Vaccine against hepatitis b virus

Abstract

The present application provides immunotherapy for hepatitis B virus infection. Provided herein are genetically modified arenavirus vectors suitable as vaccines for the prevention and treatment of hepatitis B virus infection. Also provided herein are pharmaceutical compositions and methods for the treatment of hepatitis B virus infection. In particular, provided herein are pharmaceutical compositions, vaccines, and methods of treating hepatitis B virus infection.

Description

[0001] Related applications

[0002] This application is a divisional application of Chinese invention patent application filed on November 3, 2016, entitled "Vaccine against Hepatitis B Virus" with application number 201680077751.0.

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 250,639, filed November 4, 2015, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Reference to the electronically submitted sequence list

[0005] This application incorporates by reference the sequence list created on November 2, 2016, which is 128,899 bytes in size and named "Sequence_Listing_13194-014-228.TXT" and was submitted with this application.

[0006] 1. Introduction

[0007] This document provides genetically modified arenaviruses suitable as vaccines for the prevention and treatment of hepatitis B virus infection. It also provides pharmaceutical compositions and methods for treating hepatitis B virus infection. Specifically, this document provides pharmaceutical compositions, vaccines, and methods for treating hepatitis B virus infection. Therefore, this application provides immunotherapy for hepatitis B virus infection.

[0008] 2. Background

[0009] 2.1 Pathogens and Diseases

[0010] Hepatitis B virus (HBV) is a double-stranded enveloped virus belonging to the Hepatoviridae family. The viral particle consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of proteins. The nucleocapsid surrounds the viral DNA and a DNA polymerase with reverse transcriptase activity. The outer envelope contains embedded proteins involved in viral binding and entry into susceptible cells. HBV replicates in hepatocytes of humans and other higher primates, but does not grow in artificial cell cultures.

[0011] The outcomes of HBV infection are age-dependent, including asymptomatic infection, acute hepatitis B, chronic HBV infection, cirrhosis, and hepatocellular carcinoma (HCC). Acute hepatitis B occurs in approximately 1% of perinatal infections, 10% of early childhood infections (children aged 1–5 years), and 30% of late infections (advanced age >5 years). Fulminant hepatitis occurs in 0.1–0.6% of acute hepatitis cases; the mortality rate of fulminant hepatitis B is approximately 70%. The development of chronic HBV infection is inversely related to the age of acquisition, occurring in approximately 80%–90% of perinatal infections, approximately 30% of infections in children under 6 years of age, and 5% of infections in other healthy adults (Hyams et al., 1995, Clinical Infections Diseases 20:992–1000). Complications, including concurrent HIV infection and alcohol or toxin intake, or both, may play an important role in the development of hepatitis B-related pathogenesis. It is estimated that 10% of the 40 million people living with HIV worldwide are also co-infected with HBV.

[0012] People with chronic HBV infection have a 15-25% risk of premature death from HBV-related cirrhosis and HCC (Beasley and Hwang, 1991, Proceedings of the 1990 International Symposium on Viral Hepatitis and Liver Disease: Contemporary Issues and Future Prospects 532-535). Acute HBV infection is characterized by the presence of the HBV surface antigen HBsAg and immunoglobulin M (IgM) against the core antigen HBcAg. During the initial high-replication phase of infection, patients are also seropositive for HBeAg, the extracellular and secreted form of HBcAg found in the patient's serum, which serves as a marker of active replication in chronic hepatitis. Antibodies against HBsAg (anti-HBs) become identifiable after several weeks, followed by HBsAg clearance. Chronic infection is characterized by the persistence of HBsAg (>6 months) (with or without concurrent HBeAg). The presence of HBsAg is a major biomarker for the development of chronic liver disease and the risk of late-stage hepatocellular carcinoma (HCC). The presence of HBeAg indicates that the blood and bodily fluids of the infected individual are highly infectious.

[0013] 2.2 Epidemiology and Public Health

[0014] Hepatitis B virus (HBV)-related diseases have a worldwide distribution. It is estimated that two billion people have been infected with HBV at some point in their lives. Of these, approximately 360 million are chronically infected and at risk of serious illness and death, primarily from cirrhosis and hepatocellular carcinoma (HCC). Mathematical modeling in 2000 estimated that approximately 600,000 people worldwide die annually from HBV-related diseases (Goldstein et al., 2005, International J. Epidemiology 34:1329-1339). Humans are the sole reservoir of HBV. The virus is transmitted through percutaneous and penetrating skin exposure to infected blood and other bodily fluids, primarily semen and vaginal fluid. The incubation period averages 75 days but can vary between approximately 30 and 180 days. HBV surface antigen (HBsAg) can be detected in serum 30–60 days after infection and may persist for a wide range of variable durations. The prevalence of hepatitis B is described by the prevalence of HBsAg in the general population of a defined geographic area, and varies significantly globally: an HBsAg prevalence of >8% is typical of highly prevalent areas, an incidence of 2-7% is of moderate prevalence, and in low-prevalence areas, <2% of the population is HBsAg positive.

[0015] In highly prevalent areas, the most common transmission of HBV is from mother to child at birth or from person to person in early childhood (Goldstein et al., 2005, International J. Epidemiology 34:1329-1339; Wong et al., 1984, Lancet 1:921-926; de la Hoz et al., 2008 International J. Infectious Diseases 12:183-189). Perinatal or early childhood transmission may also account for more than one-third of chronic infections in areas with low prevalence (Margolis et al., 1995, JAMA 274:1201-1208), although in these cases, sexual transmission and use of contaminated needles, particularly among injecting drug users, are the primary routes of infection (Goldstein et al., 2002, J. Infectious Diseases 185:713-719).

[0016] 2.3 Current Treatment

[0017] Universal hepatitis B vaccination has been shown to significantly reduce HBV infection and HCC rates. However, once chronic HBV infection is established, treatment remains a significant challenge because conventional therapies often fail to provide sustained control of viral replication and liver damage in most patients.

[0018] Currently approved antiviral treatments for chronic hepatitis B include pegylated (PEG) recombinant interferon-alpha and viral DNA polymerase inhibitors. These agents reduce viral replication and have been shown to delay the progression of cirrhosis, reduce the incidence of hepatocellular carcinoma (HCC), and improve long-term survival. However, treatment is complicated by the toxicity of these agents, and it only cures a small percentage of individuals with chronic infection. While viral levels in the blood are reduced to almost undetectable levels in individuals receiving standard treatment, the reduction in viral DNA in the liver is only minor. As a result, viremia rebound often occurs after treatment cessation, and people with chronic HBV infection must maintain lifelong treatment. However, even after ten years of antiviral therapy, the drugs only reduce liver failure by 40–70%, and mortality due to cirrhosis and liver cancer remains high.

[0019] 2.4 Hepatitis B and the Immune System

[0020] Chronic hepatitis B infection is characterized by dysfunction of both innate and adaptive antiviral immunity (Bertoletti & Ferrari, 2012, Gut 61:1754-1764). In contrast, HBV-specific immunity is robust and multifunctional in patients with resolved HBV infection. Several mechanisms may contribute to dysfunction of HBV-specific T-cell immunity in patients with chronic hepatitis B, including high levels of viral antigenemia and the liver's tolerance microenvironment (Jenne & Kubes, 2013, Nat. Immunol. 14:996-1006). Earlier studies have shown that suppressing viral replication can temporarily and partially restore antiviral T-cell immunity, supporting the hypothesis that prolonged exposure to high levels of antigenemia may lead to dysfunction of antiviral T cells (Boni et al., 2003, J. Hepatol. 39:595-605).

[0021] Therapeutic vaccines that can reverse the dysfunctional immune status of chronic hepatitis B and restore antiviral immunity theoretically have the potential to eliminate viremia and reduce the intrahepatic HBV DNA level to zero, thus showing great promise for curing HBV.

[0022] Recently, HBV vaccines have been considered a promising therapeutic strategy for treating and controlling HBV infection in patients with HBV carriers and persistent infection (Michel & Tiollais, 2010, Pathol. Biol. (Paris) 58:288-295; Liu et al, 2014, Virol. Sin. 29:10-16). In approximately 50% of patients with chronic active HBV, specific treatment with routine anti-HBV vaccination has effectively reduced HBV replication and suppressed immune tolerance to HBsAg protein (Couillin et al, 1999, J. Infect. Dis. 180:15-26). However, to date, monotherapy based on HBsAg vaccines has failed to produce sustained control of HBV replication and / or liver damage (Akbar et al, 2013, Hepatobiliary Pancreat. Dis. Int. 12:363-369), and new treatment strategies are needed to provide a strong and durable antiviral immune response and long-term control of HBV replication.

[0023] The failure of earlier therapeutic vaccine regimens highlights the challenges and limitations in our current understanding of the immune response in chronic HBV infection (Michel et al, 2011, J. Hepatol. 54:1286-1296). High viral load conditions, such as the combination of chronic hepatitis B and a tolerant liver microenvironment, may make it difficult to achieve complete recovery of antiviral T-cell immunity.

[0024] Current intensive research focuses on better understanding the immune response in hepatocytes, the mechanisms by which HBV evades innate immunity, and the proper selection of sensitive patients who can benefit from immunotherapy, which could improve the efficacy of therapeutic vaccination (Michel et al., 2015, Med. Microbiol. Immunol. 204:121-129). 3. Overview of the Invention

[0026] This application provides immunotherapy for hepatitis B virus infection. This document provides a viral vector for an infectious isonavirus containing nucleotide sequences selected from the following:

[0027] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0028] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0029] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0030] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0031] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0032] In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)). In some embodiments, the viral vector of the infectious, replication-defective arenavirus is bifragmented. In some embodiments, the viral vector of the infectious, replication-defective arenavirus is trifragmented. In some embodiments, the viral vector of the infectious, replication-competent arenavirus is trifragmented.

[0033] In some embodiments, this document provides a viral vector for a sand-like virus, comprising a nucleotide sequence selected from the following:

[0034] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0035] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0036] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0037] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0038] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0039] In some embodiments, the viral vector of the sand-like virus is replication-defective. In some embodiments, the viral vector of the sand-like virus is replication-competent.

