WO2024048793A1

WO2024048793A1 - Attenuated virus for treating infections

Info

Publication number: WO2024048793A1
Application number: PCT/JP2023/032142
Authority: WO
Inventors: 康宏保富; 智崇岡村
Original assignee: 国立研究開発法人医薬基盤・健康・栄養研究所
Priority date: 2022-09-02
Filing date: 2023-09-01
Publication date: 2024-03-07

Abstract

The present disclosure provides a composition for treating viral infections. In one aspect, the present disclosure provides a composition for treating viral infections in a subject, the composition comprising a nucleic acid construct which contains a nucleic acid sequence encoding an attenuated virus or an equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule. In one embodiment, a composition according to the present disclosure is characterized by being administered after viral infections. A composition according to the present disclosure completely eliminates viruses in a subject.

Description

Attenuated viruses to treat infections

The present disclosure provides compositions and related techniques for treating viral infections. More particularly, techniques are provided relating to nucleic acid constructs comprising a nucleic acid sequence encoding an attenuated virus or equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule.

Although significant resources have been invested in the development of therapeutic drugs to combat viral infections, this task has not yet been achieved for many infectious diseases. Although antiretroviral therapy has resulted in a dramatic reduction in HIV-related morbidity and mortality, it cannot cure HIV (Non-Patent Document 1). There are two cases in which HIV remission was achieved by cell transplantation. The development of an effective vaccine could prevent the spread of HIV infection, but there has been little success in vaccine development over the past 30 years. The only successful HIV vaccine to date was evaluated in the RV144 clinical trial, with an overall efficacy of only 31% (Non-Patent Document 2).

The inventors have now discovered that a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or its equivalent and a nucleic acid sequence encoding an adjuvant molecule treats a viral infection in a subject, completing the invention. reached. Any adjuvant molecule may be used as long as it has adjuvant activity. We have newly discovered that when such a nucleic acid construct is administered to a patient after infection, the virus is eliminated from the body. The idea of using such a nucleic acid construct therapeutically is unprecedented, and it is a highly versatile technology that can be used with any virus.

Therefore, the present invention provides, for example, the following items.
(Item 1)
A composition for treating a viral infection in a subject, comprising a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule.
(Item 2)
The composition according to the above item, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of accessory genes and regulatory genes.
(Item 3)
The composition according to any one of the above items, wherein the attenuated virus is deficient in an accessory gene.
(Item 4)
The composition according to any one of the above items, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of Vif, Vpr, Vpx, Vpu, and Nef or its corresponding gene.
(Item 5)
The composition according to any one of the above items, wherein the attenuated virus is a nef-deficient attenuated virus.
(Item 6)
Composition according to any of the preceding items, wherein the nucleic acid sequence encoding the adjuvant molecule is incorporated into the nucleic acid sequence encoding the attenuated virus or equivalent thereof.
(Item 7)
The composition according to any one of the above items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus.
(Item 8)
The composition according to any of the preceding items, wherein the attenuated virus is a chronically infectious virus.
(Item 9)
The attenuated virus is an attenuated virus selected from the group consisting of AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). The composition according to any one of the above items, which is an infected virus.
(Item 10)
The composition according to any one of the above items, wherein the adjuvant molecule is an adjuvant molecule derived from acid-fast bacteria.
(Item 11)
Composition according to any one of the preceding items, wherein the adjuvant molecule is selected from the group consisting of Ag85B proteins.
(Item 12)
The composition according to any one of the preceding items, wherein the composition induces a Th1 type immune response.
(Item 13)
The composition according to any one of the preceding items, wherein the composition completely eliminates the virus in the subject.
(Item 14)
Composition according to any of the preceding items, characterized in that said composition is administered after a viral infection.
(Item 15)
The composition according to any of the preceding items, wherein the nucleic acid construct is cloned.
(Item 16)
A companion reagent for use in a method for determining whether treatment with a composition according to any of the above items is required, comprising a diagnostic agent for viral infection.
(Item 17)
A kit for treating a viral infection in a subject, comprising a composition according to any one of the above items, a diagnostic agent for viral infection, and instructions.
(Item 18)
A method for treating a viral infection in a subject, the method comprising administering to the subject a composition according to any one of the preceding items.
(Item 19)
Use of a composition according to any of the above items in the manufacture of a medicament for treating a viral infection in a subject.
(Item 20)
The composition according to any one of the preceding items, wherein the subject has previously been vaccinated with a BCG vaccine.
(Item 1A)
A method of treating a viral infection in a subject, the method comprising administering an effective amount of a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or an equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule. Including, methods.
(Item 2A)
The method according to the above item, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of accessory genes and regulatory genes.
(Item 3A)
The method according to any one of the above items, wherein the attenuated virus is deficient in an accessory gene.
(Item 4A)
The method according to any one of the above items, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of Vif, Vpr, Vpx, Vpu, and Nef or its corresponding gene.
(Item 5A)
The method according to any one of the above items, wherein the attenuated virus is a nef-deficient attenuated virus.
(Item 6A)
A method according to any of the preceding items, wherein the nucleic acid sequence encoding the adjuvant molecule is incorporated into the nucleic acid sequence encoding the attenuated virus or equivalent thereof.
(Item 7A)
The method according to any one of the above items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus.
(Item 8A)
A method according to any of the preceding items, wherein the attenuated virus is a chronically infectious virus.
(Item 9A)
The attenuated virus is an attenuated virus selected from the group consisting of AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). The method according to any one of the above items, wherein the virus is a virus.
(Item 10A)
The method according to any one of the above items, wherein the adjuvant molecule is an adjuvant molecule derived from acid-fast bacteria.
(Item 11A)
A method according to any of the preceding items, wherein the adjuvant molecule is selected from the group consisting of Ag85B proteins.
(Item 12A)
The method according to any of the preceding items, wherein the nucleic acid construct induces a Th1-type immune response.
(Item 13A)
The method of any one of the preceding items, wherein the nucleic acid construct completely eliminates the virus in the subject.
(Item 14A)
Method according to any of the preceding items, characterized in that the nucleic acid construct is administered after viral infection.
(Item 15A)
The method according to any of the preceding items, wherein the nucleic acid construct is cloned.
(Item 16A)
A method of determining whether a subject requires treatment with a composition according to any one of the above items, the method comprising administering to the subject a diagnostic agent for viral infection. .
(Item 17A)
A method of treating a viral infection in a subject, the method comprising: administering to the subject a diagnostic agent for viral infection; and administering to the subject an effective amount of the composition according to any one of the above items. A method, including doing.
(Item 18A)
The method of any one of the preceding items, wherein the subject has previously been vaccinated with a BCG vaccine.
(Item 1B)
Use of a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or its equivalent and a nucleic acid sequence encoding an adjuvant molecule in the manufacture of a medicament for treating a viral infection in a subject.
(Item 2B)
The use according to the above item, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of accessory genes and regulatory genes.
(Item 3B)
The use according to any of the above items, wherein the attenuated virus is deficient in an accessory gene.
(Item 4B)
The use according to any one of the above items, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of Vif, Vpr, Vpx, Vpu, and Nef or its corresponding gene.
(Item 5B)
The use according to any one of the above items, wherein the attenuated virus is a nef-deficient attenuated virus.
(Item 6B)
Use according to any of the preceding items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated into the nucleic acid sequence encoding the attenuated virus or its equivalent.
(Item 7B)
The use according to any of the above items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus.
(Item 8B)
The use according to any of the preceding items, wherein the attenuated virus is a chronically infectious virus.
(Item 9B)
The attenuated virus is an attenuated virus selected from the group consisting of AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). The use according to any one of the above items, which is a modified virus.
(Item 10B)
The use according to any of the above items, wherein the adjuvant molecule is an adjuvant molecule derived from acid-fast bacteria.
(Item 11B)
Use according to any of the above items, wherein said adjuvant molecule is selected from the group consisting of Ag85B proteins.
(Item 12B)
The use according to any of the above items, wherein the nucleic acid construct induces a Th1 type immune response.
(Item 13B)
The use according to any of the preceding items, wherein said nucleic acid construct completely eliminates the virus in said subject.
(Item 14B)
Use according to any of the above items, characterized in that the nucleic acid construct is administered after viral infection.
(Item 15B)
The use according to any of the above items, wherein said nucleic acid construct is cloned.
(Item 16B)
The use according to any of the preceding items, wherein said subject has previously been vaccinated with a BCG vaccine.
(Item 1C)
A nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or an equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule for treating a viral infection in a subject.
(Item 2C)
The nucleic acid construct according to the above item, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of accessory genes and regulatory genes.
(Item 3C)
The nucleic acid construct according to any one of the above items, wherein the attenuated virus is deficient in an accessory gene.
(Item 4C)
The nucleic acid construct according to any one of the above items, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of Vif, Vpr, Vpx, Vpu, and Nef or its corresponding gene.
(Item 5C)
The nucleic acid construct according to any one of the above items, wherein the attenuated virus is a nef-deficient attenuated virus.
(Item 6C)
Nucleic acid construct according to any of the preceding items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated into the nucleic acid sequence encoding the attenuated virus or equivalent thereof.
(Item 7C)
The nucleic acid construct according to any one of the above items, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus.
(Item 8C)
The nucleic acid construct according to any one of the above items, wherein the attenuated virus is a chronically infectious virus.
(Item 9C)
The attenuated virus is an attenuated virus selected from the group consisting of AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). The nucleic acid construct according to any one of the above items, which is a viral virus.
(Item 10C)
The nucleic acid construct according to any one of the above items, wherein the adjuvant molecule is an adjuvant molecule derived from acid-fast bacteria.
(Item 11C)
Nucleic acid construct according to any one of the preceding items, wherein said adjuvant molecule is selected from the group consisting of Ag85B proteins.
(Item 12C)
The nucleic acid construct according to any one of the above items, wherein the nucleic acid construct induces a Th1 type immune response.
(Item 13C)
The nucleic acid construct according to any one of the preceding items, wherein the nucleic acid construct completely eliminates the virus in the subject.
(Item 14C)
Nucleic acid construct according to any one of the above items, characterized in that said nucleic acid construct is administered after viral infection.
(Item 15C)
The nucleic acid construct according to any one of the above items, wherein the nucleic acid construct is cloned.
(Item 16C)
A diagnostic agent for viral infection for use in a method for determining whether treatment with the composition according to any one of the above items is required.
(Item 17C)
The nucleic acid construct according to any one of the preceding items, wherein said subject has previously been vaccinated with a BCG vaccine.
(Item 1D)
A composition, nucleic acid molecule, method, use or nucleic acid construct according to any one of the preceding items, wherein said composition, nucleic acid molecule, method, use or nucleic acid construct is for treating a viral infection.

