0% to about 6.0% (w / v), the methionine concentration may be about 1.0 mM to about 5.0 mM, and the urea concentration may be about 0.1% to about 0.3% (w / v).In another embodiment, the stabilized formulation may include recombinant HSA, sucrose, arginine, and urea, and the recombinant HSA concentration may be 0.1% to 0.2% (w / v), the sucrose concentration may be 4% to 6% (w / v), the arginine concentration may be 10 mM to 50 mM, and the urea concentration may be 0.1% to 0.3% (w / v). Another stabilized formulation may include recombinant HSA, trehalose, arginine, and urea, and the recombinant HSA concentration is about 0.1% to 0.2% (w / v), the trehalose concentration is about 4% to 6% (w / v), the arginine concentration is about 10 mM to 50 mM, and the urea concentration is about 0.1% to 0.3% (w / v). In another embodiment, the stabilized composition may include recombinant HSA, trehalose, MSG, and urea. According to these embodiments, the recombinant HSA concentration may be from about 0.1% to about 0.2% (w / v), the trehalose concentration may be from about 4.0% to about 6.0% (w / v), the MSG concentration is from 8 mM to 12 mM, and the urea concentration is from 0.1% to 0.3% (w / v).

[0092] Some embodiments herein relate to live attenuated virus compositions that are partially or completely dehydrated for transport or other reasons. According to these embodiments, the compositions are 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more dehydrated. According to these embodiments, the pharma- ceutically acceptable compositions may be dehydrated and rehydrated in any known stabilizing composition prior to administration to a subject.

[0093] In some embodiments, the subject may be a mammal, e.g., a human or veterinarian, and / or a pet or livestock or wild animal. In some embodiments, the immunogenic compositions of the present disclosure can effectively immunize subjects who are teenagers, e.g., children aged 20 years or younger. While current dengue virus vaccine / immunogenic compositions (e.g., dengue and yellow fever chimerism) used in children have reported low efficacy and / or immunogenicity in children aged 9 years or younger, the immunogenic compositions disclosed herein generate effective immune responses in children aged about 1 year to about 17 years, or about 1 year to about 9 years, or older. Compared to the prior art, the immunogenic compositions disclosed herein exhibit superior efficacy and immunogenicity.

[0094] In some embodiments, the immunogenic composition against a flavivirus (e.g., West Nile virus) is administered at a concentration of about 1.0×10 3 ~Approx. 5×10 5 Attenuated West Nile virus, PFU, concentration approximately 1.0 × 10 3 ~Approx. 5×10 5 PFU of live West Nile virus, concentration approximately 5.0 × 10 3 ~Approx. 5×10 5 PFU of attenuated West Nile virus, and / or a concentration of about 1.0 x 10 4 ~Approx. 5×10 6 In some embodiments, the immunogenic composition against West Nile Virus may comprise one or more PFU of attenuated West Nile Virus. In some embodiments, subjects treated with the compositions and methods disclosed herein may be treated twice per year, annually, every 18 months, or on a similar schedule, for example, depending on the subject's location and travel plans. In some embodiments, the immunogenic composition against West Nile Virus may comprise one or more PFU of attenuated West Nile Virus ... 4 Attenuated West Nile virus, PFU, concentration approximately 5.0 × 10 3 Attenuated live West Nile virus (PFU), concentration approximately 1 × 10 5 PFU of attenuated West Nile virus and / or a concentration of about 3.0 x 10 5It may comprise one or more PFU of attenuated West Nile virus.

[0095] Compared with the prior art, the present disclosure has the following advantages and effects:

[0096] 1. The reverse genetics technology adopted in the construction of the attenuated Flavivirus virus (e.g., the attenuated West Nile virus strain WNV-poly(A)) of the present disclosure has the advantages of being advanced and mature, convenient and simple, and position-controllable.

[0097] 2. The attenuated Flavivirus virus (e.g., WNV-poly(A)) provided in the present disclosure can be cultured and amplified in Vero to obtain high-titer virus, and the method of obtaining it is simple, facilitating subsequent vaccine production and saving costs.

[0098] 3. The attenuated Flavivirus virus (e.g., WNV-poly(A)) provided in the present disclosure has good genetic stability during serial passage, with the 3'UTR still maintaining the deletion of SL and DB region sequences and the stable presence of the -poly(A) sequence.

[0099] 4. The attenuated Flavivirus virus (e.g., WNV-poly(A)) provided in the present disclosure does not cause disease even when infected with C57BL / 6 mice at a high dose, and is sufficiently attenuated in mice, demonstrating high safety. 4 ~10 7 A single immunization with PFU WNV-poly(A) enabled mice to produce high levels of WNV-specific IgG and neutralizing antibodies, providing complete immune protection to mice challenged with a high, lethal dose of WT WNV.

[0100] 5. Attenuated Flaviviruses (e.g., WNV-poly(A)) provided herein are effective in C57BL / 6 mouse models at low doses (10 4 PFU) or high dose (10 7After immunization with 100 PFU each, both maintained high humoral immune responses in the mice's bodies for a long period of time, and the levels of neutralizing antibodies remained stable, providing complete immune protection to the mice even after challenge with high doses of WT WNV.

[0101] The above results showed that the general preparation method of attenuated flavivirus strains provided in the present disclosure can prepare safe and effective attenuated vaccine strains, which has good application and important strategic significance in addressing the potential threat of flaviviruses in our country and global scope.