[0040] In some embodiments, the viral vectors provided herein are infectious, i.e., capable of entering host cells or injecting their genetic material into host cells. In some more specific embodiments, the viral vectors provided herein are infectious, i.e., capable of entering host cells or injecting their genetic material into host cells, and subsequently amplifying and expressing their genetic information within the host cells. In some embodiments, the viral vector is an infectious, replication-defective arenavirus viral vector engineered to contain a genome, capable of amplifying and expressing its genetic information in infected cells, but unable to produce further infectious progeny particles in normal, unengineered cells. In some embodiments, the cell lines provided herein support the growth of wild-type viruses but cannot express complementary viral proteins and therefore cannot produce further infectious progeny particles. In some embodiments, the infectious arenavirus viral vector is replication-competent, capable of producing further infectious progeny particles in normal, unengineered cells.

[0041] In some embodiments, the pre-S2 / S protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the fragment is antigenic when it is capable of (i) evoking an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey), wherein the generated antibody specifically binds to the human HBV pre-S2 / S protein; and / or (ii) evoking a specific T-cell immune response.

[0042] In some embodiments, the HBc protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the fragment is antigenic when it is capable of (i) evoking an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey), wherein the generated antibody specifically binds to human HBV HBc protein; and / or (ii) evoking a specific T-cell immune response.

[0043] In some embodiments, the fusion of the HBV HBs and HBc proteins or their antigenic fragments comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the fragment is antigenic when it is capable of (i) evoking an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey), wherein the generated antibodies specifically bind to human HBV HBs, HBc, or both HBs and HBc; and / or (ii) evoking a specific T-cell immune response.

[0044] In some embodiments, the HBe protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26. In some embodiments, the fragment is antigenic when it is capable of (i) evoking an antibody immune response in a host (e.g., mouse, rabbit, goat, or donkey), wherein the generated antibody specifically binds to the human HBV HBe protein; and / or (ii) evoking a specific T-cell immune response.

[0045] In some embodiments, the viral vector comprises at least two of the following:

[0046] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0047] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0048] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0049] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0050] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0051] In some embodiments, the viral vector comprises at least three of the following:

[0052] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0053] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0054] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0055] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0056] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0057] In some embodiments, the open reading frame (ORF) of the arenavirus is deleted or functionally inactivated and replaced with nucleic acid encoding the HBV antigen as described herein. In specific embodiments, the ORF encoding the arenavirus glycoprotein GP is deleted or functionally inactivated. In some embodiments, functional inactivation of the gene eliminates any translation product. In some embodiments, functional inactivation refers to a genetic alteration that allows for some translation, however, the translation product is no longer functional and cannot replace the wild-type protein.

[0058] In some embodiments, the viral vector can amplify and express its genetic information in cells already infected with the viral vector, but the viral vector cannot produce further infectious progeny particles in non-supplementary cells. In some embodiments, the viral vector provided herein is infectious, i.e., capable of entering host cells or injecting its genetic material into host cells. In some more specific embodiments, the viral vector provided herein is infectious, i.e., capable of entering host cells or injecting its genetic material into host cells, and subsequently amplifying and expressing its genetic information within the host cells.

[0059] In some embodiments, the genomic information encoding the infectious arenavirus particle is derived from the lymphocytic choriomeningitis virus (LCMV) clone 13 strain or the LCMV MP strain. The nucleotide sequences of the S and L fragments of clone 13 are listed in SEQ ID NO: 12 and 7, respectively.

[0060] In some embodiments, this document provides a viral vector whose genome is the genome of clone 13 (SEQ ID NOs: 12 and 7), or derived from the genome of clone 13 by deleting an ORF (e.g., the ORF of the GP protein) of the clone 13 genome and replacing it with a heterologous ORF encoding an antigen (e.g., the HBV antigen), such that the remaining LCMV genome is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the nucleotide sequence of clone 13 (SEQ ID NOs: 12 and 7) and is derived from the genome of clone 13 (SEQ ID NOs: 12 and 7).

[0061] In some embodiments, this document provides a viral vector whose genome is derived from the genome of the LCMV strain MP by deleting an ORF (e.g., the ORF of the GP protein) from the LCMV strain MP genome and replacing it with a heterologous ORF encoding an antigen (e.g., HBV antigen), such that the remaining LCMV genome is 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%, or 100% identical to the nucleotide sequence of the LCMV strain MP (SEQ ID NOs: 13 and 14).

[0062] In a more specific embodiment, the viral vector comprises a genomic fragment, wherein the genomic fragment comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotides 1639 to 3315 of SEQ ID NO: 11 or nucleotides 1640 to 3316 of SEQ ID NO: 12. In some embodiments, the viral vector comprises a genomic fragment containing a nucleotide sequence encoding an expression product, wherein at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% of the amino acid sequence encoded by nucleotides 1639 to 3315 of SEQ ID NO: 11 or nucleotides 1640 to 3316 of SEQ ID NO: 12 are identical to the amino acid sequence encoded by nucleotides 1640 to 3316 of SEQ ID NO: 12.

[0063] This document also provides isolated nucleic acids, wherein the nucleic acids are cDNA fragments of a sand-like virus genome, wherein one ORF of the genome fragment is deleted or functionally inactivated and wherein the genome fragment comprises one or any combination of the following:

[0064] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0065] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0066] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0067] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0068] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0069] In some implementations, the genomic fragment is a short fragment in which the ORF encoding GP is deleted.

[0070] In one respect, this article provides a method for generating infectious, replication-defective sand-like virus particles, including:

[0071] a. Transfecting the nucleic acids described in this article into host cells;

[0072] b. Maintain the host cell under conditions suitable for virus formation; and

[0073] c. Harvest the aforementioned infectious, replication-defective sand-like virus particles;

[0074] The host cells express an ORF that has been deleted or functionally inactivated on the genome fragment. In some embodiments, any other nucleic acids required for the rescue of the viral particles are also transfected into the host cells in step a. Such other nucleic acids may be: cDNA of a second isovirus genome fragment, nucleic acids containing LORF, and / or nucleic acids containing NPORF.

[0075] In another aspect, this document provides compositions, such as pharmaceutical compositions, immunogenic compositions, or vaccine compositions, comprising the viral vectors and pharmaceutically acceptable vectors described herein. This document also provides compositions comprising two or more different viral vectors described herein (i.e., wherein said viral vectors encode different HBV antigens) (e.g., vaccine compositions). In some embodiments, the pharmaceutical compositions comprise nucleic acids or fusion proteins described herein.

[0076] In a further aspect, this document provides methods for treating or preventing HBV infection in a patient, including administering the viral vector, pharmaceutical composition, immunogenic composition, or vaccine described herein to the patient. In yet another aspect, this document provides the use of the viral vector, pharmaceutical composition, immunogenic composition, or vaccine described herein for the treatment or prevention of HBV. In some embodiments, an infectious isovirus expressing HBV antigen or fragments thereof is capable of preventing HBV transmission and / or infection from mother to unborn fetus. In some embodiments, one or more infectious isoviruses expressing HBV antigen or fragments thereof are capable of preventing HBV transmission and / or infection from mother to unborn fetus. In some embodiments, the viral vector of the infectious isovirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious isovirus is replication-competent (see section 6.1(b)).

[0077] In some embodiments, administration of an infectious arenavirus expressing HBV antigen or fragments thereof to a patient induces a durable immune response. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)).

[0078] In some embodiments, this document provides a method for treating and / or preventing HBV infection in a patient, comprising administering to the patient two or more arenaviruses expressing HBV antigens or fragments thereof. In more specific embodiments, each arenavirus expresses a different HBV antigen or fragment thereof. In other embodiments, each arenavirus expresses an HBV antigen or a derivative thereof. In some embodiments, the derivative thereof is an HBV antigen fragment. In yet another embodiment, this document provides a composition comprising two or more arenaviruses, each expressing a different HBV antigen or fragment thereof. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)).

[0079] In some embodiments, the sand virus is lymphocytic choriomeningitis virus (LCMV) or Junin virus (JUNV).

[0080] In some embodiments, this document provides a viral vector of infectious isovirus, wherein the isovirus open reading frame is removed and replaced with a nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof. In a specific embodiment, the isovirus is lymphocytic choriomeningitis virus. In a specific embodiment, the open reading frame encoding the isovirus glycoprotein is deleted or functionally inactivated. In a specific embodiment, the viral vector is replication-defective. In a specific embodiment, the viral vector is replication-competent. In a specific embodiment, the viral vector is tri-fragmented. In some embodiments, this document provides a method for treating or preventing hepatitis B virus infection in a patient, wherein the method includes administering the viral vector to the patient, wherein the isovirus open reading frame from the viral vector is removed and replaced with a nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0081] 3.1 Conventions and Abbreviations

[0082] AFP Alpha-fetoprotein ALT Alanine transaminase APC Antigen presenting cells AST Aspartate aminotransferase C-cell Supplemental cell lines CD4 Differentiation group 4 CD8 Differentiation group 8 CMI Cell-mediated immunity GS-plasmid plasmids expressing genomic fragments HBc or HBcAg HBV core antigen HBe or HBeAg extracellular HBV core antigen HBs or HBsAg HBV (large) surface antigen HBV Hepatitis B virus HCC Hepatocellular carcinoma HRP Horseradish peroxidase IFN-γ Interferon-γ IGR Intergenic regions JUNV Hu Ning virus LCMV Lymphocytic choroid plexus meningitis virus LDH lactate dehydrogenase MHC Major Histocompatibility Complex NP nucleoprotein ORF Open reading box Pre-S2 / S HBV surface antigen TF-plasmid plasmids expressing trans-acting factors TNF-α Tumor necrosis factor-alpha UTR Non-translated area Z matrix proteins from LCMV

[0083] 4. Explanation of the sequence list

[0084] The following sequences are illustrative amino acid and nucleotide sequences that can be used with the methods and compositions described herein. In some cases, DNA sequences are used to describe RNA sequences of viral genome fragments. RNA sequences can be readily deduced from DNA sequences. The sequences themselves can also be found in Table 3 of Section 6.10.

[0085] SEQ ID NO: 1 is the nucleotide sequence of HBV pre-S2 / S ORF.

[0086] SEQ ID NO: 2 is the nucleotide sequence of HBV HBc ORF.

[0087] SEQ ID NO: 3 is the nucleotide sequence of the HBV HBs-HBc fusion protein ORF.