In the present disclosure, it is intended that the one or more features described above may be provided in further combinations in addition to the specified combinations. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, as appropriate.

FIG. 1 shows an overview of the experiment in Example 3. Figure 2 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in monkeys (#137) administered only with anti-HIV drugs. Figure 3 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in monkeys (#139) administered only with anti-HIV drugs. Figure 4 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in a monkey (#142) administered twice with low-dose SHIV-Ag85B. Figure 5 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in monkeys (#141) administered once with high dose SHIV-Ag85B. Figure 6 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in monkeys (#140) administered multiple times with high-dose SHIV-Ag85B. Figure 7 shows the dynamics of plasma viral load and CD4 ⁺ T cell number in monkeys (#138) administered once each with low and high doses of SHIV-Ag85B. FIG. 8 shows the administration schedule for each individual in Example 2. FIG. 9A shows the change in plasma virus amount in a monkey (#138) that was administered twice with low doses of SHIV-Ag85B in Example 3. FIG. 9B shows the change in plasma virus amount in a monkey (#142) that was administered once with a high dose of SHIV-Ag85B in Example 3. Figure 10 shows that in Example 3, for one monkey (#141) that was administered a dose of SHIV-Ag85B, only a short-term effect was observed at the beginning; , shows a long-term suppressive effect after the fifth vaccination. FIG. 11A shows the results of an untreated monkey (#137) that received only anti-HIV drugs in Example 4. FIG. 11B shows the results of an untreated monkey (#139) that received only anti-HIV drugs in Example 4. FIG. 12 is an experimental protocol for verifying the change in plasma virus amount when the composition according to the present disclosure is administered after inoculation with the HIV virus in Example 4. FIG. 13 shows the results of Example 4 showing that the anti-HIV drug was successful regardless of individual differences. Figure 14 shows that monkeys (#164, #165 and #167) that received high doses (5.0 x ¹⁰⁴ _TCID50 ) of SHIV-Ag85B were treated with high doses of SHIV-Ag85B four times at one week intervals. It is a figure showing the amount of virus in plasma when administered. FIG. 15 shows that monkeys (#162, #166, and #168) that received high doses (5.0×10 ⁴ TCID ₅₀ ) of SHIV-NI in Example 5 were treated with high doses of SHIV-NI four times. These are the results of plasma virus amount at the time of administration. FIG. 16 shows the results of the plasma virus amount in the control group not containing Ag85B in Example 5, 2 weeks after discontinuation of the drug.

The present disclosure will be described below. Throughout this specification, references to the singular should be understood to include the plural unless specifically stated otherwise. Accordingly, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to also include the plural concept, unless specifically stated otherwise. Further, it should be understood that the terms used herein have the meanings commonly used in the art unless otherwise specified. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

(Definition of terms)
Definitions of terms specifically used in this specification are listed below.

As used herein, "about" means ±10% of the indicated value.

As used herein, "attenuated virus" refers to a virus that has reduced pathogenicity compared to the parent virus but retains the ability to induce an immune response. Attenuation can be confirmed by the toxicity to cells and/or pathogenicity to animals. It is preferably determined by animal experiments or the like. Viruses to be attenuated include, but are not limited to, chronically infectious viruses such as AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, and hepatitis C virus (HCV). Generally, the term "attenuation" refers to artificially reducing the virulence of a pathogen by mutating its genes so that the pathogen loses virulence but retains immunogenicity. Generally, attenuation of pathogen toxicity is achieved by UV irradiation, chemical treatment, or in vitro continuous high-order subculturing. Genes are artificially modified, for example by deleting specific nucleotides in a known sequence, to reduce toxicity.

As used herein, the term "chronically infectious virus" refers to an infectious virus that exhibits lifelong infection.

As used herein, "adjuvant molecule" refers to one or more substances that cause stimulation of the immune system. The adjuvant molecule can be a protein derived from mycobacteria, for example Ag85B protein.

As used herein, "nucleic acid construct" or "nucleic acid construct" refers to a DNA or RNA molecule that includes a nucleotide sequence that encodes a protein.

As used herein, "subject" refers to mammals, including human patients, who are the targets of the treatment of the present disclosure.

As used herein, "viral infection" refers to the entry of a virus into a cell such that the encoded protein product is expressed.

As used herein, the term "accessory gene" refers to a gene that is not essential for virus proliferation. Examples include, but are not limited to, Vif, Vpr, Vpx, Vpu, and Nef.

nef (negative factor) is a protein of approximately 27 kDa that is encoded on the 3' side of the HIV-1 gene. Although it was originally reported as a factor that suppresses virus proliferation, it has since been found to actually accelerate virus proliferation in primary cultured cells and in vivo, and to be associated with HIV-1 pathogenicity. HIV-1 Nef protein, anchor domain superfamily (IPR027480), HIV-1 Nef protein, core domain superfamily (IPR027481) etc. are known.

Nucleic acid constructs comprising a nucleic acid sequence encoding an attenuated virus or its equivalent of the present disclosure and a nucleic acid sequence encoding an adjuvant molecule unexpectedly exhibit increased safety, reduced toxicity, and effective targeting. For the first time, we have been able to provide a practical therapeutic and prophylactic vaccine against a viral disease.

As used herein, the term "regulatory gene" refers to a gene that controls gene expression.

As used herein, "deficient" refers to the loss of a gene. Defect also includes the fact that a part of the gene has disappeared and is no longer functioning.

As used herein, "corresponding genes" refer to genes that have different names between different species but are functionally equivalent.

As used herein, "incorporated" refers to the addition of one or more nucleic acids into a nucleic acid construct.

As used herein, "cloned" refers to producing and/or isolating a population with the same genetic makeup.

As used herein, the term "companion reagent" refers to a reagent used to test whether a therapeutic agent is effective before treatment.

As used herein, the term "kit" refers to parts that should be provided (e.g., vaccines, test reagents, diagnostic reagents, therapeutic reagents, antibodies, labels, instructions, etc.) usually divided into two or more compartments. unit. This kit format is preferable when the purpose is to provide a composition that should not be provided mixed for reasons of stability etc., but is preferably mixed immediately before use. Such kits preferably include the parts provided (e.g., instructions or instructions describing how to use the test, diagnostic, or therapeutic agent, or how to process the reagents). When the kit is used as a reagent kit herein, the kit usually includes instructions on how to use the vaccine, test agent, diagnostic agent, therapeutic agent, antibody, etc. This includes books, etc.

As used herein, "Ag85B" refers to an immunogenic protein secreted by acid-fast bacteria. When bacteria are dyed with a pigment, acid-fast bacteria exhibit a property that the pigment is not bleached by acid, that is, they are resistant to acid. Acid-fast bacteria (mycobacteria) are broadly classified into Mycobacterium tuberculosis, Mycobacterium leprae, and non-tuberculous mycobacteria. A typical source of Ag85B is Mycobacterium Tuberculosis. Ag85B is antigen 85-B, 85B, Extracellular alpha-antigen, Antigen 85 complex B, Ag85B, Mycolyl transfera se 85B, EC 2.3.1. -, Fibronectin-binding protein B, 30 kDa extracellular protein, fbpB, A85B, Major Secretory Prote Also referred to as in Antigen 85B. A typical access number is Q847N4. https://www. uniprot. See org/uniprotkb/Q847N4/entry. A typical nucleic acid sequence ID is AY207396g, and a typical protein sequence ID is AAO62005.1. In this specification, it is shown as SEQ ID NO: 1 (nucleic acid sequence) and SEQ ID NO: 2 (amino acid sequence), but is not limited to this, and any immunogenic protein secreted by acid-fast bacteria is within the scope of the present disclosure. It is understood that there is As indicated in this disclosure, Ag85B is an immunoadjuvant. Antigen 85B (Ag85B) is considered an immunogenic protein that induces a strong Th1 immune response in hosts sensitized with Bacillus Calmette-Guerin. Ag85B belongs to the Ag85 family and is one of the most predominant proteins secreted by most mycobacterial species.

