Vaccine against hepatitis b virus

Abstract

To provide immunotherapy against hepatitis B virus infection.SOLUTION: Provided are genetically modified arenavirus vectors suitable as vaccines for the prevention and treatment of hepatitis B virus infection. Also provided are pharmaceutical compositions and methods for the treatment of hepatitis B virus infection. In particular, pharmaceutical compositions, vaccines and methods for treating hepatitis B virus infection. In one aspect, the arenavirus viral vector is an infectious replication-competent tri-segmented arenavirus viral vector comprising one L segment and two S segments.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application was filed on November 4, 2015, the disclosure of which is incorporated herein by reference in its entirety. The benefit of filed U.S. Provisional Patent Application No. 62 / 250,639 is claimed.

[0002] (Reference to electronically submitted sequence listing) This application is based on a file created on November 2, 2016, entitled "Sequence _Listing_13194-014-228.TXT" along with this application. The submitted sequence listing is incorporated by reference. [Background technology]

[0003] (1. Introduction) Provided herein are vaccines and methods for the prevention and treatment of hepatitis B virus infection. Also provided herein are genetically modified arenavirus vectors suitable for use in the Pharmaceutical compositions and methods for the treatment of hepatitis B virus infection. Provided are pharmaceutical compositions, vaccines, and methods for treating hepatitis B virus infection. Thus, the present application provides an immunotherapy for hepatitis B virus infection.

[0004] (2.Background) 2.1 Pathogens and Diseases Hepatitis B virus (HBV) is a double-stranded enveloped virus of the Hepadnaviridae family. Virus particles are icosahedral nuclei composed of an outer lipid envelope and proteins. The nucleocapsid contains the viral DNA and reverse transcriptase activity. The outer envelope contains the DNA polymerase that is responsible for the delivery of the virus to susceptible cells. HBV contains embedded proteins involved in the binding and entry of HBV. It replicates in monkey liver cells but does not grow in artificial cell cultures.

[0005] The outcome of HBV infection is age-dependent and includes asymptomatic infection, acute hepatitis B, chronic HBV infection, and liver cirrhosis. Acute hepatitis B accounts for approximately 1% of perinatal infections and 1% of childhood infections (ages 1-5 years). Fulminant hepatitis occurs in 10% of cases (children under 5 years of age) and 30% of cases of late infection (people over 5 years of age). It occurs in 0.1-0.6% of cases; the mortality rate from fulminant hepatitis B is approximately 70%. The incidence of HIV infection is inversely related to the age of acquisition, with approximately 80-90% of those infected perinatally and 90% of those infected before age 6. It occurs in approximately 30% of children with HIV infection, and less than 5% of infections occur in otherwise healthy adults (Hyams (1995, Clinical Infections Diseases 20:992-1000). HIV co-infection and alcohol Comorbidities, including ingestion of flu or aflotoxin or both, are associated with hepatitis B. It may play an important role in the development of associated pathologies. It is estimated that 10% of the 10 million people are co-infected with HBV.

[0006] Individuals with chronic HBV infection have a 15-20% increased risk of premature death due to HBV-related cirrhosis and HCC. 5% (Beasley and Hwang, 1991, 1990 National Survey of Viral Hepatitis and Liver Disease) Proceedings of the 1990 International Symposium: Current Issues and Future Prospects nal Symposium on Viral Hepatitis and Liver Disease: Contemporary Issues and Futu re Prospects), 532-535). Acute HBV infection is characterized by the surface antigen HBsAg and the core antigen HB It is characterized by the presence of immunoglobulin M (IgM) antibodies against cAg. During the replication phase, patients produce extracellular and secreted forms of HBcAg, which can be found in the patient's serum. They are also seropositive for HBeAg, a marker of active replication in chronic hepatitis. Antibodies to HBsAg (anti-HBs) are detected after a few weeks, and Chronic infection is characterized by persistence of HBsAg (>6 months). Persistence of HBsAg is associated with chronic liver disease and HBV later in life. The presence of HBeAg is a primary marker of risk for developing HBeAg in the blood and body of infected individuals. This indicates that the fluid is highly contagious.

[0007] (2.2 Epidemiology and Public Health) The disease caused by the hepatitis B virus is distributed worldwide. There are 2 billion people Of these, approximately 360 million people are chronically infected. This puts them at risk of serious illness and death, primarily from cirrhosis of the liver and hepatocellular carcinoma (HCC). Mathematical modelling for the year 2000 has estimated that the number of deaths from HBV-related diseases worldwide is It is estimated that there are approximately 600,000 cases each year (Goldstein et al., 2005, International J. Epidemiology gy 34:1329-1339). Humans are the only reservoir for HBV. The virus spreads through infected blood. Transmission is by percutaneous and mucosal exposure to semen and other body fluids, primarily semen and vaginal fluids. The average duration is 75 days, but can vary from approximately 30 to 180 days. The HBV surface antigen (HBsAg) is expressed during infection. It can be detected in serum 30-60 days after administration and persists for a variable period of time. Hepatitis B epidemics are characterized by the prevalence of HBsAg in the general population of a particular geographic area. This is explained by the rate, which varies considerably across the world: HBsAg prevalence of over 8% The prevalence is typical of high-incidence areas, with a prevalence of 2-7% seen in moderate-incidence areas. However, in low-incidence areas, less than 2% of the population is HBsAg positive.

[0008] In areas with high incidence, HBV is transmitted from mother to child at birth or from person to person during early childhood. spread is most common (Goldstein et al., 2005, International J. Epidemiology y 34:1329-1339; Wong et al., 1984, Lancet 1:921-926; de la Hoz et al., 2008 In International J. Infectious Diseases 12:183-189). Perinatal or early childhood transmission is the most frequent. may account for over one-third of chronic infections in low-risk areas (Margolis et al., 1999 5, JAMA 274:1201-1208), these settings are prone to sexually transmitted infections and, particularly, to the risk of infection among injecting drug users. The primary route of infection is through the use of contaminated needles between patients (Goldstein et al., 2002, J.I. nfectious Diseases 185:713-719).

[0009] (2.3 Current Treatment) Widespread hepatitis B vaccination significantly reduces rates of HBV infection and HCC However, once chronic HBV infection is established, conventional therapy does not usually result in sustained control of viral replication and liver damage in most patients, Treatment remains a challenge.

[0010] Currently approved antiviral treatments for chronic hepatitis B include pegylated (PEG) recombinant interferon. These drugs include steroid alpha and viral DNA polymerase inhibitors. This may reduce liver cirrhosis progression, reduce the incidence of HCC, and improve long-term survival. However, treatment is complicated by drug toxicity and is difficult for chronically infected individuals. The virus level in the blood is low even after standard treatment. In individuals with HIV infection, the viral DNA in the liver is reduced to an almost undetectable level, but the decrease in the viral DNA in the liver is only slight. As a result, relapse of viremia often occurs after treatment interruption, leading to chronic HBV Those infected must continue lifelong treatment. Even after 10 years of treatment, the drugs only reduced liver failure by 40-70% and cirrhosis and mortality from liver cancer remains high.

[0011] (2.4 Hepatitis B and the Immune System) Chronic hepatitis B infection is characterized by dysfunction of innate and adaptive antiviral immunity (Bertoletti and Ferrari, 2012, Gut 61:1754-1764). In contrast, in patients with resolved HBV infection, HBV-specific immunity in the human body is strong and multifunctional. Several mechanisms contribute to the production of high levels of viral resistance. Dysfunction of HBV-specific T-cell immunity and hepatic tolerance in patients with chronic hepatitis B, including primary hematopoietic disease This may contribute to the immune microenvironment (Jenne and Kubes, 2013, Nat. Immunol. 14:996-1006). Previous studies have shown that suppression of viral replication induces a transient and partial increase in antiviral T cell immunity. It has been shown that the immune system can recover from high levels of antigenemia, which may contribute to long-term survival. This supports the hypothesis that early exposure can cause antiviral T cell dysfunction (Boni et al. References, 2003, J. Hepatol. 39:595-605).

[0012] It can reverse the impaired immune state of chronic hepatitis B and restore antiviral immunity. A therapeutic vaccine would theoretically eliminate viremia and reduce intrahepatic HBV DNA levels to zero. and therefore may hold considerable promise for the treatment of HBV.

[0013] Recently, HBV vaccines have been developed for the treatment and control of HBV infection in HBV carriers and persistently infected patients. has been identified as a promising treatment strategy for rheumatoid arthritis (Michel and Tiollais, 2010, Pathol. Biol. (Paris) 58:288-295; Liu et al., 2014, Virol. Sin. 29:10-16). chronic active HB In approximately 50% of patients with HBV, specific therapy with conventional anti-HBV vaccination effectively inhibits HBV replication. effectively reduced the immune tolerance to HBsAg protein (Couillin et al., 1999). , J. Infect. Dis. 180:15-26). However, to date, no HBsAg-based vaccines have been used. Monotherapy with HBV did not result in durable control of HBV replication and / or liver damage (Akbar et al., 2014). 13, Hepatobiliary Pancreat. Dis. Int. 12:363-369), new therapeutic strategies are powerful and long-lasting. There is a need to provide a sustained antiviral immune response and long-term control of HBV replication.

[0014] The failure of past therapeutic vaccine approaches has undermined current knowledge about immune responses in chronic HBV infection. highlighting the challenges and limitations of this knowledge (Michel et al., 2011, J. Hepatol. 54:1286-1296). The combination of a high viral load state, such as chronic hepatitis B, and a permissive liver microenvironment may contribute to the development of antiviral T cells. This may make it difficult to achieve complete restoration of immune immunity.

[0015] Intensive research is currently underway to better understand the immune response in hepatocytes, how HBV circumvents innate immunity, and The focus is on the mechanisms of immune defence and the appropriate selection of patients likely to benefit from immunotherapy. This may increase the efficacy of therapeutic vaccination (Michel et al., 2015, Med. Mich. Crobiol. Immunol. 204:121-129). Summary of the Invention

[0016] (3. Summary of the Invention) The present application provides immunotherapy for hepatitis B virus infection. Here are: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof an infectious arenavirus virus vector comprising a nucleotide sequence selected from the group consisting of: In certain embodiments, the infectious arenavirus viral vector is replication-deficient. (See Section 6.1(a)). In certain embodiments, the infectious arenavirus virus The vector is replication-competent (see Section 6.1(b)). In certain embodiments, the infectious, replication-defective vector In one embodiment, the infectious arenavirus viral vector is bipartite. The replication-deficient arenavirus viral vector is tripartite. Infectious replication-competent arenavirus viral vectors are tripartite.

[0017] In some embodiments, provided herein are: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof an arenavirus virus vector comprising a nucleotide sequence selected from the group consisting of do.

[0018] In certain embodiments, the arenavirus viral vector is replication-deficient. In embodiments, the arenavirus viral vector is replication competent.

[0019] In certain embodiments, the viral vectors provided herein are infectious, i.e., That is, they can invade host cells or inject their genetic material into host cells. In a more specific embodiment, the viral vectors provided herein are infectious. that is, by invading or injecting its genetic material into a host cell and then The genetic information can be amplified and expressed within the host cell. In this case, the viral vector has the ability to amplify and express its genetic information in infected cells. In normal cells that have not been genetically modified, but have been modified to contain a genome with Infectious replication-deficient arenaviruses that are unable to produce further infectious progeny particles In some embodiments, provided herein are wild-type viruses. support viral growth but do not express complementary viral proteins and therefore In one embodiment, the cell line is incapable of producing infectious viral progeny particles. The infectious arenavirus viral vector is replication competent and genetically modified. The virus can then produce additional infectious progeny particles in normal cells that are not infected with the virus.

[0020] In one embodiment, the pre-S2 / S protein or antigenic fragment thereof comprises the nucleotide sequence of SEQ ID NO: 1. The amino acid sequence encoded by the nucleotide sequence is 80%, 81%, 82%, 83%, 84%, and 85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%, In some embodiments, the fragment comprises an amino acid sequence that is 99% or 100% identical to the amino acid sequence of the fragment. (i) induce an antibody immune response in a host (e.g., a mouse, rabbit, goat, or donkey); Induce (wherein the resulting antibodies specifically bind to human HBV pre-S2 / S protein); and and / or (ii) is antigenic if it is capable of eliciting a specific T cell immune response.

[0021] In certain embodiments, the HBc protein or antigenic fragment thereof comprises the nucleotide sequence of SEQ ID NO:2. The amino acid sequence encoded by the nucleotide sequence is 80%, 81%, 82%, 83%, 84%, 85%, and 86%. ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% identical. In some embodiments, the fragment comprises an amino acid sequence that is identical to ( i) induce an antibody immune response in a host (e.g., a mouse, rabbit, goat, or donkey) wherein the resulting antibody specifically binds to human HBV HBc protein; and / or (ii) specifically binds to human HBV HBc protein. A substance is antigenic if it is capable of eliciting a specific T cell immune response.

[0022] In one embodiment, the HBV HBs protein and the HBV HBc protein or their antigenicity The fusion of the fragments is carried out by combining the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 with the 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence. In certain embodiments, the fragment is intended to be used in a manner that allows it to be used in (i) a host (e.g., a mouse, a rabbit, a goat, or Inducing an antibody immune response in donkeys (wherein the resulting antibodies are capable of inhibiting human HBV, HBs, HBc, or (ii) specifically binds to both HBs and HBc; and / or (ii) elicits a specific T cell immune response. If it can be detected, it is antigenic.

[0023] In certain embodiments, the HBe protein or antigenic fragment thereof comprises the nucleotide sequence of SEQ ID NO:26. The amino acid sequence encoded by the nucleotide sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% In some embodiments, the fragment comprises an amino acid sequence that is 100% identical to or 100% identical to the amino acid sequence of the fragment. (i) elicit an antibody immune response in a host (e.g., a mouse, rabbit, goat, or donkey) (wherein the resulting antibody specifically binds to human HBV HBe protein); and / or (ii) ) A substance is antigenic if it is capable of eliciting a specific T cell immune response.

[0024] In one embodiment, the viral vector comprises: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. A nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof. Includes at least two of the following:

[0025] In one embodiment, the viral vector comprises: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. A nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof. Includes at least three of the following:

[0026] In certain embodiments, the open reading frame (ORF) of the arenavirus is HBV antigens that are deleted or functionally inactivated and that contain the HBV antigens described herein. In a specific embodiment, the glycoprotein of the arenavirus is replaced with a nucleic acid that encodes the glycoprotein. The ORF encoding the protein GP is deleted or functionally inactivated. In an embodiment, functional inactivation of a gene results in the abolishment of the translation product. In this context, functional inactivation refers to a genetic modification that allows some translation, but no translation product. It is no longer functional and cannot replace the wild-type protein.

[0027] In certain embodiments, the viral vector is administered to cells infected with the viral vector. The viral vector can amplify and express its genetic information in cells, but the viral vector can Complementing cells are unable to produce additional infectious progeny particles. The viral vectors provided herein are infectious, i.e., capable of entering host cells. Alternatively, the genetic material can be injected into a host cell. In the present invention, the viral vectors provided herein are infectious, i.e., capable of infecting host cells. Invades or injects its genetic material into a host cell and then transmits the genetic material inside the host cell. It can amplify information and express it.

[0028] In certain embodiments, the genomic information encoding the infectious arenavirus particles is It is derived from the clone 13 strain of lymphocytic choriomeningitis virus (LCMV) or the MP strain of LCMV. Clone 13 The nucleotide sequences of the S and L segments are shown in SEQ ID NOs: 12 and 7, respectively. It has been done.

[0029] In one embodiment, provided herein is a clone 13 genomic ORF (e.g., The ORF for the GP protein was deleted, and the remaining LCMV genome was inserted into the nucleotide sequence of clone 13. and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least The non-homologous ORF encoding the antigen (e.g., HBV antigen) is replaced with a non-homologous ORF so that it is 99% or 100% identical. By rearranging the genome, the genome is derived from the genome of clone 13 (SEQ ID NOs: 12 and 7). or a viral vector derived therefrom.

[0030] In some embodiments, provided herein are ORFs of the LCMV strain MP genome (e.g., GP The ORF for the LCMV protein was deleted, and the remaining LCMV genome was compared to the nucleotide sequence of LCMV strain MP. Sequences (SEQ ID NOs: 13 and 14) and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99% , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, at least 99.9 100% or 100% identical to a non-homologous ORF encoding an antigen (e.g., HBV antigen). By using the LCMV strain MP genome (SEQ ID NOs: 13 and 14), It is an illus vector.

[0031] In a more specific embodiment, the viral vector comprises nucleotide 16 of SEQ ID NO:11. 39 to 3315 or the sequence of 1640 to 3316 of SEQ ID NO: 12, and at least 80%, 81%, 82%, 83%, or 84% ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%, Genomic segments containing nucleotide sequences that are 98%, at least 99%, or 100% identical In one embodiment, the viral vector comprises a vector having an amino acid sequence set forth in SEQ ID NO: The amino acid sequence encoded by 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12 is at least Both are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, and 92%. , 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical expression product. It includes a genomic segment that contains a nucleotide sequence that encodes a product.

[0032] Also provided herein is an isolated nucleic acid, wherein the nucleic acid is an arenavirus genome. cDNA of a genome segment, wherein one ORF of the genome segment is deleted or or functionally inactivated, and the genome segment comprises: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof and an isolated nucleic acid comprising one or any combination of:

[0033] In certain embodiments, the genome segment is a short segment, wherein the GP The coding ORF is deleted.

[0034] In one aspect, provided herein are methods for producing infectious, replication-defective arenavirus particles. 1. A method of making a polymeric material comprising:

[0035] a. transfecting a host cell with a nucleic acid described herein;

[0036] b. maintaining the host cells under conditions favorable for virus formation; and

[0037] c. recovering the infectious replication-defective arenavirus particles;

[0038] wherein the host cell is In one embodiment, the method further comprises: Any additional nucleic acid required for skewing is also transfected into the host cell in step a. Such additional nucleic acid may be: a cDNA of a second arenavirus genome segment; , a nucleic acid comprising the L ORF, and / or a nucleic acid comprising the NP ORF.

[0039] In another aspect, provided herein is a viral vector described herein. and a pharmaceutically acceptable carrier, e.g., a pharmaceutical composition, an immunogenic composition, Also provided herein is a vaccine composition comprising two or more of the vaccines described herein. The above viral vectors contain different HBV antigens (i.e., the viral vectors contain different HBV antigens). In certain embodiments, the pharmaceutical composition is a composition (e.g., a vaccine composition) that The product includes a nucleic acid or a fusion protein described herein.

[0040] In a further aspect, provided herein are methods for treating or preventing HBV infection in a patient. A method of preventing the disease, comprising administering to the patient a viral vector, a pharmaceutical composition, or a combination thereof, as described herein. In yet another aspect, the method comprises administering a medicament for the treatment of a mammalian animal, an immunogenic composition, or a vaccine. In accordance with the present invention, provided herein is a method for treating or preventing HBV comprising administering to a subject a therapeutically effective amount of a compound according to the present invention, ... The present invention relates to the use of a viral vector, pharmaceutical composition, immunogenic composition, or vaccine in a mammalian animal. In some embodiments, infectious arenaviruses expressing HBV antigens or fragments thereof are transferred from the mother to the fetus. In one embodiment, the anti-HBV drug can prevent the transmission and / or infection of HBV to the infant. One or more infectious arenaviruses expressing the HBV gene or fragments thereof are involved in the transmission of HBV from mother to fetus. and / or infection can be prevented. In certain embodiments, the infectious arenavirus In certain embodiments, the viral vector is replication-deficient (see Section 6.1(a)). Infectious arenavirus viral vectors are replication competent (see Section 6.1(b)).

[0041] In one embodiment, the patient is administered an infectious arenavirus that expresses an HBV antigen or a fragment thereof. Administration of the infectious agent induces a long-lasting immune response. Lenavirus viral vectors are replication-deficient (see Section 6.1(a)). wherein the infectious arenavirus viral vector is replication competent (see Section 6.1(b)). .

[0042] In certain embodiments, provided herein are methods for treating and administering HBV infection in a patient. a method for preventing or preventing HBV infection, comprising administering to the patient two or more ArenA viruses expressing HBV antigens or fragments thereof; In a more specific embodiment, each of the alleles In another embodiment, each of the HBV antigens or fragments thereof is expressed by a different HBV antigen. The arenavirus expresses an HBV antigen or a derivative thereof. The derivative thereof is an HBV antigen fragment. The virus is a combination of two or more arenaviruses, each expressing a different HBV antigen or fragment thereof. In certain embodiments, the infectious arenavirus viral vector is replication-defective. In certain embodiments, the infectious arenavirus virus is The vector is replicable (see Section 6.1(b)).

[0043] In certain embodiments, the arenavirus is lymphocytic choriomeningitis virus (LCMV) or is Junin virus (JUNV).

[0044] In some embodiments, provided herein arenavirus open read sequences. The coding frame is removed, and the HBV HBs protein and HBV HBc protein or their antigens are Infectious arenaviruses that have been replaced with nucleotide sequences encoding fusions of the original fragments In a specific embodiment, the arenavirus is a lymphocyte-specific arenavirus vector. In a specific embodiment, the glycoprotein of the arenavirus is a choriomeningitis virus. The open reading frame encoding the protein is deleted or functionally inactivated. In a specific embodiment, the viral vector is replication-deficient. In a specific embodiment, the viral vector is replication competent. In one embodiment, the viral vector provided herein is tripartite. The present invention relates to a method of treating or preventing hepatitis B virus infection in a patient, the method comprising administering to the patient In this case, the arenavirus open reading frame is removed and the HBV HBs protein and and a nucleotide sequence encoding a fusion of the HBV HBc protein or an antigenic fragment thereof. administering the replaced viral vector. 3.1 Conventions and Abbreviations [Table 1]

[0045] 4. Explanation of Sequence Listing The following sequences are examples of sequences that can be used with the methods and compositions described herein. In some cases, DNA sequences are used to identify the amino acid and nucleotide sequences of the target gene. Describe the RNA sequence of a viral genome segment. The RNA sequence can be easily deduced from the DNA sequence. The sequences themselves can also be found in Table 3 in Section 6.10.

[0046] SEQ ID NO: 1 is the nucleotide sequence of the HBV pre-S2 / S ORF.

[0047] SEQ ID NO: 2 is the nucleotide sequence of the HBV HBc ORF.

[0048] SEQ ID NO: 3 is the nucleotide sequence of the HBV HBs-HBc fusion protein ORF.

[0049] SEQ ID NO: 4 is the nucleic acid sequence of the LCMV S segment expressing the HBV HBs-HBc fusion protein in cDNA form. The genome segment is RNA, and the sequence in SEQ ID NO: 4 is DNA. However, all thymidines ("T") in SEQ ID NO:4 are replaced with uridine. When replaced with an arginine ("U"), an RNA sequence is provided.

[0050] SEQ ID NO:5 is the nucleotide sequence of the LCMV S segment expressing the HBc ORF in cDNA form. The genome segment is RNA, and the sequence in SEQ ID NO: 5 is shown for DNA. However, replacing all thymidines ("T") in SEQ ID NO:5 with uridines ("U") and the RNA sequence is provided.

[0051] SEQ ID NO: 6 is the nucleotide sequence of the LCMV S segment expressing the pre-S2 / S ORF in cDNA form. The genome segment is RNA, and the sequence in SEQ ID NO: 6 is shown for DNA. However, all thymidines ("T") in SEQ ID NO:6 are replaced with uridines ("U"). In other words, an RNA sequence is provided.

[0052] SEQ ID NO: 7 is the complete sequence of lymphocytic choriomeningitis virus clone 13 segment L (GenBa nk: DQ361066.1). The genome segment is RNA, and the sequence in SEQ ID NO: 7 corresponds to DNA. However, all thymidines ("T") in SEQ ID NO:7 are replaced with uridine. When replaced with an arginine ("U"), an RNA sequence is provided.

[0053] SEQ ID NO: 8 is the amino acid sequence of an epitope derived from HBV HBs protein.

[0054] SEQ ID NO: 9 is the amino acid sequence of an epitope derived from HBV HBs protein.

[0055] SEQ ID NO: 10 is the amino acid sequence of an epitope derived from HBV HBc protein.

[0056] SEQ ID NO: 11 is the complete sequence of lymphocytic choriomeningitis virus segment S. The genome segment is RNA, and the sequence in SEQ ID NO: 11 is that shown for DNA; However, replacing all thymidines ("T") in SEQ ID NO: 11 with uridines ("U") results in an RNA The sequence is provided.

[0057] SEQ ID NO: 12 is the complete sequence of lymphocytic choriomeningitis virus clone 13 segment S (GenB ank: DQ361065.2). The genome segment is RNA, and the sequence in SEQ ID NO: 12 corresponds to DNA. However, all thymidines ("T") in SEQ ID NO: 12 are replaced by When replaced with a gin ("U"), an RNA sequence is provided.

[0058] SEQ ID NO: 13 is the complete sequence of lymphocytic choriomeningitis strain MP segment L. The fragment is RNA, and the sequence in SEQ ID NO: 13 is that shown for DNA; however, However, replacing all thymidines ("T") in SEQ ID NO: 13 with uridines ("U") results in the RNA sequence is provided.

[0059] SEQ ID NO: 14 is the complete sequence of lymphocytic choriomeningitis strain MP segment S. The fragment is RNA, and the sequence in SEQ ID NO: 14 is that shown for DNA; however, However, replacing all thymidines ("T") in SEQ ID NO: 14 with uridines ("U") results in the RNA sequence is provided.

[0060] SEQ ID NO: 15 is the amino acid sequence of the NP protein of the MP strain of LCMV.

[0061] SEQ ID NO: 16 is the amino acid sequence of the GP protein of the MP strain of LCMV.

[0062] SEQ ID NO: 17 is the amino acid sequence of the L protein of the MP strain of LCMV.

[0063] SEQ ID NO: 18 is the amino acid sequence of the Z protein of the MP strain of LCMV.

[0064] SEQ ID NO: 19 is the complete sequence of Junin virus Candid #1 strain segment L.

[0065] SEQ ID NO: 20 is the complete sequence of Junin virus Candid #1 strain segment S.

[0066] SEQ ID NO: 21 is the amino acid sequence of the NP protein of the clone 13 strain of LCMV.

[0067] SEQ ID NO: 22 is the amino acid sequence of the GP protein of the clone 13 strain of LCMV.

[0068] SEQ ID NO: 23 is the amino acid sequence of the L protein of the clone 13 strain of LCMV.

[0069] SEQ ID NO: 24 is the amino acid sequence of the Z protein of the clone 13 strain of LCMV.

[0070] SEQ ID NO: 25 is the amino acid sequence of the GP protein of the WE strain of LCMV.

[0071] SEQ ID NO: 26 is the nucleotide sequence of the HBV HBe antigen. [Brief explanation of the drawings]

[0072] (5. Brief description of the drawings) [Figure 1] Figure 1: The genome of wild-type arenaviruses consists of a short (1; ~3.4 kb) RNA segment and a large (2; ~7.2 kb) RNA segment. The short segment carries ORFs encoding the nucleoprotein (3) and glycoprotein (4). The large segment encodes the RNA-dependent RNA polymerase L (5) and matrix protein Z (6). Wild-type arenaviruses can be made into replication-deficient vaccine vectors by deleting the glycoprotein gene and replacing it with an antigen of choice against which an immune response will be induced (7).

[0073] [Figure 2] Figure 2A-C: Schematic diagram of the genome organization of bipartite and tripartite LCMV. The bipartite genome of wild-type LCMV consists of one S segment encoding GP and NP and one L segment encoding Z and L proteins (A). Both segments are flanked by their respective 5' and 3' UTRs. The genome of recombinant tripartite LCMV (r3LCMV) consists of one L segment and two S segments, with a position for inserting a gene of interest (here, GFP) into each of the S segments. While r3LCMV-GFPnatural (nat) has all viral genes in their natural positions (B), the GP ORF in r3LCMV-GFPartificial (art) is artificially juxtaposed to and expressed under the control of the 3' UTR (C).

[0074] [Figure 3] Figure 3: Hepatitis B virus-specific CD8+ T cells expressed as a percentage of the total CD8+B220- T cell pool in the peripheral blood of C57BL / 6 mice (5 mice per group) 10 days after intravenous immunization with 105 FFU of rLCMV / HBs-HBc (group 1), rLCMV / HBc (group 3), rLCMV / pre-S2 (group 4), or 104 FFU of rLCMV / HBs-HBc (group 2). Control mice were left untreated.