[0102] The test methods, such as plasmid linearization, RNA in vitro transcription, transfection, and virus titer assay, related to this part are conventional in the art unless otherwise specified. Listed below are only some specific embodiments of the present disclosure. Obviously, the present disclosure is not limited to the following embodiments, and many variations are possible. Therefore, any modifications or improvements made by those skilled in the art based on this disclosure shall be included in the scope of protection claimed for this disclosure.

[0103] Example 1: Construction of an attenuated flavivirus clone and virus rescue 1. Construction of attenuated flavivirus clones A schematic diagram of the construction of an attenuated flavivirus strain clone is shown in FIG. The plasmid DNA of an infectious clone of the wild-type (WT) West Nile virus (WNV) virulent strain was used as the backbone, and the sequence from SLI to CS2 in the 3' untranslated region (3'UTR) of WNV (5'-terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS3), dumbbell region 3 (DB4), dumbbell region 4 (DB5), dumbbell region 5 (DB6), dumbbell region 6 (DB7), dumbbell region 7 (DB8), dumbbell region 8 (DB9), dumbbell region 10 (DB10), dumbbell region 11 (DB11), dumbbell region 12 (DB12), dumbbell region 13 (DB13), dumbbell region 14 (DB14), dumbbell region 15 (DB15), dumbbell region 16 (DB16), dumbbell region 17 (DB17), dumbbell region 18 (DB18), dumbbell region 19 (DB19), dumbbell region 20 (DB19), dumbbell region 21 (DB19), dumbbell region 22 (DB19), dumbbell region 23 (DB19), dumbbell region 24 (DB19), dumbbell region 25 (DB19), dumbbell region 26 (DB19), dumbbell region 27 (DB19), dumbbell region 28 (DB19), dumbbell region 29 (DB19), dumbbell region 30 (DB19), dumbbell region 31 (DB19), dumbbell region 32 (DB19), dumbbell region 33 (DB19), dumbbell region 34 (DB The 3' untranslated region was deleted from the plasmid pG1001, and the 3' untranslated region was deleted from the plasmid pG1001, and the 3' untranslated region was deleted from the plasmid pG1001. The .... The 3' untranslated region was deleted from the plasmid pG1001. The 3' untranslated region was deleted from the plasmid pG1001. The 3' untranslated region was deleted from the plasmid pG1001. The 3' untranslated region was deleted from the plasmid pG1001. The 3' untranslated region was deleted from the plasmid pG1001. The 3' untranslated region was deleted from the plasmid pG1001.

[0104] Using the plasmid DNA of an infectious clone of wild-type (WT) Japanese encephalitis virus (JEV) virulent strain as a backbone, the sequence from SLI to CS2 in the 3' untranslated region (3'UTR) of JEV (including SLI, SLII, RCS3, SLIII, SLIV, CS3, DB1, RCS2, DB2, and CS2) was deleted, the circularization sequence region 1 (CS1) of the 3' untranslated region and the short hairpin-3' stem-loop region (sHP-3'SL) following the circularization sequence region 1 were retained, and a poly(A) sequence (150 adenylic acids) was inserted into the deletion site resulting from the deletion of the sequence from SLI to CS2 to obtain a JEV-poly(A) virus attenuated strain plasmid, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0105] Using the plasmid DNA of an infectious clone of wild-type (WT) dengue virus type 2 New Guinea Island strain (DENV-2 (NGC)), the sequence from SLI to CS2 in the 3' untranslated region (3'UTR) of DENV-2 (NGC) (including SLII, RCS3, SLIV, CS3, DB1, RCS2, DB2, and CS2) was deleted, the circularization sequence region 1 (CS1) of the 3' untranslated region and the short hairpin-3' end stem loop region (sHP-3'SL) following the circularization sequence region 1 were retained, and a poly(A) sequence (140 adenylic acids) was inserted into the deletion site resulting from the deletion of the sequence from SLI to CS2 to obtain a DENV-2 (NGC)-poly(A) virus attenuated strain plasmid, the nucleotide sequence of which is shown in SEQ ID NO. 3.

[0106] Using the plasmid DNA of an infectious clone of wild-type (WT) yellow fever virus (YFV) virulent strain as a backbone, the sequence from SLI to CS2 (including SLII, RCS3, and DB) in the 3' untranslated region (3'UTR) of YFV was deleted, the stem-loop region (SL) of the 3' untranslated region, the circularization sequence region 1 (CS1), and the short hairpin-3' end stem-loop region (sHP-3'SL) following the circularization sequence region 1 were retained, and a poly(A) sequence (130 adenylic acids) was inserted into the deletion site resulting from the deletion of the sequence from SLI to CS2 to obtain a YFV-poly(A) virus attenuated strain plasmid, the nucleotide sequence of which is shown in SEQ ID NO. 4.

[0107] All of the above virulent strain plasmids were corresponding poly(A) virus attenuated strain plasmids obtained by replacing all or part of the 5'-terminal stem-loop region (SL) sequence and all of the DB sequence in the 3'UTR region with poly(A) sequences, and retaining at least the circularization sequence region 1 (CS1) in the 3' untranslated region and the short hairpin-3'-terminal stem-loop region (sHP-3'SL) following the circularization sequence region 1.