As used herein, "operably" refers to a state in which transcription or translation of a nucleic acid sequence is under the control of an expression control element, transcription or translation of a nucleic acid sequence is appropriately controlled, and functional expression is achieved. refers to something in

As used herein, "Th1 type immune response" is a cellular immune response mediated by T lymphocytes, and refers to a response by cytokines and/or chemokines produced by activated T cells.

As used herein, "Th2-type immune response" refers to a humoral immune response mediated by secreted antibodies produced by B cells.

As used herein, "simian/human immunodeficiency (chimeric) virus (SHIV)" refers to a simian/human immunodeficiency chimeric virus in which at least some genes have been replaced with those of the HIV-1 gene. In SHIV, in addition to the env gene, the vpr, rev, tat, and vpu genes can be further replaced with those of HIV-1.

As used herein, "completely eliminating the virus" refers to a state in which the virus is completely removed from the living body. A state in which the virus has been completely removed from the living body means that it is below the detection limit by highly sensitive detection methods such as PCR, and it does not show any clinical symptoms.

As used herein, "vaccine" refers to a substance containing or encoding an antigen that provides active immunity against the substance containing the antigen but does not cause disease. As used herein, vaccine refers to one used prophylactically. As used herein, "DNA vaccine" means a nucleic acid encoding a vaccine antigen, and this general name is used because DNA (particularly plasmid DNA) is mainly used. In addition, as an embodiment using a nucleic acid, there is also an embodiment in which it is incorporated into a viral vector or the like and delivered into a living body, and in this case, it is understood that the nucleic acid may be provided in a form other than DNA. In such cases, it is also called a "nucleic acid vaccine." DNA vaccines usually take the form of plasmid DNA, but when a DNA vaccine in the form of plasmid DNA is subcutaneously administered, the plasmid DNA is taken up into subcutaneous cells and produces the target antigen protein within the cells.

As used herein, the term "antigen" (also referred to as Ag) refers to any substrate that can be specifically bound by an antibody molecule. As used herein, "immunogen" refers to an antigen capable of initiating lymphocyte activation resulting in an antigen-specific immune response.

As used herein, "nucleic acid construct," "construct," "construct," or "gene construct" are used interchangeably and are isolated from naturally occurring genes or combined in a non-naturally occurring manner; A nucleic acid molecule that includes a plurality of juxtaposed collections of nucleic acids.

Amino acids may be referred to herein by either their commonly known three-letter symbol or the one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly recognized one-letter codes. As used herein, comparisons of similarity, identity, and homology of amino acid sequences and base sequences are calculated using default parameters using BLAST, a sequence analysis tool. The identity search can be performed using, for example, NCBI's BLAST 2.2.28 (published on April 2, 2013) (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). The identity value in this specification usually refers to the value obtained when alignment is performed using the above-mentioned BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value is taken as the identity value. When identity is evaluated in multiple areas, the highest value among them is taken as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity. When comparing amino acid sequences with BLAST, Blastp can be used as an algorithm with default settings. The measurement results are quantified as Positives or Identities. Homology of amino acid sequences and base sequences can be determined by the algorithm BLAST by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, score=100 and wordlength=12. Furthermore, when an amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score=50 and wordlength=3. BLAST and Gapped When using BLAST programs, use the default parameters for each program. Specific techniques for these analysis methods are publicly known (http://www.ncbi.nlm.nih.gov.).

The nucleic acid or protein used in the present disclosure may include a target amino acid or base sequence in which one or more amino acids or nucleotides are substituted, deleted, and/or added. Here, in the full-length chimeric protein amino acid sequence, "one or more" usually means within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 1 amino acid). In addition, in the amino acid sequence of a domain, "one or more" usually means 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (for example, 3 amino acids or less, 2 amino acids or less, 1 amino acid or less). amino acids). In order to maintain the biological activity of the present disclosure of the chimeric protein, it is desirable that the amino acid residue to be mutated be mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side chains Examples include amino acids (H, F, Y, W) having the following (each in parentheses represents a one-letter code for an amino acid). These are also referred to herein as "conservative substitutions." It is known that proteins having amino acid sequences modified by deletion, addition and/or substitution of one or more amino acid residues with other amino acids maintain their biological activity. (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M.J. & Smith, M. Nucleic Acids Research (1982) 10, 6487 - 6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Therefore, in one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be less than or equal to any of these values. Chimeric proteins with deletions etc. can be produced, for example, by site-directed mutagenesis, random mutagenesis, biopanning using an antibody phage library, or the like. In the present disclosure, "70% or more" may be, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more, and "80% or more" may be, for example, 80% or more. , 85, 90, 95, 96, 97, 98, 99% or more. "90% or more" may be, for example, 90, 95, 96, 97, 98, 99% or more; It may be within the range of any two values. "Homology" may be calculated as the ratio of the number of homologous amino acids in two or more amino acid sequences according to a method known in the art. Before calculating the ratio, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and gaps are introduced into part of the amino acid sequences if necessary to maximize the ratio of identical amino acids. Methods for alignment, ratio calculation, comparison, and related computer programs are conventionally well known in the art (eg, BLAST, GENETYX, etc.). In the case of "identity", the proportion of identical amino acids is calculated, and in the case of "similarity", the proportion of similar amino acids is calculated. Similar amino acids include, but are not limited to, amino acids that allow conservative substitutions.

Some of the constructs specifically shown in the present disclosure are also included within the scope of the present disclosure. As used herein, "part", "fragment", or "fragment" refers to a polypeptide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). or polynucleotide. The length of the fragment can be changed as appropriate depending on the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids; lengths expressed in integers not specifically listed here (e.g., 11, etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides, which are specifically listed here. A length expressed as an integer (eg, 11, etc.) may also be suitable as a lower limit. It is understood herein that such fragments fall within the scope of the disclosure, for example, if the full-length version functions as a vaccine, so long as the fragment itself also functions as a vaccine. "Functional equivalents" of those specifically shown in this disclosure (such as constructs) are also encompassed by this disclosure. As used herein, the term "functional equivalent" refers to any object that has the same intended function but a different structure with respect to the original entity.

In accordance with the present disclosure, the term "activity" herein refers to the function of a molecule in the broadest sense. Activity generally includes, but is not intended to be limiting, a biological, biochemical, physical, or chemical function of the molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules. stability, and the ability to localize to specific subcellular locations. Where applicable, the term also relates to the function of protein complexes in the broadest sense. As used herein, "biological activity" includes activation of photoreactions and the like.

As used herein, the term "functional equivalent" refers to any object that has the same intended function but a different structure with respect to the original entity. Therefore, an "HIV virus-encoded protein" or a chimeric functional equivalent thereof is not the HIV virus-encoded protein or chimera thereof itself, but a variant or variant of the HIV virus-encoded protein or chimera thereof ( (e.g., amino acid sequence variants, etc.) which have the biological effects of the protein encoded by the HIV virus or chimera thereof, and the protein encoded by the HIV virus or its antibody itself or its chimera at the time of action. that can be changed into a protein encoded by the HIV virus or a chimeric variant or variant thereof (e.g., a protein encoded by the HIV virus or a chimera thereof or a protein encoded by the HIV virus or a chimeric variant or variant thereof); It is understood that the invention encompasses the encoding nucleic acids, as well as vectors, cells, etc. that contain the nucleic acids. Functional equivalents of the present disclosure may include insertions, substitutions and/or deletions of one or more amino acids, or additions to one or both ends of the amino acid sequence. As used herein, "insertion, substitution, and/or deletion of one or more amino acids, or addition to one or both ends of an amino acid sequence" refers to the well-known method such as site-directed mutagenesis. It means that a modification has been made by a technical method or by natural mutation, such as substitution of a plurality of amino acids to the extent that they can occur naturally. The modified amino acid sequence may include insertions, substitutions, or deletions of, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, particularly preferably 1 to 2 amino acids. It can be deleted or added to one or both ends. The modified amino acid sequence preferably has one or more (preferably one or several or one, two, three, or four) conservative substitutions in the amino acid sequence of the protein encoded by the HIV virus. It may be an amino acid sequence having

As used herein, "treatment" refers to preventing the worsening of a disease or disorder (e.g., due to a viral infection), preferably maintaining the status quo, if such a disease or disorder occurs. , more preferably alleviation, and even more preferably eradication, and includes the ability to exhibit a symptom-improving effect or preventive effect on a patient's disease or one or more symptoms associated with the disease. Performing a diagnosis in advance and providing appropriate treatment is called "companion treatment," and the diagnostic agent used for this purpose is sometimes called a "companion diagnostic agent."