[0075] [Figure 4] Figure 4A-B: Hepatitis B virus-specific CD8+ T cells, expressed as (A) a percentage of the total CD8+B220- T cell pool in peripheral blood or (B) a percentage of circulating lymphocytes in blood, in C57BL / 6 mice (5 mice per group) 8 days after intravenous immunization with 105 FFU of r3LCMV / HBs-HBc (group 1), r3LCMV / HBc (group 2), r3LCMV / pre-S2 (group 3), or 105 FFU of rLCMV / HBs-HBc (group 4). Control mice were left untreated. DETAILED DESCRIPTION OF THE INVENTION

[0076] (6. Detailed Description of the Invention) The present application provides immunotherapy for hepatitis B virus infection. Provided are methods and compositions for treating or preventing HBV infection in a subject. Provided herein are infectious antigens comprising nucleotide sequences encoding HBV antigens. In certain embodiments, the infectious arenavirus is replication-deficient. In certain embodiments, the infectious arenaviruses are replication competent. The compounds can be administered to a subject to treat or prevent HBV infection. The generation of infectious arenavirus vectors for the production of virions is described in more detail in Section 6.3. do.

[0077] Provided herein are genetically modified arenaviruses, wherein the arenavirus Russ is: It is infectious; Non-complementing cells (i.e., those that express functionality missing from replication-deficient arenaviruses) to form infectious progeny virus in cells that are unable to replicate, rendering the virus replication-deficient Unable to; capable of replicating its genome and expressing its genetic information; and It encodes an HBV antigen or a fragment thereof.

[0078] The genetically modified arenaviruses described herein are infectious, i.e., they: It is capable of attaching to a host cell and releasing its genetic material into the host cell. The genetically modified arenavirus can be replication-deficient, i.e., the arenavirus are unable to produce further infectious progeny particles in non-complementing cells. To generate an arenavirus, the genome of the arenavirus may be modified to so that the virus can no longer produce infectious progeny virus (e.g., ORF Non-complementing cells are modified by modifications of the viral genome. Therefore, the cells do not provide the functionality that has been removed from the replication-deficient arenavirus (e.g., , when the ORF encoding the GP protein is deleted or functionally inactivated, (Non-complementing cells do not provide GP proteins). However, the genes provided herein Engineered replication-defective arenaviruses can produce infectious progeny virus in complementation cells Complementation cells have been eliminated from replication-deficient arenaviruses by modifying the viral genome. cells that provide (in trans) the functionality required for the GP protein (e.g., cells lacking the ORF encoding the GP protein). Complementing cells can indeed express GP proteins even when they are missing or functionally inactivated. Expression of complementary functionality (e.g., GP protein) can be achieved by any method known to those skilled in the art ( This can be achieved, for example, by transient or stable expression. Genetically modified arenaviruses amplify and express their genetic information in infected cells. The genetically modified arenaviruses provided herein can be used to express HBV antigens, e.g. Examples include, but are not limited to, nucleotide sequences encoding HBV antigens described in Section 6.2. .

[0079] In certain embodiments, provided herein are methods for expressing the resulting virus in non-complementing cells. The arenavirus genome is then transfected so that it is unable to produce further infectious progeny virus particles. A genetically modified arenavirus in which the ORF of the virus is deleted or functionally inactivated. arena containing genetically modified genomes in which ORFs are deleted or functionally inactivated. The viral particles are then transformed into complementing cells (i.e., deleted or functionally inactivated). can be produced in cells expressing the arenavirus ORF (see Section 6.3). The genetic material of arenavirus particles can be transferred into host cells upon infection. There, the genetic material can be expressed and amplified. The genome of the genetically modified arenavirus particle is capable of expressing HBV antigens in host cells. Code the following.

[0080] In one embodiment, the ORF encoding the glycoprotein (GP) of the arenavirus is deleted. to generate replication-deficient arenaviruses for use with the present invention. In embodiments, the replication-deficient arenavirus comprises a nucleotide sequence encoding an HBV antigen. Thus, in some embodiments, the genome segments provided herein include The genetically modified arenavirus particles to be produced contain a) the ORFs present in the genome segments in the wild-type form; and b) HBV antigen (either sense or antisense). Contains the genomic segment encoding (see Section 6.3).

[0081] In some embodiments, the nucleic acid encoded by the nucleic acid inserted into the genome of the arenavirus The antigens encoded can, for example, encode an HBV antigen or a combination of HBV antigens, including: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof These include, but are not limited to:

[0082] In certain embodiments, the infectious arenavirus viral vector is replication-deficient. (See Section 6.1(a)). In certain embodiments, the infectious arenavirus virus vector is replicable (see Section 6.1(b)).

[0083] A detailed description of the antigens described herein is provided in Section 6.2.

[0084] In certain embodiments, the arenaviruses used in accordance with the invention described herein The virus can be an Old World virus, e.g., lymphocytic choriomeningitis virus (LCMV). A more detailed description of the arenaviruses described herein is provided in Section 6.1. In certain embodiments, the arenaviruses used in accordance with the methods described herein It could be a New World virus.

[0085] Provided herein are nucleic acids comprising the genomes of such replication-deficient arenaviruses. In some embodiments, the infectious replication-deficient arenavirus particles are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or a genomic segment comprising the nucleotide sequence of SEQ ID NO:3.

[0086] Provided herein are the components required for the production of the viral vectors described herein. Specifically, the present invention provides an expression plasmid encoding one or more components to be used in the present invention. Contained is an expression vector encoding the LCMV S segment, wherein the GP protein The ORF is deleted from the S segment and (e.g., in the nucleotide sequence of SEQ ID NO: 1) or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The ORF is replaced with the ORF of the human HBV pre-S2 / S protein (which has an amino acid sequence similar to that of the human HBV pre-S2 / S protein).

[0087] Provided herein are the components required for the production of the viral vectors described herein. Specifically, the present invention provides an expression plasmid encoding one or more components to be used in the present invention. Contained is an expression vector encoding the LCMV S segment, wherein the GP protein The ORF is deleted from the S segment and (e.g., in the nucleotide sequence of SEQ ID NO:2) or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2 The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The ORF is replaced with the ORF of human HBV HBc protein (which has an amino acid sequence similar to that of the human HBV HBc protein).

[0088] Provided herein are the components required for the production of the viral vectors described herein. Specifically, the present invention provides an expression plasmid encoding one or more components to be used in the present invention. Contained is an expression vector encoding the LCMV S segment, wherein the GP protein The ORF is deleted from the S segment and (e.g., in the nucleotide sequence of SEQ ID NO: 3) or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical The ORF of human HBV HBs (having the amino acid sequence corresponding to the HBV HBc ORF) is replaced by the ORF of human HBV HBs. .

[0089] Provided herein is one or more of the vector plasmids described herein. In one embodiment, provided herein is a kit comprising: a) LCMV; a) an expression plasmid containing the nucleotide sequence of the S segment of the vector; b) an expression plasmid containing the nucleotide sequence of the L segment of the LCMV vector and c) an expression plasmid containing the nucleotide sequence of the complementary functional fragment. In a specific embodiment, provided herein is a kit comprising a plasmid. a) an expression vector containing the nucleotide sequence of the LCMV S segment (wherein the O RF is deleted from the S segment and (e.g., in the nucleotide sequence of SEQ ID NO: 1) or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical a) the ORF of the human HBV pre-S2 / S protein (having an amino acid sequence corresponding to the LC c) an expression plasmid containing the nucleotide sequence of the L segment of the LCMV vector; and A plasmid containing an expression plasmid encoding the protein (or a cell line expressing the LCMV GP protein) It is a .

[0090] Provided herein is one or more of the vector plasmids described herein. In one embodiment, provided herein is a kit comprising: a) LCMV; a) an expression plasmid containing the nucleotide sequence of the S segment of the vector; b) an expression plasmid containing the nucleotide sequence of the L segment of the LCMV vector and c) an expression plasmid containing the nucleotide sequence of the complementary functional fragment. In a specific embodiment, provided herein is a kit comprising a plasmid. a) an expression vector containing the nucleotide sequence of the LCMV S segment (wherein the O The RF is deleted from the S segment (e.g., by the nucleotide sequence of SEQ ID NO:2). or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:2. Amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical a) the ORF of the human HBV HBc protein (containing the amino acid sequence); b) the LCMV vector c) an expression plasmid containing the nucleotide sequence of the L segment of LCMV; and A kit containing an expression plasmid encoding the LCMV GP protein (or a cell line expressing the LCMV GP protein) is also provided. do.

[0091] Provided herein is one or more of the vector plasmids described herein. In one embodiment, provided herein is a kit comprising: a) LCMV; a) an expression plasmid containing the nucleotide sequence of the S segment of the vector; b) an expression plasmid containing the nucleotide sequence of the L segment of the LCMV vector and c) an expression plasmid containing the nucleotide sequence of the complementary functional fragment. In a specific embodiment, provided herein is a kit comprising a plasmid. a) an expression vector containing the nucleotide sequence of the LCMV S segment (wherein the O RF is deleted from the S segment and (e.g., in the nucleotide sequence of SEQ ID NO: 3) or the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:3 The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical (having an amino acid sequence corresponding to the ORF of human HBV HBs) and the ORF of human HBV HBc) b) an expression plasmid containing the nucleotide sequence of the L segment of an LCMV vector; and c) an LCMV G containing an expression plasmid encoding the P protein (or a cell line expressing the LCMV GP protein). It is a kit.

[0092] Also provided herein are cell lines, cultures, and nucleic acids provided herein. and methods for culturing cells infected with the vectors and compositions. A more detailed description of vector systems and cell lines is provided in Section 6.4.

[0093] In one aspect, provided herein are such genes suitable as vaccines. Modified replication-defective arenaviruses and their use in vaccination and treatment or prevention of HBV infection and methods of using such arenaviruses in the context of such arenaviruses described herein. A more detailed description of methods for using lenaviruses is provided in Section 6.5.

[0094] In one embodiment, an infectious HBV virus expressing an HBV antigen or fragment thereof described herein is Immunization with arenaviruses provides a long-lasting immune response. In one embodiment, Maximum antibody levels can be achieved after two immunizations. A third immunization can be administered to achieve a booster effect. In the present specification, a vaccine for the treatment and / or prevention of HBV infection is provided. A dosing schedule using infectious arenaviruses in inoculation. A more detailed description of administration schedules using infectious arenaviruses is provided in Section 6.6. In one embodiment, the infectious arenavirus viral vector is replicative. In certain embodiments, the infectious arenavirus virus is defective (see Section 6.1(a)). The vector is replication-competent (see Section 6.1(b)).

[0095] In certain embodiments, a seronegative subject is administered an HBV antigen or fragment thereof described herein. Administration of an infectious arenavirus expressing the α-amyloid β ... In another embodiment, a subject infected with HBV infection is administered the HBV-associated antibody therapy described herein. Administration of infectious arenaviruses expressing the HBV antigens or fragments thereof increases antibody titers. At least 100%, at least 200%, at least 300%, at least 400%, at least 50 In one embodiment, the HBV antigen-expressing infectious The primary antigen exposure from the first immunization with arenaviruses was from human subjects who were immune to the infection. At least 50%, at least 100%, at least 200%, at least 30% of the previous average control serum 0%, at least 400%, at least 500%, or at least 1000% functional (neutralizing) ) and induce minimal antibody titers. In a more specific embodiment, the primary neutralization geometry The antibody titer should be at least 1:50, at least 1:100, or at least 1:100 within at least 4 weeks after immunization. In another embodiment, the β-glucan concentration is increased to a peak value of at least 1:200, or at least 1:1000. Immunization with infectious arenaviruses expressing HBV antigens or fragments thereof described herein. The epidemic lasts for at least four weeks and at least eight weeks after immunization and subsequent vaccination. for at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, produce high-titer antibodies that persist for at least 3 years, at least 4 years, or at least 5 years. In certain embodiments, the infectious arenavirus viral vector is replication-deficient. (See Section 6.1(a)). In certain embodiments, the infectious arenavirus virus vector is replicable (see Section 6.1(b)).

[0096] In yet another embodiment, an infectious arenavirus expressing an HBV antigen or a fragment thereof. Secondary antigen exposure with a second immunization with HIV increases antibody titers by at least 100%, at least 20%. 0%, at least 300%, at least 400%, at least 500%, or at least 1000% increase In another embodiment, the secondary antigen exposure is an average antibody from a human subject who is immune to the infection. At least 50%, at least 100%, at least 200%, at least 300%, or less than the control serum at least 400%, at least 500%, or at least 1000% functional (neutralizing); and In a more specific embodiment, the secondary neutralizing geometric mean antibody titer is The titer should be at least 1:50, at least 1:100, or at least 1: In another embodiment, the present invention provides a method for detecting the increase in the β-glucan concentration of the β-glucan in the presence of β-glucan. A second immunization with an infectious arenavirus expressing an HBV antigen or a fragment thereof as described in the document. at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months after immunization , at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least In one embodiment, the infectious allele produces high titers of antibodies that last for at least five years. The navirus viral vector is replication-deficient (see Section 6.1(a)). In this case, the infectious arenavirus viral vector is replication competent (see Section 6.1(b)).

[0097] In yet another embodiment, the third booster immunization increases antibody titers by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least In another embodiment, the booster is derived from a human subject who is immune to the infection. At least 50%, at least 100%, at least 200%, at least 30% of the previous average control serum 0%, at least 400%, at least 500%, or at least 1000% functional (neutralizing) ) and induces minimal antibody titers. In a more specific embodiment, a third booster Neutralizing geometric mean antibody titers after immunization should be at least 1:50, at least 1:10, within at least 4 weeks after immunization. The increase in the concentration of β-glucan increases to a peak value of at least 1:100, at least 1:200, or at least 1:1000. In an embodiment, the third booster immunization is administered to the patient to increase antibody titers at least 4 weeks after immunization. at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, Extend for at least three years, at least four years, or at least five years.

[0098] In certain embodiments, the infectious arenavirus expressing an HBV antigen or a fragment thereof is expressed in T cells. In another embodiment, the HBV antigen or fragment thereof induces a T cell-independent or T cell-dependent response. Infectious arenaviruses expressing the fragments elicit T cell responses. Infectious arenaviruses expressing the HBV antigens or fragments thereof described herein are T helper In another embodiment, an HBV antigen or fragment thereof described herein induces a vasoconstriction response. Infectious arenaviruses expressing the IL-1 agonist induce either a Th1- or a Th2-directed response. In embodiments, the infectious arenavirus viral vector is replication-deficient (see Section 6.1 In certain embodiments, the infectious arenavirus viral vector is replicative. Yes, it is possible (see Section 6.1(b)).

[0099] In a more specific embodiment, a Th1-directed response is characterized by a predominance of IgG1 antibodies over IgG2. In other embodiments, the ratio of IgG1:IgG2 is greater than 1:1, greater than 2:1, or In another embodiment, the ratio of the β-amino acid to the β-amino acid is greater than 3:1, or greater than 4:1. Infectious arenaviruses expressing HBV antigens or their fragments are inhibited by IgG3 antibodies. In one embodiment, the infectious arenavirus viral vector is replication-deficient. (See Section 6.1(a)). In certain embodiments, the infectious arenavirus virus The vector is replicable (see Section 6.1(b)).

[0100] In some embodiments, the infectious arenavirus expressing an HBV antigen or a fragment thereof. In another embodiment, the antibody expressing an HBV antigen or a fragment thereof induces a CD8+ T cell response. The infectious arenavirus induces a regulatory T cell response. Thus, the regulatory T cell response maintains immune tolerance. Infectious arenaviruses expressing the fragments elicit both CD4+ and CD8+ T cell responses. In certain embodiments, the infectious arenavirus viral vector is replication-deficient. (See Section 6.1(a)). In certain embodiments, the infectious arenavirus vector The target is replicable (see Section 6.1(b)).

[0101] In certain embodiments, the HBV antigens express one or more of the HBV antigens or fragments thereof described herein. Infectious arenaviruses induce high titers of neutralizing antibodies. Infectious arenaviruses expressing two or more HBV antigens or fragments thereof described in the document are proteins. In some embodiments, the protein complex elicits higher titers of neutralizing antibodies than the individual expression of the protein complex components. Thus, the infectious arenavirus viral vector is replication-deficient (see Section 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication competent (e.g., (see paragraph .1(b)).

[0102] In other embodiments, the two or more infectious arenaviruses expressing HBV antigens are In a more specific embodiment, two or more infectious HBV antigen-expressing viruses elicit neutralizing antibodies. Arenaviruses express a single HBV antigen or a fragment thereof, but are more highly infectious than infectious arenaviruses expressing a single HBV antigen or a fragment thereof. In one embodiment, the infectious arenavirus vector induces a high titer of neutralizing antibodies. The vector is replication-deficient (see Section 6.1(a)). In certain embodiments, the infectious arena Viral viral vectors are replication competent (see Section 6.1(b)).

[0103] In another embodiment, the HBV antigens express two, three, four, five, or more HBV antigens. Infectious arenaviruses expressing a single HBV antigen or a fragment thereof In one embodiment, the infectious arenavirus virus induces higher titers of neutralizing antibodies than the In certain embodiments, the infectious vector is replication-deficient (see Section 6.1(a)). Arenavirus viral vectors are replication competent (see Section 6.1(b)).

[0104] 6.1 Arenavirus vectors expressing HBV antigens Arenaviruses for use with the methods and compositions provided herein include those derived from ancient Kingdom viruses, e.g., Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Mobara virus virus, Mopeia virus, or Yippie virus, or a New World virus, e.g. Amapari virus, Flexar virus, Guanarito virus, Junin virus, Latino virus Viruses, Machupo virus, Oliveros virus, Parana virus, Pichinde virus , Piritar virus, Sabia virus, Tacaribe virus, Tamiami virus, Bearca virus It can be a neon virus or a whitewater arroyo virus. The progeny modified arenaviruses can be generated as described in Section 6.3.

[0105] The wild-type arenavirus genome consists of a short (~3.4 kb) RNA segment and a large (~7.2 kb) RNA segment. The short segment is composed of the nucleoprotein NP gene and the glycoprotein GP gene. The large segment contains the ORF encoding the RNA-dependent RNA polymerase L gene. and matrix protein Z gene.

[0106] (a) Replication-deficient arenavirus vector In certain embodiments, the arenavirus vector is a replication-deficient, bipartite arenavirus vector. In certain embodiments, the arenavirus vector is replication-deficient. It is a tripartite arenavirus vector that encodes a glycoprotein gene to target the immune response to the protein. By substituting one or more HBV antigens that induce a response, wild-type ArenA virus Viruses can be made replication-deficient to generate vaccine vectors.

[0107] Infectious arenavirus vectors expressing an HBV antigen or combination of HBV antigens described herein. The vectors can be used to immunize (prophylactically) or treat (immunotherapeutically) subjects against HBV infection. In a specific embodiment, a combination of HBs and HBc is used.

[0108] Arenavirus disease and immunosuppression in wild-type arenavirus infections are unchecked. It is known that they arise from viral replication without any specific mechanism for the release of particles from their genome, e.g. By deleting the Z gene, which is required for the infection of target cells, or the GP gene, which is required for the infection of target cells, Thus, replication of the arenavirus vector, i.e., production of infectious progeny virus particles, By disabling the ability to vaccinate, the total number of infected cells can be reduced, for example, by administering the vaccine to the recipient. inocula used in or inadvertently used on people or animals involved in medical or biotechnological applications. Therefore, the inoculum to be propagated can be limited. Disabling replication of vector particles is a consequence of intentional or unintentional propagation of vector particles. Provided herein is an important embodiment of a method for preventing HBV disease by administering to a subject a HBV-positive cell line expressing an HBV antigen. The object of this invention is to take advantage of the need for copy invalidation in a beneficial way. In embodiments, arenavirus particles are made replication-deficient by genetic modification of their genome. Such modifications to the Nomu include: deletion of an ORF (e.g., an ORF encoding a GP, NP, L, or Z protein); Functional inactivation of an ORF (e.g., an ORF encoding a GP, NP, L, or Z protein). For example, This can be achieved by introducing missense or nonsense mutations. ; Altering the sequence of the ORF (e.g., replacing the S1P cleavage site with the cleavage site of another protease); Mutagenesis of either the 5' or 3' end of one of the genome segments; Mutagenesis of intergenic regions (i.e., intergenic regions of L or S genome segments) Examples include:

[0109] In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein The virus is lymphocytic choriomeningitis virus (LCMV), wherein the S segment of the virus was modified by replacing the ORF encoding the GP protein with the ORF encoding the HBV antigen. It has been done.

[0110] In one embodiment, the wild-type arenavirus vector genome (FIG. 1) contains both segments. At least the necessary 5' and 3' untranslated regions (UTRs) and / or intergenic regions (IGRs) of the Without wishing to be bound by theory, the gene can be designed to retain the necessary regulatory elements. However, minimal trans-acting factors for gene expression in infected cells are expressed. These remain in the vector genome as ORFs that can be used to and can be under the control of a promoter other than the native one, or can be expressed from an internal ribosome entry site. The nucleic acid encoding the antigen is driven by an endogenous arenavirus promoter (i.e., the promoter of the S segment The 5'UTR, 3'UTR, 5'UTR, 3'UTR of the L segment are transcribed from one of the following: In the HBV genome, the nucleic acid encoding the HBV antigen is synthesized by the viral RNA-dependent RNA polymerase. , read by cellular RNA polymerase I, RNA polymerase II, or RNA polymerase III A heterologous promoter sequence can be introduced, for example, a viral UTR. Duplicate viral promoter sequences found naturally in the 28S ribosomal RNA promoter, It is expressed from the β-actin promoter or the 5S ribosomal RNA promoter. In some embodiments, the ribonucleic acid encoding the HBV antigen is used alone or in combination with an arenavirus protein. The protein is transcribed and translated as a read-through by fusion with the protein ORF, and then expressed in the host cell. Expression of the ribosome may involve the insertion of one or more, e.g., two, three, or four, internal ribosome entry sites at appropriate locations. This can be enhanced by introducing it into the viral transcript sequence.

[0111] In certain embodiments, the compositions and methods provided herein are (i) the ORF is in a position other than the wild-type position of the ORF; and (ii) the resulting virus is more GP or NP encoding the virus so that it is unable to produce infectious progeny virus particles consisting of One L segment and two S segments, in which the ORFs have been deleted or functionally inactivated In a specific embodiment, the virus particle is a tripartite arenavirus particle comprising one OR segment. F is removed and non-arenaviruses derived from organisms other than arenaviruses (e.g., encoding HBV antigens) are used. In another specific embodiment, two ORFs are removed and replaced with homologous ORFs. The heterologous ORF may be replaced with a heterologous ORF from an organism other than an arenavirus. In this case, three ORFs have been removed and a non-arenavirus-derived ORF (e.g., HBV antigen) has been added. In a specific embodiment, the GP-encoding ORF is replaced with a heterologous ORF (encoding the original gene). The ORFs encoding the antigens of non-arenaviruses have been removed, and the ORFs encoding the antigens of non-arenaviruses (e.g., HBV antigens) have been removed. In another specific embodiment, the ORF encoding NP is replaced with a heterologous ORF. non-correlated antigens (e.g., encoding HBV antigens) derived from organisms other than arenaviruses In an even more specific embodiment, the ORF encoding NP and The ORF encoding the GP has been removed, and the virus particles are derived from organisms other than arenavirus particles (e.g., one or more The HBV antigens are replaced with one or two heterologous ORFs (encoding two HBV antigens). In one embodiment, the tripartite arenavirus particle comprises (i) one L segment and two S segments. (ii) an ORF at a position other than the wild-type position of the ORF; (iii) an ORF derived from an organism other than an arenavirus It contains one or more heterologous ORFs (e.g., encoding one or more HBV antigens).

[0112] In certain embodiments, the compositions and methods provided herein are (i) the ORF is in a position other than the wild-type position of the ORF; and (ii) the resulting virus is more and / or the Z protein, so that they are unable to produce infectious progeny virus particles consisting of In the two cases, the ORF encoding the L protein has been deleted or functionally inactivated. It is a tripartite arenavirus particle containing one L segment and one S segment. In an embodiment, one ORF is removed and the vector is derived from an organism other than an arenavirus (e.g., In another specific embodiment, the sequence of the HBV antigen is replaced with a heterologous ORF (e.g., encoding an HBV antigen). In this case, two ORFs have been removed and the antigen is derived from an organism other than an arenavirus (e.g., HBV antigen). In a specific embodiment, the Z protein is replaced with a heterologous ORF. The ORFs encoding the antigens of non-arenaviruses have been removed, and the ORFs encoding the antigens of non-arenaviruses (e.g., HBV antigens) have been removed. In another specific embodiment, the L protein is replaced with a heterologous ORF encoding the L protein. The ORF encoding the virus has been removed, and the virus is derived from an organism other than an arenavirus (e.g., HBV antigen encoding the virus). In an even more specific embodiment, the Z protein is replaced with a heterologous ORF (which encodes the The ORF encoding the protein and the ORF encoding the L protein are deleted, and the arenavirus particles It is replaced with a heterologous ORF (e.g., encoding an HBV antigen) from an organism other than the host. Thus, in certain embodiments, the tripartite arenavirus particle comprises: (i) two L segments; and one S segment; (ii) an ORF at a position other than the wild-type position of the ORF; (iii) a non-arenavirus The present invention also includes heterologous ORFs (e.g., encoding HBV antigens) derived from other organisms.

[0113] Thus, in certain embodiments, for use with the compositions and methods provided herein, Tripartite arenavirus particles for use in the present invention may be: i) engineered to carry an ORF in a non-native position; ii) the ORF encoding the GP, NP, Z protein, or L protein is deleted; iii) deleted The ORFs to be used are derived from organisms other than arenaviruses (e.g., encoding one or more HBV antigens). A tripartite arenavirus particle (i.e., one L segment) that replaces one or more non-homologous ORFs. It contains either a nucleotide and two S segments or two L segments and one S segment.

[0114] In certain embodiments, the vectors constructed to encode one or more HBV antigens are LC The method can be based on a specific strain of LCMV, such as Clone 13, MP strain, or Arm CA 137. 1, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009 , 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN In one embodiment, a HBV antigen encoding one or more HBV antigens is provided. The vectors constructed can be based on clone 13 of LCMV. In the present invention, a vector constructed to encode one or more HBV antigens is based on the MP strain of LCMV. The sequence of the S segment of LCMV clone 13 is listed as SEQ ID NO: 12. In one embodiment, the sequence of the S segment of LCMV clone 13 is SEQ ID NO: 11. The sequence of the L segment of LCMV clone 13 is listed as SEQ ID NO: 7. The sequence of the S segment of LCMV strain MP is listed as SEQ ID NO: 14. LCMV The sequence of the L segment of strain MP is listed as SEQ ID NO:13.

[0115] In certain embodiments, the vectors constructed to encode one or more HBV antigens are The vaccine can be based on a specific strain of Junin virus. strains XJ13, XJ#44, and Candid#1, as well as the human isolate IV4454. In the method, a vector constructed to encode one or more HBV antigens is used to Based on ndid #1 strain.

[0116] In some embodiments, described herein is a sequence selected from SEQ ID NO: 13, SEQ ID NO: 14. or a fragment thereof, or a combination thereof. It is a virus particle.

[0117] In some embodiments, described herein are: Nucleotide sequence encoding the hepatitis B virus pre-S2 / S protein or an antigenic fragment thereof column; a nucleotide sequence encoding the hepatitis B virus HBc protein or an antigenic fragment thereof; · a nucleotide sequence encoding the hepatitis B virus HBs protein or an antigenic fragment thereof; Hepatitis B virus HBs protein and HBc protein or a fusion of these antigenic fragments coding nucleotide sequence; A nucleotide sequence encoding the hepatitis B virus HBe protein or an antigenic fragment thereof an infectious agent comprising a nucleotide sequence or a combination of nucleotide sequences selected from the group consisting of Replication-deficient arenavirus particles.

[0118] In certain embodiments, the infectious replication-defective arenavirus vector is tripartite.