[0088] SEQ ID NO: 4 is the nucleotide sequence of the LCMV S fragment expressing the HBV HBs-HBc fusion protein in cDNA form. The genomic fragment is RNA, and the sequence in SEQ ID NO: 4 is shown as DNA; however, replacing all thymine nucleotides (“T”) in SEQ ID NO: 4 with uracil (“U”) provides the RNA sequence.

[0089] SEQ ID NO: 5 is the nucleotide sequence of an LCMV S fragment expressing HBc ORF in cDNA form. The genomic fragment is RNA, and the sequence in SEQ ID NO: 5 is shown as DNA; however, replacing all thymine nucleotides (“T”) in SEQ ID NO: 5 with uracil (“U”) provides the RNA sequence.

[0090] SEQ ID NO: 6 is the nucleotide sequence of the LCMV S fragment expressing pre-S2 / S ORF in cDNA form. The genomic fragment is RNA, and the sequence in SEQ ID NO: 6 is shown as DNA; however, replacing all thymine nucleotides (“T”) in SEQ ID NO: 6 with uracil (“U”) provides the RNA sequence.

[0091] SEQ ID NO: 7 is the full sequence of clone 13 L of lymphocytic choriomeningitis virus (GenBank: DQ361066.1). The genomic fragment is RNA, and the sequence in SEQ ID NO: 7 is shown as DNA; however, replacing all thymine nucleosides (“T”) in SEQ ID NO: 7 with uracil (“U”) provides the RNA sequence.

[0092] SEQ ID NO: 8 is the amino acid sequence of an epitope derived from HBV HBs protein.

[0093] SEQ ID NO: 9 is the amino acid sequence of an epitope derived from HBV HBs protein.

[0094] SEQ ID NO: 10 is the amino acid sequence of an epitope derived from HBV HBc protein.

[0095] SEQ ID NO: 11 is fragment S of lymphocytic choriomeningitis virus, full sequence. The genomic fragment is RNA, and the sequence in SEQ ID NO: 11 is shown as DNA; however, replacing all thymine nucleosides (“T”) in SEQ ID NO: 11 with uracil (“U”) provides the RNA sequence.

[0096] SEQ ID NO: 12 is the full sequence of clone 13 of lymphocytic choriomeningitis virus (GenBank: DQ361065.2). The genomic fragment is RNA, while the sequence in SEQ ID NO: 12 is shown as DNA; however, replacing all thymine nucleosides (“T”) in SEQ ID NO: 12 with uracil (“U”) provides the RNA sequence.

[0097] SEQ ID NO: 13 is the full sequence of MP fragment L of the lymphocytic choroid plexus meningitis virus strain. The genomic fragment is RNA, and the sequence in SEQ ID NO: 13 is shown as DNA; however, replacing all thymine nucleosides (“T”) in SEQ ID NO: 13 with uracil (“U”) provides the RNA sequence.

[0098] SEQ ID NO: 14 is the full sequence of MP fragment S of the lymphocytic choroid plexus meningitis virus strain. The genomic fragment is RNA, and the sequence in SEQ ID NO: 14 is shown as DNA; however, replacing all thymine nucleosides (“T”) in SEQ ID NO: 14 with uracil (“U”) provides the RNA sequence.

[0099] SEQ ID NO: 15 is the amino acid sequence of the NP protein of the MP strain of LCMV.

[0100] SEQ ID NO: 16 is the amino acid sequence of the GP protein of the MP strain of LCMV.

[0101] SEQ ID NO: 17 is the amino acid sequence of the L protein of the MP strain of LCMV.

[0102] SEQ ID NO: 18 is the amino acid sequence of the Z protein of the MP strain of LCMV.

[0103] SEQ ID NO: 19 is fragment L of the Candid#1 strain of the Huning virus, the complete sequence.

[0104] SEQ ID NO: 20 is fragment S of the Candid#1 strain of Huning virus, the complete sequence.

[0105] SEQ ID NO: 21 is the amino acid sequence of the NP protein of LCMV clone 13.

[0106] SEQ ID NO: 22 is the amino acid sequence of the GP protein of LCMV clone 13.

[0107] SEQ ID NO: 23 is the amino acid sequence of the L protein of LCMV clone 13.

[0108] SEQ ID NO: 24 is the amino acid sequence of the Z protein of LCMV clone 13.

[0109] SEQ ID NO: 25 is the amino acid sequence of the GP protein of the WE strain of LCMV.

[0110] SEQ ID NO: 26 is the nucleotide sequence of the HBV HBe antigen.

[0111] 5. Brief description of the attached drawings

[0112] Appendix Figure 1 The genome of wild-type arenavirus consists of a short RNA fragment (1-3.4 kb) and a large RNA fragment (2-7.2 kb). The short fragment carries an ORF encoding a nucleoprotein (3) and a glycoprotein (4). The large fragment encodes an RNA-dependent RNA polymerase L (5) and a matrix protein Z (6). By deleting the glycoprotein gene and replacing it with a selected antigen (7) to induce an immune response, wild-type arenavirus can be used as a replication-defective vaccine vector.

[0113] Figures 2A-C: Schematic diagrams of the genome architecture of bifragmented and trifragmented LCMVs. The bifragmented genome of wild-type LCMV consists of an S segment encoding GP and NP, and an L segment encoding Z and L proteins (A). Both segments are flanked by their respective 5' and 3' UTRs. The genome of the recombinant trifragmented LCMV (r3LCMV) consists of one L segment and two S segments, with a location for inserting the target gene (in this case, GFP) into each S segment. r3LCMV-GFP natural (nat) possesses all viral genes (B) in their natural positions, while r3LCMV / GFP artificial The GP ORF in (art) was artificially placed in the 3'UTR and expressed under the control of the 3'UTR (C).

[0114] Appendix Figure 3 Hepatitis B virus-specific CD8+ T cells, expressed as those obtained using 10 5 FFU rLCMV / HBs-HBc (group 1), rLCMV / HBc (group 3), rLCMV / Pre-S2 (group 4) or with 10 4Ten days after intravenous immunization with FFU-rLCMV / HBs-HBc (group 2), the total CD8+ B220 in the peripheral blood of C57BL / 6 mice (n=5 per group) was [data missing]. - Percentage of T cell pool. Control mice were untreated.

[0115] Figures 4A-B: Hepatitis B virus-specific CD8+ T cells, represented by 10... 5 FFU r3LCMV / HBs-HBc (Group 1), r3LCMV / HBc (Group 2), r3LCMV / Pre-S2 (Group 3) or with 10 5 The percentage of circulating lymphocytes in the blood of C57BL / 6 mice (n=5 per group) eight days after intravenous immunization with FFU-rLCMV / / HBs-HBc (group 4). Control mice were untreated.

[0116] 6. Detailed Description of the Invention

[0117] This application provides immunotherapy for hepatitis B virus infection: methods and compositions for treating or preventing HBV infection are provided herein. More specifically, infectious isoviruses comprising a nucleotide sequence encoding an HBV antigen are provided herein. In some embodiments, the infectious isoviruses are replication-defective. In some embodiments, the infectious isoviruses are replication-competent. These viruses can be administered to subjects for treating or preventing HBV infection. The generation of the infectious isoviruses used in this invention is described in more detail in section 5.3. Genetically modified isoviruses are provided herein, wherein the isoviruses:

[0118] It is contagious;

[0119] Infectious progeny viruses cannot be formed in non-supplementary cells (i.e., cells that do not express the functionality that the replication-defective isovirus lacks, thus making the isovirus replication-defective).

[0120] It can replicate its genome and express its genetic information; and

[0121] Encodes HBV antigen or fragments thereof.

[0122] The genetically modified arenaviruses described herein are infectious, meaning they can attach to host cells and release their genetic material into the host cells. The genetically modified arenaviruses described herein can be replication-deficient, meaning they cannot produce further infectious progeny particles in non-supplementary cells. Specifically, to create replication-deficient arenaviruses, the genome of the arenavirus is modified (e.g., by deletion or functional inactivation of the ORF) such that the virus carrying the modified genome no longer produces infectious progeny viruses. A non-supplementary cell is a cell that does not provide the functionality eliminated from replication-deficient arenaviruses by modifying the viral genome (e.g., if the ORF encoding the GP protein is deleted or functionally inactivated, the non-supplementary cell does not provide the GP protein). However, the genetically modified replication-deficient arenaviruses provided herein are capable of producing infectious progeny viruses in supplementary cells. Supplementary cells are a class of cells that (in turn) provide the functionality eliminated from said replication-deficient arenaviruses by modifying the viral genome (e.g., if the ORF encoding the GP protein is deleted or functionally inactivated, the supplementary cell provides the GP protein). The expression of supplemental functionalities (e.g., GP proteins) can be achieved by any method known to those skilled in the art (e.g., transient or stable expression). The genetically modified arenaviruses described herein can be amplified and express their genetic information in cells infected with the virus. The genetically modified arenaviruses provided herein contain nucleotide sequences encoding HBV antigens, such as, but not limited to, the HBV antigens described in Section 6.2.

[0123] In some embodiments, this document provides a genetically modified arenavirus in which the ORF of the arenavirus genome is deleted or functionally inactivated, such that the resulting virus cannot generate further infectious progeny viruses in non-supplementary cells. Arenavirus particles containing a genetically modified genome can be generated in supplementary cells (i.e., in cells expressing a deleted or functionally inactivated arenavirus ORF in which the ORF is deleted or functionally inactivated (see Section 6.3). The genetic material of the generated arenavirus particles can be transferred to host cells upon infection, where the genetic material can be expressed and amplified. Furthermore, the genome of the genetically modified arenavirus particles provided herein encodes HBV antigens that can be expressed in the host cells.

[0124] In some embodiments, the ORF encoding the arenavirus glycoprotein (GP) is deleted to produce a replication-defective arenavirus for use in this invention. In a specific embodiment, the replication-defective arenavirus comprises a genome fragment containing a nucleotide sequence encoding an HBV antigen. Thus, in some embodiments, the genetically modified arenavirus particles provided herein comprise a genome fragment a) having an ORF deletion or functional inactivation present in the wild-type form of the genome fragment; and b) encoding (antisense or sense) an HBV antigen (see section 6.3).

[0125] In some embodiments, the antigen encoded by the nucleic acid inserted into the genome of the isovirus can encode, for example, HBV antigen or a combination of HBV antigens, including but not limited to:

[0126] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0127] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0128] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0129] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or their antigenic fragments;

[0130] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0131] In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)).