As used herein, the term "therapeutic drug (agent)" in a broad sense refers to any drug that can treat a target condition (eg, retinal degenerative disease, etc.). In one embodiment of the present disclosure, a "therapeutic agent" may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced, for example, by mixing the active ingredient and the carrier described above, and by any method known in the technical field of pharmaceutical science. The therapeutic agent is not limited in its usage form as long as it is used for treatment, and may be an active ingredient alone or a mixture of an active ingredient and any other ingredient. Further, the shape of the carrier is not particularly limited, and may be, for example, solid or liquid (eg, buffer).

As used herein, "prevention" refers to preventing a certain disease or disorder (for example, retinal degenerative disease) from developing into such a condition before it occurs. Diagnosis can be performed using the drug of the present disclosure, and if necessary, the drug of the present disclosure can be used to prevent, for example, retinal degenerative diseases, or to take preventive measures. In the present specification, the term "preventive drug (agent)" broadly refers to any drug that can prevent a desired condition (eg, vesicular transport disorder, apoptosis, etc.).

The present disclosure may be provided as a kit. As used herein, the term "kit" refers to parts that are usually divided into two or more compartments and provided (e.g., multiple nucleic acid constructs, lyophilized drugs and administration buffer, instructions, etc.) ) is the unit provided. Such kits preferably include provided parts, such as instructions or instructions describing how to use the nucleic acid construct or how to treat the reagents. Advantageously, the specification also includes instructions for use and the like.

The term "active ingredient" as used herein refers to an ingredient that is contained in the amount necessary for the composition of the present disclosure to achieve the desired therapeutic, preventive, or progression-suppressing effect, and the effect is less than the desired level. Other ingredients may also be included as long as they do not impair the quality. Furthermore, the medicament, composition, etc. of the present disclosure may be formulated. Furthermore, the route of administration of the medicament, composition, etc. of the present disclosure may be either oral or parenteral, and can be appropriately determined depending on the form of the preparation and the like. Constructs of the present disclosure can be used as active ingredients.

As used herein, "instructions" (including package inserts, labels used by the U.S. FDA, etc.) describe instructions to a physician or other user on how to use the present disclosure. This instruction sheet contains words instructing the administration of the medicament of the present disclosure. In addition, the instructions may include language instructing intravenous administration (for example, by injection) as the administration site. This instruction is prepared in accordance with the format prescribed by the regulatory authority of the country where the disclosure will be made (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and approved by that regulatory authority. It will be clearly stated that it was received. The instruction sheet is a so-called package insert or label, and is usually provided in paper media, but is not limited to this, and may also be provided in electronic media (e.g., a homepage provided on the Internet, PDF, etc.). It may also be provided in a format such as email).

(Preferred embodiment)
Although a description of preferred embodiments is provided below, it should be understood that this embodiment is an illustration of the present disclosure and that the scope of the present disclosure is not limited to such preferred embodiments. It should also be understood that those skilled in the art can readily make modifications, changes, etc. within the scope of the present disclosure with reference to the following preferred embodiments. Those skilled in the art can combine any of these embodiments as appropriate.

(Attenuated virus adjuvant complex molecule)
The present disclosure provides attenuated viral adjuvant complex molecules that exhibit breakthrough effects against viral infections. The molecule of the present disclosure, which shows potential as a new modality, offers the possibility of eradicating viral infection, and can be used in various ways.

In one aspect, the present disclosure provides a composition for treating a viral infection in a subject, comprising a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or an equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule. We provide related technologies such as therapeutic or preventive methods and uses that utilize this.

The complex molecule of an attenuated virus and an adjuvant molecule used in the present disclosure can be provided in both a protein form and a nucleic acid form, or can be provided in a form other than these. Preferably, a nucleic acid construct is provided that includes a nucleic acid sequence encoding an attenuated virus or equivalent thereof and a nucleic acid sequence encoding an adjuvant molecule. Such nucleic acid constructs can be provided in a form that incorporates adjuvant molecules in an attenuated virus.

Without wishing to be bound by theory, such attenuated viral adjuvant molecule complex molecules are superior to conventional therapeutic agents in that they can sense the targeted viral disease and prevent recurrence. It is attracting attention as having a unique effect.

Here, the attenuated virus may be deleted in one or more genes such as accessory genes and regulatory genes. In the present disclosure, deletion refers to a gene that has been modified in such a way that it no longer performs its original function. Refers to the fact that the person is no longer able to perform his or her functions.

In one embodiment, the genes that can be deleted in the attenuated virus may advantageously be accessory genes, such as Vif, Vpr, Vpx, Vpu, and Nef or their equivalents (e.g., corresponding genes). be able to. Even if such a gene is deleted, it may still function as a virus, but it may be advantageous to have reduced toxicity. In one preferred embodiment, the attenuated virus may be an attenuated nef-deficient virus. This is because while the toxicity is reduced, it is also effective in treating viruses.

Nucleic acid sequences encoding adjuvant molecules used in the present disclosure may advantageously be incorporated into nucleic acid sequences encoding attenuated viruses or equivalents thereof.

(Construct manufacturing method)
Constructs of the present disclosure may be produced as follows. Representative examples thereof are described in the Examples.

A molecular clone is created from the gene using the virus isolated from the patient. A gene, such as Nef of a virus (for example, HIV), which induces attenuation when deleted, is removed from a molecular clone by gene editing technology. An adjuvant gene (eg, Ag85B gene) is inserted into the removal site to produce an attenuated virus expressing the adjuvant gene.

In one embodiment, it may be advantageous that a nucleic acid sequence encoding an adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus. Since the nef gene is a gene that controls immune evasion of the AIDS virus, deletion of this portion results in attenuation by the body's immune function. Incorporation of adjuvant molecules induces strong cellular immunity, induces a strong immune response, and further attenuates the virus.

The attenuated virus used can be, but is not limited to, a chronically infectious virus. The compositions of the present disclosure are applicable to any virus, not just chronically infected viruses.

Attenuated viruses that may be utilized in the present disclosure include attenuated viruses such as AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). could be. These attenuated viruses may be prepared by using known attenuated viruses or by isolating the virus from a patient and deleting the gene that induces attenuation.

In the examples of the present disclosure, any adjuvant molecule can be used, and for example, an adjuvant molecule derived from acid-fast bacteria (eg, Ag85B) can be used. This is because many people worldwide, including in Japan, have been vaccinated with BCG vaccine, and BCG vaccination is expected to enhance the adjuvant effect of Ag85B. One specific exemplary adjuvant molecule may be Ag85B protein. Without wishing to be bound by theory, the addition of Ag85B induces strong cellular immunity, induces a strong immune response, and further attenuates the virus.

In a preferred embodiment, the subject has previously been vaccinated with a BCG vaccine. Without wishing to be bound by theory, since BCG inoculation enhances the effect of the adjuvant of the present disclosure such as Ag85B, it is expected that in the BCG inoculated group, the therapeutic effect of HIV-Ag85B against HIV will be high. . There are some countries (such as the United States) where the BCG vaccine is not implemented due to contraindications, so the BCG vaccine is not necessarily required. Sufficient effects are expected even in the BCG non-inoculated group.

One of the characteristics of the attenuated virus adjuvant molecule complex of the present disclosure may be that it induces a Th1 type immune response. Additionally, the attenuated virus adjuvant molecule complexes of the present disclosure can substantially eliminate, and preferably completely eliminate, the virus in a subject.

The attenuated virus adjuvant molecule complex of the present disclosure is advantageous in that it can burn out the virus even when administered after viral infection. Without wishing to be bound by theory, this is because inducing a Th1 type immune response has been shown to be effective in substantially eliminating viruses.

In one embodiment, the nucleic acid construct used may be one that has been cloned.

(companion reagent)
In one aspect, the present disclosure provides companion reagents, including diagnostic agents for viral infection, for use in methods of determining whether treatment according to the present disclosure is required. If the presence of viruses is tested in advance using a companion reagent, the effectiveness of treatment with the composition of the present disclosure can be diagnosed. In this diagnosis, if a positive result is obtained, it can be determined that treatment with the composition of the present disclosure is effective. In one embodiment of the present invention, "companion diagnosis" includes diagnosis performed for the purpose of assisting in optimal medication administration by predicting individual patient differences in drug effects and side effects through testing. Companion reagents include diagnostic agents for viral infection for detecting viruses in subjects suspected of infection, such as antibodies against viral antigens, antigen-binding proteins or peptides, nucleic acids that bind to viral nucleic acids, and variants thereof. These include, but are not limited to:

(kit)
The present disclosure provides a kit for treating a viral infection in a subject, comprising a composition of the present disclosure, a diagnostic agent for a viral infection, and instructions. This kit includes diagnostic agents for viral infection for detecting the virus in a subject suspected of infection, such as antibodies against viral antigens, antigen-binding proteins or peptides, nucleic acids that bind to viral nucleic acids, and modified versions thereof. These include, but are not limited to: The kit further includes a composition of the present disclosure, embodiments of which can be used alone or in combination with any of the embodiments of the present disclosure.