[0119] (b) Replication-competent tripartite arenavirus vector In certain embodiments, the compositions and methods provided herein are In one embodiment, the vector is a replication-competent tripartite arenavirus vector. The arenavirus vector must recombine into a replication-competent bipartite arenavirus particle. One L segment and two S segments or two L segments and one S segment It is a tripartite arenavirus particle containing

[0120] In certain embodiments, for use with the compositions and methods described herein Infectious arenaviruses expressing HBV antigens contain viral ORFs at positions other than the wild-type position of the ORF. In some embodiments, the arenavirus genome sequence is modified to carry the The segments are: (i) the S segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; i) the S segment in which the ORF encoding the Z protein is under the control of the arenavirus 5'UTR; (iii) (iv) the S segment, in which the ORF encoding the L protein is under the control of the arenavirus 5'UTR; (v) an S segment in which the ORF encoding the L protein is under the control of the arenavirus 3'UTR; (vi) an S segment whose encoding ORF is under the control of the arenavirus 3'UTR; (vii) an S segment in which the ORF encoding the GP is under the control of the arenavirus 3'UTR; (viii) the L segment under the control of the arenavirus 5'UTR; and (viii) the ORF encoding NP. (ix) the L segment under the control of the virus 5'UTR; (ix) the ORF encoding the L protein is (x) The L segment is under the control of the virus 5'UTR; (x) the ORF encoding GP is under the control of the arenavirus 3'UTR (xi) the L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR and (xii) an L segment in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR. segments.

[0121] In some embodiments, the arenavirus 3'UTR comprises an arenavirus S segment. or the 3'UTR of an arenavirus L segment. The 5'UTR is the 5'UTR of an arenavirus S segment or an arenavirus L segment.

[0122] For use with the compositions and methods, provided herein are reproducible versions of the ORFs. In one embodiment, the composition and method are tripartite arenavirus particles with sequences. For use with the present invention, one L segment and two S segments are provided. are tripartite arenavirus particles containing two or two L segments and one S segment. In certain embodiments, the tripartite arenavirus particle is a replication-competent bipartite arenavirus. In a specific embodiment, the tripartite arenavirus does not recombine into a viral particle. The particle comprises an ORF at a position other than the wild-type position of the ORF. , the tripartite arenavirus particle contains all four arenavirus ORFs. In certain embodiments, the tripartite arenavirus particles are replication competent and infectious. shows an exemplary schematic diagram of the genome organization of a replication-competent tripartite LCMV vector (Figure 2B-C). Figure 2C shows replication-competent arenaviruses that cannot recombine into replication-competent bipartite arenavirus particles. 2A shows an exemplary schematic diagram of the genome organization of a tripartite LCMV vector that can be used. indicates the wild-type bipartite LCMV vector.

[0123] In one embodiment, the GP, NP, Z of the tripartite arenavirus particles described herein The ORF encoding the L protein is located in the arenavirus 3'UTR or In a more specific embodiment, the tripartite allele can be under the control of the 5' UTR. The arenavirus 3'UTR is the 3'UTR of an arenavirus S segment. wherein the tripartite arenavirus 3'UTR is the 3'UTR of an arenavirus L segment. In a more specific embodiment, the tripartite arenavirus 5'UTR is In another specific embodiment, the 5'UTR is a 5'UTR of an arenavirus segment. This is the 5'UTR of the L segment.

[0124] In other embodiments, the GP, NP, Z of the tripartite arenavirus particles described herein The ORF encoding the L protein contains the conserved terminal sequence of arenaviruses. It can be under the control of a 19- to 20-nt region of the 5'- and 3'-ends (e.g., Pere z and de la Torre, 2003, J Virol. 77(2): 1184-1194).

[0125] In certain embodiments, the GP, NP, Z protein, or L protein of the tripartite arenavirus particle The protein-encoding ORF can be under the control of a promoter element in the 5'UTR. (See, e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In the method, the GP, NP, Z protein, or L protein of the tripartite arenavirus particle is The loading ORF can be under the control of a promoter element in the 3'UTR (e.g., A (See Ibarino et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, The promoter element of the 5'UTR is a 5'UTR promoter element of the S segment or the L segment. In another specific embodiment, the promoter element of the 3'UTR The promoter element is the 3'UTR promoter element of the S or L segment.

[0126] In certain embodiments, the GP, NP, Z protein, or L protein of the tripartite arenavirus particle The protein-encoding ORF may be a truncated arenavirus 3'UTR or a truncated arenavirus 5' It can be under the control of a UTR (see, e.g., Perez and de la Torre, 2003, J Virol. 77(2): 1184-1194; see Albarino et al., 2011, J Virol., 85(8):4020-4). In a specific embodiment, the truncated 3'UTR is an arenavirus S segment or an L segment. In a more specific embodiment, the truncated 5' UTR is the 3' UTR of an arenavirus S The 5'UTR of the L segment or the L segment.

[0127] In one aspect, the compositions and methods provided herein are is a tripartite arenavirus particle containing one L segment and two S segments. In one embodiment, a tripartite Arenavirus comprising one L segment and two S segments. Propagation of the virus particles does not result in replication-competent bipartite virus particles. In the same manner, tripartite arenavirus particles containing one L segment and two S segments The proliferation of the virus is at least 10 days, at least 20 days, at least 30 days, or at least 10 days after persistent infection. At least 40 days later, at least 50 days later, at least 60 days later, at least 70 days later, at least After 80 days, at least 90 days, or at least 100 days, type I interferon receptor, type II lacking the type 1 interferon receptor and recombination activating gene (RAG1), and 4 PFU's 3 minutes Replication-competent bipartite virus particles in mice infected with segmented arenavirus particles. In another embodiment, the amino acid sequence of the present invention is a sequence comprising one L segment and two S segments. The propagation of tripartite arenavirus particles was confirmed after at least 10 passages and at least 20 passages. after at least 30 passages, after at least 40 passages, or after at least 50 passages , does not produce replication-competent bipartite virus particles.

[0128] Tripartite arenavirus particles containing all viral genes in their respective wild-type locations Such antibodies are known in the art (see, e.g., Emonet et al., 2011 J. Virol., 85(4):1473; P Opkin et al., 2011, J. Virol. 85(15):7928). In particular, the tripartite arenavirus genome consists of one L segment and two S segments, where a heterologous ORF (e.g., GFP) is inserted at one position in each S segment. More specifically, The S segment encodes GP and GFP, respectively. The other S segment encodes GFP and NP, respectively. The L segment encodes the L protein and the Z protein. The nucleotides are flanked by the respective 5'UTR and 3'UTR.

[0129] In certain embodiments, for use with the compositions and methods provided herein Intersegmental recombination of two S segments of a tripartite arenavirus particle, resulting in the formation of two arenaviruses. The leukemia virus ORFs are grouped together on one segment rather than on two separate segments. Intersegment recombination occurs when a non-functional promoter (i.e., structure: 5'UTR---------- -5'UTR or 3'UTR------------3'UTR genomic segment), wherein the genomic Each UTR forming one end of a genome is an inverted repeat of the other end of the same genome. be.

[0130] In one embodiment, a tripartite arena comprising one L segment and two S segments. Virus particles are engineered to carry arenavirus ORFs at positions other than the wild-type position of the ORF In another embodiment, a tripartite fragment containing one L segment and two S segments is used. Nodal arenavirus particles contain two arenavirus ORFs or three arenavirus ORFs at positions other than the wild-type position. one arenavirus ORF, or four arenavirus ORFs, or five arenavirus ORFs, or modified to carry six arenavirus ORFs. Therefore, tripartite arenavirus particles containing one L segment and two S segments are necessary. Thus, in some embodiments, the Tripartite arenavirus particles are infectious and replication-competent tripartite arenavirus particles In a specific embodiment, the two S segments of the tripartite arenavirus particle are It has been modified to carry one of these ORFs at a position other than the native position. In a specific embodiment, the two S segments comprise the required number of S segment ORFs. In a specific embodiment, the L segment carries an ORF at a position other than the wild-type position. or the L segment is a wild-type genomic segment. can.

[0131] In some embodiments, one of the two S segments is: (i) Arenaviruses in which the ORF encoding the Z protein is under the control of the arenavirus 5'UTR S segment; (ii) Arenavirus, in which the ORF encoding the L protein is under the control of the arenavirus 5'UTR S segment; (iii) The ORF encoding NP is located in the arenavirus S segment under the control of the arenavirus 5'UTR. nt; (iv) The ORF encoding GP is under the control of the arenavirus 3'UTR in the arenavirus S segment. nt; (v) The L-encoding ORF is under the control of the arenavirus 3'UTR. and (vi) Arenavirus in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR S segment It can be.

[0132] In one embodiment, a tripartite arena comprising one L segment and two S segments. The viral particles contain overlapping ORFs (i.e., two wild-type S segment ORFs, e.g., GP or N). In a specific embodiment, the nucleotide sequence may comprise one L segment and two S segments. Tripartite arenavirus particles containing the nucleotide sequence may contain one overlapping ORF (e.g., (GP, GP)) or two overlapping ORFs. The sequence may include overlapping ORFs (e.g., (GP, GP) and (NP, NP)).

[0133] Table 1A below shows a tripartite arenavirus particle containing one L segment and two S segments. 1 is an exemplary illustration of the genome organization of a tripartite arenavirus genome, wherein two Intersegmental recombination of the S segments does not result in replication-competent bipartite virus particles, abolish arenavirus promoter activity (i.e., the resulting recombinant S segment , composed of two 3'UTRs rather than a 3'UTR and a 5'UTR). Table 1A Tripartite arenavirus particles containing one L segment and two S segments. Position 1 is under the control of the arenavirus S segment 5'UTR; position 2 is under the control of the arenavirus S segment 5'UTR position 3 is under the control of the arenavirus S segment 5'UTR; position 4 is under the control of the arenavirus S segment 5'UTR; position 5 is under the control of the arenavirus S segment 5'UTR Position 4 is under the control of the arenavirus S segment 3'UTR; position 5 is under the control of the arenavirus L segment position 6 is under the control of the arenavirus L segment 3'UTR. * The ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted. is doing. [Table 2] TIFF2026036693000004.tif201170

[0134] In certain embodiments, the IGR between positions 1 and 2 is an arenavirus S segment or L segment. the IGR between positions 2 and 3 can be an arenavirus S segment or the L segment IGR; and the IGR between positions 5 and 6 can be an arenavirus L In a specific embodiment, the IGR between positions 1 and 2 is can be an arenavirus S segment IGR; an IGR between positions 2 and 3 can be an arenavirus S segment IGR; and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In some embodiments, other combinations are possible. For example, a tripartite arenavirus particle containing one L segment and two S segments, Intersegmental recombination of the two S segments in the tripartite arenavirus genome results in multiple Neutralizes arenavirus promoter activity without producing producible bipartite virus particles (i.e., the resulting recombinant S segment consists of two 5'UTRs rather than a 3'UTR and a 5'UTR. (Consists of:

[0135] In one embodiment, a tripartite arena comprising one L segment and two S segments. Intersegmental recombination of the S and L segments in the virus particle results in the division of the two viruses A functional segment in which the gene is located on only one segment, rather than on two separate segments. In another embodiment, one L segment and two S segments are restored. Intersegmental recombination of S and L segments in tripartite arenavirus particles containing does not produce replication-competent bipartite virus particles.

[0136] Table 1B below shows a tripartite arenavirus particle containing one L segment and two S segments. 1 is an exemplary illustration of the genome organization of the S segment in the tripartite arenavirus genome. Intersegmental recombination of the L and L segments does not produce replicative bipartite virus particles. , abolishing arenavirus promoter activity (i.e., the resulting recombinant S segment consists of two 3'UTRs rather than a 3'UTR and a 5'UTR). Table 1B Tripartite arenavirus particles containing one L segment and two S segments. Position 1 is under the control of the arenavirus S segment 5'UTR; position 2 is under the control of the arenavirus S segment 5'UTR position 3 is under the control of the arenavirus S segment 5'UTR; position 4 is under the control of the arenavirus S segment 5'UTR; position 5 is under the control of the arenavirus S segment 5'UTR Position 4 is under the control of the arenavirus S segment 3'UTR; position 5 is under the control of the arenavirus L segment position 6 is under the control of the arenavirus L segment 3'UTR. * The ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted. is doing. [Table 3]

[0137] In one embodiment, the IGR between position 1 and position 2 is an arenavirus S segment or L segment. the IGR between positions 2 and 3 can be an arenavirus S segment or and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In a specific embodiment, the IGR between positions 1 and 2 can be an allele. the IGR between positions 2 and 3 can be an arenavirus S segment IGR; and the IGR between positions 5 and 6 is an arenavirus L segment IGR. In some embodiments, other combinations are possible. For example, a tripartite arenavirus particle comprising one L segment and two S segments, wherein the three Intersegmental recombination of two S segments in a segmented arenavirus genome results in replication-competent It prevents the production of bipartite virus particles and disables arenavirus promoter activity (i.e., That is, the resulting recombinant S segment consists of two 5'UTRs rather than a 3'UTR and a 5'UTR. (Can be).

[0138] In one aspect, the compositions and methods provided herein are is a tripartite arenavirus particle containing two L segments and one S segment. In one embodiment, a tripartite Arenavirus comprising two L segments and one S segment. Propagation of the virus particles does not result in replication-competent bipartite virus particles. In the same manner, tripartite arenavirus particles containing two L segments and one S segment The proliferation of the virus is at least 10 days, at least 20 days, at least 30 days, or at least 10 days after persistent infection. At least 40 days later, at least 50 days later, at least 60 days later, at least 70 days later, at least After 80 days, at least 90 days, or at least 100 days, type I interferon receptor, type II lacking the type 1 interferon receptor and recombination activating gene (RAG1), and 4 PFU's 3 minutes Replication-competent bipartite virus particles in mice infected with segmented arenavirus particles. In another embodiment, the amino acid sequence of the present invention is a sequence comprising two L segments and one S segment. The propagation of tripartite arenavirus particles was confirmed after at least 10 passages and at least 20 passages. after at least 30 passages, after at least 40 passages, or after at least 50 passages , does not produce replication-competent bipartite virus particles.

[0139] In certain embodiments, for use with the compositions and methods provided herein Intersegmental recombination of two L segments of a tripartite arenavirus particle, resulting in the formation of two arenaviruses. The leukemia virus ORFs are grouped together on one segment rather than on two separate segments. Intersegment recombination occurs when a non-functional promoter (i.e., structure: 5'UTR---------- -5'UTR or 3'UTR------------3'UTR genomic segment), wherein the genomic Each UTR forming one end of a genome is an inverted repeat of the other end of the same genome. be.

[0140] In one embodiment, a tripartite arena comprising two L segments and one S segment. Virus particles are engineered to carry arenavirus ORFs at positions other than the wild-type position of the ORF In another embodiment, a tripartite fragment containing two L segments and one S segment is used. Nodal arenavirus particles contain two arenavirus ORFs or three arenavirus ORFs at positions other than the wild-type position. one arenavirus ORF, or four arenavirus ORFs, or five arenavirus ORFs, or modified to carry six arenavirus ORFs. Therefore, tripartite arenavirus particles containing two L segments and one S segment are necessary. Thus, in some embodiments, the Tripartite arenavirus particles are infectious and replication-competent tripartite arenavirus particles In a specific embodiment, the two L segments of the tripartite arenavirus particle are It has been modified to carry one of these ORFs at a position other than the native position. In a specific embodiment, the two L segments comprise the total number of L segment ORFs. In a specific embodiment, the S segment has the sequence of the S sequence of the ORF at a position other than the wild-type position. or the S segment is modified to have one of the S segments of the wild-type genome. It can be a ment.

[0141] In some embodiments, one of the two L segments is: (i) the L segment, in which the ORF encoding GP is under the control of the arenavirus 5′UTR; (ii) the L segment, in which the ORF encoding NP is under the control of the arenavirus 5′UTR; (iii) the L segment, in which the ORF encoding the L protein is under the control of the arenavirus 5′UTR; (iv) the L segment, in which the ORF encoding GP is under the control of the arenavirus 3′UTR; (v) an L segment in which the ORF encoding NP is under the control of the arenavirus 3'UTR; and (vi) the L segment, in which the ORF encoding the Z protein is under the control of the arenavirus 3'UTR; It can be.

[0142] In one embodiment, a tripartite arena comprising one L segment and two S segments. The virus particles contain overlapping ORFs (i.e., two wild-type L segment ORFs, e.g., Z protein In a specific embodiment, the polypeptide may comprise two L segments. Tripartite arenavirus particles containing one S segment and one overlapping ORF (e.g., , (Z protein, Z protein)) or two overlapping ORFs (e.g., (Z protein, Z protein) It may contain (L protein) and (L protein, L protein).

[0143] Table 2A below shows a tripartite arenavirus particle containing two L segments and one S segment. 1 is an exemplary illustration of the genome organization of a tripartite arenavirus genome, wherein two Intersegmental recombination of the L segment of does not produce replication-competent bipartite virus particles, abolishes arenavirus promoter activity (i.e., the S segment is located between the 3'UTR and 5'UTR) Based on Table 3, rather than a 3'UTR and a 5'UTR, Similar combinations are predicted to generate arenavirus particles composed of two 5'UTRs. It is possible. Table 2A Tripartite arenavirus particles containing two L segments and one S segment. * Position 1 is under the control of the arenavirus L segment 5'UTR; position 2 is under the control of the arenavirus L segment position 3 is under the control of the arenavirus L segment 5'UTR; position 4 is under the control of the arenavirus L segment 5'UTR; position 5 is under the control of the arenavirus L segment 5'UTR Position 4 is under the control of the arenavirus L segment 3'UTR; position 5 is under the control of the arenavirus S segment position 6 is under the control of the arenavirus S segment 3'UTR; * The ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted. is doing. [Table 4] TIFF2026036693000007.tif106170

[0144] In one embodiment, the IGR between position 1 and position 2 is an arenavirus S segment or L segment. the IGR between positions 2 and 3 can be an arenavirus S segment or and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In a specific embodiment, the IGR between positions 1 and 2 can be an allele. the IGR between positions 2 and 3 can be an arenavirus L segment IGR; and the IGR between positions 5 and 6 is an arenavirus S segment IGR. In some embodiments, other recombinations are possible.

[0145] In one embodiment, a tripartite arena comprising two L segments and one S segment. Intersegmental recombination of the L and S segments from the virus particles results in the formation of two viruses. The functional gene is located on only one segment, rather than on two separate segments. In another embodiment, two L segments and one S segment are restored. Intersegmental recombination of L and S segments in tripartite arenavirus particles containing does not produce replication-competent bipartite virus particles.

[0146] Table 2B below shows a tripartite arenavirus particle containing two L segments and one S segment. 1 is an exemplary illustration of the genome organization of a tripartite arenavirus genome, wherein the L segment in the tripartite arenavirus genome is Intersegmental recombination of the SV40 and S segments generates replication-competent bipartite virus particles. This prevents the arenavirus promoter from interacting with the ribosomal RNA, thereby neutralizing the arenavirus promoter activity (i.e., the resulting recombinant S (The fragment consists of two 3'UTRs, rather than a 3'UTR and a 5'UTR.) Table 2B Tripartite arenavirus particles containing two L segments and one S segment. * Position 1 is under the control of the arenavirus L segment 5'UTR; position 2 is under the control of the arenavirus L segment position 3 is under the control of the arenavirus L segment 5'UTR; position 4 is under the control of the arenavirus L segment 5'UTR; position 5 is under the control of the arenavirus L segment 5'UTR Position 4 is under the control of the arenavirus L segment 3'UTR; position 5 is under the control of the arenavirus S segment position 6 is under the control of the arenavirus S segment 3'UTR; * The ORF indicates that a heterologous ORF, for example, a heterologous ORF encoding an HBV antigen, has been inserted. is doing. [Table 5]

[0147] In one embodiment, the IGR between position 1 and position 2 is an arenavirus S segment or L segment. the IGR between positions 2 and 3 can be an arenavirus S segment or and the IGR between positions 5 and 6 can be an arenavirus L segment IGR. In a specific embodiment, the IGR between positions 1 and 2 can be an allele. the IGR between positions 2 and 3 can be an arenavirus L segment IGR; and the IGR between positions 5 and 6 is an arenavirus S segment IGR. In some embodiments, other recombinations are possible.

[0148] In certain embodiments, the tripartite arenavirus particles described herein are infectious. and produce replication-competent arenavirus particles. In certain embodiments, the arenavirus particles described in arenavirus particles are attenuated. Nodal arenavirus particles allow the virus to remain, at least partially, replication-competent. and can replicate in vivo but produce only low viral loads; Attenuated to produce subclinical infections that are non-pathogenic. In another embodiment, the antigenic virus can be used as an immunogenic composition. Although lenavirus particles are infectious, they do not produce additional infectious progeny in non-complementing cells. cannot be done.

[0149] In some embodiments, the arenavirus genome segments and their respective arenaviruses are The virus particle or tripartite arenavirus particle may comprise heterologous ORFs. In the arenavirus genome segment and the respective arenavirus particle or tripartite The nodal arenavirus particle can include a gene of interest. In a more specific embodiment, the heterologous ORF or gene of interest encodes an antigen. Therefore, the heterologous ORF or gene of interest may encode an HBV antigen or an antigenic fragment thereof (see Section 6.2). do.

[0150] In certain embodiments, an arenavirus genome segment, an arenavirus particle, or Tripartite arenavirus particles may contain one or more heterologous ORFs or one or more genes of interest. In other embodiments, arenavirus genome segments, arenavirus particles, or tripartite arenavirus particles contain at least one heterologous ORF, at least two heterologous ORFs, It may contain one homologous ORF, at least three heterologous ORFs, or more heterologous ORFs. In other embodiments, the arenavirus particle or tripartite arenavirus particle comprises at least At least one gene of interest, at least two genes of interest, at least three genes of interest In a more specific embodiment, the gene comprises one or more non-target genes. The homologous ORF or gene of interest encodes one or more HBV antigens or antigenic fragments thereof (see Section 6.2). Code.

[0151] In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein are tripartite arenavirus particles containing one L segment and two S segments. In certain embodiments, an infectious arenavirus expressing an HBV antigen described herein are tripartite arenavirus particles containing two L segments and one S segment. .

[0152] (6.2 HBV antigen) In certain embodiments, for use with the methods and compositions described herein The antigen is HBV antigen.

[0153] In one embodiment, the ORFs of two or more of the listed HBV antigens are transcribed as a single transcript. It is photographed.

[0154] In certain embodiments, any genotype or subgenotype of human HBV or any subtype of human HBV Any clinical isolate may be used in conjunction with the present invention to express the arenavirus vectors described herein. Such HBV genotypes and subgenotypes include: Genotypes A to J, as well as subgenotypes A1 to A6, B1 to B4, C1 to C6, D1 to D7, and F1 to F4 are included. do.

[0155] In certain embodiments, the HBV antigen is an HBV antigen ortholog, e.g., a mammalian (i.e., The antigen may be a non-human primate, porcine, canine, feline, or equine HBV antigen.

[0156] (a) Pre-S2 / S protein antigen In certain embodiments, the antigen is HBV pre-S2 / S protein or a fragment thereof. In an embodiment, the antigen comprises at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250 , 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acid fragments In certain embodiments, the antigen is an antigenic fragment of the HBV pre-S2 / S protein. In one embodiment, the antigen is a sequence identical to SEQ ID NO: 1 and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 10 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% identical to the nucleic acid sequence of the antibody. The original sequence is 80%, 81%, and 82% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. 2%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence.

[0157] ((b) HBc protein antigen) In certain embodiments, the antigen is HBV HBc protein or a fragment thereof. In embodiments, the antigen comprises at least 10, 15, 20, 25, 50, 75, 100, or more of the HBV HBc protein. In certain embodiments, the antibody is a fragment of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 The antigen is an antigenic fragment of HBc. In one embodiment, the antigen is a fragment of SEQ ID NO: 2 and 80%, 81% or more of SEQ ID NO: 3. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 100% identical to In one embodiment, the antigen is encoded by the nucleotide sequence of SEQ ID NO:2. The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical Contains the amino acid sequence.

[0158] ((c) HBs antigen) In certain embodiments, the antigen is HBV HBs protein or a fragment thereof. In embodiments, the antigen comprises at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, In certain embodiments, the antigen is a fragment of 45, 50, or more amino acids. is an antigenic fragment of HBs.

[0159] In certain embodiments, the antigen is HBV HBs small polypeptide (e.g., "S") or a fragment thereof. In certain embodiments, the antigen is an HBV HBs polypeptide (e.g., a "pre-S" fragment). 2 / S") or a fragment thereof. In one embodiment, the antigen is HBV HBs large polypeptide ( For example, "pre-S1 / pre-S2 / S") or a fragment thereof. In some embodiments, the antigen is , HBV HBs small polypeptide at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 8 In some embodiments, the fragment is 0, 90, 100, 150, or more amino acids. The antigen is at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of a polypeptide in HBV HBs. , 60, 70, 80, 90, 100, 150, or more amino acids. In such a case, the antigen is at least 10, 15, 20, 25, 30, 35, 40 or 50% of the HBV HBs large polypeptide. a fragment of 0, 45, 50, 100, 150, 200, 250, 300, 350, or more amino acids .

[0160] (d) HBs and HBc fusion proteins In certain embodiments, the antigen is HBV HBs protein and HBV HBc protein or both. In one embodiment, the antigen is a fusion protein of antigenic fragments of HBs and HBc. at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or a fragment of more amino acids. In one embodiment, the antigen is 3 and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleic acid sequence. In certain embodiments, the antigen is encoded by the nucleotide sequence of SEQ ID NO:3. The amino acid sequence encoded by the , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It contains an amino acid sequence that is identical to the amino acid sequence of the amino acid sequence.

[0161] ((e) HBe protein antigen) In certain embodiments, the antigen is HBV HBe protein or a fragment thereof. In embodiments, the antigen comprises at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, , 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids. In certain embodiments, the antigen is an antigenic fragment of HBe. The original sequence was sequence number 26 and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nuclei In one embodiment, the antigen is encoded by the nucleotide sequence of SEQ ID NO:26. The amino acid sequence encoded by the nucleotide sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or an amino acid sequence that is 100% identical.

[0162] (f) Polymerase protein antigen In certain embodiments, the antigen is an HBV polymerase protein or an antigenic fragment thereof. In one embodiment, the antigen comprises at least 10, 100, or 1000 of the HBV polymerase protein. 5, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700 pieces , or a fragment of more amino acids.

[0163] The nucleic acid sequences encoding HBV antigens include glycoprotein GP, ​​matrix protein Z, and nucleoprotein Substitution of the nucleic acid sequence of the ORF of the protein NP or the polymerase protein L results in the production of infectious arena In another embodiment, a nucleic acid encoding an HBV antigen can be introduced into the viral genome. The nucleic acid sequence may be a glycoprotein GP, ​​a matrix protein Z, a nucleoprotein NP, or a polyprotein. The nucleotide sequence encoding the HBV antigen is fused to the ORF of the HBV fusion protein L. Once inserted into the genome of an infectious arenavirus, the four arenavirus promoters - (5'UTR and 3'UTR of the S segment, and 5'UTR and 3'UTR of the L segment), as well as viral RNA-dependent RNA polymerase, cellular RNA polymerase I, and RNA polymerase Regulatory elements that can be read by RNA polymerase II or RNA polymerase III, e.g., These sequences are the duplicated viral promoter sequences naturally found in viral UTRs, the 28S ribosomal promoter sequences, and the 28S ribosomal promoter sequences. ribosomal RNA promoter, β-actin promoter, or 5S ribosomal RNA promoter The ribonucleic acid can be inserted into a nucleic acid molecule that can be transcribed and / or expressed under the control of the ribonucleic acid. The nucleic acid encoding the HBV antigen may be used alone or in combination with the arenavirus ORF and gene, respectively. as a read-through by fusion of, and / or one or more, e.g., two, three, or four The nucleic acid sequence can be transcribed and / or expressed in combination with one internal ribosome entry site.

[0164] In one embodiment, the antigen is an antigen that is useful in the prevention and / or treatment of an infectious disease. In a specific embodiment, the antigen is derived from HBV. The ORFs encoding arenavirus glycoproteins encode the HBV pre-S2 / S proteins. In one embodiment, the glycoprotein of the arenavirus is replaced by a nucleic acid sequence comprising the The ORF encoding the HBV HBc protein is replaced by a nucleic acid sequence encoding the HBV HBc protein. In one embodiment, the ORF encoding the glycoprotein of an arenavirus is the HBV HBs protein. In one embodiment, the arenavirus is replaced by a nucleic acid sequence encoding the The ORF encoding the glycoprotein may be the HBV HBs protein and the HBV HBc protein or these. is replaced by a nucleic acid sequence encoding a fusion of an antigenic fragment of

[0165] ((g) Replacement of ORFs encoding arenavirus glycoproteins) In certain embodiments, the ORF encoding the glycoprotein of an arenavirus is HBV antigens as set forth in claim 1, wherein the nucleic acid sequence encoding the HBV antigen is replaced by a nucleic acid sequence encoding one, two, or more of the HBV antigens as set forth in claim 1. will be done.