[0108] 2. In vitro transcribed RNA with poly(A) The poly(A) virus attenuated strain plasmid obtained in step 1 was linearized, and complete linearization was confirmed by 0.8% agarose gel electrophoresis, followed by phenol-chloroform extraction. Finally, 11 μL of RNAase-free water was added to dissolve the plasmid, and the DNA concentration was measured using a Thermo Scientific NanoDrop 2000. The DNA mass was detected by electrophoresis. Using 1 μg of DNA after phenol-chloroform extraction as a template, the plasmid was in vitro transcribed using an in vitro transcription kit T7 mMESSAGE mMACHINE kit (Ambion) according to the kit instruction manual to obtain recombinant poly(A) RNA, i.e., genomic RNA of the WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) virus attenuated strains. The RNA concentration was measured using a Thermo Scientific NanoDrop 2000 and the RNA mass was detected by electrophoresis on a freshly prepared 0.8% agarose gel and stored at -80°C until use.

[0109] 3. Poly(A) virus rescue The study was divided into two arms: (1) Test group: The recombinant poly(A) RNA obtained by in vitro transcription in step 2 was transfected into BHK-21 cells by liposome transfection. (2) Control group: As a positive control, wild-type viral RNA was transfected into BHK-21 cells by liposome transfection.

[0110] The specific test procedure was as follows: On the day before transfection, 2 × 10 cells were cultured in a 5% CO2-free medium to allow the cells to reach approximately 80% on the day of transfection. 510 BHK-21 cells were seeded in a 35 mm cell culture dish, and three 10 mm × 10 mm cover glasses were placed on each dish. During transfection, the medium in the culture dish was first discarded, washed once with 1 ml of Opti-MEM, and then 1 ml of Opti-MEM was added (to make the cells in an infiltrated state). 1 ml of Opti-MEM was added to a 1.5 ml EP tube, and 4 μl of DMRIE-C (mix well before using DMRIE-C) was first mixed evenly by inverting upside down. 1 μg of in vitro transcribed recombinant poly(A) RNA was added to the test group, and wild-type viral RNA was added to the control group. 1 μg was added and mixed evenly by inverting, the Opti-MEM in the culture dish was quickly discarded, the mixture was added to the dish (without blowing on the cells, but by gently moving), and the cells were cultured at 37°C in a CO2 incubator for 4 h. After that, the culture was discarded, and 2 mL of DMEM medium containing 2% FBS was added to each dish. The cell status of the test group and the control group was observed under a microscope. One slide glass was taken at 24h, 48h, and 72h after transfection. The cells were fixed with 5% acetone fixative (purchased from Sinopharm Chemical Reagent Co., Ltd.) at room temperature for 15 minutes, washed with PBS three times, and then stored at 4℃. When the IFA detection positive rate was high, the supernatant was collected as attenuated flavivirus poly(A) P0 generation virus, that is, four kinds of P0 generation WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) attenuated strains were obtained and stored at -80℃.

[0111] 4. Detection of viral protein expression of recombinant poly(A) by indirect immunofluorescence (IFA) The slides stored at 4 °C in step 3 above were incubated with the primary antibody at room temperature, and the flavivirus attenuated strain antibody was 4G2 monoclonal antibody (a universal antibody against flavivirus E protein) diluted 1:500. After incubating the primary antibody at room temperature for 1-2 h and rinsing 10 times with PBS, the secondary antibody was incubated at room temperature in the dark, and the antibody was fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody diluted 1:125. After incubating the secondary antibody for 1 h, it was rinsed 10 times with PBS, the slides were removed, marked, 95% glycerol was dropped on each mark, and the cover glass was placed on the glycerol drop with the cell side facing down, and observed under a fluorescent microscope at a magnification of 200 times (Figure 1).

[0112] The results in Figure 1 show that the positivity rates of the attenuated strains of WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) were all lower than that of the WT at each time point. However, as the culture time after transfection was prolonged, the number of positive cells gradually increased with the same trend as the WT, indicating that WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) all rescued infectious virus particles.

[0113] Example 2: Construction of clones of other attenuated West Nile virus strains and virus rescue 1. Construction of other attenuated West Nile virus clones A schematic diagram of the construction of other attenuated West Nile virus strain clones is shown in FIG. Using the plasmid DNA of the infectious clone of the wild-type (WT) West Nile virus (WNV) virulent strain in Example 1 as a backbone, the sequence from SLI to CS3 (including SLI, SLII, RCS3, SLIII, SLIV, and CS3) in the 3' untranslated region (3'UTR) of WNV was deleted, and dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region 2 (DB2), circularization sequence region 2 (CS2), circularization sequence region 1 (CS1), and the short hairpin-3' end stem-loop region (sHP-3'SL) following circularization sequence region 1 in the 3' untranslated region were retained, and a poly(A) sequence (130 adenylic acids) was inserted into the deletion site resulting from the deletion of the sequence from SLI to CS3, thereby obtaining a stem-loop region (SL)-deleted West Nile virus SL-del-poly(A) attenuated strain plasmid.

[0114] Using the plasmid DNA of the infectious clone of the wild-type (WT) West Nile virus (WNV) virulent strain in Example 1 as a backbone, the sequence from DB1 to CS1 (including DB1, RCS2, DB2, CS2, and CS1) in the 3' untranslated region (3'UTR) of WNV was deleted, and the 5'-terminal stem-loop region I (SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), and short hairpin-3' terminal stem-loop region (sHP-3'SL) in the 3' untranslated region were retained, and a poly(A) sequence (130 adenylic acids) was inserted into the deletion site resulting from the deletion of the sequence from DB1 to CS1, thereby obtaining a dumbbell region (DB)-deleted West Nile virus DB-del-poly(A) attenuated strain plasmid.

[0115] 2. In vitro transcribed RNA with poly(A) The transfer method was as described in Example 1, step 2.

[0116] 3. Poly(A) virus rescue The rescue method was as described in Example 1, step 3.