[0132] Detailed descriptions of the antigens described in this article are provided in section 6.2.

[0133] In some embodiments, the arenaviruses used according to the invention described herein may be Old World viruses, such as lymphocytic choriomeningitis virus (LCMV). A more detailed description of the arenaviruses described herein is provided in section 6.1. In some embodiments, the arenaviruses used according to the invention described herein may be New World viruses.

[0134] This document provides nucleic acids containing the genome of such a replication-defective arenavirus. In some respects, infectious, replication-defective arenavirus particles contain genome fragments containing the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0135] This document provides expression plasmids that encode one or more components required to produce the viral vectors described herein. Specifically, this document provides expression vectors that encode the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORF of the human HBV pre-S2 / S protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1).

[0136] This document provides expression plasmids that encode one or more components required to produce the viral vectors described herein. Specifically, this document provides expression vectors that encode the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORF of the human HBV HBc protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2).

[0137] This document provides expression plasmids that encode one or more components required to produce the viral vectors described herein. Specifically, this document provides expression vectors that encode the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORFs of human HBV HBs and human HBV HBc (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3).

[0138] This document provides kits containing one or two of the vector plasmids described herein. In some embodiments, this document provides kits containing a) an expression plasmid containing the nucleotide sequence of the S fragment of an LCMV vector; b) an expression plasmid containing the nucleotide sequence of the L fragment of an LCMV vector; and c) an expression plasmid encoding supplementary functionality. In a specific embodiment, this document provides a kit comprising: a) an expression vector containing the nucleotide sequence of the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORF of the human HBV pre-S2 / S protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1); b) an expression plasmid containing the nucleotide sequence of the L fragment of the LCMV vector; and c) an expression plasmid encoding the LCMV GP protein (or a cell line expressing the LCMV GP protein).

[0139] This document provides kits containing one or two of the vector plasmids described herein. In some embodiments, this document provides kits containing a) an expression plasmid containing the nucleotide sequence of the S fragment of an LCMV vector; b) an expression plasmid containing the nucleotide sequence of the L fragment of an LCMV vector; and c) an expression plasmid encoding supplementary functionality. In a specific embodiment, this document provides a kit comprising: a) an expression vector containing the nucleotide sequence of the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORF of the human HBV HBc protein (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2); b) an expression plasmid containing the nucleotide sequence of the L fragment of the LCMV vector; and c) an expression plasmid encoding the LCMV GP protein (or a cell line expressing the LCMV GP protein).

[0140] This document provides kits containing one or two of the vector plasmids described herein. In some embodiments, this document provides kits containing a) an expression plasmid containing the nucleotide sequence of the S fragment of an LCMV vector; b) an expression plasmid containing the nucleotide sequence of the L fragment of an LCMV vector; and c) an expression plasmid encoding supplementary functionality. In a specific embodiment, this document provides a kit comprising: a) an expression vector containing the nucleotide sequence of the LCMV S fragment, wherein the ORF of the GP protein has been deleted from the S fragment and replaced with the ORF of human HBV HBs and human HBV HBc (e.g., having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3, or an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3); b) an expression plasmid containing the nucleotide sequence of the L fragment of the LCMV vector; and c) an expression plasmid encoding the LCMV GP protein (or a cell line expressing the LCMV GP protein).

[0141] This article also provides cell lines, cultures, and methods for culturing cells infected with the nucleic acids, vectors, and compositions provided herein. More detailed descriptions of the nucleic acid, vector systems, and cell lines described herein are provided in Section 6.4.

[0142] In one respect, this article provides information on genetically modified replication-defective arenaviruses suitable as vaccines, and methods for using such arenaviruses in vaccination and in the treatment or prevention of HBV infection. More detailed instructions on the methods of using such arenaviruses described herein are provided in section 6.5.

[0143] In some embodiments, immunization with infectious arenavirus expressing HBV antigen or fragments thereof, as described herein, provides a durable immune response. In some embodiments, maximum antibody levels can be achieved after two immunizations. In another embodiment, a third immunization can be administered to enhance the effect. In more specific embodiments, this document provides an administration schedule for the use of the infectious arenavirus for the treatment and / or prevention of HBV infection. A more detailed description of the administration schedule for the infectious arenavirus described herein is provided in section 6.6. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)).

[0144] In some embodiments, administration of an infectious isovirus expressing HBV antigen or fragments thereof, as described herein, to a seronegative subject induces a detectable antibody titer that persists for at least 4 weeks. In another embodiment, administration of an infectious isovirus expressing HBV antigen or fragments thereof, as described herein, to an HBV-infected subject increases the antibody titer by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. In some embodiments, primary antigen exposure to an initial immunization with an infectious isovirus expressing HBV antigen induces a functional (neutralizing) and minimal antibody titer, which is at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the mean control serum from an infected-immunized human subject. In a more specific embodiment, the initial neutralizing geometric mean antibody titer increases to a peak of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 at least 4 weeks post-immunization. In another embodiment, immunization with an infectious arenavirus expressing HBV antigen or fragments thereof, as described herein, produces high titers of antibodies that persist for at least 4 weeks, 8 weeks, 12 weeks, 6 months, 12 months, 2 years, 3 years, 4 years, or 5 years following a single dose of vaccine. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0145] In yet another embodiment, the antibody titer is increased by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% by a second antigen exposure to an infectious isovirus expressing HBV antigen or fragments thereof. In another embodiment, the second antigen exposure induces a functional, (neutralizing), and minimum antibody titer that is at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the mean control serum from infected-immunized human subjects. In a more specific embodiment, the second neutralizing geometric mean antibody titer increases to a peak of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 within at least 4 weeks post-immunization. In another embodiment, a second immunization with an infectious arenavirus expressing HBV antigen or fragments thereof, as described herein, produces high-titer antibodies for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years following continuous immunization. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0146] 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 1000%. In another embodiment, the booster immunization induces functional, (neutralizing), and minimum antibody titers, which are at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% of the mean control serum from infected-immunized human subjects. In a more specific embodiment, the neutralizing geometric mean antibody titer following the third booster immunization increases to a peak of at least 1:50, at least 1:100, at least 1:200, or at least 1:1000 within at least 4 weeks post-immunization. In another embodiment, the third booster immunization prolongs antibody titers to at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, or at least 5 years post-immunization.

[0147] In some embodiments, infectious isavirus expressing HBV antigen or fragments thereof elicits a T cell-independent or T cell-dependent response. In other embodiments, infectious isavirus expressing HBV antigen or fragments thereof elicits a T cell response. In other embodiments, the infectious isavirus expressing HBV antigen or fragments thereof described herein elicits a T helper cell response. In yet another embodiment, the infectious isavirus expressing HBV antigen or fragments thereof described herein elicits a Th1-directed or Th2-directed response. In some embodiments, the viral vector of the infectious isavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious isavirus is replication-competent (see section 6.1(b)).

[0148] In a more specific embodiment, the Th1-directed response is indicated by the dominance of IgG1 antibody against IgG2. In other embodiments, the IgG1:IgG2 ratio is greater than 1:1, greater than 2:1, greater than 3:1, or greater than 4:1. In yet another embodiment, the infectious arenavirus expressing HBV antigen or fragments thereof described herein is indicated by the dominance of IgG3 antibody. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see section 6.1(b)).

[0149] In some embodiments, infectious isoflavones expressing HBV antigen or fragments thereof elicit a CD8+ T cell response. In other embodiments, infectious isoflavones expressing HBV antigen or fragments thereof elicit a regulatory T cell response. In a more specific embodiment, the regulatory T cell response maintains immune tolerance. In yet another embodiment, infectious isoflavones expressing HBV antigen or fragments thereof elicit both CD4+ and CD8+ T cell responses. In some embodiments, the viral vector of the infectious isoflavones is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious isoflavones is replication-competent (see section 6.1(b)).

[0150] In some embodiments, the infectious arenaviruses described herein expressing one or more HBV antigens or fragments thereof elicit high titers of neutralizing antibodies. In another embodiment, the infectious arenaviruses described herein expressing two or more HBV antigens or fragments thereof elicit even higher titers of neutralizing antibodies compared to expressing a protein complex component alone. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0151] In other embodiments, two or more infectious arenaviruses expressing HBV antigens elicit high titers of neutralizing antibodies. In more specific embodiments, two or more infectious arenaviruses expressing HBV antigens elicit higher titers of neutralizing antibodies compared to infectious arenaviruses expressing one HBV antigen or a fragment thereof. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0152] In another embodiment, infectious arenaviruses expressing two, three, four, five, or more HBV antigens elicit higher titers of neutralizing antibodies compared to infectious arenaviruses expressing one HBV antigen or a fragment thereof. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0153] 6.1 Arenavirus vector expressing HBV antigen

[0154] The arenavirus used in the methods and compositions provided herein can be an Old World virus, such as Lassa virus, lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, or Ippy virus, or a New World virus, such as Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear Canyon virus, or Whitewater Arroyo virus. Genetically modified arenaviruses can be generated as described in Section 6.3.

[0155] The genome of wild-type arenavirus consists of a short RNA fragment (~3.4 kb) and a large RNA fragment (~7.2 kb). The short fragment carries ORFs encoding the nucleoprotein NP and the glycoprotein GP genes. The large fragment contains the RNA-dependent RNA polymerase L and the matrix protein Z genes.

[0156] (a) Replication-defective arenavirus vector

[0157] In some embodiments, the arenavirus vector is a replication-defective, two-fragmented arenavirus vector. In some embodiments, the arenavirus vector is a replication-defective, three-fragmented arenavirus vector. Wild-type arenavirus can be made replication-defective to produce a vaccine vector by replacing the glycoprotein gene with one or more HBV antigens to induce an immune response.

[0158] Infectious arenaviruses expressing HBV antigens, or combinations thereof, as described herein, can be used to immunize (in a preventative manner) or treat (in an immunotherapy manner) individuals against HBV infection. In a specific implementation, a combination of HBs and HBc is used.