[0166] In one embodiment, the ORF encoding the glycoprotein of an arenavirus encodes an HBV antigen. In one embodiment, the glycoprotein of an arenavirus is replaced by a nucleic acid sequence encoding the glycoprotein of the arenavirus. The ORF encoding the protein is the gene product of the gene for the HBV pre-S2 / S protein or its fragment. of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids of In one embodiment, the allele is replaced by a nucleic acid sequence encoding an antigen that is a fragment thereof. The ORF encoding the nasal virus glycoprotein contains nucleic acids encoding the pre-S2 / S antigenic fragment. In one embodiment, the glycoprotein of an arenavirus is replaced by a The ORF encoding the antigen includes, but is not limited to, pre-S2 / S or a fragment of pre-S2 / S. The nucleic acid sequence is replaced by a nucleic acid sequence corresponding to the

[0167] In one embodiment, the ORF encoding the glycoprotein of an arenavirus is the HBc glycoprotein of HBV. At least 10, 15, 20, 25, 50, 75, 10 to nucleic acid sequences encoding antigens that are fragments of 0, 125, 150, or more amino acids. In one embodiment, the arenavirus glycoprotein is substituted with The ORF is replaced by a nucleic acid sequence encoding an antigenic fragment of HBc. The ORF encoding the glycoprotein of an arenavirus may be, but is not limited to, HBc or HBc-like The nucleic acid sequence encoding the antigen, including the fragment, is replaced.

[0168] In one embodiment, the ORF encoding the glycoprotein of an arenavirus is the HBs of HBV. At least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 2 by nucleic acid sequences encoding antigens that are fragments of 45, 50, or more amino acids. In one embodiment, the ORF encoding the glycoprotein of the arenavirus is replaced by is replaced by a nucleic acid sequence encoding an antigenic fragment of HBs. The ORF encoding the glycoprotein of an arenavirus may be, but is not limited to, an antigen such as HBs or HBs is replaced by a nucleic acid sequence encoding an antigen, including a fragment of

[0169] In certain embodiments, the ORF encoding the glycoprotein of an arenavirus is selected from two or more HBV-specific glycoproteins. V protein or at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 22 replaced by a nucleic acid sequence encoding a fragment of five or more amino acids In certain embodiments, the ORF encoding the glycoprotein of an arenavirus is a glycoprotein encoding HBs and HBV. c is replaced by a nucleic acid sequence encoding

[0170] In certain embodiments, the ORF encoding the glycoprotein of the arenavirus is pre-S2 / S protein or its antigenic fragment, HBc protein or its antigenic fragment, HBs protein or and a nucleic acid encoding one or more of the Hbe protein or antigenic fragments thereof. The nucleic acid sequence is replaced by a nucleic acid sequence corresponding to the

[0171] 6.3 Generation of infectious arenaviruses expressing HBV antigens Typically, arenavirus particles are generated using standard reverse genetic techniques, as described for LCMV. It can be produced recombinantly by conventional techniques (L. Flatz, A. Bergthaler, J.C. de la To rre, and D.D. Pinschewer, Proc Natl Acad Sci USA 103:4663-4668, 2006; A. B. Sanchez and JC de la Torre, Virology 350:370, 2006; E. Ortiz-Riano, B References Y. Cheng, JC de la Torre, and L. Martinez-Sobrido, J Gen Virol. 94:1175-88. , 2013).

[0172] (a) Replication-deficient arenavirus To generate infectious, replication-deficient arenaviruses for use with the present invention, Although techniques such as these can be used, the genome of the rescued virus should be analyzed as described in Section 6.1. These modifications can be: i) Four arenavirus ORFs (glycoprotein (GP); nucleoprotein (NP); matrix protein Z) ; RNA-dependent RNA polymerase L) one or more, for example, two, three, or four It activates or functionally inactivates the virus, preventing the formation of infectious particles in normal cells, but still permitting gene expression in host cells infected with the virus vector; and and ii) the ability to introduce nucleic acids encoding HBV antigens. The replication-deficient viruses are described in International Patent Publication No. 2004 / 0022994, each of which is incorporated herein by reference in its entirety. Patent application publication WO 2009 / 083210 (application number PCT / EP2008 / 010994) and international patent application publication WO 20 and can be produced as described in PCT Application No. PCT / EP2014 / 055144. do.

[0173] Once generated from the cDNA, the infectious, replication-deficient arenaviruses provided herein can be propagated in complementing cells, which are replication-deficient due to modifications of their genomes. A cell that provides functionality that has been removed from the arenavirus (e.g., GP protein) Complementing cells can also express the nucleotide sequence of a gene encoding the nucleotide sequence of a ... , certainly providing GP proteins).

[0174] Have one or more of the viral genes in the arenavirus vector been removed? or functionally inactivated (here, deletion of glycoprotein GP is taken as an example), The viral vector is then transfected with a deleted viral gene, e.g., GP, in this example. Such complementing cell lines (hereafter referred to as C cells) can be generated and propagated in the cells provided. The cells are derived from mammalian cell lines such as BHK-21, HEK 293, VERO, or other cells (referred to herein as BHK -21) contains one or more plasmids (C-plasmids) for the expression of the viral gene of interest. It is produced by transfecting a complementary plasmid called a C-plasmid. The smid contains one or more expression vectors suitable for expression in mammalian cells, accompanied by a polyadenylation signal. The cassette may contain, for example, a mammalian polymerase II promoter, such as a CMV or EF1α promoter. The missing viral genes are expressed in arenavirus vectors generated under the control of a human virion gene promoter. Furthermore, the complementing plasmid contains an expression cassette suitable for gene expression in mammalian cells. a mammalian selection vector under the control of a polymerase II expression cassette, such as those described above. Markers, e.g., characterized by puromycin resistance or by the subsequent transcription of viral genes. In particular, internal ribosome entry sites, such as the internal ribosome entry site of the encephalomyocarditis virus, followed by a mammalian resistance marker. The plasmid further features a bacterial selection marker, such as an ampicillin resistance cassette. do.

[0175] Cells that can be used include, for example, BHK-21, HEK 293, MC57G, or others. They are maintained in culture and this is achieved by commonly used strategies, e.g., calcium phosphate, liposomes, Complementation plus fusion was performed using either a fusion-based protocol or electroporation. After several days, a suitable selection agent, e.g., puromycin, is gradually introduced. Surviving clones are isolated and subcloned according to standard procedures. and then analyzed by Western blotting or flow cytometry with an antibody against the viral protein of interest. High-expressing C-cell clones are identified using a cytometry method. As an alternative to the use of transfected C cells, transient transfection of normal cells can be used to can complement missing viral genes in each of the following steps: Additionally, helper viruses can be used to provide missing functionality in trans. do.

[0176] The plasmids that can be used are of two types: i) in this example, e.g., LCMV Minimal trans-acting factors of arenaviruses derived from NP and L proteins ii) two plasmids called TF-plasmids for intracellular expression of the alleles in C cells; and Transformation of viral vector genome fragments, e.g., segments with engineered modifications, into C cells For intracellular expression, a plasmid called a GS-plasmid can be used. The smids deliver the NP and L proteins of each arenavirus vector to mammalian cells. An expression cassette suitable for protein expression in animal cells, typically e.g., a mammalian polymerase Either of these may be under the control of a gene II promoter, such as a CMV or EF1α promoter. The GS-plasmid expresses either one of the two in combination with a polyadenylation signal. , expressing the small (S) and large (L) genome segments of the vector. Typically, a polymerase I-driven expression cassette or a T7 bacteriophage RNA polymerase (T7- ) driving expression cassette can be used, the latter incorporating a 3'-end ribozyme into the primary transcript. It is used preferentially for processing to generate accurate ends. When using a T7-based system Expression of T7 in C cells requires the use of a further plasmid that provides T7, constructed in a similar manner to the TF-plasmid. This must be achieved by including an expression plasmid containing the Alternatively, the C cells are engineered to further express T7 in a stable manner. In this case, the TF plasmid and the GS plasmid can be the same, i.e., the genomic sequence and trans-acting factors are expressed on one plasmid by T7, polI, and polII promoters. can be transcribed from

[0177] For recovery of the arenavirus vectors, the following procedure can be used: : In a 6-well plate, C cells (usually 80% confluent) were transfected with two TF-plasmids and two GS-plasmids. In one embodiment, a mixture of TF and G plasmids is transfected. The S plasmid can be the same, i.e., the genomic sequence and trans-acting factors are It can be transcribed from one plasmid by the T7, polI, and polII promoters. For this reason, commonly used strategies, such as calcium phosphate and liposome-based Either a direct transfer protocol or electroporation can be utilized.

[0178] After 3-5 days: Collect the culture supernatant (arenavirus vector preparation), dispense it, and incubate it in an aliquot. Store at 4°C, -20°C, or -80°C depending on how long the viral vector will be stored. The infectious titer of the arenavirus vector preparation was then determined by immunofocal assay on C cells. The results are evaluated by a ELISA.

[0179] The present invention further provides a cell culture infected with an infectious arenavirus that expresses an HBV antigen. When used to express HBV antigen in cultured cells, Two procedures can be used: i) injecting a cell type of interest with one or more of the arenavirus vector preparations described herein; For example, by infecting at a multiplicity of infection (MOI) of 2, 3, or 4, all cells were infected with HBV immediately after infection. Resulting in the production of V antigen. ii) Alternatively, a lower MOI can be used, allowing individual cell clones to express their viral load. The arenavirus vector can then be used to select for a dynamic HBV antigen expression level. Due to the non-cytolytic nature of the cloning medium, individual clones can be propagated indefinitely. Regardless of the method, HBV antigens are then extracted from the culture supernatant or the culture medium depending on the characteristics of the HBV antigens produced. However, the present invention provides a method for the preparation of a protein comprising: These two strategies are not limited to the above, and infectious replication-deficient arenavirus vectors may also be used. Other methods of using HBV antigens to drive expression can be considered.

[0180] Alternatively, a rescue system consisting of three plasmids can be used: (1) the first plasmid The smid is transfected by transcription with polymerase II and subsequent translation. (2) the second plasmid expresses the protein NP by transcription with polymerase I; This not only generates the (minus-strand) L-segment of the LCMV genome, but also The L protein is produced by transcription from the same template by polymerase II in the opposite direction to the promoter. (3) The third plasmid expresses the S sequence of the LCMV genome by transcription with polymerase I. -segment (encoding antigen coding sequences in place of LCMV glycoproteins). μg of each plasmid was used for electroporation of C-cells, and the cells were then cultured for 6 weeks. The cells are seeded onto well plates and incubated at 37°C. After incubation, the cells are transferred to the well plates. The cells and supernatant from the transfection were mixed with freshly plated C-cells and incubated at defined time points post-infection. Once the vector is produced, it is harvested and cleared from cells and debris. , antigens of oncogenic viruses and / or immunomodulatory peptides, polypeptides, or proteins The nucleic acid encoding the protein (see Section 6.2) can be inserted into a plasmid, from which The genome segment of the infectious replication-defective vector is transcribed by any technique known to those skilled in the art. will be done.

[0181] Have one or more of the viral genes in the arenavirus vector been removed? or functionally inactivated (here, deletion of glycoprotein GP is taken as an example), The viral vector can be engineered to contain a deleted or functionally inactivated viral gene (e.g., GP). The resulting virus can be produced and propagated in cells that provide the virus in trans. The body is infectious but contains deleted or functionally inactivated viral genes (e.g., GP). Complementation is a process whereby the host cells lack the necessary complementation to produce additional infectious progeny particles. Cells may be stably transfected, transiently transfected, or transfected with The missing functionality can be provided by infection with a helper virus that expresses the missing functionality. This can be done.

[0182] In certain embodiments, the complementing cell is deleted from the arenavirus vector genome. In a specific embodiment, the viral gene is functionally inactivated. , Complementation cells used to generate arenavirus vector genomes In another embodiment, the complementing cell is The virus strain used to generate the genome of the arenavirus vector is different from that used to generate the genome of the arenavirus vector. Viral genes from the genus virulence strain are provided, e.g., viral genes provided in complementation cells. is obtained from the MP strain of LCMV and contains a protein having the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18. In another example, the viral genes provided in the complementing cell encode a clone of LCMV. A protein obtained from the Lone 13 strain and having the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In another example, the viral genes provided in the complementation cell encode the WE strain of LCMV. and encodes a protein having the amino acid sequence of SEQ ID NO:25.

[0183] In a specific embodiment, the complementing cell provides the GP of the MP strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the MP strain of LCMV. The arenavirus vector was derived from LCMV clone 13 and encodes the GP protein. Instead of the ORF for the human HBV antigen, the ORF for the human HBV antigen described herein is included. In one embodiment, the GP protein has at least 90%, 91%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 16. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical do.

[0184] In a specific embodiment, the complementing cells provide the GP of the clone 13 strain of LCMV, and The virus vector contains the human GP protein described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises an ORF of a clone HBV antigen of LCMV. The GP of the LCMV strain 13 was provided, and the arenavirus vector was derived from the MP strain of LCMV, which encodes the GP protein. In place of the ORF encoding the antigen, it contains an ORF for a human HBV antigen as described herein. In a more specific embodiment, the GP protein has at least the amino acid sequence of SEQ ID NO:22. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% %Identical.

[0185] In a specific embodiment, the complementing cells provide the GP of the WE strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the WE strain of LCMV. The arenavirus vector was derived from LCMV clone 13 and encodes the GP protein. Instead of the ORF for the human HBV antigen, the ORF for the human HBV antigen described herein is included. In one embodiment, the GP protein has at least 90%, 91%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:25. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical do.

[0186] In a specific embodiment, the complementing cells provide the GP of the WE strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the WE strain of LCMV. The arenavirus vector is derived from the MP strain of LCMV and encodes the GP protein. In place of F, the ORF of a human HBV antigen described herein is included. In embodiments, the GP protein has at least 90%, 91%, 92%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:25. %, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical.

[0187] In certain embodiments, the infectious replication-defective arenavirus is tripartite.

[0188] (b) Replication-competent tripartite arenavirus For use with the present methods and compositions, provided herein are replicable alleles. The present invention relates to a method for producing an infectious, replication-competent, tripartite viral vector. No. 62 / 079,493, which is incorporated herein by reference in its entirety. It can be produced as described.

[0189] In one embodiment, the method for producing tripartite arenavirus particles comprises: (i) infecting a host cell with the , one L segment and two S segments or two L segments and one S segment (ii) minimal transactivation of arenaviruses into host cells; (iii) transfecting the host cells with a plasmid expressing factors NP and L; (iv) maintaining the arenavirus particles under conditions favorable for arenavirus formation; and (iv) recovering the arenavirus particles. Includes:

[0190] Once generated from cDNA, tripartite arenavirus particles (i.e., infectious and multimeric) In one embodiment, tripartite arenaviruses (those that can be produced) can be propagated. The particles are then grown to a titer that allows the virus to be used as described herein. The host cell may be propagated in any host cell that allows the host cell to grow. The chief cells showed that the tripartite arenavirus particles were expressed at titers similar to those determined for the corresponding wild-type virus. This allows the bacteria to grow to a certain potency.

[0191] In certain embodiments, the tripartite arenavirus particles can be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO In a specific embodiment, a tripartite arenavirus is The particles can be grown in a cell line.

[0192] In one embodiment, host cells are maintained in culture and the cells are transfected with one or more plasmids. The plasmid is transfected into a vector suitable for expression in mammalian cells, e.g., poly(A)-. Produced under the control of one or more expression cassettes consisting of the ferrocene I promoter and terminator The vector expresses an arenavirus genome segment to be transfected.

[0193] In a specific embodiment, host cells are maintained in culture and the cells are transfected with one or more plasmids. The plasmid is suitable for expression in mammalian cells, e.g. under the control of one or more expression cassettes consisting of a polymerase I promoter and terminator It expresses the viral genes that will be produced.

[0194] To generate tripartite arenaviruses containing one L segment and two S segments, The plasmids that can be used for this purpose include: i) two plasmids each encoding an S genome segment; ii) a plasmid expressing the L genome segment, e.g., a pol-I-driven S segment expression plasmid; and plasmids encoding the L segment, such as pol-I-driven L segment expression plasmids. It is required for tripartite arenaviruses that contain two L segments and one S segment. The plasmids referred to are: i) a plasmid encoding an L genome segment, e.g., pol-L, ii) a plasmid encoding an S genome segment, e.g., pol-I S.

[0195] In one embodiment, arenaviruses that direct intracellular synthesis of the viral L and S segments. A plasmid encoding the spolymerase can be incorporated into the transfection mixture. For example, a plasmid encoding the L protein and a plasmid encoding NP (or The L protein and NP are the most important proteins required for viral RNA transcription and replication. Alternatively, the intracellular synthesis of the viral L and S segments is regulated by NP. and L and S segments of two separate plasmids, respectively, from opposite sides along with the L protein. The expression was carried out using an expression cassette with both pol-I and pol-II promoters driving the cDNA of the target gene. It is possible to do so.

[0196] Additionally, the plasmid may contain an expression cassette suitable for gene expression in mammalian cells, e.g. A mammalian selectable marker, such as a marker for the gene encoding the polymerase II gene, under the control of a polymerase II expression cassette as described above. For example, they are characterized by puromycin resistance or have an internal linker after the viral gene transcript. There is an internal ribosome entry site, e.g., the internal ribosome entry site of the encephalomyocarditis virus, followed by In addition, there is a mammalian resistance marker. For production in E. coli, the plasmid further , featuring a bacterial selection marker such as an ampicillin resistance cassette.

[0197] Transfection of BHK-21 cells with plasmids can be achieved using commonly used strategies, e.g. , calcium phosphate, liposome-based protocols, or electroporation. After a few days, the cells are cultured using a suitable selection agent, e.g., Puromycin. Increasing concentrations of cysteine ​​are added. Surviving clones are isolated and subcultured according to standard procedures. Cloning and Western blotting with antibodies against viral proteins of interest Alternatively, high-expressing clones are identified using flow cytometry.

[0198] Typically, an RNA polymerase I-driven expression cassette, an RNA polymerase II-driven cassette, or The latter can use a T7 bacteriophage RNA polymerase-driven cassette. , 3'-end ribozymes are preferentially used to process primary transcripts to generate precise ends. In one embodiment, a plasmid encoding an arenavirus genome segment is The genome sequence and trans-acting factors can be the same, i.e., T7, polI, , and pol II promoters can be transcribed from one plasmid.

[0199] For the recovery of tripartite arenavirus vectors, the following procedure is envisioned: Day 1: M6 In a well plate, cells are typically 80% confluent, and the plasmid mixture is added as above. For this purpose, any commonly used strategy, for example, calcium phosphate transfection, Lipoprotein-, liposome-, or electroporation-based protocols can be used. Cut.

[0200] After 3-5 days: Collect the culture supernatant (arenavirus vector preparation), dispense it, and incubate it in an aliquot. Store at 4°C, -20°C, or -80°C depending on how long the viral vector will be stored. The infectious titer of arenavirus vector preparations was assessed by immunofocus assay. Alternatively, the transfected cells and supernatants were cultured 3 to 5 days after transfection. The cells may then be subcultured into larger vessels (e.g., T75 tissue culture flasks), and the culture supernatants may be , and harvest up to 5 days after passaging.

[0201] The present application further relates to the expression of heterologous ORFs (e.g., HBV antigens), wherein The plasmid encoding the genome segment is modified to incorporate the heterologous ORF. The homologous ORF can be incorporated into the plasmid using restriction enzymes. In one embodiment, the heterologous ORF encodes an HBV antigen. The loading plasmid is modified to incorporate one or more heterologous ORFs. In this case, the heterologous ORF encodes one or more HBV antigens.

[0202] 6.4 Nucleic Acids, Vector Systems, and Cell Lines In one embodiment, described herein is an infectious arena a nucleic acid sequence that is the cDNA of the large genome segment (L segment) of the virus, One ORF of the genome segment is deleted or functionally inactivated, or and the genome segment comprises a nucleotide sequence encoding an HBV antigen. wherein the infectious arenavirus viral vector is replication-deficient (see section 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication competent. (See Section 6.1(b)).

[0203] In one embodiment, described herein is an infectious arena a nucleic acid sequence encoding the short genome segment (S segment) of the virus, wherein One ORF of the genome segment is deleted or functionally inactivated, and The short genome segment comprises a nucleotide sequence encoding an HBV antigen. In the same manner, described herein are the infectious arenaviruses described herein. A nucleic acid sequence encoding a short genome segment (S segment), where the glycoprotein The ORF of the gene is deleted or functionally inactivated, and the short genomic sequence The fragment comprises a nucleotide sequence encoding an HBV antigen. In the above, the HBV antigen is an antigen described in Section 6.2.

[0204] In certain embodiments, the nucleic acid sequences provided herein are derived from specific strains of LCMV. LCMV strains include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, and UB. C, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927, 810362 , 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and their derivatives In a specific embodiment, the nucleic acid is derived from clone 13 of LCMV. In other specific embodiments, the nucleic acid is derived from the MP strain of LCMV.

[0205] In more specific embodiments, provided herein are SEQ ID NO: 1, SEQ ID NO: 2, or a sequence identical to SEQ ID NO: 3 by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 2 Arenavirus genome segments containing sequences that are 8%, at least 99%, or 100% identical In another embodiment, provided herein is a nucleic acid comprising: (i) SEQ ID NO: 11 The sequence of nucleotides 1639 to 3315 of the %, 97%, 98%, at least 99%, or 100% identical nucleotide sequence; and (ii) HBV a nucleic acid comprising an arenavirus genome segment containing a nucleotide sequence encoding an antigen; be.

[0206] In another embodiment, provided herein is a nucleic acid sequence comprising: (i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: Identifies at least 90%, 91%, 92%, or 93% of the amino acid sequence encoded by 1639-3315 of No. 11 %, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical expression products. and (ii) an allele containing a nucleotide sequence encoding an HBV antigen. It is a nucleic acid comprising a segment of the flu virus genome.

[0207] In another embodiment, provided herein is (i) nucleotide 164 of SEQ ID NO: 12. Sequences of 0 to 3316 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, a nucleotide sequence that is at least 99% or 100% identical; and (ii) encodes an HBV antigen. A nucleic acid comprising an arenavirus genome segment comprising a nucleotide sequence.

[0208] In another embodiment, provided herein is a nucleic acid sequence comprising: (i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: Identifies at least 90%, 91%, 92%, or 93% of the amino acid sequence encoded by sequences 1640 to 3316 of No. 12 %, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical expression products. and (ii) an allele containing a nucleotide sequence encoding an HBV antigen. It is a nucleic acid comprising a segment of the flu virus genome.

[0209] In one embodiment, described herein is an infectious arena A vector system that contains one or more vectors that collectively contain the genome of a viral particle. Additionally, provided herein are vectors, wherein one or more vectors are selected from the group consisting of an infectious allele as described herein. Two arenavirus genome segments, namely the L segment and the S segment, are Such a vector system may comprise (one or more separate) on a DNA molecule) can contain:

[0210] Arenavirus particles carrying this modified S genome segment produce infectious progeny viruses. Arenavirus S genome segments that have been modified so that they are unable to produce serotype particles. and an array containing a nucleotide sequence encoding (sense or antisense) an HBV antigen. navirus L genome segment;

[0211] Arenavirus particles carrying this modified L genome segment produce infectious progeny viruses. Arenavirus L genome segments that have been modified so that they are unable to produce virus particles. and an array containing a nucleotide sequence encoding (sense or antisense) an HBV antigen. navirus S genome segment;

[0212] Arenavirus particles carrying this modified S genome segment produce infectious progeny viruses. It has been modified so that it is unable to produce HBV particles and has the ability to express HBV antigens (sense or anti-HBV). a nucleotide sequence encoding a wild-type arenavirus L genome segment (in both senses) an arenavirus S genome segment, including a ment; or

[0213] Arenavirus particles carrying this modified L genome segment produce infectious progeny viruses. It has been modified so that it is unable to produce HBV particles and has the ability to express HBV antigens (sense or anti-HBV). a nucleotide sequence encoding a wild-type arenavirus S genome segment (in both senses) ment, arenavirus L genome segment.

[0214] In one embodiment, described herein is a vector encoding the GP of the S genome segment. The ORFs are: a nucleotide sequence encoding a hepatitis B pre-S2 / S protein or an antigenic fragment thereof; a nucleotide sequence encoding the hepatitis B virus HBc protein or an antigenic fragment thereof; a nucleotide sequence encoding the hepatitis B virus HBs protein or an antigenic fragment thereof; Hepatitis B virus HBs protein and HBc protein or a fusion of these antigenic fragments coding nucleotide sequence; Nucleotide sequences encoding hepatitis B virus HBe protein or antigenic fragments thereof a nucleotide sequence comprising: It is a nucleic acid sequence containing

[0215] In one embodiment, described herein is a vector encoding the GP of the S genome segment. The ORF encoding one or more HBV antigens (e.g., one or more of those listed in the paragraph above) may be a nucleotide sequence encoding ... It is a nucleic acid sequence containing a nucleotide segment.

[0216] In another embodiment, provided herein is a nucleic acid described above in this section. or cells containing the vector system. Cell lines derived from such cells, Cultures containing the nucleic acid or vector system and methods for culturing such cells infected with the nucleic acid or vector system are also described herein. In some embodiments, provided herein are methods for treating a variety of conditions, including the use of a compound of formula (I) or (II) as described herein. Cells containing nucleic acid containing the large genome segment (L segment) of the infectious arenavirus wherein one ORF of the genome segment is deleted or functionally inactive. and the genome segment contains a nucleotide sequence encoding an HBV antigen. nothing.

[0217] In other embodiments, provided herein are methods for administering the infectious alleles described herein. a cell containing a nucleic acid sequence containing a short genome segment (S segment) of the influenza virus, wherein one ORF of the genome segment is deleted or functionally inactivated. and the short genome segment encodes an HBV pre-S2 / S protein or an antigenic fragment thereof. It comprises a nucleotide sequence

[0218] In other embodiments, provided herein are methods for administering the infectious alleles described herein. a cell containing a nucleic acid sequence containing a short genome segment (S segment) of the influenza virus, wherein one ORF of the genome segment is deleted or functionally inactivated. and the short genome segment encodes the HBV HBc protein or an antigenic fragment thereof. It contains a nucleotide sequence.

[0219] In other embodiments, provided herein are methods for administering the infectious alleles described herein. a cell containing a nucleic acid sequence containing a short genome segment (S segment) of the influenza virus, wherein one ORF of the genome segment is deleted or functionally inactivated. and the short genome segment encodes the HBV HBs protein or an antigenic fragment thereof. It contains a nucleotide sequence.

[0220] In other embodiments, provided herein are methods for administering the infectious alleles described herein. a cell containing a nucleic acid sequence containing a short genome segment (S segment) of the influenza virus, wherein one ORF of the genome segment is deleted or functionally inactivated. and the short genome segment contains a small number of HBV HBs protein and HBV HBc protein. The nucleotide sequence encoding the fusion protein includes at least one domain.

[0221] In other embodiments, provided herein are methods for administering the infectious alleles described herein. a cell containing a nucleic acid sequence containing a short genome segment (S segment) of the influenza virus, wherein one ORF of the genome segment is deleted or functionally inactivated. and the short genome segment contains nucleotides encoding one or more of the HBV antigens. Contains code arrays.

[0222] In another embodiment, provided herein is a nucleic acid sequence comprising two nucleic acids described herein. or cells containing the vector system. Cell lines derived from such cells, Cultures containing the nucleic acid or vector system and methods for culturing such cells infected with the nucleic acid or vector system are also described herein. provided in the book.

[0223] In certain embodiments, provided herein are sequences having at least SEQ ID NO:13 or SEQ ID NO:14. At most 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequences In some embodiments, provided herein are nucleic acids comprising SEQ ID NO: 13 or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In one embodiment, the expression vector comprises a nucleotide sequence as provided herein. Provided are those having at least 80%, 81%, 82%, 83%, 84%, or 85% of SEQ ID NO: 13 or SEQ ID NO: 14. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% identical to the nucleotide sequence of the host cell.