(Treatment)
The present disclosure is a method for treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of a composition or nucleic acid construct of the present disclosure to the subject in need thereof. I will provide a. In one aspect, the present disclosure provides a method of prophylaxis by administering a composition or medicament of the present disclosure. In one embodiment, a composition or nucleic acid construct of the present disclosure is administered by injection. In another embodiment, a composition or nucleic acid construct of the present disclosure is administered internally. In certain embodiments, a composition or nucleic acid construct of the present disclosure may be provided with a preservation solution. In some embodiments, the storage solution can be a buffer. In other embodiments, a composition or nucleic acid construct of the present disclosure may be provided in a container. In certain embodiments, a container containing a composition or nucleic acid construct of the present disclosure can be a syringe.

In another aspect, the disclosure provides the use of a composition or nucleic acid construct of the disclosure in the manufacture of a medicament for treating a viral infection in a subject.

Administration of agents associated with the attenuated viruses of the present disclosure can be carried out by any suitable means that, in combination with other ingredients, provides concentrations of therapeutic agent effective in preventing, ameliorating, or mitigating the virus. The agent may be contained in any suitable carrier material in any suitable amount and is generally present in an amount from 1 to 95% by weight of the total weight of the composition. The compositions may be provided in dosage forms suitable for parenteral (eg, subcutaneous, intravenous, intramuscular, or intraperitoneal) routes of administration. Pharmaceutical compositions can be formulated according to conventional pharmaceutical practice (eg, Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York. ). Pharmaceutical compositions of the present disclosure can be formulated to release the active compound substantially immediately after administration, or at a predetermined time or after a predetermined time after administration.

When the present disclosure is used for parenteral administration, it may be formulated in unit-dose ampoules or multi-dose containers or tubes, and may include stabilizers, buffers, preservatives, isotonic agents, etc. It may also contain additives such as agents. In addition, the preparation for parenteral administration may be formulated into a powder that can be redissolved in an appropriate carrier (sterile water, etc.) at the time of use. Examples of parenteral administration include intravenous administration, intramuscular administration, and subcutaneous administration, with intravenous administration being preferred. By administering the composition of the present disclosure to humans in the manner described above, it can be used for vaccine effects, particularly for the purpose of inducing a Th1 type immune response in a subject infected with a virus. The constructs described herein, active ingredients, may be administered together with a carrier. A carrier includes a diluent, adjuvant, excipient, or vehicle. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, as desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, combinations thereof, and the like.

A pharmaceutically acceptable carrier as used in the present disclosure refers to a vehicle containing the constructs or agents described herein that can be injected into a subject without side effects. Pharmaceutically acceptable carriers include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil. , sesame oil, and combinations thereof. Suitable pharmaceutically acceptable carriers include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, Includes glycerol, propylene, glycols, water, ethanol, combinations thereof, and the like. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Administration of the vaccine may be by any route typically used for vaccination, including topical, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, oral, inhalation routes, or combinations thereof. It may be due to the route.

The compositions described herein can be formulated into vaccines. The vaccines described herein comprise a suitably formulated full-length genomic RNA molecule of an infectious, non-pathogenic and/or attenuated virus operably linked to a suitable promoter for expression in eukaryotic cells. including the vectors described herein that include DNA encoding.

In one particular embodiment, the constructs, medicaments, etc. of the present disclosure are administered one or more times during a treatment period. As described in the Examples, it has been confirmed that the medicine of the present disclosure is effective when administered at least once, and patient compliance is considered to be good.

(General technology)
The molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the field, and can be found, for example, in Current Protocols in Molecular Biology (http://onlinelibrary. wiley.com/book/10.1002/0471142727) and Molecular Cloning: A Laboratory Manual (Fourth Edition) (http://www.molecularcloning.com), etc. , and these are the relevant parts of this specification. (which may be all) are incorporated by reference.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

(Example 1) Construct production method SHIV-NM3rN having the HIV-1 NL432 gene in the SIVmac239 background was used as a starting material. Recombinant SHIV was constructed according to previously reported methods (15, 16). The SHIV-nef vector (SHIV-NI) was constructed from an infectious molecular clone of SHIV-NM3rN (48). The source of the SHIV-NI env gene was HIV-1 NL432, an X4-tropic virus. In SHIV-NI, the nef gene was replaced with unique restriction enzyme sites such as ClaI and ApaI. The Ag85B gene was generated using Mycobacterium kansasii as a template, 5'-ATATCGATAACCATGTTCTCCCGTCCCGGGCT-3' (ClaI) (SEQ ID NO: 5) and 5'-AGGGCCCCTAGCGGGCGCCCAGGCTGG-3' (ApaI). Amplified by PCR using primers (SEQ ID NO: 6) . The PCR product was then digested with restriction enzymes at ClaI and ApaI sites. This plasmid is called pSHIV-Ag85B. SHIV-Ag85B was prepared by transfecting 293T cells with pSHIV-Ag85B using FuGENE 6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN), and the culture supernatant 48 hours after transfection was stored in liquid nitrogen until use. Medium Saved with. The sequence of SHIV-Ag85B is shown in SEQ ID NO:3.

(Example 2) Performance confirmation of construct (Detection of Ag85B protein)
M8166 cells were infected with SHIV-Ag85B at an MOI of 0.1 and cultured for 1 hour. After washing the cells three times with phosphate buffered saline (PBS), they were further cultured in culture medium for 48 hours. After three additional washes with PBS, cells were lysed with PBS containing 1.5 M urea, 2% NP-40 and 5% 2-mercaptoethanol. They were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose membranes, and blocked with 5% nonfat dry milk in PBS containing 0.01% Tween 20 (PBST). After washing three times with PBST, the membrane was incubated with rabbit anti-Ag85B polyclonal antibody for 2 hours. The membrane was washed three times with NBT/BCIP (Roche Diagnostics, Mannheim, Germany) and then incubated with alkaline phosphatase-labeled anti-rabbit IgG (New England Biolabs, Beverly, MA).

(In vivo stability of inserted Ag85B gene)
Proviral DNA was extracted from 1×10 ⁶ PBMC of inoculated macaque monkeys. CD8 ⁺ depleted PBMCs co-cultured with M8166 cells were also monitored if the virus was re-isolated. Cellular DNA was extracted using the DNeasy tissue kit (QIAGEN). To confirm the stability of the inserted Ag85B gene in SHIV-Ag85B, a proviral DNA fragment containing the inserted Ag85B gene in SHIV-Ag85B was PCR amplified using primers. The sequences of the primers are as described above.

(Example 3) Evaluation of SHIV-Ag85B therapeutic vaccine The outline of the experiment of this example is shown in FIG. 1. In this example, changes in plasma virus amount and CD4 ⁺ T cell count were examined when the composition according to the present invention was administered after inoculation with HIV virus.

(material and method)
(virus strain)
In this study, SHIV-Ag85B, SHIV-NI, and SHIV89.6P were used. These virus strains were grown in cynomolgus monkey PBMC. PBMCs separated by standard Ficoll density gradient separation were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 100 units/ml IL-2 (Shionogi & Co., Ltd.), and stimulated with phytohemagglutinin for 72 hours. , cells were infected with SHIV-Ag85B, SHIV-NI, or SHIV89.6P at a multiplicity of infection (MOI) of 0.1. Half of the culture medium was replaced with fresh culture medium every 3 days, and cell-free supernatants were collected 6 to 9 days after infection. The TCID ₅₀ of each SHIV was measured using M8166 cells. The TCID ₅₀ values of the virus stocks were 5×10 ⁴ for SHIV-Ag85B, 4.7×10 ⁴ for SHIV-NI, and 3×10 ⁵ for SHIV89.6P.

(animal)
Adult monkeys (from Indonesia, the Philippines, and Malaysia) that were negative for simian SIV, type D retrovirus, T-cell lymphoma virus, simian foamy virus, Epstein-Barr virus, cytomegalovirus, and B virus were all used. Seven monkeys were inoculated intravenously with SHIV89.6P50 at 10 ⁴ TCID ₅₀ . Then, 1 or 2 weeks after SHIV89.6P infection (after 5 weeks for #137), anti-HIV drugs (tenofovir 20 mg/kg, emtricitabine 40 mg/kg or dolutegravir 2.5 mg/kg) were administered subcutaneously once a day. administered. Five monkeys were inoculated intravenously with SHIV-Ag85B (high-dose SHIV-Ag85B inoculation; 8x10 ⁴ TCID ₅₀ , low-dose SIV-Ag85B inoculation; 10 ⁴ TCID ₅₀ ) after stopping anti-HIV drug dosing. Blood was collected periodically with sodium citrate as an anticoagulant and used for measurement of CD4 ⁺ T cell counts, quantification of plasma viral load, and immunological analysis. Lymph tissue samples were obtained by biopsy and used for proviral DNA content determination and pathological examination.