[0117] 4. Detection of viral protein expression of recombinant poly(A) by indirect immunofluorescence (IFA) The detection method was as described in step 4 of Example 1. The results of the fluorescence microscopy are shown in Figure 2. The results in Figure 2 show that viruses obtained by deleting a portion of the nucleotide sequence of the 3' untranslated region (3' UTR) of a Flavivirus virus, without deleting the entire 5' SL and DB, and inserting a poly(A) sequence into the deleted site, produce viruses in the same way.

[0118] Example 3: Comparison of growth curves of attenuated flavivirus strains and wild-type viruses 1. Measurement of Viral Titer The virus titer was measured by plaque assay, and the specific steps were as follows: 1 x 10 cells in each well of a 24-well cell culture plate 5 100μl of poly(A)P0 generation virus of attenuated flavivirus collected in step 3 of Example 1 diluted 10-fold with 2% FBS-containing DMEM medium was added, and the wells were shaken thoroughly once every 15 minutes. After the adsorption was completed, the virus solution was extracted from each well and discarded, and 2% FBS-containing DMEM medium and 2% methylcellulose coating were added, and the wells were cultured in an incubator at 37℃ and 5% CO2 for 3 days. After plaques were formed, the wells were stained with a staining solution containing 1% crystal violet and 3.7% formaldehyde, and treated at room temperature for 30 min. The staining solution in the wells was extracted, and the bottom of the wells was washed with running water, dried, counted, and converted to virus titer.

[0119] 2. Measurement of viral growth curve 2 x 10 cells in a 35 mm cell culture dish 5Vero cells were inoculated, and when the cell confluence reached 60% under 37°C and 5% CO2 culture conditions, the poly(A)P0 generation virus of the attenuated flavivirus collected in step 3 of Example 1 and the corresponding wild-type (WT) virus were added separately to each dish at an infection efficiency of MOI of 0.1. After adsorption for 2 h in a 5% CO2 incubator at 37°C, the virus solution was discarded, 2 ml of DMEM medium containing 2% fetal bovine serum was added to each well, and the wells were cultured under 37°C and 5% CO2 culture conditions. After infection, 400 μl of the virus supernatant was collected every 12 h, and 400 μl of DMEM medium containing 2% fetal bovine serum was added. The collected viruses were used as virus samples at different time points with the same infection efficiency of the poly(A) virus and the corresponding WT virus, and were stored at -80°C. The titers of the viruses collected at different time points were measured by the plaque assay method, and the growth curves were plotted (Figure 3). As a result, the attenuated strains of WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) showed similar growth trends compared to the corresponding WT strains, but the titers were all 10- to 100-fold lower than those of the wild type.

[0120] Example 4: Verification of genetic stability of attenuated flavivirus strains 1. The poly(A) P0 generation virus of the attenuated flavivirus obtained by rescue in Example 1 was infected into Vero cells, and after 3 days, cytopathic effect (CPE) was evident. The cell supernatant (P1 generation) was collected, and each generation was infected with three strains of virus in parallel. After 10 consecutive blind passages, viral RNA was extracted from the virus-infected cell samples collected from the P1, P5, and P10 generations according to the Trizol kit instructions, and stored at -80°C for use. RT-PCR was performed using the PrimeScript One Step RT-PCR Kit to detect changes in the 3'UTR, and sequenced. As a result of sequencing, the polyA insertion position and the number of insertions of about 130 to 150A were almost unchanged for WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A), and YFV-poly(A) even after 10 consecutive passages (Figure 4).

[0121] 2. The supernatant virus fluids collected at the P1 and P10 generations were taken and the growth curves in Vero cells were compared at an infection efficiency MOI of 0.1 (the method was the same as in Example 2). As a result, as shown in poly(A)-P10 in Figure 4, the WNV-poly(A), JEV-poly(A), DENV-2(NGC)-poly(A) and YFV-poly(A) virus strains had similar growth curves at the P1 and P10 generations before and after 10 generations of passage, further indicating that the poly(A) virus has good genetic stability.

[0122] Example 5: Pathogenicity of attenuated West Nile virus In this example, the pathogenicity of the attenuated West Nile virus (WNV) strain WNV-poly(A) in C57BL / 6 was examined.

[0123] Six groups of 4-week-old female C57BL / 6 mice (5 mice / group) were selected, and each group was infected with 10% of the P1 generation WNV-poly(A) attenuated strain obtained in Example 3. 4 PFU, 10 5 PFU, 10 6 PFU or 10 7 PFU and corresponding wild-type West Nile virus (WT WNV) 10 4 Mice were intraperitoneally infected with PFU and injected with an equal volume of PBS as controls. Virus-containing blood was measured by detecting the virus titer in mouse serum on the first, second, and third days after infection. Specifically, about 100 μl of blood was collected from the orbit of each mouse into a 1.5 ml EP tube, left at 4° C. for 3 hours, and then centrifuged at 5000 rpm for 5 minutes to collect serum, which was used to measure the virus titer. The measurement method was the same as that of the virus titer measurement in Example 1. At the same time, the weight of the mice was measured for 14 consecutive days after infection, and the survival status of the mice for 21 days was observed.

[0124] As a result, all mice inoculated with the attenuated WNV-poly(A) strain survived (Fig. 5A). 7In mice inoculated with the attenuated PFU WNV-poly(A) strain, low virulence was detected only on day 1, but no viremia was detected in any of the mice inoculated with the other doses (Fig. 5B). The changes in body weight were consistent with those of the PBS-immunized mice (Fig. 5C), and no other pathological conditions were observed. However, WT WNV10 4 Mice inoculated with PFU showed viremia (Fig. 5B), lost weight significantly from day 5 (Fig. 5C), and all died by day 8 (Fig. 5A). This indicates that the virulence of the attenuated WNV-poly(A) strain is sufficiently low, it is non-pathogenic in C57BL / 6 mice, has high safety, and can be used as a potential live attenuated vaccine.