[0224] In certain embodiments, provided herein are at least one of SEQ ID NOs: 15, 16, 17, or 18. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In one embodiment, the nucleic acid is a nucleic acid comprising a nucleotide sequence encoding the Provided are sequences that are at least 80%, 81%, 82%, 83%, 84% identical to SEQ ID NO: 15, 16, 17, or 18. ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%, nucleotide sequences encoding amino acid sequences that are 98%, 99%, or 100% identical In some embodiments, provided herein are vectors represented by SEQ ID NOs: 15, 16, , 17, or 18 and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical A host cell containing a nucleotide sequence encoding a certain amino acid sequence.

[0225] In certain embodiments, provided herein are at least one of SEQ ID NOs: 15, 16, 17, or 18. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In one embodiment, provided herein is an isolated protein comprising: , SEQ ID NO: 15, 16, 17, or 18 at least 80%, 81%, 82%, 83%, 84%, 85%, 86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or a host cell expressing a protein containing an amino acid sequence that is 100% identical to the target gene. In an embodiment, the host cells are cultured in a cell culture medium.

[0226] In some embodiments, provided herein are sequences comprising SEQ ID NO: 12 or 7 and at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical nucleotide sequence In some embodiments, provided herein are nucleic acids having at least one of SEQ ID NO: 12 or 7. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Nucleotides that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In some embodiments, provided herein is an expression vector comprising the sequence. Column number 12 or 7 and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical A host cell containing a nucleotide sequence.

[0227] In certain embodiments, provided herein are at least one of SEQ ID NOs: 21, 22, 23, or 24. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In one embodiment, the nucleic acid is a nucleic acid comprising a nucleotide sequence encoding the Provided are sequences that are at least 80%, 81%, 82%, 83%, 84% identical to SEQ ID NO: 21, 22, 23, or 24. ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%, nucleotide sequences encoding amino acid sequences that are 98%, 99%, or 100% identical In some embodiments, provided herein are vectors represented by SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5 , 23, or 24 and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical A host cell containing a nucleotide sequence encoding a certain amino acid sequence.

[0228] In certain embodiments, provided herein are at least one of SEQ ID NOs: 21, 22, 23, or 24. At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical In one embodiment, provided herein is an isolated protein comprising: , SEQ ID NO: 21, 22, 23, or 24 at least 80%, 81%, 82%, 83%, 84%, 85%, 86% ,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or a host cell expressing a protein containing an amino acid sequence that is 100% identical to the target gene. In an embodiment, the host cells are cultured in a cell culture medium.

[0229] (6.5 How to use) Provided herein is an immunotherapy for hepatitis B virus infection. In accordance with the present invention, provided herein is a method of treating an infection in a subject, the method comprising: The subject is administered one or more infectious arenaviruses expressing HBV antigens described herein or their In one embodiment, the infectious arenavirus is a viral vector. In certain embodiments, the infectious arenavirus is replication-competent. In a specific embodiment, the methods of treating an infection described herein include administering Administering to a subject in need thereof one or more infectious arenaviruses expressing HBV antigens as described herein. The subject may be, but is not limited to, a human, mouse, or the like. mice, rats, guinea pigs, livestock, including but not limited to cattle, horses, sheep, and pigs The animal may be a mammal, such as a goat, cat, dog, hamster, or donkey. In an embodiment, the subject is a human.

[0230] In another embodiment, provided herein is a method for determining an immune response to HBV in a subject. The method comprises administering to the subject an infectious arenavirus expressing an HBV antigen or an antibody therefor. The method comprises administering a composition of formula (I) to a subject.

[0231] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to whom the drug or composition thereof is administered may have, be susceptible to, or be a patient with HBV infection. In another specific embodiment, the HBV-related markers described herein are at risk. The subject to which the infectious arenavirus expressing the V antigen or a composition thereof is administered is susceptible to HBV infection. infected, susceptible to, or at risk of contracting the disease.

[0232] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to whom the drug or composition thereof is administered may be infected with, for example, HBV infection in the liver. In a specific embodiment, An infectious arenavirus expressing an HBV antigen described herein or a composition thereof is administered. The subject to be treated is infected with or has HBV infection in one or more organs of the body, e.g., the liver. susceptible to or at risk of

[0233] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to be administered the drug or composition thereof has test results (e.g., blood test results) indicative of liver damage. In certain embodiments, the subject has a blood alanine aminotransferase level indicative of liver damage. In one embodiment, the subject has a blood alcohol level indicative of liver damage. In one embodiment, the patient has a steroid aminotransferase (AST) level. The subject has a blood alkaline phosphatase level indicative of liver damage. In one embodiment, the subject has a blood lactate dehydrogenase (LDH) level indicative of liver damage. In embodiments, the subject is evaluated for blood levels of ALT, AST, alkaline phosphatase, and erythrocyte sedimentation (ESS), which are indicative of liver damage. LDH levels.

[0234] In certain embodiments, the subject is diagnosed with liver cancer or a susceptibility to liver cancer. In one embodiment, the subject has a blood test that indicates liver damage. In some embodiments, the subject has an intermediate bilirubin (e.g., conjugated bilirubin) level. have blood albumin levels indicative of liver damage.

[0235] In some embodiments, the subject has abdominal ultrasound results indicating liver damage. In some embodiments, the subject has a CAT scan showing liver damage. wherein the subject has MRI results showing liver damage.

[0236] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to whom the drug or composition thereof is administered has a detectable level of HBs antigen (HBsAg) in the blood. In certain embodiments, the subject has a detectable level of HBc antigen (HBcAg) in the blood. In one embodiment, the subject has a detectable level of IgM antibodies in the blood. In one embodiment, the patient has an HBe antigen (HBeAg, extracellular / secretory form of HBc protein). The subject has detectable levels of antibodies to HBsAg in the blood.

[0237] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to whom the drug or composition thereof is administered has persistent levels of HBsAg indicative of chronic hepatitis. In certain embodiments, the subject has persistent levels of HBeAg indicative of chronic hepatitis. In an embodiment, the subject has persistent levels of HBsAg and HBeAg indicative of chronic hepatitis.

[0238] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subject to which the compound or a composition thereof is administered may suffer from, but is not limited to, anorexia, fatigue, nausea, vomiting, Suffering from symptoms of HBV infection, including itching, abdominal pain, bloating, or jaundice.

[0239] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The subjects to which the drug or composition thereof is administered include, but are not limited to, those suffering from acute hepatitis B, chronic HBV infection, liver cirrhosis, and the like. In another embodiment, the subject is suffering from symptoms of HBV, including cirrhosis, and hepatocellular carcinoma (HCC). The infectious arenaviruses expressing the HBV antigens described herein or compositions thereof can be used to treat asymptomatic HBV infections. It is administered to subjects with active HBV.

[0240] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The drug or composition thereof is intended to be used by individuals infected with, susceptible to, or at risk of HBV infection. In a specific embodiment, the method of the present invention is administered to a subject of any age who has been exposed to a steroid. The infectious arenaviruses expressing the HBV antigens described herein or compositions thereof are useful in treating immune system deficiencies. subjects with HIV, pregnant subjects, subjects who have undergone organ or bone marrow transplantation, subjects with immunosuppression Subjects taking anticoagulant medications, undergoing hemodialysis, having cancer, or having HBV infection Administered to a subject who is infected, susceptible to, or at risk for infection In a more specific embodiment, an infected person expressing an HBV antigen described herein is Arenaviruses or compositions thereof infect or predispose to HBV infection? administered to a subject who has or is at risk of having an immune system compromised due to HIV infection. In yet another specific embodiment, a HBV antigen-expressing HBV antigen described herein is The infectious arenavirus or composition thereof is intended to be used in the treatment of HBV infection or in the treatment of HBV-infected or susceptible individuals. Watermelon or at risk of it, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, The subject is administered to a child who is 13, 14, 15, 16, or 17 years of age. an infectious arenavirus or composition thereof expressing an HBV antigen as described herein, The person is infected with, susceptible to, or at risk of HBV infection. In yet another specific embodiment, the method is administered to a subject who is an infant. Infectious arenaviruses expressing the HBV antigens or compositions thereof are used to treat HBV infection. or susceptible to or at risk for it, 0, 1, 2, 3, 4, 5, 6, 7 In yet another specific embodiment, the subject is administered a steroid drug to a subject who is a toddler, 8, 9, 10, 11, or 12 months of age. In embodiments, an infectious arenavirus expressing an HBV antigen as described herein or its The composition may be administered to individuals infected with, susceptible to, or at risk of HBV infection. It is administered to elderly subjects who are

[0241] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The drug or composition thereof is administered to a subject at increased risk of disseminated HBV infection. In certain embodiments, an infectious arenavirus or The composition is administered to a neonatal subject having an immature neonatal immune system. In such a manner, an infectious arenavirus or a combination thereof expressing an HBV antigen as described herein. The composition is administered to subjects who are at increased risk of HBV infection and who use intravenous drugs.

[0242] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is The drug or composition thereof is administered to a subject infected with one or more genotypes or subgenotypes of HBV. In certain embodiments, the genotype is one of genotypes A to J, or another genotype. In some embodiments, the subgenotype is one or more of subgenotypes A1 to A6, B 1 to B4, C1 to C6, D1 to D7, F1 to F4, or another subgenotype.

[0243] In another embodiment, an infectious arenavirus expressing an HBV antigen described herein is When administered to a subject, the compound or a composition thereof confers cell-mediated immunity (CMI) against HBV infection. Without being bound by theory, in another embodiment, the HBV Infectious arenaviruses or compositions thereof expressing antigens infect the host and stimulate the major histocompatibility complex (M HC) for direct presentation of antigens of interest on class I and II antigen-presenting cells of the host. In another embodiment, the antibodies described herein are expressed in antigen-specific receptors (APCs) (e.g., macrophages). When an infectious arenavirus expressing an HBV antigen or a composition thereof is administered to a subject, the virus Large-scale, multifunctional IFN-γ and TNF-α co-producing HBV-specific C antigens for treating or preventing infection CD4+ and CD8+ T cell responses are induced (IFN-γ is produced by CD4+ and CD8+ T cells and TN F-α is produced by CD4+ T cells).

[0244] In another embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. When administered, the risk of an individual acquiring HBV infection is reduced to the same level as the individual who acquires HBV infection in the absence of such treatment. At least about 10%, at least about 20%, at least about 25%, or less than the risk of at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least About 60%, at least about 70%, at least about 80%, at least about 90%, or more Decreases.

[0245] In another embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. When administered, the symptoms of HBV infection are similar to those that occur in the absence of such treatment. Compared to at least about 10%, at least about 20%, at least about 25%, at least about 30% , at least about 35%, at least about 40%, at least about 50%, at least about 60%, or less Both are reduced by about 70%, at least about 80%, at least about 90%, or more.

[0246] In another embodiment, a subject with an immature neonatal immune system expressing an HBV antigen is Administration of an infectious arenavirus or a composition thereof enhances cell-mediated immunity (CM) against HBV infection. I) The response is compared with the cell-mediated immune (CMI) response to HBV infection in the absence of such treatment. At least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least Induction may be about 70%, at least about 80%, at least about 90%, or greater.

[0247] In one embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. In one embodiment, administration of the compound reduces ALT levels in the blood. Administration of infectious arenaviruses or compositions thereof reduces AST levels in the blood. In one embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. In one embodiment, the HBV anti-cancer drug reduces alkaline phosphatase levels in the blood. Administration of an infectious arenavirus expressing the antigen or a composition thereof increases blood LDH levels. In one embodiment, an infectious arenavirus or a combination thereof expressing an HBV antigen is When the compound was administered, the levels of ALT, AST, alkaline phosphatase, and LDH in the blood increased. One or more of the following is reduced.

[0248] In one embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. In one embodiment, administration of the antibody reduces AFP levels in the blood. Administration of infectious arenaviruses or compositions thereof increases the amount of bilirubin (e.g., conjugated bilirubin) in the blood. In one embodiment, the level of lirubin is reduced in an infectious adenomatous virus expressing an HBV antigen. Administration of the virus or a composition thereof increases albumin levels in the blood.

[0249] In one embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. In one embodiment, administration of the antibody reduces HBsAg levels in the blood. Administration of infectious arenaviruses or compositions thereof increases the IgM antibody level against HBcAg in the blood. In one embodiment, an infectious arenavirus expressing an HBV antigen or an Administration of the composition reduces HBeAg levels in the blood. Administration of an infectious arenavirus expressing the antigen or a composition thereof results in an increase in the activity of HBsAg in the blood. The antibody levels are reduced.

[0250] In one embodiment, an infectious arenavirus expressing an HBV antigen or a composition thereof is administered. Administration of 100 mg ... In embodiments, administration of an infectious arenavirus expressing an HBV antigen or a composition thereof results in: The number of anti-HBV antibodies detected in a patient's blood sample is reduced. In one embodiment, the HBV antigen When an infectious arenavirus expressing the The amount of HBV detected in semen or breast milk is reduced. Upon administration of an infectious arenavirus or composition thereof, the virus can be detected in urine, throat swabs, and bronchial washes. In one embodiment, the level of virus cultured from a fluid or tissue sample is reduced. Administration of an infectious arenavirus expressing an HBV antigen or a composition thereof results in quantitative or qualitative This reduces the levels of virus detected by standard PCR tests.

[0251] administering an infectious arenavirus or composition thereof that expresses an HBV antigen in a subject The alteration of cellular immune (CMI) response function against HBV infection induced by It can be measured by any assay, including flow cytometry ( See, e.g., Perfetto SP et al., Nat Rev Immun. 2004; 4(8):648-55), lymphocytes Proliferation assays (e.g., Bonilla FA et al., Ann Allergy Asthma Immunol. 2008; 101 :101-4; and Hicks MJ et al., Am J Clin Pathol. 1983; 80:159-63), T lymphocytes This included determining changes in surface marker expression after activation with respect to cytokine measurements. Assays for measuring lymphocyte activation (e.g., Caruso A. et al., Cytometry). 1997;27:71-6), ELISPOT assays (see, e.g., Czerkinsky CC et al., J Immunol Methods. 1983; 65:109-121; and Hutchings PR et al., J Immunol Methods. 1989; 120:1-8), or natural killer cell cytotoxicity assays (e.g., Bonilla FA Ann Allergy Asthma Immunol. 2005 May; 94(5 Suppl 1):S1-63). Examples include, but are not limited to:

[0252] In another embodiment, described herein is a method for treating HBV infection using an HBV antigen described herein. and methods for use with infectious arenaviruses (e.g., LCMV) expressing S genotypes. The ORF encoding the GP of the rRNA segment is: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof is replaced with a nucleotide sequence comprising:

[0253] In another embodiment, provided herein is a method for preventing maternal-fetal HBV transmission and / or or a method for preventing infection, comprising administering to a subject of childbearing age an HBV anti-HBV drug described herein. and administering an infectious arenavirus expressing a gene. See Section 6.2. In a specific embodiment, provided herein is a method for the transmission of HB from mother to fetus. A method for preventing transmission and / or infection of V, comprising administering to a seronegative subject of childbearing age a vaccine as described herein. and administering an infectious arenavirus expressing an HBV antigen as described in the document. In yet another embodiment, provided herein is a method for the transmission of HBV from mother to fetus. 1. A method for preventing transmission and / or infection of a viral load comprising administering to a subject of childbearing age who intends to bear a child: administering an infectious arenavirus expressing an HBV antigen as described herein; It is a method.

[0254] In another embodiment, provided herein is a method for preventing maternal-fetal HBV transmission and / or or a method for preventing infection, comprising administering to a subject of childbearing age an HBV anti-HBV drug described herein. Section 6.2. In a specific embodiment, provided herein is a method for the transfer of a fetus from a mother to a fetus. 1. A method for preventing transmission and / or infection of HBV to a seronegative subject of childbearing age, comprising administering to the subject: administering one or more infectious arenaviruses expressing the HBV antigens described herein. In yet another embodiment, provided herein is a method for administering a steroid hormone to a mammal. A method for preventing the transmission and / or infection of HBV to a fetus, comprising administering to a woman of childbearing age who intends to bear a child. administering to a subject one or more infectious arenaviruses expressing an HBV antigen described herein. The method includes:

[0255] In another embodiment, provided herein is a method for preventing maternal-fetal HBV transmission and / or or a method for preventing infection, comprising administering to a pregnant subject an HBV antigen as described herein. Specific embodiments include administering an infectious arenavirus expressing In the present invention, provided are methods for preventing the transmission and / or infection of HBV from mother to fetus. The method comprises administering to a pregnant subject an HBV-infected virus expressing an HBV antigen as described herein. The method comprises administering an effective amount of a virulent arenavirus.

[0256] In another embodiment, provided herein is a method for preventing maternal-fetal HBV transmission and / or or a method for preventing infection, comprising administering to a pregnant subject an HBV antigen as described herein. Specific examples of methods include administering one or more infectious arenaviruses expressing In embodiments, provided herein are methods for preventing maternal-fetal HBV transmission and / or infection. A method for preventing HBV infection comprising administering to a pregnant subject a HBV antigen-expressing HBV antigen described herein. The method comprises administering an effective amount of one or more infectious arenaviruses to the subject.

[0257] In another embodiment, administration of an infectious arenavirus expressing an HBV antigen results in the development of a congenital In another embodiment, one or more infectious arenas expressing HBV antigens are reduced. Administration of the virus reduces congenital HBV infection.

[0258] In another embodiment, administration of an infectious arenavirus expressing an HBV antigen results in the development of a congenital The incidence of sexual HBV infection is at least about 10%, at least about 20%, at least 25%, or at least At least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60% , at least about 70%, at least 80%, at least 90%, or more. In another specific embodiment, administering an infectious arenavirus that expresses an HBV antigen , reducing neonatal mortality due to congenital HBV infection.

[0259] In another embodiment, administration of one or more infectious arenaviruses expressing HBV antigens results in , the occurrence of congenital HBV infection is at least about 10%, at least about 20%, at least 25%, or at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least A decrease of about 60%, at least about 70%, at least 80%, at least 90%, or more In another specific embodiment, the one or more infectious arenaviruses express an HBV antigen. Administration of HBV reduces neonatal mortality due to congenital HBV infection.

[0260] Such congenital HBV manifestations include acute hepatitis B, chronic HBV infection, cirrhosis, and hepatocellular carcinoma (HCC). HCC) are included, but are not limited to:

[0261] 6.6 Compositions, Administration, and Dosages The present invention further provides vaccines, including the genetically modified arenaviruses described herein. Such vaccines and pharmaceutical compositions are well known in the art. The formulation can be carried out according to standard procedures in the art.

[0262] In another embodiment, provided herein is a method for treating an infectious allergy as described herein. Such compositions are useful in methods of treating and preventing disease. In a specific embodiment, the compositions described herein can be used in It is used in the treatment of subjects infected with or susceptible to HBV infection. In a specific embodiment, the immunogenic compositions provided herein are used to The immunogens described herein are capable of inducing an immune response in a host to which they are administered. The original composition may be used as a vaccine and, if appropriate, formulated as a pharmaceutical composition. In a specific embodiment, the immunogenic compositions described herein can comprise: It is used in preventing HBV infection in a subject (e.g., a human subject). Thus, the infectious arenavirus viral vector is replication-deficient (see Section 6.1(a)). In certain embodiments, the infectious arenavirus viral vector is replication competent (e.g., (see paragraph .1(b)).

[0263] In some embodiments, provided herein are any of the arenaviruses described herein. and immunogenic compositions comprising a virus vector (or a combination of various arenavirus vectors). In certain embodiments, such immunogenic compositions contain a pharmaceutically acceptable excipient. In certain embodiments, such immunogenic compositions further comprise an adjuvant. Adjuvants for administration in combination with the compositions described herein include those It can be administered before, simultaneously with, or after the administration of the composition. In one embodiment, the term "adjuvant" refers to a compound used in combination with the compositions described herein. When administered in combination with or as part of a vaccine, it stimulates an immune response to infectious arenavirus particles. enhance, potentiate, and / or enhance the infectious arena It refers to a compound that does not generate an immune response against viral particles. Therefore, adjuvants generate an immune response against infectious arenavirus particles, but Adjuvants have the potential to, for example, stimulate lymphocyte recruitment, B and / or other adverse reactions. Immune responses are mediated by several mechanisms, including stimulation of T cells and macrophages. The vaccine or immunogenic composition of the present invention may contain an adjuvant. or when administered together with one or more adjuvants, ajuva Examples of adjuvants include inorganic salt adjuvants or inorganic salt gel adjuvants, particulate adjuvants, These include particulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., For example, aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-de-O-acid Monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), hKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas), Dazopyridine compounds (International Application PCT / US2007 / 064, published as International Publication WO2007 / 109812) 857), imidazoquinoxaline compounds (see International Publication WO2007 / 109813 See International Application No. PCT / US2007 / 064858), and saponins, e.g., QS21 (Vaccine Design Kensil et al. in Cancer Design: Subunit and Adjuvant Approaches (Powell and Newman, eds., Plenum Press, NY, 1995); In some embodiments, the present invention provides a method for producing a fluororesin-based polymeric polymer, including but not limited to, a fluororesin-based polymeric polymer (see U.S. Pat. No. 5,057,540). In this case, the adjuvant is Freund's adjuvant (complete or incomplete). Bant is an oil-in-water emulsion optionally combined with an immunostimulant such as monophosphoryl lipid A. ols (e.g., squalene or peanut oil) (Stoute et al., N. Engl. J. Med. 336, 86-91 (1997).

[0264] The compositions may contain the infectious arenaviruses described herein alone or in combination with pharmaceutically acceptable carriers. A suspension or dispersion of the genetically modified arenavirus, particularly an isotonic suspension or dispersion, is included together with a carrier that provides Aqueous suspensions or dispersions can be used. The pharmaceutical compositions can be sterile and and / or excipients, such as preservatives, stabilizers, wetting agents, and / or emulsifiers, solubilizers, Salts to adjust osmolality and / or buffers may be included, and may be used in the preparation of, for example, conventional separators. In one embodiment, the composition is prepared by a dispersion and suspension process in a manner known per se. Such dispersions or suspensions may contain viscosity modifiers. Maintain the temperature at around 2-8°C, or freeze for long-term storage and then store immediately after use. For injection, the vaccine or immunogenic preparation is prepared in an aqueous solution. Preferably, physiologically compatible buffers, such as Hanks' solution, Ringer's solution, or in a saline buffer. The solution may be formulated in a formulation agent, e.g., a suspending agent. The composition may contain a dispersing agent, a stabilizer, and / or a dispersing agent.

[0265] In certain embodiments, the compositions described herein do not contain preservatives, such as mercury derivatives. In a specific embodiment, the pharmaceutical compositions described herein further comprise thimerosal. In another embodiment, the composition comprises 0.001% to 0.01% thimerosal. The pharmaceutical compositions described do not contain preservatives.

[0266] The pharmaceutical composition comprises about 10 3 ~about 10 11 Genetically modified arenavirus containing focus-forming units Unit dosage forms for parenteral administration are, for example, ampoules or vials, e.g., containing about 10 3 ~10 10 Focus forming unit or 10 5 ~10 15 Genetically modified arenaviruses of physical particles It is a vial containing

[0267] In another embodiment, the vaccine or immunogenic composition provided herein is a , including but not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal, and inhalation routes, and by scarification (e.g., by using a bifurcated needle to It is administered to the subject by scraping from the top layer. Specifically, it can be administered subcutaneously, intramuscularly, or intravenously. An intravenous route may be used.

[0268] For administration intranasally or by inhalation, the preparations for use according to the invention may be prepared in a suitable Propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane Use a pressurized pack or nebulizer with trifluoroethane, carbon dioxide, or other suitable gas. The composition can be conveniently delivered in the form of an aerosol spray presentation from a pressurized aerosol. In the case of a drug, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, e.g., gelatin, for use in inhalers or insufflators, can be used. The cartridge may comprise a powder mix of the compound and a suitable powder base such as lactose or starch. It can be formulated as follows.

[0269] The dosage of the active ingredient depends on the type of vaccination and on the subject and its age, weight, It depends on the individual condition, the individual pharmacokinetic data, and the mode of administration.

[0270] Also provided herein are pharmaceuticals comprising genetically modified arenaviruses as active ingredients. Method for the production of a vaccine in the form of a preparation and genetically modified arenaviruses for said production The pharmaceutical composition of the present invention can be prepared, for example, by conventional mixing and / or dispersion processes. It is prepared by a method known per se.

[0271] 6.7 Optimization of LCMV Vector Construction Have one or more of the viral genes in the arenavirus vector been removed? or functionally inactivated (here, deletion of glycoprotein GP is taken as an example), The viral vector can be engineered to contain a deleted or functionally inactivated viral gene (e.g., GP). The resulting virus can be produced and propagated in cells that provide the virus "in trans." The body is infectious but contains deleted or functionally inactivated viral genes (e.g., GP). Complementation is a process whereby the host cells lack the necessary complementation to produce additional infectious progeny particles. Cells may be stably transfected, transiently transfected, or transfected with The missing functionality can be provided by infection with a helper virus that expresses the missing functionality. This can be done.

[0272] In certain embodiments, the complementing cell is deleted from the arenavirus vector genome. In a specific embodiment, the viral gene is functionally inactivated. , Complementation cells used to generate arenavirus vector genomes In another embodiment, the complementing cell is The virus strain used to generate the genome of the arenavirus vector is different from that used to generate the genome of the arenavirus vector. Viral genes from the genus virulence strain are provided, e.g., viral genes provided in complementation cells. is obtained from the MP strain of LCMV and contains a protein having the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18. In another example, the viral genes provided in the complementing cell encode a clone of LCMV. A protein obtained from the Lone 13 strain and having the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In another example, the viral genes provided in the complementation cell encode the WE strain of LCMV. and encodes a protein having the amino acid sequence of SEQ ID NO:25.

[0273] In a specific embodiment, the complementing cell provides the GP of the MP strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the MP strain of LCMV. The arenavirus vector was derived from LCMV clone 13 and encodes the GP protein. Instead of the ORF for the human HBV antigen, the ORF for the human HBV antigen described herein is included. In one embodiment, the GP protein has at least 90%, 91%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 16. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical do.

[0274] In a specific embodiment, the complementing cells provide the GP of the clone 13 strain of LCMV, and The virus vector contains the human GP protein described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises an ORF of a clone HBV antigen of LCMV. The GP of the LCMV strain 13 was provided, and the arenavirus vector was derived from the MP strain of LCMV, which encodes the GP protein. In place of the ORF encoding the antigen, it contains an ORF for a human HBV antigen as described herein. In a more specific embodiment, the GP protein has at least the amino acid sequence of SEQ ID NO:22. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% %Identical.

[0275] In a specific embodiment, the complementing cells provide the GP of the WE strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the WE strain of LCMV. The arenavirus vector was derived from LCMV clone 13 and encodes the GP protein. Instead of the ORF for the human HBV antigen, the ORF for the human HBV antigen described herein is included. In one embodiment, the GP protein has at least 90%, 91%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:25. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical do.

[0276] In a specific embodiment, the complementing cells provide the GP of the WE strain of LCMV and the GP of the Arenavirus vector. The vector contains the human HBV antigen described herein in place of the ORF encoding the GP protein. In an even more specific embodiment, the complementing cell comprises the GP of the WE strain of LCMV. The arenavirus vector is derived from the MP strain of LCMV and encodes the GP protein. In place of F, the ORF of a human HBV antigen described herein is included. In embodiments, the GP protein has at least 90%, 91%, 92%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO:25. %, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical.

[0277] 6.8 Combination Therapy (6.8(a) Method) In one embodiment, provided herein is a method for treating and / or administering to a subject an HBV infection. The present invention also provides a method of preventing HBV infection, comprising administering to a subject a HBV infection that expresses two or more HBV antigens as described herein. and administering a viral arenavirus to the host. See, e.g., Section 6.2. In a specific embodiment, the method of treating and / or preventing HBV infection includes, for example, administering S. The ORF encoding the GP of the genome segment was replaced with the nucleotide sequence encoding the HBV antigen. wherein the HBV antigen is: a) a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c) a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d) encoding a fusion of the HBV HBs protein and the HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e) a nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof; The vector can be, but is not limited to, a vector expressing an HBV antigen as described herein. a first infectious arenavirus, as well as an ORF encoding the GP of the S genome segment, is replaced with a nucleotide sequence encoding an HBV antigen, wherein the HBV antigen is: a) a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c) a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d) encoding a fusion of the HBV HBs protein and the HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e) a nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof The vector can be, but is not limited to, a vector expressing an HBV antigen as described herein. and administering a second infectious arenavirus to the animal.