[0159] It is known that arenavirus disease and immunosuppression in wild-type arenavirus infection are caused by unchecked viral replication. By deleting, for example, the Z gene required for particle release or the GP gene required for infecting target cells from their genome, the replication of the arenavirus vector—that is, the ability to produce infectious progeny viral particles—can be eliminated, thus limiting the total number of infected cells to the inoculum, which is administered, for example, to a vaccine recipient, or accidentally transmitted to personnel or animals involved in a medical or biotechnological application. Therefore, eliminating the replication of the arenavirus vector prevents the disease resulting from the intentional or unintentional transmission of the vector particles. An important aspect provided herein is that the necessity of eliminating replication described above is utilized in a manner beneficial to the expression of HBV antigens. In some embodiments, the arenavirus particles are made replication-deficient through genetic modifications to the genome. Such modifications to the genome may include:

[0160] Delete the ORF (e.g., the ORF encoding the GP, NP, L, or Z protein);

[0161] Functional inactivation of ORFs (e.g., ORFs encoding GP, NP, L, or Z proteins).

[0162] For example, this can be achieved by introducing missense or nonsense mutations;

[0163] Altering the sequence of the ORF (e.g., exchanging the SIP cleavage site for the cleavage site of another protease);

[0164] Mutagenesis of one of the 5' or 3' ends of a genome segment;

[0165] This is achieved by mutagenizing intergenic regions (i.e., intergenic regions of L or S genomic segments).

[0166] In some embodiments, the infectious isan virus expressing HBV antigen described herein is lymphocytic choriomeningitis virus (LCMV), wherein the S segment of said virus is modified by replacing the ORF encoding the GP protein with an ORF encoding the HBV antigen.

[0167] In some implementations, the wild-type arenavirus vector genome (attached) Figure 1 The vector genome can be designed to retain at least the essential regulatory elements on the 5' and 3' untranslated regions (UTRs) of both fragments, and / or intergenic regions (IGRs). Without being theoretically limited, minimal trans-acting factors for gene expression in infected cells are retained in the vector genome as expressible ORFs, but they can be placed differently in the genome, under the control of a promoter different from the natural promoter, or expressed from an internal ribosomal entry site. In some embodiments, the nucleic acid encoding the HBV antigen is transcribed from one of the endogenous arenavirus promoters (i.e., the 5' UTR and 3' UTR of the S fragment, and the 5' UTR and 3' UTR of the L fragment). In other embodiments, the nucleic acid encoding the HBV antigen is expressed from a heterologously introduced promoter sequence, which can be read by a viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II, or RNA polymerase III, for example, a copy of a viral promoter sequence naturally present in the viral URT, a 28S ribosomal RNA promoter, a β-actin promoter, or a 5S ribosomal RNA promoter. In some implementations, the ribonucleic acid encoding the HBV antigen is transcribed and translated on its own, or transcribed and translated as a readthrough by fusion with the arenavirus protein ORF. Protein expression in the host cell can be enhanced by introducing one or more, for example, two, three, or four internal ribosome entry sites at appropriate locations in the viral transcript sequence.

[0168] In some embodiments, for use in the compositions and methods, this document provides tri-fragmented arenavirus particles comprising one L fragment and two S fragments, wherein (i) the ORF is positioned at a location different from the wild-type position of the ORF; and (ii) the ORF encoding GP or NP has been removed or functionally inactivated, so that the resulting virus cannot produce further infectious progeny virus particles. In a specific embodiment, one ORF is removed and replaced with a heterologous ORF from an organism other than arenavirus (e.g., encoding HBV antigen). In another specific embodiment, two ORFs are removed and replaced with a heterologous ORF from an organism other than arenavirus. In other specific embodiments, three ORFs are removed and replaced with a heterologous ORF from an organism other than arenavirus (e.g., encoding HBV antigen). In a specific embodiment, the ORF encoding GP is removed and replaced with a heterologous ORF from an organism other than arenavirus (e.g., encoding HBV antigen). In other specific embodiments, the ORF encoding NP is removed and replaced with a heterologous ORF from an organism other than arenavirus (e.g., encoding HBV antigen). In a more specific embodiment, the ORF encoding NP and the ORF encoding GP are removed and replaced with one or two heterologous ORFs from an organism other than the arenavirus particle (e.g., encoding one or two HBV antigens). Thus, in some embodiments, the tri-fragmented arenavirus particle comprises (i) one L fragment and two S fragments; (ii) an ORF at a location other than the wild-type position of the ORF; and (iii) one or more heterologous ORFs from an organism other than the arenavirus (e.g., encoding one or more HBV antigens).

[0169] In some embodiments, for use in the compositions and methods, this document provides tri-fragmented arenavirus particles comprising two L fragments and one S fragment, wherein (i) the ORF is positioned at a location different from the wild-type position of the ORF; and (ii) the ORF encoding the Z protein and / or L protein has been removed or functionally inactivated, so that the resulting virus cannot produce further infectious progeny virus particles. In a specific embodiment, one ORF is removed and replaced with a heterologous ORF (e.g., encoding HBV antigen) from an organism other than arenavirus. In another specific embodiment, both ORFs are removed and replaced with a heterologous ORF (e.g., encoding HBV antigen) from an organism other than arenavirus. In a specific embodiment, the ORF encoding the Z protein is removed and replaced with a heterologous ORF (e.g., encoding HBV antigen) from an organism other than arenavirus. In other specific embodiments, the ORF encoding the L protein is removed and replaced with a heterologous ORF (e.g., encoding HBV antigen) from an organism other than arenavirus. In a more specific embodiment, the ORF encoding the Z protein and the ORF encoding the L protein are removed and replaced with a heterologous ORF from an organism other than the arenavirus particle (e.g., encoding the HBV antigen). Thus, in some embodiments, the tri-fragmented arenavirus particle comprises (i) two L fragments and one S fragment; (ii) an ORF at a location other than the wild-type position of the ORF; and (iii) a heterologous ORF from an organism other than the arenavirus (e.g., encoding the HBV antigen).

[0170] Therefore, in some embodiments, the tri-fragmented isopyrvirus particles used in the compositions and methods provided herein comprise tri-fragmented isopyrvirus particles (i.e., one L fragment and two S fragments, or two L fragments and one S fragment), which i) are engineered to carry an ORF in a non-natural position; ii) have an ORF encoding a GP, NP, Z, or L protein removed; and iii) have the removed ORF replaced by a heterologous ORF from an organism other than isopyrvirus (e.g., encoding one or more HBV antigens).

[0171] In some embodiments, the vector generated to encode one or more HBV antigens may be based on a specific strain of LCMV. LCMV strains include clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille#12, HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN, and derivatives thereof. In some embodiments, the vector generated to encode one or more HBV antigens may be based on LCMV clone 13. In other embodiments, the vector generated to encode one or more HBV antigens may be based on the LCMV MP strain. The sequence of the S fragment of LCMV clone 13 is listed in SEQ ID NO: 12. In some embodiments, the sequence of the S fragment of LCMV clone 13 is the sequence listed in SEQ ID NO: 11. The sequence of the L fragment of LCMV clone 13 is as listed in SEQ ID NO: 7. The sequence of the S fragment of LCMV strain MP is as listed in SEQ ID NO: 14. The sequence of the L fragment of LCMV strain MP is as listed in SEQ ID NO: 13.

[0172] In some embodiments, the vector generated to encode one or more HBV antigens may be based on a specific strain of Junin virus. Junin virus strains include vaccine strains XJ13, XJ#44, and Candid#1, as well as a human isolate IV4454. In some embodiments, the vector generated to encode one or more HBV antigens may be based on the Candid#1 strain of Junin virus.

[0173] In some embodiments, this document describes infectious, replication-defective sand-like virus particles that contain nucleotide sequences or fragments thereof selected from SEQ ID NO: 13, SEQ ID NO: 14, or combinations thereof.

[0174] In some embodiments, this document describes infectious, replication-defective arenavirus particles that comprise nucleotide sequences or combinations thereof selected from:

[0175] • The nucleotide sequence encoding the hepatitis B virus pre-S2 / S protein or its antigenic fragment;

[0176] • The nucleotide sequence encoding the hepatitis B virus HBc protein or its antigenic fragment;

[0177] • The nucleotide sequence encoding the hepatitis B virus HBs protein or its antigenic fragment;

[0178] • Nucleotide sequence encoding a fusion of hepatitis B virus HBs and HBc proteins or antigenic fragments thereof;

[0179] • The nucleotide sequence encoding the hepatitis B virus HBe protein or its antigenic fragments.

[0180] In some embodiments, the viral vector of the infectious, replication-defective sand-like virus is tri-fragmented.

[0181] (b) Replication of competent cells in a three-fragmented isovirus vector

[0182] In some embodiments, the compositions and methods provided herein are used with a replication-competent tri-fragmented isovirus vector. In some embodiments, the isovirus vector is a tri-fragmented isovirus particle comprising one L fragment and two S fragments, or two L fragments and one S fragment, which do not recombine into a replication-competent bi-fragmented isovirus particle.

[0183] In some embodiments, the infectious isovirus expressing HBV antigen used in the compositions and methods described herein is engineered to carry a viral ORF located outside the wild-type position of the ORF. In some embodiments, the isovirus genomic fragment is selected from: (i) an S fragment wherein the ORF encoding NP is under the control of the isovirus 5'UTR; (ii) an S fragment wherein the ORF encoding the Z protein is under the control of the isovirus 5'UTR; (iii) an S fragment wherein the ORF encoding the L protein is under the control of the isovirus 5'UTR; (iv) an S fragment wherein the ORF encoding the GP is under the control of the isovirus 3'UTR; (v) an S fragment wherein the ORF encoding the L protein is under the control of the isovirus 3'UTR; (vi) an S fragment wherein the ORF encoding the Z protein is under the control of the isovirus 5'UTR. Under the control of the 3'UTR of the virus; (vii) L fragment, wherein the ORF encoding GP is under the control of the 5'UTR of the isopyrvirus; (viii) L fragment, wherein the ORF encoding NP is under the control of the 5'UTR of the isopyrvirus; (ix) L fragment, wherein the ORF encoding L protein is under the control of the 5'UTR of the isopyrvirus; (x) L fragment, wherein the ORF encoding GP is under the control of the 3'UTR of the isopyrvirus; (xi) L fragment, wherein the ORF encoding NP is under the control of the 3'UTR of the isopyrvirus; and (xii) L fragment, wherein the ORF encoding Z protein is under the control of the 3'UTR of the isopyrvirus.