[0125] Example 6: Humoral immunity and challenge protection effects of attenuated West Nile virus in a mouse model In this example, we investigated the humoral immunity and challenge protection effects of an attenuated strain of WNV-poly(A) in a C57BL / 6 mouse model.

[0126] 1. Humoral immunity to an attenuated strain of WNV-poly(A) in a C57BL / 6 mouse model Six groups of 4-week-old female C57BL / 6 mice (5 mice / group) were selected, and each group was infected with 10% of the P1 generation WNV-poly(A) attenuated strain obtained in Example 3. 4 PFU, 10 5 PFU, 10 6 PFU or 10 7 Mice were intraperitoneally immunized with PFU, and mice immunized with an equal volume of PBS and Mock mice that received no treatment were used as controls. Blood was collected by the orbital blood collection method described in Example 4 on the 14th and 28th days after immunization, and left to stand at 4°C for 3 hours, then centrifuged to collect serum, which was inactivated at 56°C for 30 minutes and frozen and stored at -20°C for use.

[0127] IgG antibody titers were detected by ELISA, and neutralizing antibody titers were detected by PRNT50. 1) Detection of IgG antibody titers by ELISA A 96-well plate was coated with an attenuated WNV-poly(A) strain and blocked with 5% skim milk prepared in PBS. Serum was serially diluted from 1:50 to 4-fold and then incubated with the coated 96-well plate at 37°C for 2 h. The plate was washed three times with PBST, and then incubated with HRP goat anti-mouse antibody at 37°C for 1 h. Color was developed using a two-component color development kit (Proteintech). The reaction was terminated by adding 1M H2SO4, and the absorbance at 450 nm was detected using a multifunctional microplate reader. The IgG antibody titer was determined at the highest dilution when the absorbance of the immunized mouse serum was twice that of the non-immunized mouse serum.

[0128] 2) Detection of neutralizing antibody titers using PRNT50 2 x 10 cells in each well of a 12-well cell culture plate 5 When the cells reached 90% confluence, the serum was serially diluted 2-fold, and 100 μl of each diluted antibody was added to each of the same volume of 100 PFU. After homogeneously mixing with WT-WNV, the wells were incubated at 37°C for 1 h, the medium in the wells was discarded, and the incubated serum-virus mixture was added to the corresponding wells and adsorbed at 37°C for 1 h. The wells were shaken thoroughly once every 15 minutes. After adsorption was completed, the virus liquid in each well was extracted and discarded, 1 mL of 2% methylcellulose-containing coating was added, and the wells were incubated in a 5% CO2 incubator at 37°C for 72 hours. After plaques were formed, the wells were stained with a staining solution containing 1% crystal violet and 3.7% formaldehyde. After treatment at room temperature for 30 minutes, the staining solution in the wells was extracted, the bottom of the wells was washed with running water, and the wells were dried. The virus titers after serum neutralization at each dilution were counted, and the serum neutralizing antibody titer (PRNT50) was the highest dilution of serum at which the virus titer was neutralized by 50%.

[0129] The results showed that C57BL / 6 mice immunized once with different doses of the attenuated WNV-poly(A) strain developed a strong antibody response after 14 days, with both IgG and neutralizing antibody titers increasing on the 28th day after immunization. The specific IgG antibody titers produced in mice immunized with different doses did not show significant dose dependency, and the IgG antibody titers on the 28th day after immunization were all around 1:12800 (Figure 6A). The neutralizing antibody level increased with increasing immunization dose, and was higher than that of the 100% attenuated WNV-poly(A) strain. 4 Mice immunized with PFU had neutralizing antibody titers of 1:40 to 1:80 on day 28, whereas mice immunized with 100% WNV-poly(A) attenuated strain had neutralizing antibody titers of 1:40 to 1:80 on day 28. 7 Mice immunized with PFU had neutralizing antibody titers ranging from 1:160 to 1:640 (Figure 6B). The WNV-poly(A) attenuated strain was demonstrated to generate a strong antibody response against WNV after immunization of C57BL / 6 mice.

[0130] 2. Protective effect of an attenuated strain of WNV-poly(A) in a C57BL / 6 mouse model On the 30th day after immunization, the Mock group received no treatment, while the other five groups were treated with WT WNV10. 7 PFU was challenged intraperitoneally. The survival status of the mice was recorded and weighed every day, and the virus-containing blood was measured according to the method of Example 4 on the second day after the challenge. As a result, the control group mice immunized with PBS showed a significant weight loss on the fifth day after the challenge (Fig. 6D), a relatively high virus-containing blood level was detected on the second day after the challenge (Fig. 6E), and symptoms such as disheveled hair and paralysis of the hind legs appeared, and all of them died on the ninth day (Fig. 6C). All of the mice immunized with the WNV-poly(A) attenuated strain survived, and like the mice in the Mock group that were not treated, no symptoms were observed and no virus-containing blood was detected. The above test showed that the WNV-poly(A) attenuated strain can provide good immune protection to C57BL / 6 mice as an attenuated vaccine.

[0131] Example 7: CD8 in a mouse model of attenuated West Nile virus + T cell response In this example, we investigated the CD8 + T cell responses were discussed.