[0278] In certain embodiments, a first infectious arenavirus and a second infectious arenavirus In one embodiment, the first infectious arenavirus and the second infectious arenavirus are replication-deficient. In one embodiment, the first infectious arenavirus is capable of replication. Either the virus or the second infectious arenavirus is replication-deficient. Thus, the first infectious arenavirus and the second infectious arenavirus are bipartite. In some embodiments, the first infectious arenavirus and the second infectious arenavirus are 3 In some embodiments, the first infectious arenavirus or the second infectious arenavirus is segmented. One of the virus strains is bipartite, the other is tripartite.

[0279] In specific embodiments, provided herein are methods for treating and / or preventing HBV infection. The method comprises administering to a subject a subject, the subject being a patient, the subject being a patient having an HBV pre-S2 / S protein or an antigenic variant thereof, as described herein. Fragments; HBV HBc protein or its antigenic fragments, HBV HBs protein or its antigens a first HBV antigen selected from: an HBV HBe protein or an antigenic fragment thereof; a first infectious arenavirus expressing an antigen, and an HBV pre-S2 / S protein or its antigen. HBV HBc protein or its antigenic fragment, H BV HBs protein or its antigenic fragment, or HBV HBe protein or its antigenic fragment a second infectious arenavirus expressing a second HBV antigen selected from: The method includes:

[0280] In certain embodiments, provided herein are methods for treating and / or preventing an infection. The method comprises administering two arenavirus vector constructs expressing the HBV antigens described herein. In a specific embodiment, the two constructs are administered to The HBV vector constructs express different HBV antigens.

[0281] In certain embodiments, provided herein are methods for treating and / or preventing an infection. The method comprises administering to a patient a vaccine comprising administering to the patient a vaccine containing two or more arenavirus vectors expressing HBV antigens as described herein. In a specific embodiment, the method comprises administering a construct as described herein. Provided herein are methods for treating and / or preventing infection, comprising administering to a subject a subject comprising a human ... administering three or more arenavirus vector constructs expressing BV antigens. In one embodiment, the arenavirus vector construct comprises a LC It can be based on MV.

[0282] In certain embodiments, provided herein are methods for treating and / or preventing an infection. The method comprises the steps of: providing two or more arenaviruses each expressing a different HBV antigen as described herein; In a specific embodiment, the method comprises administering a vector construct to a mammal. Provided herein are methods for treating and / or preventing infection, comprising administering to a subject a therapeutically effective amount of a compound as defined herein. Three or more arenavirus vector constructs, each expressing a different HBV antigen, as described. In one embodiment, the arenavirus vector The construct may be based on LCMV.

[0283] In a specific embodiment, the antigen is HBV pre-S2 / S protein or a fragment thereof ( See, e.g., Section 6.2(a)).

[0284] In certain embodiments, the antigen is an HBV HBc protein or a fragment thereof (e.g., See Section 6.2(b)).

[0285] In certain embodiments, the antigen is an HBV HBs protein or a fragment thereof (e.g., See Section 6.2(c)).

[0286] In certain embodiments, the antigen is HBV HBs protein and HBV HBc protein or both. or a fusion of an antigenic fragment thereof (see, e.g., Section 6.2(d)).

[0287] In certain embodiments, the antigen is an HBV HBe protein or a fragment thereof (e.g., See Section 6.2(e)).

[0288] In certain embodiments, the antibody is engineered to encode one or more of the HBV antigens described herein. The vectors described include one or more nucleic acids encoding the HBV antigens described, and combinations thereof. In a specific embodiment, the HBV antigens described herein are They are separated by various linkers, spacers, and cleavage sites.

[0289] In another embodiment, the first infectious arenavirus is one or more H as described herein. The vectors constructed to encode BV antigens were based on LCMV clone 13 or the MP strain of LCMV. (See, for example, Section 7.1.)

[0290] In another embodiment, the second infectious arenavirus is one or more Hs as described herein. The vectors constructed to encode BV antigens were based on LCMV clone 13 or the MP strain of LCMV. (See, e.g., Section 7.1.) In another embodiment, the first infectious agent vectors engineered to encode one or more of the HBV antigens described herein. The target can be based on the Junin virus.

[0291] In another embodiment, the second infectious arenavirus is one or more Hs as described herein. Vectors engineered to encode BV antigens can be based on Junin virus. .

[0292] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBc protein or an antigenic fragment thereof. and administering a second infectious arenavirus to the host.

[0293] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBs protein or an antigenic fragment thereof. and subsequently administering a second infectious arenavirus that induces the first or second infectious arenavirus.

[0294] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. a first infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0295] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBs protein and HBV HBc protein or This can be achieved by sequentially administering a second infectious arenavirus expressing a fusion of these antigenic fragments. and

[0296] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and subsequently administering a second infectious arenavirus expressing a fusion of the antigenic fragments of the two. Hmm, that's the method.

[0297] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. and subsequently administering a second infectious arenavirus comprising the first infectious arenavirus.

[0298] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenaviruses expressing fusions of these antigenic fragments and HBV pre-S2 / S and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. and

[0299] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenavirus and HBV HBe protein expressing fusions of these antigenic fragments and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. Hmm, that's the method.

[0300] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenavirus expressing a fusion of these antigenic fragments and the HBV HBc protein and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. Hmm, that's the method.

[0301] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof. and administering a second infectious arenavirus to the host.

[0302] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0303] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and subsequently administering a second infectious arenavirus expressing a fusion of the antigenic fragments of the two. Hmm, that's the method.

[0304] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and subsequently administering a second infectious arenavirus expressing a fusion of the antigenic fragments of the two. Hmm, that's the method.

[0305] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. and subsequently administering a second infectious arenavirus comprising the first infectious arenavirus.

[0306] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenavirus expressing a fusion of these antigenic fragments and the HBV HBs protein and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. Hmm, that's the method.

[0307] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. and subsequently administering a second infectious arenavirus comprising the first infectious arenavirus.

[0308] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0309] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0310] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBc protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0311] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBc protein or an antigenic fragment thereof; The method comprises administering two infectious arenaviruses simultaneously.

[0312] In another embodiment, the first infectious arenavirus expressing an HBV antigen is a primary vaccine. A second infectious arenavirus expressing a different HBV antigen is used as a secondary vaccine. It is an antigen.

[0313] In one embodiment, the HBV pre-S2 / S protein or a fragment thereof or the HBV HBc protein A first infectious arenavirus expressing a protein and an HBV pre-S2 / S protein or an HBV HBc protein. When a second infectious arenavirus expressing the HBV protein is administered, the virus expresses the HBV antigen, e.g. For example, expressing only the pre-S2 / S protein (or fragments thereof) or only the HBc protein, Vaccination provides superior protection against HBV compared with single doses of infectious arenaviruses In another embodiment, the HBV pre-S2 / S protein or a fragment thereof is provided. or a first infectious arenavirus expressing HBV HBc protein and HBV pre-S2 / S protein. a second infectious arenavirus expressing the HBV HBc protein or a fragment thereof, or the HBV HBc protein; Upon administration, the HBV antigens are expressed, e.g., only the pre-S2 / S protein (or fragments thereof) or HBc protein alone. In another embodiment, the HBV pre-S2 / S protein or its derivatives induce a potent immune response. A first infectious arenavirus expressing a fragment or HBV HBc protein and an HBV pre-S2 / S tag. a second infectious arenavirus expressing the HBV HBc protein or a fragment thereof, or the HBV HBc protein; When administered, the vaccine expresses HBV antigens, such as the pre-S2 / S protein (or a fragment thereof). Rather than administering a single infectious arenavirus expressing only the HBc protein or only the HBc protein, A large CD8+ T cell response is elicited. In another embodiment, HBV pre-S2 / S proteins are also elicited. or a fragment thereof, or a first infectious arenavirus expressing HBV HBc protein and an HBV protein. a second infectious allele expressing the HBV S2 / S protein or a fragment thereof or the HBV HBc protein; When HBV is administered, it expresses HBV antigens, such as the pre-S2 / S protein (or administration of a single infectious arenavirus expressing only the HBc protein (or a fragment thereof) or only the HBc protein. Higher titers of neutralizing antibodies are induced than with other vaccines.

[0314] In one embodiment, the HBV pre-S2 / S protein or a fragment thereof or the HBV HBs protein A first infectious arenavirus expressing a protein and an HBV pre-S2 / S protein or an HBV HBs protein. When a second infectious arenavirus expressing the HBV protein is administered, the virus expresses the HBV antigen, e.g. For example, expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBs protein, Vaccination provides superior protection against HBV compared with single doses of infectious arenaviruses In another embodiment, the HBV pre-S2 / S protein or a fragment thereof is provided. or a first infectious arenavirus expressing HBV HBs protein and HBV pre-S2 / S protein a second infectious arenavirus expressing a HBV HBs protein or a fragment thereof, or an HBV HBs protein; Upon administration, the HBV antigens are expressed, e.g., only the pre-S2 / S protein (or fragments thereof) or greater than administering a single infectious arenavirus expressing only the HBs protein. In another embodiment, the HBV pre-S2 / S protein or its derivatives induce a potent immune response. A first infectious arenavirus expressing a fragment or HBV HBs protein and an HBV pre-S2 / S tag. a second infectious arenavirus expressing the HBV HBs protein or a fragment thereof, or the HBV HBs protein; When administered, the vaccine expresses HBV antigens, such as the pre-S2 / S protein (or a fragment thereof). Rather than administering a single infectious arenavirus expressing only the HBs protein or only the HBs protein, A large CD8+ T cell response is elicited. In another embodiment, HBV pre-S2 / S proteins are also elicited. a first infectious arenavirus expressing the HBV HBs protein or a fragment thereof, and an HBV protein; a second infectious allele expressing the HBV S2 / S protein or a fragment thereof or the HBV HBs protein; When HBV is administered, it expresses HBV antigens, such as the pre-S2 / S protein (or administration of a single infectious arenavirus expressing only the HBs protein (or a fragment thereof) or only the HBs protein. Higher titers of neutralizing antibodies are induced than with other vaccines.

[0315] In one embodiment, the HBV pre-S2 / S protein or a fragment thereof or the HBV HBs protein The first infectious arenavirus expressing a fusion of HBV protein and HBc protein and HBV pre-S2 / S a second infectious allele expressing a protein or a fusion of the HBV HBs and HBc proteins; When HBV is administered, it expresses HBV antigens, such as the pre-S2 / S protein (or A single HBV vector expressing only the HBs protein and HBc protein (or fragments thereof) or only a fusion of the HBV HBs protein and HBc protein. Vaccination provides better protection against HBV than administration of infectious arenaviruses In another embodiment, the HBV pre-S2 / S protein or a fragment thereof or HB V. The first infectious arenavirus expressing a fusion of the HBs and HBc proteins and HB V pre-S2 / S protein or a fragment thereof, or a fusion of HBV HBs protein and HBc protein Upon administration of a second infectious arenavirus expressing the HBV antigen, e.g., Re-S2 / S protein (or its fragment) alone or a fusion of HBV HBs protein and HBc protein This induces a greater immune response than administration of a single infectious arenavirus expressing only the complex. In another embodiment, the HBV pre-S2 / S protein or a fragment thereof or the HBV HB First infectious arenavirus expressing a fusion of the s protein and the HBc protein and HBV protein Re-S2 / S protein or a fragment thereof, or a fusion of HBV HBs protein and HBc protein Upon administration of a second infectious arenavirus that expresses HBV antigens, e.g., pre- S2 / S protein (or its fragment) alone or a fusion of HBV HBs protein and HBc protein induced a greater CD8+ T cell response than administration of a single infectious arenavirus expressing only In another embodiment, the HBV pre-S2 / S protein or a fragment thereof or HBV The first infectious arenavirus expressing a fusion of the HBs and HBc proteins and HBV Pre-S2 / S protein or a fragment thereof, or a fusion of HBV HBs protein and HBc protein Upon administration of a second infectious arenavirus expressing the HBV antigen, e.g., Re-S2 / S protein (or its fragment) alone or a fusion of HBV HBs protein and HBc protein Higher titers of neutralizing antibodies were induced by administering only the combined antigens, compared with administering a single infectious arenavirus. It is induced.

[0316] In one embodiment, the HBV pre-S2 / S protein or a fragment thereof or the HBV HBe protein A first infectious arenavirus expressing a protein and an HBV pre-S2 / S protein or an HBV HBe protein. When a second infectious arenavirus expressing the HBV protein is administered, the virus expresses the HBV antigen, e.g. For example, expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBe protein, Vaccination provides superior protection against HBV compared with single doses of infectious arenaviruses In another embodiment, the HBV pre-S2 / S protein or a fragment thereof is provided. or a first infectious arenavirus expressing HBV HBe protein and HBV pre-S2 / S protein a second infectious arenavirus expressing the HBV HBe protein or a fragment thereof, Upon administration, the HBV antigens are expressed, e.g., only the pre-S2 / S protein (or fragments thereof) or HBe protein alone. In another embodiment, the HBV pre-S2 / S protein or its derivatives induce a potent immune response. A first infectious arenavirus expressing a fragment or HBV HBe protein and an HBV pre-S2 / S tag a second infectious arenavirus expressing the HBV HBe protein or a fragment thereof, or the HBV HBe protein; When administered, the vaccine expresses HBV antigens, such as the pre-S2 / S protein (or a fragment thereof). Rather than administering a single infectious arenavirus expressing only the HBe protein or only the HBe protein, A large CD8+ T cell response is elicited. In another embodiment, HBV pre-S2 / S proteins are also elicited. or a fragment thereof, or a first infectious arenavirus expressing the HBV HBe protein and an HBV protein. a second infectious allele expressing the HBV S2 / S protein or a fragment thereof or the HBV HBe protein; When HBV is administered, it expresses HBV antigens, such as the pre-S2 / S protein (or administration of a single infectious arenavirus expressing only the HBe protein (or a fragment thereof) or only the HBe protein. Higher titers of neutralizing antibodies are induced than with other vaccines.

[0317] In yet another embodiment, provided herein is an HBV vector as described herein. A combination of a replication-defective arenavirus expressing an antigen and one or more replication-defective viral vectors. In a more specific embodiment, the replication-defective viral vector is a poxvirus. , adenovirus, alphavirus, herpes simplex virus, paramyxovirus, Viruses such as poliovirus, adeno-associated virus, and Sendai virus, as well as In a specific embodiment, the poxvirus is selected from the group consisting of: Irusu is a modified vaccine Ankara.

[0318] In yet another embodiment, provided herein is an HBV vector as described herein. Replication-deficient arenaviruses expressing HBV antigens and one or more replication-deficient viral vectors expressing HBV antigens. In a more specific embodiment, the replication-defective viral vector is Poxvirus, adenovirus, alphavirus, herpes simplex virus, paramyxovirus Xovirus, rhabdovirus, poliovirus, adeno-associated virus, and Sendai virus In a specific embodiment, the compound is selected from the group consisting of: In this case, the poxvirus is a modified vaccine Ankara.

[0319] In another embodiment, a first infectious arena expressing an HBV antigen described herein is The virus may be a second infectious arenavirus expressing an HBV antigen as described herein. For example, a first infectious arenavirus expressing an HBV antigen may be administered after a second The vaccine is administered approximately 30 to 60 minutes before or after the first dose of the infectious arenavirus.

[0320] In another embodiment, the first infectious arenavirus expressing the vaccine antigen is In one embodiment, the antibody is administered before a second infectious arenavirus expressing the antigen. and between the administration of the first infectious arenavirus and the administration of the second infectious arenavirus, approximately 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months Month, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year be.

[0321] In another embodiment, the two infectious arenaviruses are in a ratio of, among others: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ​​ratio, 1: Treatment regimens ranging from about 1:1 to 1:1000 molar ratios, including 1:700 ratio, 1:800 ratio, 1:900 ratio, and 1:1000 ratio It is administered in a dose of

[0322] In another embodiment, two or more infectious arenas expressing HBV antigens described herein are The subject to whom the virus is administered may have, be susceptible to, or be at risk of HBV infection. In another embodiment, two HBV-expressing HBV antigens described herein are The subjects to whom the infectious arenaviruses are administered are those who are infected with or suffering from HBV infection. susceptible to or at risk of developing a disease.

[0323] In another embodiment, two or more infectious arenas expressing HBV antigens described herein are The subject to whom the virus is co-administered has or is susceptible to HBV infection, or In another embodiment, a person who expresses an HBV antigen described herein is at risk. Subjects receiving two or more infectious arenaviruses simultaneously are not infected with HBV. or susceptible to or at risk of

[0324] In another embodiment, two or more infectious arenas expressing HBV antigens described herein are The subject to whom the virus is subsequently administered may have, be susceptible to, or be a patient suffering from HBV infection. In another embodiment, a patient expressing an HBV antigen described herein is at risk of HBV. Subjects receiving two or more infectious arenaviruses sequentially are either infected with HBV or , susceptible to, or at risk of.

[0325] In another embodiment, the two or more infectious alleles expressing the HBV antigens described herein The HBV may further be combined with at least one other agent for treating and / or preventing HBV. Therapeutic agents for treating and / or preventing HBV include enterovirus, Kavil (BARACLUDE®; Bristol-Myers Squibb), lamivudine (EPIVIR HBV®), GlaxoSmithKline), adefovir dipivoxil (HEPSERA®; Gilead Sciences), ), interferon alpha 2b (INTRON A®; Schering), pegylated interferon (PE GASYS®; Roche), telbivudine (TYZEKA®, Novartis), and tenofovir Examples of suitable anti-inflammatory drugs include, but are not limited to, VIREAD® (Gilead Sciences).

[0326] In another embodiment, the two or more infectious alleles expressing the HBV antigens described herein The virus may also be administered in combination with at least one other immune modulator. In a more specific embodiment, the two or more HBV antigens expressing the HBV antigens described herein The infectious arenavirus may further be combined with at least one Th1-specific adjuvant. In a more specific embodiment, the Th-1 specific adjuvant is Calmette's Bacillus guerin (BCG)

[0327] In another embodiment, the dosing regimen comprises administering to a symptomatic subject an HBV antiviral agent described herein. The method may further include administering a second infectious arenavirus expressing the antigen. In one embodiment, the administration regimen is administered to subjects with compromised immune systems, particularly those undergoing transplantation. subjects, HIV-infected subjects, pregnant subjects, subjects with cancer, with HBV anti-cancer drugs described herein. The method may further include administering a second infectious arenavirus expressing the antigen. In embodiments, two or more infectious arenaviruses expressing an HBV antigen described herein are , suffering from, susceptible to, or at risk of HBV infection Children aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years old who are It is administered to a subject.

[0328] In another embodiment, the administration regimen comprises administering to a subject who is a child a first HBV antigen-expressing and administering to the same adolescent subjects the first HBV antigen-expressing arenavirus. In a specific embodiment, the administration of a second arenavirus can include administering a second arenavirus. The dosing regimen is for ages 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years. administering to a subject a first arenavirus that expresses an HBV antigen as described herein. and the same pair who are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 years old. The method can include administering to the elephant a second infectious arenavirus that expresses an HBV antigen. do.

[0329] In another embodiment, the administration regimen comprises administering to a prepubertal subject a second HBV antigen-expressing In another embodiment, the method comprises administering an infectious arenavirus of The administration regimen is administered to adolescent males aged 12-18 years who have HBV antigen-expressing HBV strains as described herein. In another embodiment, the method comprises administering two infectious arenaviruses. The dosing regimen is designed to treat females aged 12 to 18 years with a second infectious arenavirus that expresses HBV antigen. It can include administering.

[0330] In another embodiment, administration of two or more infectious arenaviruses expressing HBV antigens results in , the risk that an individual will develop an HBV infection compared to the risk of developing an HBV infection in the absence of such treatment The risk of developing the disease is at least 10%, at least about 20%, at least about 25%, or at least about 30%. %, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least The decrease is at least about 80%, at least about 90%, or more.

[0331] In another embodiment, two or more infectious ArenA viruses expressing HBV antigens are administered separately. administration of HBV reduces the risk of developing HBV infection compared with the risk in the absence of such treatment , the individual has at least a 10%, at least about a 20%, or at least about a 2% risk of developing HBV infection 5%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, The decrease is at least about 70%, at least about 80%, at least about 90%, or more.

[0332] In another embodiment, two or more infectious arenaviruses expressing HBV antigens are administered sequentially. administration of rux reduces the risk of developing HBV infection compared with the risk in the absence of such treatment. The individual's risk of developing HBV infection is at least 10%, at least about 20%, at least about 25% %, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least The decrease is at least about 70%, at least about 80%, at least about 90%, or more.

[0333] Without being limited by theory, it is believed that the first infectious arenavirus and subsequent The administration of the second infectious arenavirus vector results in a prime-boost effect.

[0334] In certain embodiments, provided herein are methods for treating and / or preventing HBV infection. The method comprises the steps of: (a) administering two or more arenavirus vectors each expressing the same or different HBV antigens; The method comprises administering the constructs sequentially, with the time interval between each administration being about 1 minute. weeks, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months , about 18 months, or about 24 months.

[0335] In certain embodiments, a first infectious arenavirus and a second infectious arenavirus In one embodiment, the first infectious arenavirus and the second infectious arenavirus are homologous. The virus is heterologous.

[0336] In certain specific embodiments, the first infectious arenavirus is an Old World arenavirus. and the second infectious arenavirus is an Old World arenavirus. In embodiments, the first infectious arenavirus is an Old World arenavirus and the second infectious arenavirus is an Old World arenavirus. In a specific embodiment, the first arenavirus is a New World arenavirus. The first infectious arenavirus is a New World arenavirus, and the second infectious arenavirus is In a specific embodiment, the first infectious arenavirus is a New World arenavirus. Rus is a New World arenavirus, and the second infectious arenavirus is an Old World arenavirus. It is.

[0337] In certain specific embodiments, the first infectious arenavirus is derived from LCMV and the second The infectious arenavirus is derived from LCMV. In a specific embodiment, the first infectious The arenavirus is derived from LCMV, and a second infectious arenavirus is derived from Junin virus. In a specific embodiment, the first infectious arenavirus is derived from a Junin virus. The second infectious arenavirus is derived from a Junin virus. In the present study, the first infectious arenavirus is derived from Junin virus and the second infectious arenavirus is derived from Junin virus. The virus is derived from LCMV.

[0338] In certain embodiments, provided herein are methods for treating and / or preventing HBV infection. The method comprises administering a first infectious arenavirus first as a "primary immunization" and a second The first and second infectious arenaviruses are administered as a "booster". Infectious arenavirus vectors can express the same or different HBV antigens. In a specific embodiment, the "primary immunization" administration is an administration of an infectious arenavirus derived from LCMV. The "booster" is an infectious arenavirus derived from Junin virus. In a specific embodiment, the "primary immunization" administration is performed using Funingweed. The vaccine was administered using an infectious arenavirus derived from the genus leukemia virus, and the "booster" was administered using an infectious arenavirus derived from LCMV. This is performed using infectious arenaviruses.

[0339] In one embodiment, a first infectious arenavirus expressing an HBV antigen or a fragment thereof. and then administering a second infectious arenavirus expressing an HBV antigen or a fragment thereof. Thus, rather than administering a single infectious arenavirus expressing an HBV antigen or a fragment thereof, A large antigen-specific CD8+ T cell response is elicited. In one embodiment, the antigen-specific CD The number of 8+ T cells increased by 50%, 100%, 150%, or 20% after the second dose compared to the first dose. In one embodiment, a third infectious arenavirus expressing an HBV antigen is administration of two consecutive infectious arenaviruses expressing HBV antigens produces a greater effect than administration of two consecutive infectious arenaviruses expressing HBV antigens. In one embodiment, a large antigen-specific CD8+ T cell response is elicited. T cell counts increased by approximately 50%, 100%, 150%, and 100% after the third dose compared to the first dose. An increase of 200%, or about 250%.

[0340] In certain embodiments, provided herein are methods for treating and / or preventing an infection. A method comprising administering two or more arenavirus vector constructs. wherein the two or more arenavirus vector constructs are homologous; The time interval between each administration is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks. , about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.

[0341] In one embodiment, a first infectious arenavirus expressing an HBV antigen or a fragment thereof. and a second, heterologous, infectious arenavirus expressing an HBV antigen or a fragment thereof. a first infectious arenavirus expressing an HBV antigen or a fragment thereof and an HBV antigen or a fragment thereof; administration of a second homologous infectious arenavirus expressing the It is induced.

[0342] In a specific embodiment, a first infectious allele expressing an HBV pre-S2 / S protein is The HBV virus is LCMV, a second heterologous infectious allele that expresses HBV pre-S2 / S proteins. In a specific embodiment, the HBV pre-S2 / S protein is a Junin virus. The first infectious arenavirus to express the protein was Junin virus, which is a HBV pre-S2 / S protein. A second heterologous infectious arenavirus that expresses a protein is LCMV.

[0343] In certain specific embodiments, a first infectious ArenA virus expressing an HBV HBc protein is The virus is LCMV, a second heterologous infectious arenavirus that expresses the HBV HBc protein. In a specific embodiment, the virus expressing the HBV HBc protein is Junin virus. The first infectious arenavirus to be identified was Junin virus, which expresses the HBV HBc protein. A second heterologous infectious arenavirus is LCMV.

[0344] In certain specific embodiments, a first infection expressing HBV HBs and HBc fusion proteins The primary arenavirus is LCMV, a second heterologous arenavirus that expresses the HBV HBs and HBc fusion proteins. In a specific embodiment, the infectious arenavirus is Junin virus. The first infectious arenavirus to express the HBs and HBc fusion proteins was Junin virus. A second heterologous infectious arenavirus expressing the HBV HBs and HBc fusion proteins is LC It's a music video.

[0345] In certain specific embodiments, a first infectious ArenA virus expressing an HBV HBe protein is The virus is LCMV, a second heterologous infectious arenavirus that expresses the HBV HBe protein. In a specific embodiment, the virus expressing the HBV HBe protein is Junin virus. The first infectious arenavirus to be identified was Junin virus, which expresses the HBV HBe protein. A second heterologous infectious arenavirus is LCMV.

[0346] In a specific embodiment, a first infectious allele expressing an HBV pre-S2 / S protein is A second heterologous infectious arenavirus expressing HBV and HBV pre-S2 / S proteins administration of the first infectious arenavirus expressing the HBV pre-S2 / S protein and HBV administration of a second homologous infectious arenavirus expressing the pre-S2 / S protein. In a specific embodiment, a large CD8+ T cell response is elicited. A first infectious arenavirus expressing HBV protein and a second expressing HBV pre-S2 / S protein. Administration of a heterologous infectious arenavirus induces the first generation of HBV expressing the HBV pre-S2 / S protein. and a second homologous infectious arenavirus expressing HBV pre-S2 / S proteins. Approximately 20%, 40%, 60%, 80%, 100%, 120%, and 14% more effective than administering flu vaccines. 0%, about 160%, about 180%, or about 200% greater CD8+ T cell responses are elicited.

[0347] In certain specific embodiments, a first infectious ArenA virus expressing an HBV HBc protein is and a second, heterologous infectious arenavirus expressing the HBV HBc protein. The first infectious arenavirus expressing the HBV HBc protein and the first infectious arenavirus expressing the HBV HBc protein were identified. administration of a second homologous infectious arenavirus expressing the In a specific embodiment, a first infection that expresses HBV HBc protein is induced. A second, heterologous, infectious arenavirus expressing the HBV HBc protein. Upon administration of the drug, the first infectious arenavirus expressing the HBV HBc protein and the HBV HBc Approximately 20% and 40% of the cells expressing the protein were infected with a second, homologous infectious arenavirus. %, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater A rapid CD8+ T cell response is elicited.

[0348] In certain specific embodiments, a first infection expressing HBV HBs and HBc fusion proteins A second heterologous infectious arenavirus expressing the HBs and HBc fusion proteins in HBV. Administration of arenaviruses results in the production of primary infectious arenaviruses expressing the HBV HBs and HBc fusion proteins. A second homologous infectious arena expressing HBV and HBs and HBc fusion proteins. In a specific embodiment, administration of the virus induces a greater CD8+ T cell response. A first infectious arenavirus expressing HBV HBs and HBc fusion proteins and HB V. Administer a second heterologous infectious arenavirus expressing the HBs and HBc fusion proteins. and a first infectious arenavirus expressing HBV HBs and HBc fusion proteins and an HBV HB Rather than administering a second homologous infectious arenavirus expressing the HBs and HBc fusion proteins, Approximately 20%, approximately 40%, approximately 60%, approximately 80%, approximately 100%, approximately 120%, approximately 140%, approximately 160%, approximately 180%, or elicits approximately 200% greater CD8+ T cell responses.