(Preparation of DNA sample and amplification of SHIV gag gene by nested PCR)
To measure proviral DNA in monkeys vaccinated with SHIV-Ag85B, nested PCR was used to amplify fragments of the gag gene segment. Proviral DNA was extracted from PBMC of inoculated monkeys. Cellular DNA was extracted using DNeasy tissue kits (QIAGEN). Nested PCR was performed using TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan). The original protocol and the nested PCR protocol are described in references 49 and 50 (49 and 50). The primers used in this study were Outer SIVgag-F (5'-CCATTAGTGCCAACAGGCTCAG-3' (SEQ ID NO: 7)) and Outer SIVgag-R (5'-CCCCAGTTGGATCCATCTCCTG-3' (SEQ ID NO: 8)) in the first round of PCR. ), and Nested SIVgag-F (5'-ACTGTCTGCGTCATCTGGTG-3' (SEQ ID NO: 9)) and Nested SIVgag-R (5'-GTCCCAATCTGCAGCCTCCTC-3') were used in the second round of PCR. After the second amplification, 10 μl of the product amplified by nested PCR was applied to a 1.0% agarose gel and stained with ethidium bromide to visualize the DNA bands. Upon initial amplification with the outer gag primer pair, the lowest concentration of plasmid SIV DNA detected with this PCR method was 100 copies. Further amplification with nested/internal gag primers routinely detects one copy of plasmid DNA (49, 50).

(Amount of viral RNA in plasma)
SHIV infection levels were monitored by measuring the amount of viral RNA in plasma using sensitive quantitative real-time RT-PCR as previously described (23, 51, 52). Viral RNA was isolated from plasma using the MagnA PureCompact Nucleic Acid Isolation Kit (Roche Diagnostics). Real-time RT-PCR was performed using a QuantiTec Probe RT-PCR Kit (Qiagen) and a LightCycler 480 thermocycler (Roche Diagnostics, Rotkreuz, Switzerland). The gag gene of SIVmac239 was probed with probe 5'-FAM-TGTCCACCTGCCATTAAGTCCCGA-TAMRA-3' (where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine) (SEQ ID NO: 11), primer 5'- TGGAAGAAAGACCTCCAGAAAATG-3' (SEQ ID NO: 12) and 5'-CAAGTGCAGTTAGCAAGCGAGGAT-3' (SEQ ID NO: 13) were amplified. The detection limit was calculated to be 1000 viral RNA copies/ml.

(Proviral DNA amount)
DNA samples were extracted from PBMC and lymphoid tissue using the DNeasy tissue kit (QIAGEN) according to the manufacturer's protocol. Ultra-high sensitivity digital PCR was performed with QX200 Droplet Digital PCR system (Bio-Rad). A 20 μl reaction mixture was prepared containing 2 μl of DNA sample, ddPCR supermix for probes (without dUTP) (Bio-Rad), 900 nM of each primer, 200 nM of probe, and demineralized water. This mixed solution was placed in a DG8 cartridge along with 70 μl of droplet generation oil (Bio-Rad), and droplets were formed using a droplet generator (Bio-Rad). The droplets were then transferred to a 96-well microplate. PCR amplification was performed with the following program: initial denaturation and stabilization at 95°C for 10 min, 40 cycles of denaturation at 94°C for 30 s, annealing/extension at 57°C for 60 s, followed by 10 min at 98°C. Droplets were then sorted and analyzed on a QX200 droplet reader (Bio-Rad) using QuantaSoft v1.6 (Bio-Rad) software. Samples were only considered if 20,000 or more droplets were read. Cell numbers were monitored by sensitive quantitative real-time PCR as previously described (53). DNA samples were extracted from PBMC and lymphoid tissue using a DNA DNAeasy tissue kit (QIAGEN) according to the manufacturer's protocol. Cell numbers were determined using rhesus IL-4 specific primers 5'-TGTGCTCCGGCAGTTCTACA-3' (SEQ ID NO: 14) and 5'-CCGTTTCAGGAATCGGATCA-3' (SEQ ID NO: 15) and probe 5'-FAM-TGCACAGCAGTTCCACAGGCACAAG-TAMRA-3' ( The cellular IL-4 sequence was detected and confirmed using SEQ ID NO: 16).

(CD4 ⁺ T cell number)
100 μl of whole blood from each cynomolgus monkey was stained with fluorescently labeled monoclonal antibodies: anti-CD3 (clone SP34-2, Alexa700; BD), anti-CD4 (clone L200, PerCP-Cy5.5; BD). Flow cytometry was performed on a FACSCanto II flow cytometer (BD). Data were analyzed using FACSDiVa software.

(result)
(Administration of anti-HIV drugs only)
In monkeys that received only anti-HIV drugs (#137 and #139), the plasma viral load remained at a low level, but when anti-HIV drug administration was discontinued, the plasma viral load increased (Figure 2 and Figure 3). Additionally, in the monkeys (#137 and #139) that were inoculated with SHIV89.6P and administered anti-HIV drugs, the number of CD4 ⁺ T cells was very low during the observation period (Figures 2 and 3).

(Low dose SHIV-Ag85B administered twice)
In the monkey (#138) that received two low doses of SHIV-Ag85B, almost no virus was detected in the plasma even after anti-HIV drug administration was discontinued, and 14 weeks after the first administration of SHIV-Ag85B, The amount of virus in plasma reached below the detection limit (Figure 4). Furthermore, in the monkey (#138) that received low doses of SHIV-Ag85B twice, the number of CD4 ⁺ T cells remained at normal levels after SHIV-Ag85B administration (Figure 4).

(High dose SHIV-Ag85B once administered)
In the monkey (#142) that received a single high dose of SHIV-Ag85B, the plasma viral load remained below the detection limit over the observation period after anti-HIV drug administration was discontinued (Figure 5). Furthermore, in the monkey (#142) that received one high dose of SHIV-Ag85B, the number of CD4 ⁺ T cells remained at a normal level after SHIV-Ag85B administration (Figure 5).

(High dose SHIV-Ag85B 5 times administration)
In the monkey (#141) that received a high dose of SHIV-Ag85B, the plasma virus load decreased to a low level after two doses of high dose SHIV-Ag85B, but after a while the plasma virus load increased ( Figure 6). When low doses of SHIV-Ag85B were again administered (days 91, 147, and 154 post-infection), the plasma viral load decreased. Even in such cases, it is considered possible to maintain the plasma viral load at a low level by administering SHIV-Ag85B multiple times. CD4 ⁺ T cells maintained normal levels (Figure 6).

(low dose and high dose SHIV-Ag85B administered once each)
In monkeys (#140) administered low-dose SHIV-Ag85B on day 66 post-infection, the amount of virus in plasma increased from day 77 post-infection onwards. High-dose SHIV-Ag85B was administered on day 98 post-infection, but the plasma viral load remained elevated (Figure 7). CD4 ⁺ T cell numbers remained at low levels throughout the observation period (Figure 7).

The experimental method of this example was conducted according to FIG. 1.

Specifically, the details are as follows.
Start of anti-HIV drug administration:
After infection with SHIV89.6P and after the virus in plasma peaked (excluding #137).
Administration method:
Once a day; subcutaneous administration Anti-HIV drugs:
Tenofovir 20 mg/kg
Emtricitabine 40 mg/kg
Dolutegravir 2.5 mg/kg
References (Goswami R, et al.. Analytical Treatment Interruption after Short-Term Antiretroviral Therapy in a Postnatally Simian-Hum an Immunodeficiency Virus-Infected Infant Rhesus Macaque Model. mBio. 2019 Sep 5;10(5):e01971-19. doi : 10.1128/mBio.01971-19., Nishimura Y, et al., Prevention and treatment of SHIVAD8 infection in rhesus macaques by a pote nt d-peptide HIV entry inhibitor. Proc Natl Acad Sci USA. 2020 Sep 8; 117(36):22436-22442. doi: 10.1073/pnas.2009700117)

Specifically, it was carried out using substantially the same procedure as for SHIV described above. The administration schedule for each individual is as shown in FIG.

In monkeys that received two doses of low-dose SHIV-Ag85B (#138) and monkeys that received one dose of high-dose SHIV-Ag85B (#142), the plasma viral load remained low for a long time after vaccination with SHIV-Ag85B. remained below the detection limit (FIGS. 9A and 9B). In addition, for one monkey (#141) that received a high dose of SHIV-Ag85B, only a short-term effect was observed at the beginning, and when it was administered every time the virus appeared in the plasma, after the fifth vaccination, A long-term suppressive effect was observed (Figure 10).

The explanation of the above results is as follows.

The monkey (#138) received low dose SHIV-Ag85B twice, and when low dose (1x10 ⁴ TCID50) SHIV-Ag85B was administered on the 4th and 11th day after stopping the treatment, no virus was detected in the plasma. CD4 ⁺ cells have also recovered to normal levels and are maintained below the limit.