[0184] In some embodiments, the 3'UTR of the sand virus is the 3'UTR of the sand virus S fragment or the sand virus L fragment. In some embodiments, the 5'UTR of the sand virus is the 5'UTR of the sand virus S fragment or the sand virus L fragment.

[0185] The compositions and methods provided herein are trisegmented arecavirus particles with rearranged ORFs. In one aspect, the compositions and methods provided herein are trisegmented arecavirus particles comprising one L fragment and two S fragments, or two L fragments and one S fragment. In some embodiments, the trisegmented arecavirus particles are not recombined into replication-competent bifragmented arecavirus particles. In a particular embodiment, the trisegmented arecavirus particles contain ORFs at a location other than the wild-type position of the ORF. In yet another particular embodiment, the trisegmented arecavirus particles contain all four arecavirus ORFs. Thus, in some embodiments, the trisegmented arecavirus particles are replication-competent and infectious. Figure 2 shows an exemplary schematic diagram of the genomic architecture of a replication-competent trisegmented LCMV vector (Figures 2B-C). Figure 2C shows an exemplary schematic diagram of the genomic architecture of a replication-competent trisegmented LCMV vector that cannot be recombined into a replication-competent bifragmented arecavirus particle. In contrast, Figure 2A shows a wild-type bifragmented LCMV vector.

[0186] In some embodiments, the ORF encoding the GP, NP, Z, or L proteins of the tri-fragmented arenavirus particle described herein may be under the control of the arenavirus 3'UTR or the arenavirus 5'UTR. In a more specific embodiment, the tri-fragmented arenavirus 3'UTR is the 3'UTR of the arenavirus S fragment. In another specific embodiment, the tri-fragmented arenavirus 3'UTR is the 3'UTR of the arenavirus L fragment. In a more specific embodiment, the tri-fragmented arenavirus 5'UTR is the 5'UTR of the arenavirus S fragment. In other specific embodiments, the 5'UTR is the 5'UTR of the arenavirus L fragment.

[0187] In other embodiments, the ORF encoding the GP, NP, Z, or L protein of the tri-fragmented arenavirus particle described herein may be under the control of conserved terminal sequence elements of the arenavirus (5'-terminal and 3'-terminal 19-20-nt regions) (see, for example, Perez & de la Torre, 2003, J Virol. 77(2): 1184-1194).

[0188] In some embodiments, the ORF encoding the GP, NP, Z, or L proteins of the tri-fragmented arenavirus particle may be under the control of a promoter element in the 5'UTR (see, for example, Albariño et al., 2011, J Virol., 85(8):4020-4). In another embodiment, the ORF encoding the GP, NP, Z, and L proteins of the tri-fragmented arenavirus particle may be under the control of a promoter element in the 3'UTR (see, for example, Albariño et al., 2011, J Virol., 85(8):4020-4). In a more specific embodiment, the promoter element of the 5'UTR is a promoter element of the 5'URT of the S or L fragment. In another specific embodiment, the promoter element of the 3'UTR is a promoter element of the 3'URT of the S or L fragment.

[0189] In some embodiments, the ORF encoding the GP, NP, Z, or L proteins of the tri-fragmented arenavirus particle may be under the control of a truncated arenavirus 3'UTR or a truncated arenavirus 5'UTR (see, for example, Perez & dela Torre, 2003, J Virol. 77(2): 1184-1194; Albariño et al, 2011, J Virol, 85(8): 4020-4). In a more specific embodiment, the truncated 3'UTR is the 3'UTR of the arenavirus S fragment or L fragment. In a more specific embodiment, the truncated 5'UTR is the 5'UTR of the arenavirus S fragment or L fragment.

[0190] In one aspect, the compositions and methods provided herein use trifragmented arenavirus particles comprising one L fragment and two S fragments. In some embodiments, the proliferation of the trifragmented arenavirus particles comprising one L fragment and two S fragments does not produce replication-competent bifragmented virus particles. In a specific embodiment, in the absence of type I interferon receptor, type II interferon receptor, and recombinant activation gene (RAG1), using 10 4In mice infected with the tri-fragmented arenavirus particles of PFU, replication of the tri-fragmented arenavirus particles comprising one L fragment and two S fragments does not produce replication-competent bi-fragmented virus particles after at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days of sustained infection. In other embodiments, replication of the tri-fragmented arenavirus particles comprising one L fragment and two S fragments does not produce replication-competent bi-fragmented virus particles after at least 10, 20, 30, 40, or 50 passages.

[0191] Triple-fragmented arenavirus particles, with all viruses located at their respective wild-type positions, are known in the art (e.g., Emone et al, 2011 J. Virol, 85(4):1473; Popkin et al, 2011 J. Virol, 85(15):7928). Specifically, the triple-fragmented arenavirus genome consists of one L fragment and two S fragments, with a heterologous ORF (e.g., GFP) inserted at a position on each S fragment. More specifically, one S fragment encodes GP and GFP, respectively. The other S fragment encodes GFP and NP, respectively. The L fragment encodes the L protein and the Z protein. All fragments are flanked by their respective 5' and 3' UTRs.

[0192] In some embodiments, intrafragmental recombination of the two S fragments of the tri-fragmented isovirus particle used in the compositions and methods provided herein combines the two isovirus ORFs on one rather than two separate fragments to produce a nonfunctional promoter (i.e., a genomic fragment with the structure: 5'UTR----------5'UTR or 3'UTR----------3'UTR), wherein each UTR forming one end of the genome is a reversed repetitive sequence at the other end of the same genome.

[0193] In some embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments is engineered to carry isovirus ORFs at a location other than the wild-type location of the ORF. In other embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments is engineered to carry two, three, four, five, or six isovirus ORFs at a location other than the wild-type location. In a specific embodiment, the tri-fragmented isovirus particle containing one L fragment and two S fragments comprises all four types of isovirus ORFs. Thus, in some embodiments, the tri-fragmented isovirus particle is an infectious and replication-competent tri-fragmented isovirus particle. In a specific embodiment, the two S fragments of the tri-fragmented isovirus particle are engineered to carry one of their ORFs at a location other than the wild-type location. In a more specific embodiment, the two S fragments comprise the entirety of the S fragment ORFs. In some specific implementations, the L fragment has been engineered to carry the ORF at a location other than the wild-type location, or the L fragment may be a wild-type genome fragment.

[0194] In some implementations, one of the two S segments can be:

[0195] (i) The S fragment of the isoplasmosis virus, wherein the ORF encoding the Z protein is under the control of the 5'UTR of the isoplasmosis virus;

[0196] (ii) The S fragment of the isoplasmosis virus, wherein the ORF encoding the L protein is under the control of the 5'UTR of the isoplasmosis virus;

[0197] (iii) Sand virus S segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR;

[0198] (iv) Sand virus S fragment, in which the ORF encoding GP is under the control of the sand virus 3'UTR;

[0199] (v) Sand virus S segment, wherein the ORF encoding L is under the control of the sand virus 3'UTR; and

[0200] (vi) The S fragment of the isovirus, in which the ORF encoding the Z protein is under the control of the isovirus 3'UTR.

[0201] In some embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments may contain duplicate ORFs (i.e., two wild-type S fragment ORFs, such as GP or NP). In specific embodiments, the tri-fragmented isovirus particle containing one L fragment and two S fragments may contain one duplicate ORF (e.g., (GP, GP)) or two duplicate ORFs (e.g., (GP, GP) and (NP, NP)).

[0202] Table 1A below is an exemplary example of the genomic architecture of a tri-fragmented arenavirus particle containing one L fragment and two S fragments, wherein inter-segment recombination of the two S fragments in the tri-fragmented arenavirus genome does not produce a replication competent bi-fragmented virus particle and cancels arenavirus promoter activity (i.e., the resulting recombinant S fragment consists of two 3'UTRs instead of 3'UTR and 5'UTR).

[0203] Table 1A

[0204] The three-fragmented sand virus particle contains one L fragment and two S fragments. Position 1 is under the control of the 5'UTR of the sand virus S fragment; position 2 is under the control of the 3'UTR of the sand virus S fragment; position 3 is under the control of the 5'UTR of the sand virus S fragment; position 4 is under the control of the 3'UTR of the sand virus S fragment; position 5 is under the control of the 5'UTR of the sand virus L fragment; and position 6 is under the control of the 3'UTR of the sand virus L fragment.

[0205] *ORF indicates a heterologous ORF; for example, a heterologous ORF encoding the HBV antigen has been inserted.

[0206] Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 *ORF GP *ORF NP Z L *ORF NP *ORF GP Z L *ORF NP *ORF GP L Z *ORF NP *ORF Z L GP *ORF NP Z GP *ORF Z *ORF NP Z GP Z *ORF *ORF NP *ORF L Z GP *ORF L *ORF NP Z GP *ORF L Z NP *ORF GP *ORF L *ORF GP Z NP *ORF L Z GP *ORF NP *ORF Z L NP *ORF GP *ORF Z *ORF GP L NP *ORF Z L GP *ORF NP L GP *ORF NP *ORF Z L GP *ORF *ORF Z NP L GP *ORF Z *ORF NP L *ORF Z GP *ORF NP L GP *ORF NP *ORF Z L GP *ORF Z *ORF NP L GP Z NP *ORF *ORF L GP Z NP *ORF *ORF

[0207] Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 L *ORF Z NP *ORF GP L NP *ORF Z *ORF GP L NP Z *ORF GP *ORF L *ORF Z *ORF GP NP L NP Z GP *ORF *ORF L NP *ORF Z *ORF GP L *ORF Z NP *ORF GP L Z *ORF GP *ORF NP L Z *ORF NP *ORF GP Z GP *ORF NP *ORF L Z GP *ORF *ORF L NP Z GP *ORF L *ORF NP Z *ORF L GP *ORF NP Z GP *ORF NP *ORF L Z GP *ORF L *ORF NP Z GP L NP *ORF *ORF Z GP L NP *ORF *ORF Z *ORF L NP *ORF GP Z NP *ORF *ORF L GP z NP *ORF GP *ORF L z NP *ORF *ORF L GP z NP *ORF L *ORF GP z NP L GP *ORF *ORF z *ORF L GP *ORF NP z NP *ORF GP *ORF L z NP *ORF L *ORF GP z *ORF L NP *ORF GP z L *ORF GP *ORF NP

[0208] In some embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment or L-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In specific embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In some embodiments, other combinations are also possible. For example, a tri-fragmented isoprev. particle containing one L-fragment and two S-fragments, wherein inter-segment recombination of the two S-fragments in the tri-fragmented isoprev. genome does not produce a duplexed viral particle with a replication competent state, and cancels isoprev. promoter activity (i.e., the resulting recombinant S-fragment consists of two 5'UTRs instead of 3'UTR and 5'UTR).