[0349] In certain specific embodiments, a first infectious ArenA virus expressing an HBV HBe protein is and a second, heterologous infectious arenavirus expressing the HBV HBe protein. The first infectious arenavirus expressing the HBV HBe protein and the first infectious arenavirus expressing the HBV HBe protein are identified. administration of a second homologous infectious arenavirus expressing the In a specific embodiment, a first infection that expresses the HBV HBe protein is induced. arenaviruses and a second heterologous infectious arenavirus expressing the HBV HBe protein. Upon administration of the drug, the first infectious arenavirus expressing the HBV HBe protein and the HBV HBe Approximately 20% and 40% of the cells expressing the protein were infected with a second, homologous infectious arenavirus. %, about 60%, about 80%, about 100%, about 120%, about 140%, about 160%, about 180%, or about 200% greater A rapid CD8+ T cell response is elicited.

[0350] In certain embodiments, provided herein are methods for treating and / or preventing an infection. A method comprising administering two or more arenavirus vector constructs. wherein the two or more arenavirus vector constructs are heterologous; The time interval between each administration is about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, 12 months, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months.

[0351] In yet another embodiment, provided herein is an HBV vector as described herein. A combination of a replication-defective arenavirus expressing an antigen and one or more replication-defective viral vectors. In a more specific embodiment, the replication-defective viral vector is a poxvirus. , adenovirus, alphavirus, herpes simplex virus, paramyxovirus, Viruses such as poliovirus, adeno-associated virus, and Sendai virus, as well as In a specific embodiment, the poxvirus is selected from the group consisting of: Irusu is a modified vaccine Ankara.

[0352] In yet another embodiment, provided herein is an HBV vector as described herein. Replication-deficient arenaviruses expressing HBV antigens and one or more replication-deficient viral vectors expressing HBV antigens. In a more specific embodiment, the replication-defective viral vector is Poxvirus, adenovirus, alphavirus, herpes simplex virus, paramyxovirus Xovirus, rhabdovirus, poliovirus, adeno-associated virus, and Sendai virus In a specific embodiment, the compound is selected from the group consisting of: In this case, the poxvirus is a modified vaccine Ankara.

[0353] In another embodiment, a first infectious arena expressing an HBV antigen described herein is The virus may be a second infectious arenavirus expressing an HBV antigen as described herein. For example, a first infectious arenavirus expressing an HBV antigen may be administered after a second The vaccine is administered approximately 30 to 60 minutes before or after the first dose of the infectious arenavirus.

[0354] In another embodiment, the first infectious arenavirus expressing the vaccine antigen is In one embodiment, the antibody is administered before a second infectious arenavirus expressing the antigen. and between the administration of the first infectious arenavirus and the administration of the second infectious arenavirus, approximately 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months Month, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year be.

[0355] In another embodiment, the two infectious arenaviruses are in a ratio of, among others: 1:1 ratio, 1:2 ratio, 1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ​​ratio, 1: Treatment regimens ranging from about 1:1 to 1:1000 molar ratios, including 1:700 ratio, 1:800 ratio, 1:900 ratio, and 1:1000 ratio It is administered in a dose of

[0356] In another embodiment, two or more infectious arenas expressing HBV antigens described herein are The subject to whom the virus is administered may have, be susceptible to, or be at risk of HBV infection. In another embodiment, two HBV-expressing HBV antigens described herein are The subjects to whom the infectious arenaviruses are administered are those who are infected with or suffering from HBV infection. susceptible to or at risk of developing a disease.

[0357] Subjects that can be treated with the methods provided herein include those susceptible to HBV infection and those with or is at risk of

[0358] In another embodiment, the two or more infectious alleles expressing the HBV antigens described herein The virus may further comprise at least one other immunostimulatory peptide, polypeptide, or protein. In certain embodiments, the immunostimulatory peptide, polypeptide, or tag is expressed in a mammalian cell line. Proteins include calreticulin (CRT) or a fragment thereof; ubiquitin or a fragment thereof; Granulocyte-macrophage colony-stimulating factor (GM-CSF) or its fragments; invariant chain (CD74) or Its antigenic fragment: Mycobacterium tuberculosis heat shock protein 70 or or an antigenic fragment thereof; herpes simplex virus 1 protein VP22 or an antigenic fragment thereof; CD40 ligand or an antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or is its antigenic fragment.

[0359] Two infectious replication-defective arenavirus vectors are expressed from different arenaviruses (e.g., LCMV and Non-homologous expression using infectious replication-defective arenavirus vectors derived from Junin virus and Junin virus These infectious, replication-defective arenavirus vectors are also provided. Antigens, for example, antigens of HBV, can be expressed.

[0360] Two infectious replication-competent arenavirus vectors are expressed from different arenaviruses (e.g., LCMV and Non-homologous infection using infectious replication-competent arenavirus vectors derived from Junin virus and Junin virus Primer-boost methods are also provided. These infectious, replication-competent arenavirus vectors are Antigens, for example, antigens of HBV, can be expressed.

[0361] (6.8(b) Composition) The present invention further provides vaccines, including the genetically modified arenaviruses described herein. Such vaccines and pharmaceutical compositions are well known in the art. The formulation can be carried out according to standard procedures in the art.

[0362] In one embodiment, provided herein is a vaccine that expresses an HBV antigen as described herein. A composition comprising two or more infectious arenaviruses that express the virus. See, e.g., Section 6.2. In specific embodiments, the compositions described herein are administered to a subject, e.g., For example, the ORF encoding the GP of the S genome segment is identical to the nucleotide sequence encoding the HBV antigen. A first infectious arenavirus expressing a substituted HBV antigen as described herein ( The HBV antigen is: a) a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c) a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d) encoding a fusion of the HBV HBs protein and the HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e) a nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof; , and for example, the GP of the S genome segment. The ORF encoding the HBV antigen is substituted for the nucleotide sequence encoding the HBV antigen. a second infectious arenavirus composition expressing a described HBV antigen, wherein the HBV antigen is: a) a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b) a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c) a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d) encoding a fusion of the HBV HBs protein and the HBV HBc protein or antigenic fragments thereof the nucleotide sequence; e) a nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof In some embodiments, the administration of wherein the first infectious arenavirus and the second infectious arenavirus are replication-deficient. In certain embodiments, the first infectious arenavirus and the second infectious arenavirus are In certain embodiments, the first infectious arenavirus or Either the second infectious arenavirus is replication-deficient.

[0363] In specific embodiments, provided herein are methods for treating and / or preventing HBV infection. The method comprises administering to a subject a subject, the subject being a patient, the subject being a patient having an HBV pre-S2 / S protein or an antigenic variant thereof, as described herein. Fragment; HBV HBc protein or its antigenic fragment; HBV HBs protein or its antigen HBV HBs protein and HBV HBc protein or fusions of these antigenic fragments or an HBV HBe protein or an antigenic fragment thereof. and the first infectious arenavirus, and HBV pre-S2 / S protein or antigenic fragment thereof. fragment; HBV HBc protein or its antigenic fragment; or HBV HBs protein or its antigenic fragment HBV HBs protein and HBV HBc protein or fusions of antigenic fragments thereof or an HBV HBe protein or an antigenic fragment thereof. and administering a second infectious arenavirus that expresses the virus.

[0364] In certain embodiments, provided herein are methods for treating and / or preventing HBV infection. The method comprises administering to a subject a virus containing two arenavirus constructs expressing HBV antigens as described herein. In a specific embodiment, the composition is suitable for a method comprising administering to a subject Two arenavirus vector constructs express HBV antigens.

[0365] In certain embodiments, provided herein are HBV antigens described herein. The composition comprises two or more arenavirus vector constructs expressing the In embodiments, provided herein are antibodies expressing the HBV antigens described herein. In one embodiment, the composition comprises three or more arenavirus vector constructs. In the method, the arenavirus can be LCMV.

[0366] In a specific embodiment, the antigen is HBV pre-S2 / S protein or a fragment thereof ( See, e.g., Section 6.2(a)).

[0367] In certain embodiments, the antigen is an HBV HBc protein or a fragment thereof (e.g., See Section 6.2(b)).

[0368] In certain embodiments, the antigen is an HBV HBs protein or a fragment thereof (e.g., See Section 6.2(c)).

[0369] In certain embodiments, the antigen is HBV HBs protein and HBV HBc protein or both. and fusions of antigenic fragments thereof (see, e.g., Section 6.2(d)).

[0370] In certain embodiments, the antigen is an HBV HBe protein or a fragment thereof (e.g., See Section 6.2(e)).

[0371] In certain embodiments, the antibody is engineered to encode one or more of the HBV antigens described herein. The vectors described include one or more nucleic acids encoding the HBV antigens described, and combinations thereof. In a specific embodiment, the HBV antigens described herein are They are separated by various linkers, spacers, and cleavage sites.

[0372] In another embodiment, the first infectious arenavirus is one or more H as described herein. The vectors constructed to encode BV antigens were cloned into 13 strains of LCMV or the MP strain of LCMV. (See, e.g., Section 7.1).

[0373] In another embodiment, the second infectious arenavirus is one or more Hs as described herein. The vectors constructed to encode BV antigens were cloned into 13 strains of LCMV or the MP strain of LCMV. (See, e.g., Section 7.1).

[0374] In specific embodiments, provided herein are methods for treating HBV infection in a subject. and / or a method for preventing HBV infection, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. a first infectious arenavirus composition expressing a fragment thereof and an HBV HBc protein or its antigenic fragment; and administering a second infectious arenavirus composition expressing the fragment. It is a composition.

[0375] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBs protein or an antigenic fragment thereof. and a second infectious arenavirus, each of which is a virus that causes a viral infection. .

[0376] In a specific embodiment, provided herein is a composition suitable for treating an infection in a subject. a composition comprising: a first infected subject expressing an HBV HBc protein or an antigenic fragment thereof; a second infectious arenavirus expressing the HBV HBs protein or an antigenic fragment thereof; and administering a viral vector simultaneously.

[0377] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBs protein and HBV HBc protein or This can be achieved by sequentially administering a second infectious arenavirus expressing a fusion of these antigenic fragments. and

[0378] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and simultaneously administering a second infectious arenavirus expressing a fusion of these antigenic fragments. A composition suitable for the method includes:

[0379] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. The composition is suitable for a method comprising sequentially administering a second infectious arenavirus comprising administering a first infectious arenavirus to a mammalian host.

[0380] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenaviruses expressing fusions of these antigenic fragments and HBV pre-S2 / S and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. and

[0381] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenaviruses expressing fusions of these antigenic fragments, as well as HBV HBe proteins, and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. A composition suitable for the method includes:

[0382] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenaviruses expressing fusions of these antigenic fragments, as well as HBV HBc proteins, and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. A composition suitable for the method includes:

[0383] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV pre-S2 / S protein or an antigenic fragment thereof. A first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof. and administering a second infectious arenavirus to the host.

[0384] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus expressing an HBV HBs protein or an antigenic fragment thereof; The composition is suitable for methods involving the simultaneous administration of two infectious arenaviruses.

[0385] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and subsequently administering a second infectious arenavirus expressing a fusion of the antigenic fragments of the two. It is a composition suitable for the method.

[0386] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus and an HBV HBs protein and an HBV HBc protein or and subsequently administering a second infectious arenavirus expressing a fusion of the antigenic fragments of the two. It is a composition suitable for the method.

[0387] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. The composition is suitable for a method comprising sequentially administering a second infectious arenavirus comprising administering a first infectious arenavirus to a mammalian host.

[0388] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or a method for preventing, comprising administering to the subject an HBV HBs protein and an HBV HBc protein or The first infectious arenaviruses expressing fusions of these antigenic fragments, as well as HBV HBstan, and subsequently administering a second infectious arenavirus expressing the protein or an antigenic fragment thereof. A composition suitable for the method includes:

[0389] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. and a first infectious arenavirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof. The composition is suitable for a method comprising sequentially administering a second infectious arenavirus comprising administering a first infectious arenavirus to a mammalian host.

[0390] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a gene encoding an HBV HBc protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof; The composition is suitable for methods involving the simultaneous administration of two infectious arenaviruses.

[0391] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBe protein or an antigenic fragment thereof; The composition is suitable for methods involving the simultaneous administration of two infectious arenaviruses.

[0392] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject a compound expressing HBV HBs protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBc protein or an antigenic fragment thereof; The composition is suitable for methods involving the simultaneous administration of two infectious arenaviruses.

[0393] In specific embodiments, provided herein are methods for treating and administering to a subject an infection. and / or preventive methods, comprising administering to said subject an antibody expressing HBV HBe protein or an antigenic fragment thereof. a first infectious arenavirus expressing HBV HBc protein or an antigenic fragment thereof; The composition is suitable for methods involving the simultaneous administration of two infectious arenaviruses.

[0394] In another embodiment, the first infectious arenavirus composition expressing an HBV antigen is a primary A second infectious arenavirus expressing a different HBV antigen is used as a secondary vaccine. It is a genotype antigen.

[0395] In yet another embodiment, provided herein is an HBV vector as described herein. Replication-Defective Arenavirus Compositions Expressing Antigens and One or More Replication-Defective Viral Vector Compositions In a more specific embodiment, the replication-defective viral vector composition is : Poxvirus, adenovirus, alphavirus, herpes simplex virus, paravirus Myxovirus, rhabdovirus, poliovirus, adeno-associated virus, and Sendai virus The agent may be, but is not limited to, a virus, or a mixture thereof. In one embodiment, the poxvirus is a modified vaccine Ankara.

[0396] In another embodiment, the two infectious arenavirus compositions are mixed in a 1:1 ratio, a 1:2 ratio, a 1:3 ratio, a 1:4 ratio, a 1:5 ratio, a 1:6 ratio, a 1:7 ratio, a 1:8 ratio, a 1:9 ratio, a 1:10 ratio, a 1:11 ratio, a 1:20 ratio, a 1:12 ratio, a 1:13 ratio, a 1:14 ratio, a :5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200 ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 having a molar ratio ranging from about 1:1 to 1:1000, including a 1:700 ratio, a 1:800 ratio, a 1:900 ratio, and a 1:1000 ratio; do.

[0397] In another embodiment, two or more infectious arenas expressing HBV antigens described herein are The viral composition may be a composition comprising a virus that is present in a subject having, susceptible to, or at risk of HBV infection. In another embodiment, the HBV vaccine described herein is suitable for administration to a subject. Two or more infectious arenavirus compositions expressing antigens or subjects to which the compositions are administered are , infected with, susceptible to, or at risk of HBV infection.

[0398] In another embodiment, the two or more infectious arenavirus compositions are used to treat HBV infection and and / or at least one other agent for the prevention of tecavir (BARACLUDE®; Bristol-Myers Squibb), lamivudine (EPIVIR HBV®), trademark; GlaxoSmithKline), adefovir dipivoxil (HEPSERA®; Gilead Sciences es), interferon alpha 2b (INTRON A®; Schering), pegylated interferon ( PEGASYS®; Roche), telbivudine (TYZEKA®, Novartis), and tenofovir These include, but are not limited to, VIREAD® (Gilead Sciences).

[0399] In another embodiment, the composition comprises administering to a symptomatic subject an HBV antigen or It is preferred to administer a second infectious arenavirus composition expressing the fragment. In another embodiment, the composition is administered to subjects with compromised immune systems, particularly transplant recipients. a subject who is HIV-infected, a pregnant subject, or a subject with cancer, It is suitable to administer a second infectious arenavirus composition expressing the antigen or a fragment thereof. In another embodiment, two or more HBV antigens expressing the HBV antigens or fragments thereof described herein are provided. The infectious arenavirus composition is directed to a subject suffering from or susceptible to HBV infection. or at risk of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 The invention is suitable for administration to subjects who are children aged 14, 15, 16, or 17 years.

[0400] In another embodiment, the composition comprises administering to a subject, the subject being a child, a first allele expressing an HBV antigen. and administering to the same adolescent subjects a second antigen expressing HBV antigens. In a specific embodiment, the administration regimen is suitable for administering a viral vector. Subjects who are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age, administering a first arenavirus expressing an HBV antigen described herein; and , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 years of age, and HBV antigen The method can include administering a second infectious arenavirus expressing the

[0401] In another embodiment, the composition is administered to a prepubertal subject who has a second infection that expresses HBV antigens. In another embodiment, the administration regimen is suitable for administering a virulent arenavirus. Adolescent males aged 12-18 years are given a second infectious allele expressing the HBV antigens described herein. In another embodiment, the dosing regimen comprises administering a flu virus. , administering a second infectious arenavirus expressing HBV antigens to females aged 12 to 18 years. It can include.

[0402] In another embodiment, two or more HBV antigens expressing the HBV antigens or fragments thereof described herein are The infectious arenavirus composition reduces the risk of developing HBV infection in the absence of such treatment. reduce the risk of an individual developing HBV infection by at least 10%, at least about 20%, or at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least About 60%, at least about 70%, at least about 80%, at least about 90%, or more To lower.

[0403] In another embodiment, the HBV antigens or fragments thereof described herein are administered separately. and (c) a method for treating HBV infection in the absence of such treatment, comprising administering to a subject a viral load comprising administering to said ... Reduce an individual's risk of developing HBV infection by at least 10% compared with the risk of developing HBV infection. at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least About 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or a greater decrease.

[0404] In another embodiment, the HBV antigens or fragments thereof described herein are administered sequentially. and two or more infectious arenavirus compositions expressing the same or similar compounds, which are capable of inhibiting HBV infection in the absence of such treatment. Reduce an individual's risk of developing HBV infection by at least 10% compared with the risk of developing HBV. at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or will decrease it even more.

[0405] In another embodiment provided herein, the present invention provides a method for treating two or more HBV-expressing HBV antigens. A vaccine composition is provided that includes a synergistic combination of infectious, replication-deficient arenaviruses.

[0406] In another embodiment provided herein, the present invention provides a method for treating two or more HBV-expressing HBV antigens. A vaccine composition is provided that includes a synergistic combination of infectious, replication-competent arenaviruses.

[0407] (6.9 Assay) Assay for measuring the infectivity of arenavirus vectors Any assay known to one of skill in the art can be used to measure the infectivity of an arenavirus vector preparation. For example, determining the virus / vector titer can be used to This can be done by a Fully Functional Unit Assay (FFU assay). For example, HEK 293 cells expressing LCMV GP protein are plated and the virus / vector is then transfected. After an incubation period, cells form a monolayer. The plate is then covered with methylcellulose to allow the virus to attach to the cells. Upon further incubation, the originally infected cells release viral progeny. Because the cells are layered, the spread of new viruses is restricted to neighboring cells. Each infectious particle gives rise to a circular zone of infected cells called a focus. Such foci can be visualized using antibodies against LCMV-NP and an HRP-based color reaction. This allows for counting of the virus / vector titer. can be calculated in focus-forming units per milliliter (FFU / mL).

[0408] This assay was performed to determine the infectious titer (FFU / mL) of the vector carrying the transgene. , improved by using respective transgene-specific antibodies instead of anti-LCMV-NP antibodies. Change.

[0409] (Serum ELISA) Determination of humoral immune responses after vaccination of animals (e.g., mice, guinea pigs) is an antigen-specific This can be done by differential serum ELISA (enzyme-linked immunosorbent assay). The plate is coated with an antigen (e.g., a recombinant protein) to prevent non-specific binding of the antibody. To prevent this, the cells were blocked and incubated with serial dilutions of serum. After incubation, the bound serum-antibodies are separated, for example, by enzyme-conjugated (total IgG or IgG subclass) antibodies. using anti-species (e.g., mouse, guinea pig) specific antibodies (detecting the The antibody titer can be determined, for example, as the endpoint geometric mean titer. can be done.

[0410] Immunocapture ELISA (IC-ELISA) can also be performed (Shanmugham et al., 2010, Clin. Va. (See, e.g., Cancer Immunol. 17(8):1252-1260), in which case the capture agent is crosslinked to the beads. do.

[0411] (Neutralization assay in ARPE-19 cells) Determination of the neutralizing activity of induced antibodies in serum was performed using ARPE-19 cells from ATCC and GFP-tagged viruses. The following cell assays using rhesus and erythrocytes were performed. In addition, supplementation as a source of exogenous complement was performed. The assay is performed in a 384-well plate one or two days before use for neutralization. 6.5 x 10 3 Start by seeding cells / well (50 μl / well). Neutralization incubation is performed in a sterile 96-well tissue culture plate at 37°C for 1 hour. After this step, the mixture was added to the cells and incubated for another 4 days for GFP detection using a plate reader. A positive neutralizing human serum was added to each plate as an assay positive control. All results are validated using a four-parameter logistic curve fitting method. Use a fluorescent microscope to determine the potency (EC50). As an additional test, check the wells under a fluorescent microscope. .

[0412] (Plaque reduction assay) Briefly, a plaque reduction (neutralization) assay for hepatitis B virus was performed using green fluorescent This was performed using a photoprotein-tagged HBV isolate and 5% rabbit serum as exogenous Plaques were enumerated by fluorescence microscopy using the antibody as a source of complement. The maximum dilution of serum that results in a 50% reduction in plaques compared to the dilution in the (pre-immune) serum sample. It is defined as interpretation.

[0413] (Neutralization assay in guinea pig lung fibroblast (GPL) cells) Briefly, serial dilutions of test and control (pre-vaccination) sera were administered to additional rabbits. The antibodies were prepared in GPL complete medium containing 1% feline serum as a source of exogenous complement. Serum dilutions ranged from 1:5120 to 1:5120. Serum dilutions were incubated with eGFP-tagged virus (100–200 pfu per well). Incubate at 37°C for 30 min, then transfer to a 12-well plate containing confluent GPL cells. After 2 hours of incubation at 37°C, the cells were transferred to a plate. Samples were processed in triplicate. The cells were washed with PBS, refed with GPL complete medium, and incubated at 37°C / 5% CO2 for 5 days. The markers were visualized by fluorescence microscopy, counted, and compared with control wells. The serum dilution that resulted in a 50% reduction in the number of larks was designated the neutralization titer.

[0414] (qPCR) The LCMV RNA genome was isolated using QIAamp Viral RNA PCR according to the protocol provided by the manufacturer. The LCMV RNA genome equivalent is isolated using a SuperScript® mini kit (QIAGEN). ) III Platinum® One-Step qRT-PCR Kit (Invitrogen) and LCMV NP coding region Primers and probes (FAM reporter and NFQ-MGB quencher) specific to the region were used. Quantitative PCR performed on a StepOnePlus real-time PCR system (Applied Biosystems) was used. Detection by R. The reaction temperature profile was: 60°C for 30 minutes, 95°C for 2 minutes, followed by 95°C for 15 seconds. 45 cycles of 30 seconds at 56°C. Spectrophotometrically quantified in vitro transcription factors corresponding to fragments of the LCMV NP coding sequence containing the NP site. Quantitation is performed by comparison with a standard curve constructed from a log10 dilution series of the transcript RNA fragment.

[0415] (Western blotting) Infected cells grown in tissue culture flasks or in suspension were subjected to RIPA at the indicated post-infection times. Lyse with buffer (Thermo Scientific) or use directly without cell lysis. The sample was heated to 99°C for 10 minutes with a reducing agent and NuPage LDS sample buffer (NOVEX). After cooling to room temperature, the proteins are loaded onto a 4-12% SDS-gel for electrophoresis. Blotting onto membrane using Vitrogens iBlot Gel Transfer Apparatus and Ponceau staining Finally, the preparations are incubated with a primary antibody against the protein of interest and alkaline phosphatase. Probe with a phospholipase-conjugated secondary antibody, followed by 1-Step NBT / BCIP solution ( Stain with INVITROGEN.

[0416] MHC-peptide multimer staining assay for detecting antigen-specific CD8+ T cell proliferation Any assay known to one of skill in the art can be used to test for antigen-specific CD8+ T cell responses. For example, an MHC-peptide tetramer staining assay can be used (e.g., , Altman JD et al., Science. 1996; 274:94-96; and Murali-Krishna K. et al., I (See, e.g., Community. 1998; 8:177-187). Briefly, the assay involves the following steps: The tetramer assay is used to detect the presence of antigen-specific T cells. To detect specific peptides, T cells are incubated with the peptide (usually fluorescently labeled). The antigen-specific T cells must recognize both the tetramer of MHC molecules that are specifically designed for them. The tetramers are then detected by flow cytometry via the fluorescent label.

[0417] ELISPOT assay for detection of antigen-specific CD4+ T cell proliferation Any assay known to those skilled in the art can be used to test antigen-specific CD4+ T cell responses. For example, an ELISPOT assay can be used (see, for example, Czerkinsky CC et al. J Immunol Methods. 1983; 65:109-121; and Hutchings PR et al., J Immuno (See Methods. 1989; 120:1-8). Briefly, the assay involves the following steps: Coat the immunospot plate with anti-cytokine antibodies. The cells secrete cytokines, which are then washed away. The plate is then coated with a second biotinylated anti-cytokine antibody. Visualize with the Gin-HRP system.

[0418] Intracellular cytokine assay for detecting the functionality of CD8+ and CD4+ T cell responses Test the functionality of CD8+ and CD4+ T cell responses using any assay known to one of skill in the art. For example, intracellular cytokine assays combined with flow cytometry can be used. (See, for example, Suni MA et al., J Immunol Methods. 1998; 212 :89-98; Nomura LE et al., Cytometry. 2000; 40:60-68; and Ghanekar SA et al. (See Clinical and Diagnostic Laboratory Immunology. 2001; 8:628-63). In summary, this assay involves the following steps: infecting cells with specific peptides or proteins; activation of cytokines, and inhibitors of protein transport (e.g., brefeldin A) After washing, antibodies against other cell markers can be added to the cells. The cells are then fixed and permeabilized. Anti-cytokine antibodies are added, and the cells are then flow- It can be analyzed by cytometry.

[0419] Assay to confirm replication deficiency of viral vectors Any assay known to those skilled in the art that determines the concentration of infectious and replicative viral particles can be used to measure replication-deficient viral particles in a sample. For example, non-complementing cells An FFU assay using (as described in

[0408] ) can be used for this purpose. .

[0420] Additionally, plaque-based assays provide information on plaque-forming units (PFU) in virus samples. This is the standard method used to determine virus concentration. Confluent monolayers of host cells are infected with various dilutions of virus and plated onto a semi-solid medium such as agar. Cover with medium to prevent the virus from spreading uncontrollably. When the virus successfully replicates itself in cells within the cell monolayer, viral plaques are formed. (See, for example, Kaufmann, SH; Kabelitz, D. (2002). Methods in Microbiology Immunology of Infection. Academic Press. ISBN Plaque formation occurs over a period of 3 to 14 days, depending on the virus being analyzed. Plaques can be roughly counted manually and the results used to prepare plates. This was used in conjunction with the dilution factor used to determine the number of plaque formation units per unit volume of sample. Calculate the number of PFU / mL. The PFU / mL result represents the number of infectious, replicative particles in the sample.

[0421] (Measurement of viral load in blood or liver) The volume in the blood or liver can be determined using any assay known to those skilled in the art to determine viral load. The number of HBV particles per 1000 μg / ml can be detected (see, e.g., Mendy et al., 2010, J. Viral Hepat. 17(2): 115-122). Non-limiting examples of such assays include nucleic acid-based These include nucleic acid-based tests, such as PCR, and non-nucleic acid-based tests.

[0422] (liver biopsy) For example, those skilled in the art would be able to perform liver biopsies to screen patients for chronic HBV infection or liver cancer. Any known procedure can be used to determine the extent of liver damage. Typical examples include percutaneous needle biopsy, laparoscopic biopsy, and intravenous biopsy. In an embodiment, a liver biopsy is used to identify ground-glass liver cells when the cells are examined under a light microscope. The observation of ground-glass hepatocytes indicates the presence of HBsAg in the liver cells. It is something that is.

[0423] Assay for viral antigen expression Any assay known to one of skill in the art may be used to measure viral antigen expression. For example, an FFU assay (as described in

[0408] ) can be performed. For detection, monoclonal or polyclonal antibodies against each viral antigen are used. Use a whole body preparation (transgene-specific FFU).

[0424] Additionally, Western blotting (as described in

[0415] ) can be performed. can.