The explanation of the above results is as follows.

The monkey (#142) received one dose of high-dose SHIV-Ag85B, and when high-dose SHIV-Ag85B (5x10 ⁴ TCID50) was administered 7 days after treatment was stopped, the virus in plasma remained below the detection limit. However, CD4 ⁺ cells also recovered to normal values and were maintained.

The monkey (#141) that received a high dose of SHIV-Ag85B was treated with a high dose of SHIV-Ag85B (5x10 ⁴ TCID50) on the 7th day after stopping the treatment, and the virus in the plasma remained below the detection limit for a short period of time. However, when the vaccine was administered every time the virus appeared in plasma, a long-term suppressive effect was observed after the fifth vaccination. CD4 ⁺ cells also returned to normal levels and were maintained.

Example 4 Further clinical studies with SHIV-Ag85B Cynomolgus monkeys are administered high doses of SHIV-Ag85B four times weekly. Thereafter, changes in the amount of virus in plasma and the number of CD4 ⁺ T cells in the cynomolgus monkeys were measured.
(1) Effects of drug suspension Regarding the effects of drug suspension, the number of inoculations and the amount of SHIV-Ag85B inoculation were investigated, and an experiment similar to that in Example 3 was conducted. A drug holiday was performed by discontinuing the drug for 3 days after 8 weeks of administration.

The above results are shown in FIG. 11 (#137 is FIG. 11A, #139 is FIG. 11B).

The explanation of the above results is as follows.

FIG. 11A is a control untreated monkey (#137) that received anti-HIV drugs only. The virus in the plasma appeared on the 7th day after discontinuation of the drug, and then increased, and inversely proportional to this, the number of CD4 ⁺ cells decreased, and the patient died 170 days after infection.

FIG. 11B is a control untreated monkey (#139) that received anti-HIV drugs only. The virus in the plasma appeared 110 days after discontinuation of the drug, and then increased, and inversely proportional to this, the number of CD4 ⁺ cells decreased, and the patient died 500 days after infection.

(2) Optimization of treatment protocol An outline of the experiment of this example is shown in FIG. 12. In this example, the change in plasma virus amount when the composition according to the present disclosure was administered after inoculation with the HIV virus was verified.

(protocol)
Medication will begin 1 week after SHIV infection. SHIV-Ag85B (5x10 ⁴ TCID50) was administered four times at one-week intervals starting 7 days after the treatment drug was discontinued.

(Verification method)
(Amount of viral RNA in plasma)
SHIV infection levels were monitored by measuring the amount of viral RNA in plasma using sensitive quantitative real-time RT-PCR as previously described (23, 51, 52). Viral RNA was isolated from plasma using the MagnA PureCompact Nucleic Acid Isolation Kit (Roche Diagnostics). Real-time RT-PCR was performed using a QuantiTec Probe RT-PCR Kit (Qiagen) and a LightCycler 480 thermocycler (Roche Diagnostics, Rotkreuz, Switzerland). The gag gene of SIVmac239 was probed with probe 5'-FAM-TGTCCACCTGCCATTAAGTCCCGA-TAMRA-3' (where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine) (SEQ ID NO: 11), primer 5'- TGGAAGAAAGACCTCCAGAAAATG-3' (SEQ ID NO: 12) and 5'-CAAGTGCAGTTAGCAAGCGAGGAT-3' (SEQ ID NO: 13) were amplified. The detection limit was calculated to be 1000 viral RNA copies/ml.

(Result)

The results are shown in Figure 13. The anti-HIV drugs used were shown to be effective regardless of individual differences. .

(Example 5: Treatment optimization)
In this example, a test was conducted in which SHIV-Ag85B was administered four times. The protocol is essentially the same as described above.

(SHIV-Ag85B 4 times administration)
In monkeys (#164, #165, and #167) that received a high dose (5.0×10 ⁴ TCID ₅₀ ) of SHIV-Ag85B, administration of high dose SHIV-Ag85B four times at one-week intervals resulted in plasma The viral load remained below the detection limit over the observation period (Figure 14). In the control group that did not contain Ag85B, virus appeared in the plasma two weeks after the drug was discontinued, but in the SHIV-Ag85B administration group, the virus in the plasma remained below the detection limit.

(SHIV-NI 4 doses)
In monkeys (#162, #166, and #168) that received a high dose (5.0 x 10 ⁴ TCID ₅₀ ) of SHIV-NI, four doses of high-dose SHIV-NI increased the plasma concentration in #168. Although the virus amount remained below the detection limit, the plasma virus amount increased in #162 and #166 (Figure 15). In the control group that does not contain Ag85B, virus appeared in the plasma 2 weeks after drug withdrawal, but in the SHIV-Ag85B administration group, the virus in the plasma remained below the detection limit (Figure 15 for the control group that does not contain Ag85B. -Ag85B inoculated group is shown in Figure 14; untreated group is shown in Figure 16).

(Example 6) Attenuated HCV incorporating adjuvant molecules
(material and method)
(Construct manufacturing method)
HCV-Ag85B is produced according to Example 1. A gene of HCV that induces attenuation when deleted is replaced with an adjuvant molecule (eg, Ag85B).

(virus strain)
In this example, HCV virus strains are grown in cynomolgus monkey PBMC. PBMCs separated by standard Ficoll density gradient separation were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 100 units/ml IL-2 (Shionogi & Co., Ltd.), and stimulated with phytohemagglutinin for 72 hours. , cells are infected with HCV at a multiplicity of infection (MOI) of 0.1. Half of the culture medium is replaced with fresh culture medium every 3 days, and cell-free supernatants are collected 6-9 days post-infection. The TCID ₅₀ of HCV is measured using M8166 cells.

(animal)
Adult monkeys (from Indonesia, the Philippines, and Malaysia) that are negative for simian SIV, type D retrovirus, T-cell lymphoma virus, simian foamy virus, Epstein-Barr virus, cytomegalovirus, and B virus are all used. Seven monkeys are inoculated intravenously with HCV at 10 ⁴ TCID ₅₀ . Thereafter, HCV-Ag85B is inoculated intravenously. Blood is collected periodically with sodium citrate as an anticoagulant and used for determination of CD4 ⁺ T cell counts, quantification of plasma viral load, and immunological analyses. Lymphoid tissue samples are obtained by biopsy and used for proviral DNA content determination and pathological examination.

(Preparation of DNA sample and amplification of HCV virulence gene by nested PCR)
To measure proviral DNA in monkeys inoculated with HCV-Ag85B, nested PCR is used to amplify fragments of HCV-specific gene segments. Proviral DNA is extracted from PBMC of inoculated monkeys. Cellular DNA was extracted using DNeasy tissue kits (QIAGEN). Nested PCR is performed using TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan).

(Amount of viral RNA in plasma)
HCV infection levels are monitored by measuring the amount of viral RNA in plasma using sensitive quantitative real-time RT-PCR, as described above. Viral RNA is isolated from plasma using the MagnA PureCompact Nucleic Acid Isolation Kit (Roche Diagnostics). Real-time RT-PCR is performed using a QuantiTec Probe RT-PCR Kit (Qiagen) and a LightCycler 480 thermocycler (Roche Diagnostics, Rotkreuz, Switzerland). HCV-specific genes are amplified using probes and primers.

(Proviral DNA amount)
DNA samples are extracted from PBMC and lymphoid tissue using the DNeasy tissue kit (QIAGEN) according to the manufacturer's protocol. Ultra-high sensitivity digital PCR is performed with QX200 Droplet Digital PCR system (Bio-Rad). Prepare a 20 μl reaction mixture containing 2 μl of DNA sample, ddPCR supermix for probes (no dUTP) (Bio-Rad), 900 nM of each primer, 200 nM of probe, and demineralized water. This mixed solution is placed in a DG8 cartridge along with 70 μl of droplet generation oil (Bio-Rad), and droplets are formed using a droplet generator (Bio-Rad). The droplets were then transferred to a 96-well microplate. PCR amplification was performed with the following program: initial denaturation and stabilization at 95°C for 10 min, 40 cycles of denaturation at 94°C for 30 s, annealing/extension at 57°C for 60 s, followed by 10 min at 98°C. Droplets are then sorted and analyzed on a QX200 droplet reader (Bio-Rad) using QuantaSoft v1.6 (Bio-Rad) software. A sample is only considered if 20,000 or more droplets are read. Cell numbers are monitored by sensitive quantitative real-time PCR as described above. DNA samples are extracted from PBMC and lymphoid tissue using a DNA DNAeasy tissue kit (QIAGEN) according to the manufacturer's protocol.

(CD4 ⁺ T cell number)
100 μl of whole blood from each cynomolgus monkey is stained with fluorescently labeled monoclonal antibodies: anti-CD3 (clone SP34-2, Alexa700; BD), anti-CD4 (clone L200, PerCP-Cy5.5; BD). Flow cytometry is performed on a FACSCanto II flow cytometer (BD). Data are analyzed using FACSDiVa software.