[0132] In anti-West Nile virus (WNV) infection, CD8 + T cell responses play an important role. After immunization of mice with an attenuated strain of WNV-poly(A), CD8 + To detect whether the mice could induce T cell responses, two groups of 6-8 week-old female C57BL / 6 mice (3 mice / group) were used. One group was immunized with 100 μg / mL of the attenuated WNV-poly(A) strain. 7 One group was immunized intraperitoneally with PFU, and the other group was immunized with an equal volume of PBS as a control. On the 7th day after immunization, the spleens of the mice were excised and stimulated in vitro with specific polypeptides (WNV E protein and NS4B protein) and, as a positive control, with nonspecific stimulators phorbol myristate acetate (PMA) and ionomycin (IONO). The spleens were then stimulated in vitro with CD8+ / -100% spleen activity in the ELISPOT assay. + IFN-γ + The T cell proliferation status was detected. The results are shown in Figure 7. Mice immunized with the attenuated strain of WNV-poly(A) showed a CD8 + IFN-γ + T cells showed significant activation in the presence of specific polypeptides and nonspecific stimuli, whereas in the mock group, stimulation with specific polypeptides did not activate CD8 + IFN-γ + The above results show that after immunization of C57BL / 6 mice with the rescued WNV-poly(A) attenuated strain, the CD8 T cell activation was more potent against the WNV E protein and NS4B epitope specific. + IFN-γ + It has been shown that it can generate T cell responses.

[0133] Example 8: Long-term immune protection in a mouse model of attenuated West Nile virus In this example, we investigated the long-term immune protective effect of an attenuated WNV-poly(A) strain in C57BL / 6 mice.

[0134] Three groups of 4-week-old female C57BL / 6 mice (5 mice / group) were selected and each group was incubated for 10 4 PFU and 10 7 Mice were immunized intraperitoneally with PFU of the attenuated strain of WNV-poly(A), and mice immunized with PBS served as controls. Blood was collected from the orbit on days 28, 56, 112, and 168 after immunization, and specific IgG and neutralizing antibody titers were detected by the method of Example 5. On day 170 after immunization, WT WNV10 7 The mice were intraperitoneally infected with PFU and challenged. Two days after the challenge, the blood containing the virus was measured by the method of Example 4 to determine the survival rate of the mice. 4 PFU and 10 7 The specific IgG and neutralizing antibody titers induced in mice immunized with PFU of the attenuated WNV-poly(A) strain remained essentially stable within 168 days (Figures 8A and B), and the survival rate of mice after challenge was 100%, with no virus-containing blood detected on day 2 after challenge (Figures 8C and D). The above studies demonstrated that the attenuated WNV-poly(A) strain could provide long-term immune protection to mice.

[0135] Example 9: Pathogenicity of attenuated dengue virus In this example, the pathogenicity of the attenuated DENV strain DENV-poly(A) in AG129 was examined.

[0136] Five groups of 4-week-old female AG129 mice (5 mice / group) were selected and each group was incubated for 10 3 PFU, 10 4 PFU or 10 5 PFU of DENV-poly(A) attenuated strain and 10 4Mice were intraperitoneally infected with PFU WT DENV and injected with an equal volume of PBS as controls. Virus-containing blood was measured by detecting the virus titer of mouse serum on the second and third days after infection. Specifically, about 100 μl of orbital blood was collected from each mouse in a 1.5 ml EP tube, left at 4° C. for 3 h, then centrifuged at 5000 rpm for 5 min, and serum was collected and used to measure the virus titer. The measurement method was the same as that of the virus titer measurement in Example 1. At the same time, the body weight of the mice was measured for 7 consecutive days after infection, and the survival status of the mice within 27 days was observed.

[0137] As a result, all mice inoculated with the DENV-poly(A) attenuated strain survived (Fig. 9A), viremia was not detected at each dose (Fig. 9B), the changes in body weight were consistent with those of the mice immunized with PBS (Fig. 9C), and no other symptoms were observed. 4 Mice inoculated with PFU showed viremia (Fig. 9B), began to lose weight significantly from day 10 (Fig. 9C), and all died by day 21 (Fig. 9A). The virulence of the DENV-poly(A) attenuated strain was sufficiently reduced, indicating that it is non-pathogenic in AG129 mice, has high safety, and can be used as a potential live attenuated vaccine.

[0138] Example 10: Humoral immunity and challenge protection effects in a mouse model of attenuated dengue virus This example examined the humoral immunity and challenge protection effects of an attenuated DENV-poly(A) strain in the AG129 mouse model.

[0139] 1. Humoral immunity in the AG129 mouse model with an attenuated DENV poly(A) strain Five groups of 4-week-old female AG129 mice (5 mice / group) were selected, and each group was infected with 10 of the P1 generation DENV-poly(A) attenuated strains obtained in Example 3. 3 PFU, 10 4 PFU or 10 5Mice were intraperitoneally immunized with PFU and immunized with an equal volume of PBS, and Mock mice that received no treatment were used as controls. On the 28th day after immunization, blood was collected by the orbital blood collection method described in Example 4, and left to stand at 4°C for 3 hours, then centrifuged to collect serum, which was inactivated at 56°C for 30 minutes and frozen and stored at -20°C for use.