[0425] (microparticle enzyme immunoassay) AXSYM® HbsAg (Abbott) is a sera or antibody prepared from adult, pediatric, and neonatal samples, including those from pregnant women. This is a microparticle enzyme immunoassay (MEIA) for the detection of HBsAg in plasma. It can be used as a diagnostic aid for acute or chronic HBV. It can also be used to check the presence of

[0426] To perform the assay, a sample of the patient's blood is subjected to a test for detection antibodies and a test for HBV (e.g., The blood sample is placed in a reaction well containing microparticles coated with antibodies (anti-HBV antigen). If HBV proteins (e.g., HBsAg) are included, they will bind to the microparticles in the reaction wells. This reaction is detected by another substance that emits light, and then this light is measured to If the initial test is positive, the patient's blood is then The fluid is retested to confirm the presence of HBV (e.g., HBV antigen). Enzyme immunoassays can be used to measure the presence of HBsAg or other HBV antigens.

[0427] (Other HBV assays) A sample of the patient's blood is contacted with either HBV antibodies or HBV antigens. Antibodies against HBsAg and HBeAg, antibodies against HBsAg, IgM antibodies against HBeAg and HBcAg These include antibodies against the HBV antibody, and HBcAg. If a patient is infected with HBV, The antigens and / or antibodies that react with the test will cause a chemical reaction when the test is being performed. This assay identifies which HBV antigens and / or antibodies are present in the patient's blood. This allows for the detection of the stage of HBV.

[0428] Assessing levels of HBV, HBV antigen, or HBV antibody using any assay known to those of skill in the art For non-limiting examples of such assays, see, for example, Mayer et al. Reference, 2012, BMC Clin. Pathol. 12:8; Reference, Van Helden et al., 2004, Clin. Lab. 50(1-2) :63-73, and Villar et al., 2011, J. Med. Virol. 83(9):1522-1529.

[0429] (Animal model) A vaccine comprising an infectious arenavirus expressing an HBV antigen as described herein or The safety, tolerability, and immunogenic effects of the compositions can be tested in animal models. In certain embodiments, the safety, tolerability, and safety of the vaccines and compositions thereof used herein are evaluated. Animal models that can be used to test immunogenic effects include mice, These include guinea pigs, rats, monkeys, and chimpanzees. The safety, tolerability, and immunogenic efficacy of the vaccines and compositions thereof used herein are Animal models that can be used for testing include mice.

[0430] In a specific example, a transgenic mouse model is used to develop immunotherapy or vaccines. To evaluate the antiviral potential of pharmacological agents such as steroids and to assess physiological responses, including immune responses. The process can be evaluated (see, e.g., Guidotti et al., 1995, J. Virol. 69(10): Such transgenic mouse models have been shown to mimic human class I and II HL. A molecule and / or hepatitis B surface antigen (HBsAg) (e.g., See, for example, Bourgine et al., 2012, Virology 430(1):10-9).

[0431] In another specific example, woodchucks (Marmota monax) are Developing and testing therapeutic and prophylactic approaches for chronic hepadnavirus infections, such as chronic hepatitis B. can be used as an animal model for the treatment of rheumatoid arthritis (see, e.g., Kosinska et al., Hepat. Re. (See S. Treat. 2010:817580). The woodchuck model is a promising tool for identifying potential immunotherapies, such as vaccines. The method is applicable to the evaluation of immunogenicity and other immune responses (e.g., Vaccine 27(25-26):327 1-3275).

[0432] (6.10 Arrays) The sequences in Table 3 are examples of sequences that can be used with the methods and compositions described herein. In some cases, DNA sequences are used to identify the amino acid and nucleotide sequences of the target gene. The RNA sequences of viral genome segments are listed. The RNA sequences can be easily deduced from the DNA sequences. It is possible. Table 3. Exemplary amino acid sequences [Table 6] TIFF2026036693000010.tif215170TIFF2026036693000011.tif216170TIFF2026036693000012.tif213170TIFF2026036693000013.tif219170TIFF2026036693000014.tif219170TIFF2026036693000015.tif219170TIFF2026036693000016.tif216170TIFF2026036693000017.tif219170TIFF2026036693000018.tif219170TIFF2026036693000019.tif220170TIFF2026036693000020.tif214170TIFF2026036693000021.tif219170TIFF2026036693000022.tif219170TIFF2026036693000023.tif220170TIFF2026036693000024.tif219170TIFF2026036693000025.tif219170TIFF2026036693000026.tif214170TIFF2026036693000027.tif219170TIFF2026036693000028.tif217170TIFF2026036693000029.tif216170TIFF2026036693000030.tif216170TIFF2026036693000031.tif216170TIFF2026036693000032.tif218170TIFF2026036693000033.tif216170TIFF2026036693000034.tif216170TIFF2026036693000035.tif216170TIFF2026036693000036.tif216170TIFF2026036693000037.tif216170TIFF2026036693000038.tif219170TIFF2026036693000039.tif216170TIFF2026036693000040.tif216170TIFF2026036693000041.tif219170TIFF2026036693000042.tif217170TIFF2026036693000043.tif218170TIFF2026036693000044.tif218170TIFF2026036693000045.tif216170TIFF2026 036693000046.tif216170TIFF2026036693000047.tif216170TIFF2026036693000048.tif216170TIFF2026036693000049.tif2 17170TIFF2026036693000050.tif216170TIFF2026036693000051.tif216170TIFF2026036693000052.tif217170TIFF20260366 93000053.tif219170TIFF2026036693000054.tif217170TIFF2026036693000055.tif214170TIFF2026036693000056.tif51170. [Example]

[0433] 7. Working Example 7.1 Arenavirus Vector Genome Design / Vector Construction Established techniques (U.S. Patent Application Publication No. US 2010 / 0297172 A1; and Flatz L. et al., Nat Med. 2010 March; 16(3): 339-345), each HBV antigen or its specific Designing LCMV- and Junin virus (JUNV)-based vaccine vectors expressing the domains (Figure 1).

[0434] (7.2 Vaccine against Hepatitis B Virus) Candidate vaccines against hepatitis B virus (HBV) include rLCMV systems expressing pre-S2 / S and rJUN. Junin virus strain Candid#1 vectors (rLCMV / pre-S2 / S, rJUNV / pre-S2 / S), expressing HBc The vectors (rLCMV / HBc, rJUNV / HBc) that produce HBs and HBc ORF-containing fusion proteins The vector expressing HBsHBc (rLCMV / HBs), and the vector expressing HBe (rLCMV / HBe, rJUNV / HBe). The vector contains a replication-deficient construct (rLCMV, also known as r2LCMV, rJUNV, and Also called r2JUNV) and replication-competent tripartite constructs (r3LCMV, r3JUNV; e.g., Emon et al., 2009, PNAS, 106(9):3473-3478), where the transgene is So-called "artificial" arrangement (r3LCMV art , r3JUNV art ). Mouse (e.g., C 57BL / 6 mice) in a homologous or heterologous primary-boost vaccination The animals are immunized with one or a combination of the constructs. Administration can be intraperitoneal, intramuscular, or intravenous. It is administered by intravenous route. The dose is 10 4 ~10 7 Within the scope of the focus forming unit (FFU) HBV-specific CD8+ T cells were measured in the blood and / or spleen at time points ranging from 7 to 100 days after immunization. For example, to determine the magnitude of CD8+ T cell responses to HBV-derived epitopes, Measure T cells using HC class I tetramers in combination with anti-CD8 antibodies can be done.

[0435] In a complementary approach, synthetic peptides are used in intracellular cytokine assays to Directly and selectively stimulates ex vivo blood and / or spleen-derived CD8+ T cells. The cytokine assays include interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and / or It measures the frequency of interleukin (IL)-2-producing CD8+ T cells. Surface expression of CD107a serves as a marker of cytolytic degranulation in flow cytometry (FACS). HBs-derived epitope [ka] , HBs-derived epitopes [ka] , and HBc-derived epitopes [ka] The peptide specificity is analyzed, including:

[0436] 7.3 Immunogenicity of replication-deficient arenavirus-based vectors expressing HBV antigens C57BL / 6 mice (5 mice per group) were administered 10 5 FFU of rLCMV / HBs-HBc (Group 1), rLCMV / HBc (Group 3), rLCMV / pre-S2 (Group 4), or 10 4 FFU of rLCMV / HBs-HBc (second Control mice were left untreated. Ten days after immunization, MHC class II antibodies were detected. CD8+ T cells were measured in blood using HBs-derived epitopes. [ka] H-2K complexed with b Dextramers and HBc-derived epitopes [ka] H-2K complexed with b Dextramer in combination with anti-CD8α antibody was used to treat hepatitis B. Virus-specific CD8+ T cells were identified. The enumerated cells were compared with the total CD8 + B220 - T cells Expressed as a percentage of the pool.

[0437] The results shown in Figure 3 show that the vaccines with rLCMV / HBs-HBc, rLCMV / HBc, and rLCMV / pre-S2 Vaccination resulted in substantial antigen-specific CD8+ responses to the antigens expressed by each vector. These results suggest that vaccination with rLCMV / HBs-HBc induces T cell responses. The induced anti-HBs and anti-HBc CD8+ T cell responses showed a clear dose-dependence. The higher frequency of anti-HBc CD8+ T cells with rLCMV / HBs-HBc immunization compared with HBs-HBc immunization was associated with a higher incidence of HBs-HBc CD8+ T cells. These results indicate that fusion of the two results in enhanced immunogenicity of HBc.

[0438] The frequency of anti-HBs CD8+ T cells was higher after immunization with rLCMV / pre-S2 than after immunization with rLCMV / HBs-HBc. The level was slightly higher after the first 24 h, suggesting that the anti-HBc CD8+ T cell response may have competed with the anti-HBs response for antigen utilization. It's happening.

[0439] 7.4 Immunogenicity of Attenuated Replication-Competent Arenavirus-Based Vectors Expressing HBV Antigens C57BL / 6 mice (5 mice per group) were administered 10 5 FFU r3LCMV / HBs-HB c (group 1), r3LCMV / HBc (group 2), r3LCMV / pre-S2 (group 3), or 10 5 FFU of rLCMV / HBs-HBc( Control mice were left unvaccinated. Eight days after the first injection, HBs and HBc epitope-specific CD8+ T cells were identified using MHC class I multimers. HBs-derived epitopes were measured in blood. [ka] H-2K complexed with b Dextramers and HBc-derived epitopes [ka] H-2K complexed with b Dextramer in combination with anti-CD8α antibody was used to treat hepatitis B. Virus-specific CD8+ T cells were identified. The enumerated cells were compared with the total CD8 + B220 - T cells as a percentage of the pool (Figure 4A) or as a percentage of circulating lymphocytes in the blood ( Figure 4B).

[0440] The results shown in Figure 4 demonstrate that all r3LCMV-based constructs and the replication-deficient rLCMV / H Bs-HBc reference vectors are immunogenic and, respectively, specific for their vectored antigens. Furthermore, the results show that epitope-specific CD8+ T cells were generated. When target CD8+ T cells were enumerated as a percentage of circulating lymphocytes, replicating r3LCMV / HBs -HBc is shown to be more immunogenic than its replication-deficient counterpart, rLCMV / HBs-HBc.

[0441] (Equivalents and Incorporation by Reference): The embodiments described herein are intended to be merely exemplary and will be readily apparent to those skilled in the art. Those skilled in the art will recognize, or be able to use routine experimentation to identify, many equivalents to the specific procedures described herein. All such equivalents are within the scope of the present invention. It is believed to be within the scope of the invention and is encompassed by the following embodiments. All references (including patent applications, patents, and publications) are hereby incorporated by reference as if they were each individual publication or The patent or patent application is specifically and individually incorporated by reference in its entirety for all purposes. incorporated herein by reference to the same extent as if the reference were made in whole and for all purposes. No. 6,299,499, filed on Oct. 1, 2003, and incorporated herein by reference. The present application provides the following inventions. (Configuration 1) the arenavirus open reading frame is removed, and a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof an infectious arenavirus having a nucleotide sequence selected from the group consisting of: Rus virus vector. (Configuration 2) The pre-S2 / S protein or an antigenic fragment thereof is identified by the nucleotide sequence of SEQ ID NO: 1. The amino acid sequence encoded by the , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2. The viral vector of claim 1, comprising an amino acid sequence identical to that of the viral vector of claim 1. (Configuration 3) The HBc protein or antigenic fragment thereof is encoded by the nucleotide sequence of SEQ ID NO:2. The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical 2. The viral vector of claim 1, comprising an amino acid sequence (Configuration 4) The fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof is represented by SEQ ID NO: The amino acid sequence encoded by the nucleotide sequence of No. 3 and 80%, 81%, 82%, 83%, and 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 100%, 98%, 99%, or 100% identical amino acid sequence to the viral vector of claim 1. -. (Configuration 5) The HBe protein or antigenic fragment thereof is encoded by the nucleotide sequence of SEQ ID NO:26. The amino acid sequence and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical 2. The viral vector of claim 1, comprising an amino acid sequence. (Configuration 6) a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof 2. The viral vector of claim 1, comprising at least two of: (Configuration 7) a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof 2. The viral vector of claim 1, comprising at least three of the following: (Configuration 8) Expression of the nucleotide sequence results in higher titers than expression of the protein complex components individually. The viral vector according to claim 6 or 7, which produces an antigenic protein complex that induces neutralizing antibodies. Tar. (Configuration 9) Any one of aspects 1 to 8, wherein the arenavirus is lymphocytic choriomeningitis virus. The viral vector described. (Configuration 10) The open reading frame encoding the glycoprotein of said arenavirus is deleted The viral vector according to any one of configurations 1 to 9, which is functionally inactivated or inactivated. Tar. (Configuration 11) The genomic information encoding the infectious arenavirus viral vector is expressed in the lymphocytic context. The viral vector according to any one of configurations 1 to 10, which is derived from meningitis virus clone 13 strain. Tar. (Configuration 12) The genomic information encoding the infectious arenavirus viral vector is expressed in the lymphocytic context. 12. The viral vector according to any one of configurations 1 to 11, which is derived from the MP strain of meningitis virus. (Configuration 13) The viral vector comprises a genome segment, wherein the genome segment is At least 90% identical to nucleotides 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical 13. The viral vector according to any one of configurations 1 to 12, comprising a nucleotide sequence which is (Configuration 14) The viral vector has an amino acid sequence of 1639 to 3315 of SEQ ID NO: 11 or The amino acid sequence encoded by 1640 to 3316 of 12 is at least 90%, 91%, 92%, or 93% , 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical expression products. 14. The method of claim 1, further comprising: The viral vectors described above. (Configuration 15) 15. The virus according to any one of aspects 1 to 14, wherein the arenavirus is a Junin virus. Specter. (Configuration 16) The genome information encoding the infectious arenavirus vector is 16. The viral vector of construct 15, derived from the Candid #1 strain. (Configuration 17) Aspects 1 to 16, wherein the proliferation or infectivity of the arenavirus is not affected by heterologous nucleic acid. The viral vector according to any one of the preceding claims. (Configuration 18) A method for producing a pharmaceutical composition comprising the viral vector according to any one of aspects 1 to 17 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: (Configuration 19) A method for producing a pharmaceutical composition comprising the viral vector according to any one of aspects 1 to 17 and a pharmaceutically acceptable carrier. An immunogenic composition. (Configuration 20) A method for producing a pharmaceutical composition comprising the viral vector according to any one of aspects 1 to 17 and a pharmaceutically acceptable carrier. Vaccine. (Configuration 21) A method of treating or preventing hepatitis B virus infection in a patient, comprising administering to the patient a composition comprising: the viral vector according to any one of claims 1 to 17, the pharmaceutical composition according to claim 18, and the immunoglobulin according to claim 19. 21. The method comprising administering an immunogenic composition or a vaccine according to claim 20. (Configuration 22) Any one of claims 1 to 17 for the treatment or prevention of hepatitis B virus infection in a patient. 19. A viral vector according to claim 18, a pharmaceutical composition according to claim 19, an immunogenic composition according to claim 20, or 21. Use of the vaccine according to claim 20. (Configuration 23) The viral vector according to any one of configurations 1 to 17, the pharmaceutical composition according to configuration 18, and configuration 19. 22. The immunogenic composition of claim 20, or the vaccine of claim 21, wherein the immunogenic composition of claim 20 is suitable for intramuscular injection. Use of the above. (Configuration 24) The viral vector according to any one of configurations 1 to 17, the pharmaceutical composition according to configuration 18, and configuration 19. 22. The immunogenic composition according to claim 20, or the vaccine according to claim 21, wherein the immunogenic composition according to claim 20 is suitable for intravenous injection. Use of the above. (Configuration 25) 1. An isolated nucleic acid, the nucleic acid comprising an arenavirus genome segment, wherein: One open reading frame of the genome segment is deleted or functional and the genome segment is inactivated by: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof The isolated nucleic acid comprising one or more of: (Configuration 26) The genome segment is a short segment, in which an open loop encoding a GP is 26. The isolated nucleic acid of claim 25, wherein the nucleic acid sequence is deleted. (Configuration 27) 1. A method of producing an infectious replication-deficient arenavirus viral vector, comprising: a. transfecting a host cell with the nucleic acid of embodiment 25 or 26; b. maintaining the host cells under conditions favorable for virus formation; and c. recovering the infectious replication-deficient arenavirus viral vector; Including, wherein the host cell is a cell in which the genome segment is deleted or functionally inactivated. The method further comprises expressing an open reading frame comprising: (Configuration 28) The arenavirus open reading frame is a glycoprotein (GP) open reading frame. The arenavirus virus vector of embodiment 1, which is a flanking frame. (Configuration 29) It is modified to contain a genome capable of amplifying and expressing its genetic information in infected cells. The modified virus produces additional infectious progeny particles in normal, unaltered cells. an infectious replication-deficient arenavirus vector that is unable to transmit a single antigen; The HBV open reading frame is removed and the HBV antigen or its antigenic fragment is a nucleotide sequence encoding a fragment thereof, wherein the allenavir nucleotide sequence is replaced with a nucleotide sequence encoding a fragment thereof, wherein the allenavir nucleotide sequence is administered to the subject. administration of the HBV vector results in long-lasting immunity to the HBV antigen or an antigenic fragment thereof. The infectious, replication-deficient arenavirus vector induces a response. (Configuration 30) The long-lasting immune response produces detectable antibody titers against HBV antigens or antigenic fragments thereof. 29. The arenavirus vector of construct 29, (Configuration 31) The long-lasting immune response comprises detectable antibodies against the HBV antigen or antigenic fragment thereof. 30. The arenavirus vector of claim 29, wherein titers are induced for at least a minimum of 4 weeks. . (Configuration 32) The long-lasting immune response increases antibody titers against the HBV antigen or antigenic fragment thereof by at least at least 100%, at least 200%, at least 300%, at least 400%, at least 500 %, or at least 1000% increase in the arenavirus viral vector of configuration 30 or 31. Tar. (Configuration 33) It is modified to contain a genome capable of amplifying and expressing its genetic information in infected cells. The modified virus produces additional infectious progeny particles in normal, unaltered cells. a first infectious replication-deficient arenavirus vector that is unable to Two arenavirus open reading frames were removed, and: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. a nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof; wherein the first nucleotide sequence is replaced with a first nucleotide sequence selected from the group consisting of: Infectious replication-deficient arenavirus vectors and their ability to amplify their genetic information in infected cells , modified to contain a genome capable of expressing it, but not genetically modified A second infectious complex that cannot produce further infectious progeny particles in normal cells that do not contain the virus. A defective arenavirus viral vector comprising one arenavirus open read sequence. The ping frame is removed, and: a. a nucleotide sequence encoding the HBV pre-S2 / S protein or an antigenic fragment thereof; b. a nucleotide sequence encoding the HBV HBc protein or an antigenic fragment thereof; c. a nucleotide sequence encoding the HBV HBs protein or an antigenic fragment thereof; d. Encoding a fusion of HBV HBs protein and HBV HBc protein or antigenic fragments thereof a nucleotide sequence corresponding to the e. Nucleotide sequence encoding HBV HBe protein or an antigenic fragment thereof wherein the second nucleic acid sequence is replaced with a second nucleotide sequence selected from the group consisting of: A pharmaceutical composition comprising a virulent replication-deficient arenavirus vector. (Configuration 34) 34. The pharmaceutical composition of claim 33, wherein the first nucleic acid sequence and the second nucleic acid sequence are different. (Configuration 35) the first nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof. 35. The method of claim 33 or 34, wherein the nucleic acid sequence encodes the HBV HBc protein or a fragment thereof. Pharmaceutical composition. (Configuration 36) the first nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof. The nucleic acid sequence of the present invention is a fusion of the HBV HBs protein and the HBV HBc protein or fragments thereof. 35. The pharmaceutical composition according to claim 33 or 34, encoding (Configuration 37) The first nucleic acid sequence encodes the HBV HBc protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV HBc protein or a fragment thereof. The nucleic acid sequence encodes a fusion of the HBV HBs protein and the HBV HBc protein or fragments thereof. 35. The pharmaceutical composition according to claim 33 or 34, (Configuration 38) the first nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof. 35. The pharmaceutical composition of claim 33 or 34, wherein the nucleic acid sequence encodes the HBV HBe protein or a fragment thereof. Pharmaceutical composition. (Configuration 39) The first nucleic acid sequence encodes the HBV HBe protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV HBe protein or a fragment thereof. The nucleic acid sequence encodes a fusion of the HBV HBs protein and the HBV HBc protein or fragments thereof. 35. The pharmaceutical composition according to claim 33 or 34, (Configuration 40) the first nucleic acid sequence encodes the HBV HBs protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV HBs protein or a fragment thereof. 35. The pharmaceutical composition according to claim 33 or 34, wherein the nucleic acid sequence encodes the HBV HBe protein or a fragment thereof. Finished product. (Configuration 41) the first nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof, and the second nucleic acid sequence encodes the HBV pre-S2 / S protein or a fragment thereof. 35. The method of ...

Claims

1. An infectious, replication-competent tripartite arenavirus containing one L segment and two S segments A viral vector, wherein the two S segments are a first S segment and a second S segment. segment, a. the first S segment is an open S segment encoding a first HBV antigen or an antigenic fragment thereof The open reading frame (ORF) is under the control of the arenavirus 5'UTR and The S segment contains an ORF encoding the arenavirus glycoprotein (GP) under the control of the arenavirus 3'UTR. It is a b. The second S segment comprises an ORF encoding a second HBV antigen or an antigenic fragment thereof. It is under the control of the arenavirus 5'UTR and encodes the arenavirus nucleoprotein (NP). the ORF is an S segment under the control of the arenavirus 3'UTR; and c. the L segment encodes arenavirus matrix protein Z (Z protein). The ORF encoding the arenavirus 5'UTR is under the control of the arenavirus RNA-dependent RNA. The ORF encoding polymerase L (L protein) is under the control of the arenavirus 3'UTR, L segment, and the arenavirus is lymphocytic choriomeningitis virus (LCMV), The arenavirus vector.

2. a. The first HBV antigen is HBV pre-S2 / S protein or an antigenic fragment thereof, HBV HBc protein HBV HBs protein or an antigenic fragment thereof, the HBV HBs protein and HBV HBc protein or a fusion protein of their antigenic fragments, H BV HBe protein or an antigenic fragment thereof, or HBV polymerase protein or an antigenic fragment thereof, and b. The second HBV antigen is HBV pre-S2 / S protein or an antigenic fragment thereof, HBV HBc protein, or HBV HBs protein or an antigenic fragment thereof, the HBV HBs protein and HBV HBc protein or a fusion protein of their antigenic fragments, H BV HBe protein or an antigenic fragment thereof, or HBV polymerase protein or 2. The arenavirus virus vector of claim 1, which is an antigenic fragment thereof.

3. 3. The method according to claim 1, wherein the first HBV antigen and the second HBV antigen are the same HBV antigen. Arenavirus viral vectors.

4. The pre-S2 / S protein or an antigenic fragment thereof is identified by the nucleotide sequence of SEQ ID NO:

1. The amino acid sequence encoded by the 99% identical or 100% identical amino acid sequence according to any one of claims 1 to 3 The viral vector described.

5. The HBc protein or antigenic fragment thereof is encoded by the nucleotide sequence of SEQ ID NO:

2. The amino acid sequence to be encoded is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

4. The method according to claim 1, wherein the amino acid sequence of the nucleotide ... Viral vector.

6. The fusion of the HBV HBs protein and the HBV HBc protein or antigenic fragments thereof is The amino acid sequence encoded by the nucleotide sequence of sequence number 3 is at least 90%, 91%, or 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or 100% identical amino acid sequences The viral vector of any one of claims 1 to 3, comprising:

7. The HBe protein or antigenic fragment thereof is encoded by the nucleotide sequence of SEQ ID NO:

26. The amino acid sequence to be loaded is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1101%, 1112%, 1123%, 113%, 114 4. The method of claim 1, comprising an amino acid sequence that is 9% identical or 100% identical. viral vectors.

8. The first HBV antigen is different from the second HBV antigen, and the first HBV antigen and The expression of two HBV antigens or their fragments is higher than the individual expression of the protein complex components.

8. A method according to claim 1, which produces an antigenic protein complex that induces a high titer of neutralizing antibodies. The viral vector according to item 1.

9. The lymphocytic choriomeningitis virus (LCMV) is lymphocytic choriomeningitis virus clone 13 The virus according to any one of claims 1 to 8, which is a lymphocytic choriomeningitis virus strain or a lymphocytic choriomeningitis virus MP strain. Russ Vector.

10. The viral vector comprises a genome segment, the genome segment comprising: a. a sequence identical to at least nucleotides 1639 to 3315 of SEQ ID NO:11 or 1640 to 3316 of SEQ ID NO:12 a nucleotide sequence that is at least 90% identical to b. an expression product whose amino acid sequence is nucleotides 1639 to 3315 of SEQ ID NO: 11; or At least 90% identical to the amino acid sequence encoded by 1640 to 3316 of SEQ ID NO: 12 , a nucleotide sequence encoding said expression product The viral vector of any one of claims 1 to 9, comprising:

11. administering the arenavirus viral vector to a subject comprises administering the first HBV antigen and / or or a second HBV antigen or antigenic fragment thereof, Item 11. The viral vector according to any one of Items 1 to 10.

12. (i) the long-lasting immune response is directed against the first HBV antigen and / or the second HBV antigen, or induces detectable antibody titers against the antigenic fragment; or (ii) the long-lasting immune response is sustained for at least a minimum of four weeks by the first HBV antigen and / or HBV antigen or a second HBV antigen or an antigenic fragment thereof.

1. A viral vector according to claim 1.

13. the long-lasting immune response is directed against the first HBV antigen and / or the second HBV antigen or antigens thereof; Increase the antibody titer against the antigenic fragment by at least 100%, at least 200%, at least 300%, or 13. The method of claim 11 or 12, wherein the α-amino acid α-glucan is increased by at least 400%, at least 500%, or at least 1000%. viral vectors.

14. A pharmaceutical composition, an immunogenic composition, or a combination thereof, comprising the viral vector according to any one of claims 1 to 13. composition, or vaccine.

15. A method according to any one of claims 1 to 13 for the treatment or prevention of hepatitis B virus infection in a patient. a viral vector according to claim 14, or a pharmaceutical composition or immunogenic composition according to claim 15, is a vaccine.

16. A viral vector according to any one of claims 1 to 13 for intramuscular or intravenous injection.

15. A pharmaceutical composition, immunogenic composition or vaccine according to claim 14.

17. Any of claims 1 to 13 for the treatment of cancer in a patient, administered by intravenous injection. The viral vector according to any one of claims 1 to 14, or the pharmaceutical composition or immunogenic composition according to claim 14. Or a vaccine.

18. A virus according to any one of claims 1 to 13 for the treatment of cancer in a patient, the cancer being liver cancer. A vector or a pharmaceutical composition or immunogenic composition or vaccine according to claim 14.

19. (a) a first nucleic acid comprising the cDNA of the first S segment of claim 1; (b) a second nucleic acid comprising the cDNA of the second S segment of claim 1; and (c) a third nucleic acid comprising the cDNA of the L segment of claim 1; A set of nucleic acids comprising:

20. The nucleic acid of claim 19 for producing an infectious, replication-competent arenavirus viral vector. Use of a set of a. transfecting a host cell with the set of nucleic acids of claim 19; b. Inducing in said host cell the expression of the minimal arenavirus transactivator proteins NP and L. transfecting a plasmid c. maintaining the host cells under conditions favorable for virus formation; and d. Recovering the infectious, replication-competent arenavirus particles. The use.

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