[0209] In some embodiments, the intersegmental recombination of the S and L fragments in the tri-fragmented arenavirus particle containing one L fragment and two S fragments restores a functional fragment having two viral genes on only one fragment, rather than two separate fragments. In other embodiments, the intersegmental recombination of the S and L fragments in the tri-fragmented arenavirus particle containing one L fragment and two S fragments does not produce a replication-competent bi-fragmented virus particle.

[0210] Table 1B below is an example of the genomic architecture of a tri-fragmented arenavirus particle containing one L fragment and two S fragments, wherein inter-fragment recombination of the S and L fragments in the tri-fragmented arenavirus genome does not produce a replication competent bi-fragmented virus particle and cancels arenavirus promoter activity (i.e., the resulting recombinant S fragment consists of two 3'UTRs instead of 3'UTR and 5'UTR).

[0211] Table 1B shows tri-fragmented sand virus particles containing one L fragment and two S fragments.

[0212] Location 1 is under the control of the 5'UTR of the S segment of the sand-like virus; Location 2 is under the control of the 3'UTR of the S segment of the sand-like virus; Location 3 is under the control of the 5'UTR of the S segment of the sand-like virus; Location 4 is under the control of the 3'UTR of the S segment of the sand-like virus; Location 5 is under the control of the 5'UTR of the L segment of the sand-like virus; Location 6 is under the control of the 3'UTR of the L segment of the sand-like virus.

[0213] *ORF indicates a heterologous ORF; for example, a heterologous ORF encoding the HBV antigen has been inserted.

[0214] Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 L GP *ORF NP Z *ORF L GP Z *ORF *ORF NP L GP *ORF NP Z *ORF

[0215] Position 1 Position 2 Position 3 Position 4 Position 5 Position 6 L GP Z *ORF *ORF NP L NP *ORF GP Z *ORF L NP Z *ORF *ORF GP L NP *ORF GP Z *ORF L NP Z *ORF *ORF GP Z GP *ORF NP L *ORF Z GP L *ORF *ORF NP Z GP *ORF NP L *ORF Z NP L *ORF *ORF GP Z NP *ORF GP L *ORF Z NP L *ORF *ORF GP

[0216] In some embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment or L-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In specific embodiments, the IGR between position one and position two can be an isoprev. S-fragment or L-fragment IGR; the IGR between position two and position three can be an isoprev. S-fragment IGR; and the IGR between position five and position six can be an isoprev. L-fragment IGR. In some embodiments, other combinations are also possible. For example, a tri-fragmented isoprev. particle containing one L-fragment and two S-fragments, wherein inter-segment recombination of the two S-fragments in the tri-fragmented isoprev. genome does not produce a duplexed viral particle with a replication competent state, and cancels isoprev. promoter activity (i.e., the resulting recombinant S-fragment consists of two 5'UTRs instead of 3'UTR and 5'UTR).

[0217] In one aspect, the compositions and methods provided herein are used with trifragmented arenavirus particles comprising two L fragments and one S fragment. In some embodiments, the proliferation of the trifragmented arenavirus particles comprising two L fragments and one S fragment does not produce replication competent bifragmented virus particles. In a specific embodiment, in the absence of type I interferon receptor, type II interferon receptor, and recombinant activation gene (RAG1), using 10 4 In mice infected with the trisegmented arenavirus particles of PFU, replication of the trisegmented arenavirus particles comprising two L fragments and one S fragment does not produce replication-competent bisegmented virus particles after at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days of sustained infection. In other embodiments, replication of the trisegmented arenavirus particles comprising two L fragments and one S fragment does not produce replication-competent bisegmented virus particles after at least 10, 20, 30, 40, or 50 passages.

[0218] In some embodiments, intrafragmental recombination of the two L fragments of the tri-fragmented isovirus particle used in the compositions and methods provided herein combines the two isovirus ORFs on one rather than two separate fragments to produce a nonfunctional promoter (i.e., a genomic fragment with the structure: 5'UTR----------5'UTR or 3'UTR----------3'UTR), wherein each UTR forming one end of the genome is a reversed repetitive sequence promoter of the other end of the same genome.

[0219] In some embodiments, the tri-fragmented isovirus particle containing two L fragments and one S fragment is engineered to carry isovirus ORFs at a location other than the wild-type location of the ORF. In other embodiments, the tri-fragmented isovirus particle containing two L fragments and one S fragment is engineered to carry one, three, four, five, or six isovirus ORFs at a location other than the wild-type location. In a specific embodiment, the tri-fragmented isovirus particle containing two L fragments and one S fragment contains all four types of isovirus ORFs. Thus, in some embodiments, the tri-fragmented isovirus particle is an infectious and replication-competent tri-fragmented isovirus particle. In a specific embodiment, the two L fragments of the tri-fragmented isovirus particle are engineered to carry one of their ORFs at a location other than the wild-type location. In a more specific embodiment, the two L fragments contain all L fragment ORFs. In some specific embodiments, the S fragment is engineered to carry one of its ORFs at a location other than the wild-type location, or the S fragment may be a wild-type genome fragment. In some embodiments, one of the two L fragments may be:

[0220] (i)L segment, in which the ORF encoding GP is under the control of the sand virus 5'UTR;

[0221] (ii) L segment, in which the ORF encoding NP is under the control of the sand virus 5'UTR;

[0222] (iii) The L fragment, in which the ORF encoding the L protein is under the control of the 5'UTR of the isopyrvirus;

[0223] (iv)L segment, in which the ORF encoding GP is under the control of the sand virus 3'UTR;

[0224] (v)L segment, in which the ORF encoding NP is under the control of the sand virus 3'UTR; and

[0225] (vi)L fragment, in which the ORF encoding the Z protein is under the control of the arena virus 3'UTR;

[0226] In some embodiments, the tri-fragmented arenavirus particle containing one L fragment and two L fragments may contain duplicate ORFs (i.e., two wild-type L fragment ORFs, such as Z protein or L protein). In specific embodiments, the tri-fragmented arenavirus particle containing two L fragments and one S fragment may contain one duplicate ORF (e.g., (Z protein, Z protein)) or two duplicate ORFs (e.g., (Z protein, Z protein) and (L protein, L protein)).

[0227] Table 2A below provides an exemplary example of the genomic architecture of a tri-fragmented arenavirus particle containing two L segments and one S segment, wherein intersegmental recombination of the two L segments in the tri-fragmented arenavirus genome does not produce a replication-competent bi-fragmented viral particle and cancels arenavirus promoter activity (i.e., the resulting recombinant S segment consists of two 3'UTRs instead of a 3'UTR and a 5'UTR). Based on Table 3, similar combinations can be predicted for producing arenavirus particles consisting of two 5'UTRs instead of a 3'UTR and a 5'UTR.

[0228] Table 2A shows tri-fragmented sand virus particles containing two L fragments and one S fragment.

[0229] *Location 1 is under the control of the 5'UTR of the L segment of the sand virus; Location 2 is under the control of the 3'UTR of the L segment of the sand virus; Location 3 is under the control of the 5'UTR of the L segment of the sand virus; Location 4 is under the control of the 3'UTR of the L segment of the sand virus; Location 5 is under the control of the 5'UTR of the S segment of the sand virus; Location 6 is under the control of the 3'UTR of the S segment of the sand virus.

[0230] *ORF indicates a heterologous ORF; for example, a heterologous ORF encoding the HBV antigen has been inserted.

[0231]

[0232]

[0233] In some embodiments, the IGR between positions one and two can be an S-fragment or L-fragment IGR of isoprene virus; the IGR between positions two and three can be an S-fragment or L-fragment IGR of isoprene virus; and the IGR between positions five and six can be an L-fragment IGR of isoprene virus. In specific embodiments, the IGR between positions one and two can be an L-fragment IGR of isoprene virus; the IGR between positions two and three can be an L-fragment IGR of isoprene virus; and the IGR between positions five and six can be an S-fragment IGR of isoprene virus. In some embodiments, other combinations are also possible.

[0234] In some embodiments, the intersegmental recombination of the L and S fragments in a tri-fragmented arenavirus particle containing two L fragments and one S fragment restores the functional fragment, which has two viral genes located on only one fragment rather than two separate fragments. In other embodiments, the intersegmental recombination of the L and S fragments in a tri-fragmented arenavirus particle containing two L fragments and one S fragment does not produce a duplexed viral particle with a replication competent state.

[0235] Table 2B below is an example of the genomic architecture of a tri-fragmented arenavirus particle containing two L fragments and one S fragment, wherein inter-fragment recombination of the L and S fragments in the tri-fragmented arenavirus genome does not produce a replication competent bi-fragmented virus particle and cancels arenavirus promoter activity (i.e., the resulting recombinant S fragment will consist of two 3'UTRs instead of 3'UTR and 5'UTR).

[0236] Table 2B

[0237] Tri-fragmented sand virus particles containing two L fragments and one S fragment

[0238] *Location 1 is under the control of the 5'UTR of the L segment of the sand virus; Location 2 is under the control of the 3'UTR of the L segment of the sand virus; Location 3 is under the control of the 5'UTR of the L segment of the sand virus; Location 4 is under the control of the 3'UTR of the L segment of the sand virus; Location 5 is under the control of the 5'UTR of the S segment of the sand virus; Location 6 is under the control of the 3'UTR of the S segment of the sand virus.

[0239] *ORF indicates a heterologous ORF; for example, a heterologous ORF encoding the HBV antigen has been inserted.