[0140] IgG antibody titers were detected by ELISA, and neutralizing antibody titers were detected by PRNT50. 1) Detection of IgG antibody titers by ELISA A 96-well plate was coated with an attenuated DENV-poly(A) strain and blocked with 5% skim milk prepared in PBS. Serum was serially diluted from 1:50 to 4-fold, then incubated with the coated 96-well plate at 37°C for 2 h. The plate was washed three times with PBST, and then incubated with HRP goat anti-mouse antibody at 37°C for 1 h. Color development was performed using a two-component color development kit (Proteintech). The reaction was terminated by adding 1M H2SO4, and the absorbance at 450 nm was detected using a multifunctional microplate reader. The IgG antibody titer was determined at the highest dilution when the absorbance of the immunized mouse serum was twice that of the non-immunized mouse serum.

[0141] 2) Detection of neutralizing antibody titers using PRNT50 2 x 10 cells in each well of a 12-well cell culture plate 5When the cells reached 90% confluence, the serum was serially diluted 2-fold, and 100 μl of each diluted antibody was added to an equal volume of WT-DENV 100 μl of each antibody. After uniformly mixing with PFU, incubate at 37°C for 1h, discard the medium in the well, add the serum-virus mixture after incubation to the corresponding well, adsorb at 37°C for 1h, shake thoroughly once every 15 minutes, extract and discard the virus liquid from each well after adsorption is completed, add 1mL of 2% methylcellulose-containing coating, and culture in a 5% CO2 incubator at 37°C for 72h. After plaque formation, stain with a staining solution containing 1% crystal violet and 3.7% formaldehyde. After treatment at room temperature for 30min, extract the staining solution in the well, wash the bottom of the well with running water, dry, and count the virus titer after neutralization of the serum at each dilution. The neutralizing antibody titer of the serum (PRNT50) was the highest dilution of the serum when the virus titer was neutralized by 50%.

[0142] As a result, AG129 mice immunized once with different doses of the attenuated DENV-poly(A) strain developed strong antibody responses after 28 days. The IgG antibody titers were all around 1:3200 to 1:12800 (Figure 10A). The neutralizing antibody level increased with increasing immunization dose, and was higher than that of the attenuated WNV-poly(A) strain at 100 mg / kg. 3 Mice immunized with PFU had neutralizing antibody titers ranging from 1:40 to 1:80 on day 28, whereas mice immunized with the 10-μg WNV-poly(A) attenuated strain had neutralizing antibody titers ranging from 1:40 to 1:80 on day 28. 5 Mice immunized with PFU had neutralizing antibody titers ranging from 1:80 to 1:160 (Figure 6B). The above study demonstrated that the DENV-poly(A) attenuated strain elicited a strong antibody response against DENV after immunization of AG129 mice.

[0143] 2. Protective effect of DENV-poly(A) attenuated strain against challenge in the AG129 mouse model On the 30th day after immunization, except for the Mock group, which received no treatment, the other four groups of mice were infected with WT DENV 10 6PFU was challenged intraperitoneally. The survival status of the mice was recorded and their body weights were measured every day, and on the third day after the challenge, the virus-containing blood was measured according to the method of Example 4. As a result, the control group mice immunized with PBS started to lose weight significantly from the fourth day after the challenge (Fig. 10D), a relatively high level of virus-containing blood was detected on the third day after the challenge (Fig. 10E), and symptoms such as standing hair on the back appeared, and all of them died on the 16th day (Fig. 10C). All of the mice immunized with the DENV-poly(A) attenuated strain survived, similar to the mice in the mock group that received no treatment, and no symptoms of the disease were observed, and no virus-containing blood was detected. The above test demonstrated that the DENV-poly(A) attenuated strain can provide good immune protection to AG129 mice as an attenuated vaccine.

Claims

1. A 5'-terminal stem-loop region I (5'SLI), stem-loop region II (SLII), repeat circularized sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularized sequence region 3 (CS3), dumbbell region 1 (DB1), repeat circularized sequence region 2 (RCS2), dumbbell region (DB2), and circularized sequence region 2 (CS2) in the 3' untranslated region (3'UTR) of a wild-type Flavivirus virus. a sequence comprising: a 3' untranslated region (3'UTR) that is attenuated by deleting a sequence comprising the sequence listed above; a 3' untranslated region (3'UTR) that is attenuated by deleting a sequence comprising the sequence listed above; a 3' untranslated region (3'UTR) that is attenuated by deleting a sequence comprising the sequence listed above; a 3' untranslated region (3'UTR) that is attenuated by deleting a sequence comprising the sequence listed above;

2. An attenuated Flavivirus obtained by deleting sequences including stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region (DB2), and circularization sequence region 2 (CS2) in the 3' untranslated region (3'UTR) of a wild-type Flavivirus, maintaining circularization sequence region 1 (CS1) and short hairpin-3' end stem-loop region (sHP-3'SL) in the 3' untranslated region (3'UTR), and inserting a poly(A) sequence consisting of 130 to 150 adenylic acids at the site of the deletion, wherein the attenuated Flavivirus is an attenuated dengue virus (DENV).

3. An attenuated Flavivirus obtained by deleting sequences including stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), and dumbbell region (DB) in the 3' untranslated region (3'UTR) of a wild-type Flavivirus, while maintaining the stem-loop region (SL), circularization sequence region 1 (CS1), and short hairpin-3' end stem-loop region (sHP-3'SL) in the 3' untranslated region (3'UTR), and inserting a poly(A) sequence consisting of 130 to 150 adenylic acids at the site of the deletion, wherein the attenuated Flavivirus is an attenuated yellow fever virus (YFV).