(Example 7) Attenuated HTLV incorporating adjuvant molecules
(material and method)
(Construct manufacturing method)
HTLV-Ag85B is produced according to Example 1. Genes that induce attenuation when deleted in HTLV are replaced with adjuvant molecules (eg, Ag85B).

(virus strain)
In this example, an HTLV virus strain is grown in cynomolgus monkey PBMC. PBMCs separated by standard Ficoll density gradient separation were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 100 units/ml IL-2 (Shionogi & Co., Ltd.), and stimulated with phytohemagglutinin for 72 hours. , cells are infected with HCV at a multiplicity of infection (MOI) of 0.1. Half of the culture medium is replaced with fresh culture medium every 3 days, and cell-free supernatants are collected 6-9 days post-infection. The TCID ₅₀ of HCV is measured using M8166 cells.

(animal)
Adult monkeys (from Indonesia, the Philippines, and Malaysia) that are negative for simian SIV, type D retrovirus, T-cell lymphoma virus, simian foamy virus, Epstein-Barr virus, cytomegalovirus, and B virus are all used. Monkeys are inoculated intravenously with HTLV at 10 ⁴ TCID ₅₀ . Thereafter, HTLV-Ag85B is inoculated intravenously. Blood is collected periodically with sodium citrate as an anticoagulant and used for determination of CD4 ⁺ T cell counts, quantification of plasma viral load, and immunological analyses. Lymphoid tissue samples are obtained by biopsy and used for proviral DNA content determination and pathological examination.

(Preparation of DNA sample and amplification of HTLV virulence gene by nested PCR)
To measure proviral DNA in monkeys inoculated with HTLV-Ag85B, nested PCR is used to amplify fragments of HTLV-specific gene segments. Proviral DNA is extracted from PBMC of inoculated monkeys. Cellular DNA was extracted using DNeasy tissue kits (QIAGEN). Nested PCR is performed using TaKaRa Ex Taq (Takara Bio Inc., Shiga, Japan).

(Amount of viral RNA in plasma)
HTLV infection levels are monitored by measuring the amount of viral RNA in plasma using sensitive quantitative real-time RT-PCR, as described above. Viral RNA is isolated from plasma using the MagnA PureCompact Nucleic Acid Isolation Kit (Roche Diagnostics). Real-time RT-PCR is performed using a QuantiTec Probe RT-PCR Kit (Qiagen) and a LightCycler 480 thermocycler (Roche Diagnostics, Rotkreuz, Switzerland). HCV-specific genes are amplified using probes and primers.

(Example 8) Other attenuated viruses incorporating adjuvant molecules (measles virus, rubella virus, etc.)
Similar to Examples 6 and 7, an attenuated virus incorporating an adjuvant is produced and a similar experiment is performed. Genes whose deletion induces attenuation in measles and rubella viruses are replaced with adjuvant molecules (eg, Ag85B). Alternatively, adjuvants are added to known attenuated measles and rubella viruses to create attenuated viruses incorporating adjuvants.

(Example 9) Clinical trial with HIV-Ag85B, HCV-Ag85B, HTLV-Ag85B In this example, HIV-Ag85B (SEQ ID NO: 4), HCV-Ag85B, or HTLV-Ag85B was treated with the corresponding therapeutic agent. The drug is administered subcutaneously or intravenously 4 to 8 times at intervals of 1 to 2 weeks after cessation of the corresponding treatment to virus-infected humans (BCG vaccinated and non-BCG vaccinated groups) who have received or have not received the drug. The therapeutic effect of HIV-Ag85B, HCV-Ag85B, or HTLV-Ag85B is confirmed.

Based on Examples 1 to 6, the plasma viral load and CD4 ⁺ cell count are confirmed. Since BCG vaccination is expected to enhance the adjuvant effect of Ag85B, it is expected that the BCG vaccinated group will have a high therapeutic effect against viruses caused by HIV-Ag85B, HCV-Ag85B, and HTLV-Ag85B.

(Example 10) Formulation Example When forming a formulation, it can be manufactured by the following manufacturing method.

(Example 1) An appropriate volume of physiological saline can be directly added to an appropriate amount of lyophilized nucleic acid construct to prepare a solution for injection.

(Example 2) An appropriate volume of isotonic 5% glucose solution can be added to an appropriate amount of the lyophilized nucleic acid construct to prepare a solution for injection.

(Example 3) An appropriate volume of electrolyte correction solution Otsuka Salt Injection 10% or the like is added to an appropriate amount of the nucleic acid construct dissolved in water to adjust the concentration to 0.9% NaCl to prepare a solution for injection.

(Example 4) An appropriate amount of the nucleic acid construct dissolved in water can be lyophilized to obtain a lyophilized preparation of the sodium salt of the nucleic acid construct.

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(Note)
As described above, although the present disclosure has been illustrated using the preferred embodiment of the present disclosure, the present disclosure should not be construed as being limited to this embodiment. It is understood that the scope of this disclosure is to be construed solely by the claims. It is understood that those skilled in the art can implement the present invention to an equivalent extent based on the description of the present disclosure and common general technical knowledge from the description of the specific preferred embodiments of the present disclosure. The patents, patent applications, and publications cited herein are hereby incorporated by reference to the same extent as if the contents were specifically set forth herein. be understood. This application claims priority to Japanese Patent Application No. 2022-140223 filed on September 2, 2022 in Japan, and the contents thereof are all cited as reference in this application as necessary.

The present disclosure is useful in the pharmaceutical industry.

SEQ ID NO: 1: Nucleic acid sequence of Ag85B SEQ ID NO: 2: Amino acid sequence of Ag85B SEQ ID NO: 3: Nucleic acid sequence of SHIV-Ag85B SEQ ID NO: 4: Nucleic acid sequence of HIV-Ag85B SEQ ID NO: 5: ClaI primer SEQ ID NO: 6: ApaI primer SEQ ID NO: 7: Outer SIVgag-F primer SEQ ID NO: 8: Outer SIVgag-R primer SEQ ID NO: 9: Nested SIVgag-F primer SEQ ID NO: 10: Nested SIVgag-R primer SEQ ID NO: 11: Probe for SIVmac239 gag SEQ ID NO: 12: SIVmac239 gag Primer 1 for
SEQ ID NO: 13: Primer 2 for gag of SIVmac239
SEQ ID NO: 14: Primer 1 for IL-4
SEQ ID NO: 15: Primer 2 for IL-4
SEQ ID NO: 16: Probe for IL-4

Claims

A composition for treating a viral infection in a subject, comprising a nucleic acid construct comprising a nucleic acid sequence encoding an attenuated virus or its equivalent and a nucleic acid sequence encoding an adjuvant molecule.
The composition according to claim 1, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of accessory genes and regulatory genes.
The composition according to claim 1 or 2, wherein the attenuated virus is deficient in an accessory gene.
The composition according to any one of claims 1 to 3, wherein the attenuated virus is deficient in at least one gene selected from the group consisting of Vif, Vpr, Vpx, Vpu, and Nef or its corresponding gene. .
The composition according to any one of claims 1 to 4, wherein the attenuated virus is a nef-deficient attenuated virus.
A composition according to any one of claims 1 to 5, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated into the nucleic acid sequence encoding the attenuated virus or its equivalent.
The composition according to claim 5, wherein the nucleic acid sequence encoding the adjuvant molecule is integrated at the position of the deleted nef gene in the nef-deficient attenuated virus.
The composition according to any one of claims 1 to 7, wherein the attenuated virus is a chronically infectious virus.
The attenuated virus is an attenuated virus selected from the group consisting of AIDS virus, human T-cell leukemia virus (HTLV), measles virus, rubella virus, hepatitis C virus (HCV), and hepatitis B virus (HBV). 9. The composition according to any one of claims 1 to 8, which is an infected virus.
The composition according to any one of claims 1 to 9, wherein the adjuvant molecule is an adjuvant molecule derived from acid-fast bacteria.
The composition according to any one of claims 1 to 10, wherein the adjuvant molecule is selected from the group consisting of Ag85B proteins.
The composition according to any one of claims 1 to 11, wherein the composition induces a Th1 type immune response.
The composition according to any one of claims 1 to 12, wherein the composition completely eliminates the virus in the subject.
The composition according to any one of claims 1 to 13, characterized in that the composition is administered after a viral infection.
The composition according to any one of claims 1 to 14, wherein the nucleic acid construct is cloned.
A companion reagent for use in a method for determining whether treatment with the composition according to any one of claims 1 to 15 is necessary, comprising a diagnostic agent for viral infection.
A kit for treating a viral infection in a subject, comprising the composition according to any one of claims 1 to 15, a diagnostic agent for viral infection, and instructions.
A method for treating a viral infection in a subject, said method comprising administering to said subject a composition according to any one of claims 1 to 15.
Use of a composition according to any one of claims 1 to 15 in the manufacture of a medicament for treating a viral infection in a subject.
The composition according to any one of claims 1 to 15, wherein the subject has previously been vaccinated with a BCG vaccine.