4. A method for producing a virus of the genus Flavivirus comprising deleting sequences including 5'-terminal stem-loop region I (5'SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), and circularization sequence region 3 (CS3) in the 3' untranslated region (3'UTR) of a wild-type Flavivirus, and 1. An attenuated Flavivirus obtained by maintaining the deleted region 2 (RCS2), dumbbell region (DB2), circularization sequence region 2 (CS2), circularization sequence region 1 (CS1), and short hairpin-3'-end stem-loop region (sHP-3'SL) and inserting a poly(A) sequence consisting of 130 to 150 adenylic acids at the deletion site, wherein the attenuated Flavivirus is an attenuated West Nile virus (WNV).

5. A method for producing a virus comprising the steps of: deleting a dumbbell region 1 (DB1), a repeat circularization sequence region 2 (RCS2), a dumbbell region (DB2), a circularization sequence region 2 (CS2), and a circularization sequence region 1 (CS1) in the 3' untranslated region (3'UTR) of a wild-type Flavivirus; and isolating a 5'-terminal stem-loop region I (5'SLI), a stem-loop region II (SLII), a repeat circularization sequence region 3 (RCS3), and a 5'-terminal stem-loop region II (SLII) in the 3' untranslated region (3'UTR). An attenuated Flavivirus obtained by maintaining stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), and short hairpin-3'-end stem-loop region (sHP-3'SL) and inserting a poly(A) sequence consisting of 130 to 150 adenylic acids at the deletion site, the attenuated Flavivirus being an attenuated West Nile virus (WNV).

6. The attenuated Flavivirus virus according to any one of claims 1 to 5, wherein the plaque titer of the attenuated Flavivirus virus is 5 x 10 4 to 5 x 10 6 PFU / ml.

7. An attenuated Flavivirus virus described in any one of claims 1 to 5, wherein the virulence of the attenuated Flavivirus virus is reduced by 10 to 100 times compared to that of the parent wild-type virus.

8. An attenuated Flavivirus virus described in any one of claims 1 to 5, wherein the attenuated Flavivirus virus is rescued in baby hamster kidney cells BHK-21 and / or the attenuated Flavivirus virus is amplified in Vero cells, which are African green monkey kidney cells.

9. The attenuated Flavivirus virus described in claim 2, wherein the dengue virus is selected from dengue virus types 1, 2, 3 and 4.

10. The attenuated Flavivirus of claim 1, wherein the nucleotide sequence of the attenuated West Nile virus is SEQ ID NO.

11. The attenuated Flavivirus virus according to claim 1, wherein the nucleotide sequence of the attenuated Japanese encephalitis virus is SEQ ID NO.

2.

12. The attenuated Flavivirus of claim 2, wherein the nucleotide sequence of the attenuated dengue virus is SEQ ID NO.

13. The attenuated Flavivirus of claim 3, wherein the nucleotide sequence of the attenuated yellow fever virus is SEQ ID NO.

14. A DNA capable of being transcribed to generate the RNA genome of the attenuated Flavivirus virus according to any one of claims 1 to 13.

15. The DNA described in claim 14, wherein the DNA is an infectious clone of an attenuated strain of a Flavivirus virus.

16. The DNA described in claim 15, wherein the infectious clone is a plasmid.

17. A cell comprising the attenuated Flavivirus virus according to any one of claims 1 to 13, or the DNA according to any one of claims 14 to 16.

18. A vaccine comprising the attenuated Flavivirus virus according to any one of claims 1 to 13, the DNA according to any one of claims 14 to 16, and / or the cell according to claim 17.

19. A pharmaceutical composition comprising an attenuated Flavivirus virus according to any one of claims 1 to 13, a DNA according to any one of claims 14 to 16, a cell according to claim 17 and / or a vaccine according to claim 18, and a pharma- ceutically acceptable carrier.

20. A method for preparing the attenuated Flavivirus virus according to claim 1, comprising replacing a part of a nucleotide sequence of a 3' untranslated region (3'UTR) of a wild-type virus with a poly(A) sequence and maintaining a circularization sequence region 1 (CS1) and a short hairpin-3' terminal stem-loop region (sHP-3'SL) of the 3' untranslated region (3'UTR), The portion of the nucleotide sequence includes 5'-terminal stem-loop region I (5'SLI), stem-loop region II (SLII), repeat circularization sequence region 3 (RCS3), stem-loop region III (SLIII), stem-loop region IV (SLIV), circularization sequence region 3 (CS3), dumbbell region 1 (DB1), repeat circularization sequence region 2 (RCS2), dumbbell region (DB2), and circularization sequence region 2 (CS2); The method.

21. The method described in claim 20, wherein the attenuated Flavivirus virus is rescued in baby hamster kidney cells BHK-21 and / or the attenuated Flavivirus virus is obtained by amplified in African green monkey kidney cells Vero cells.

22. A vaccine for use in stimulating an immune response against a Flavivirus virus, the vaccine comprising an attenuated Flavivirus virus described in any one of claims 1 to 13, a DNA described in any one of claims 14 to 16, or a cell described in claim 17.

23. The vaccine of claim 22, wherein the vaccine is a live attenuated West Nile virus vaccine, a live attenuated Japanese encephalitis virus vaccine, a live attenuated Dengue virus vaccine, a live attenuated Yellow Fever virus vaccine, or a live attenuated Zika virus vaccine.

24. 20. The vaccine according to claim 18 or the pharmaceutical composition according to claim 19 for use in the treatment or prevention of a disease caused by a Flavivirus virus.

25. The vaccine or drug composition of claim 24, wherein the attenuated Flavivirus virus is an attenuated West Nile virus, an attenuated Japanese encephalitis virus, an attenuated Dengue virus, an attenuated yellow fever virus, or an attenuated Zika virus.

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