[0240] Position 1 Location Position 3 Position 4 Position 5 Position 6 NP Z *ORF GP L *ORF NP Z GP *ORF *ORF L NP Z *ORF GP L *ORF NP Z GP *ORF *ORF L NP L *ORF GP Z *ORF NP L GP *ORF *ORF Z NP L *ORF GP Z *ORF NP L GP *ORF *ORF Z GP Z *ORF NP L *ORF GP Z NP *ORF *ORF L GP Z *ORF NP L *ORF GP L NP *ORF *ORF Z GP L *ORF NP Z *ORF GP L NP *ORF *ORF Z

[0241] In some embodiments, the IGR between positions one and two can be an S-fragment or L-fragment IGR of isoprene virus; the IGR between positions two and three can be an S-fragment or L-fragment IGR of isoprene virus; and the IGR between positions five and six can be an L-fragment IGR of isoprene virus. In specific embodiments, the IGR between positions one and two can be an L-fragment IGR of isoprene virus; the IGR between positions two and three can be an L-fragment IGR of isoprene virus; and the IGR between positions five and six can be an S-fragment IGR of isoprene virus. In some embodiments, other combinations are also possible.

[0242] In some embodiments, the tri-fragmented isoflavone virus particles described herein produce infectious and replication-competent isoflavone virus particles. In specific embodiments, the isoflavone virus particles described herein are attenuated. In particular embodiments, the tri-fragmented isoflavone virus particles are attenuated such that the virus retains, at least partially retains, replication competence and can replicate in vivo, but only produces a low viral load, resulting in a non-pathogenic subclinical level of infection. Such attenuated virus can be used as an immunogenic composition. In other embodiments, the isoflavone virus particles are infectious but cannot produce further infectious progeny in non-complement cells.

[0243] In some embodiments, the arenavirus genome fragment and the corresponding arenavirus particle or tri-fragmented arenavirus particle may contain a heterologous ORF. In other embodiments, the arenavirus genome fragment and the corresponding arenavirus particle or tri-fragmented arenavirus particle may contain a gene of interest. In a more specific embodiment, the heterologous ORF or gene of interest encodes an antigen. In a more specific embodiment, the heterologous ORF or target gene encodes an HBV antigen or an antigenic fragment thereof (see Section 6.2).

[0244] In some embodiments, the arenavirus genome fragment, the arenavirus particle, or the tri-fragmented arenavirus particle may contain one or more heterologous ORFs or one or more target genes. In other embodiments, the arenavirus genome fragment, the arenavirus particle, or the tri-fragmented arenavirus particle may contain at least one heterologous ORF, at least two heterologous ORFs, at least three heterologous ORFs, or more heterologous ORFs. In other embodiments, the arenavirus particle or the tri-fragmented arenavirus particle contains at least one target gene, at least two target genes, at least three target genes, or more target genes. In a more specific embodiment, one or more heterologous ORFs or target genes encode one or more HBV antigens or antigenic fragments thereof (see Section 6.2).

[0245] In some embodiments, the infectious arenavirus expressing HBV antigen described herein is a trifragmented arenavirus particle comprising one L fragment and two S fragments.

[0246] 6.2 HBV antigen

[0247] In some embodiments, the antigen used in the methods and compositions described herein is the HBV antigen.

[0248] In some implementations, the ORFs of two or more HBV antigens described are transcribed as a single transcript.

[0249] In some embodiments, any genotype or subgenotype of human HBV or any clinical isolate of human HBV can be used in this invention to obtain antigens for producing the arenavirus vectors described herein. Such HBV genotypes and subgenotypes include genotypes AJ and subgenotypes A1-A6, B1-B4, C1-C6, D1-D7, and F1-F4.

[0250] In some embodiments, the HBV antigen may be an ortholog of the HBV antigen, such as mammalian (i.e., non-human primate, pig, dog, cat or horse) HBV antigen.

[0251] (a) pre-S2 / S protein antigen

[0252] In some embodiments, the antigen is an HBV pre-S2 / S protein or a fragment thereof. In some embodiments, the antigen is a fragment of the HBV pre-S2 / S protein containing at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids. In some embodiments, the antigen is an antigenic fragment of the HBV pre-S2 / S protein. In some embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

[0253] (b) HBc protein antigen

[0254] In some embodiments, the antigen is HBV HBc protein or a fragment thereof. In some embodiments, the antigen is a fragment of HBV HBc protein containing at least 10, 15, 20, 25, 50, 75, 100, 125, 150 or more amino acids. In some embodiments, the antigen is an antigenic fragment of HBc. In some embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

[0255] (c) HBs protein antigen

[0256] In some embodiments, the antigen is HBV HBs protein or a fragment thereof. In some embodiments, the antigen is a fragment of HBV HBs protein containing at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In some embodiments, the antigen is an antigenic fragment of HBs.

[0257] In some embodiments, the antigen is a small HBV HBs polypeptide (e.g., "S") or a fragment thereof. In some embodiments, the antigen is a medium HBV HBs polypeptide (e.g., "pre-S2 / S") or a fragment thereof. In some embodiments, the antigen is a large HBV HBs polypeptide (e.g., "pre-S1 / pre-S2 / S") or a fragment thereof. In some embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids of a small HBV HBs polypeptide. In some embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids of a polypeptide of HBV HBs. In some embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350 or more amino acids of HBV HBs macropeptide.

[0258] (d) HBs and HBc fusion protein

[0259] In some embodiments, the antigen is a fusion protein of HBV HBs and HBc proteins or antigenic fragments thereof. In some embodiments, the antigen is a fragment of at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225 or more amino acids of a fusion protein of HBs and HBc. In some embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.

[0260] (e) HBe protein antigen

[0261] In some embodiments, the antigen is HBV HBe protein or a fragment thereof. In some embodiments, the antigen is a fragment of HBV HBe protein containing at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150 or more amino acids. In some embodiments, the antigen is an antigenic fragment of HBe. In some embodiments, the antigen is encoded by a nucleic acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, the antigen comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26.

[0262] (f) Polymerase protein antigen

[0263] In some embodiments, the antigen is an HBV polymerase protein or an antigenic fragment thereof. In some embodiments, the antigen is a fragment of an HBV polymerase protein containing at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700 or more amino acids.

[0264] The nucleic acid sequence encoding the HBV antigen can be introduced into the genome of infectious arenavirus by replacing the nucleic acid sequence of the ORF of glycoprotein GP, ​​matrix protein Z, nucleoprotein NP, or polymerase protein L. In other embodiments, the nucleic acid sequence encoding the HBV antigen is fused to the ORF of glycoprotein GP, ​​matrix protein Z, nucleoprotein NP, or polymerase protein L. Once inserted into the genome of infectious arenavirus, the nucleotide sequence encoding the HBV antigen can be transcribed and / or expressed under the control of four arenavirus promoters (5'UTR and 3'UTR of the S fragment, 5'UTR and 3'UTR of the L fragment) and one of the ribonucleic acids that can be inserted along with regulatory elements that can be read by viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II, or RNA polymerase III, for example, copies of viral promoter sequences naturally present in viral UTRs, 28S ribosomal RNA promoters, β-actin promoters, or 5S ribosomal RNA promoters. The nucleic acid encoding the HBV antigen can be transcribed and / or expressed on its own, or transcribed and / or expressed in a readthrough manner by fusing with the arenavirus ORF and gene, and / or by combining with one or more, such as two, three, or four internal ribosome entry sites.

[0265] In one embodiment, the antigen is an antigen useful for the prevention and / or treatment of infectious diseases. In a specific embodiment, the antigen is derived from HBV. In some embodiments, the ORF encoding a isopyrvirus glycoprotein is replaced by a nucleic acid sequence encoding the HBV pre-S2 / S protein. In some embodiments, the ORF encoding a isopyrvirus glycoprotein is replaced by a nucleic acid sequence encoding the HBV HBc protein. In some embodiments, the ORF encoding a isopyrvirus glycoprotein is replaced by a nucleic acid sequence encoding the HBV HBs protein. In some embodiments, the ORF encoding a isopyrvirus glycoprotein is replaced by a nucleic acid sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0266] (g) Substitution of the ORF encoding the glycoprotein of arenavirus

[0267] In some implementations, the ORF encoding the glycoprotein of the sand virus is replaced by a nucleic acid sequence encoding one, two or more HBV antigens as described herein.

[0268] In one embodiment, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an HBV antigen. In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigen, said antigen being a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids of the gene product of the gene or a fragment of the HBV pre-S2 / S protein. In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigenic fragment of pre-S2 / S. In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigen, said antigen including, but not limited to, pre-S2 / S or fragments of pre-S2 / S.

[0269] In some embodiments, the ORF encoding the isovirus glycoprotein is replaced by a nucleic acid sequence encoding an antigen, said antigen being a fragment of the gene product of the HBV HBc protein gene or a fragment thereof, comprising at least 10, 15, 20, 25, 50, 75, 100, 125, 150, or more amino acids. In some embodiments, the ORF encoding the isovirus glycoprotein is replaced by a nucleic acid sequence encoding an antigenic fragment of HBc. In some embodiments, the ORF encoding the isovirus glycoprotein is replaced by a nucleic acid sequence encoding an antigen, said antigen including, but not limited to, HBc or fragments of HBc.

[0270] In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigen, said antigen being a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids of the gene product of the HBs protein gene or a fragment thereof. In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigenic fragment of HBs. In some embodiments, the ORF encoding a glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding an antigen, said antigen including, but not limited to, HBs or fragments of HBs.

[0271] In some embodiments, the ORF encoding the glycoprotein of arenavirus is replaced by a nucleic acid sequence encoding two or more HBV proteins or fragments of at least 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225 or more amino acids thereof. In some embodiments, the ORF encoding the arenavirus sugar and HBc is replaced by a nucleic acid sequence encoding HBs and HBc.

[0272] In some embodiments, the ORF encoding the glycoprotein of the sand virus is replaced by a nucleic acid sequence encoding one or more of the pre-S2 / S protein or an antigenic fragment thereof, the HBc protein or an antigenic fragment thereof, the HBs protein or an antigenic fragment thereof, and the Hbe protein or an antigenic fragment thereof.

[0273] 6.3 Production of infectious arenaviruses expressing HBV antigens

[0274] Generally, arenavirus particles can be recombinantly generated using standard reverse genetic techniques described for LCMV (L. Flatz, A. Bergthaler, JC de la Torre, and DDPinschewer, Proc Natl Acad SciUSA 103:4663-4668, 2006; ABSanchez and JC de la Torre, Virology 350:370, 2006; E. Ortiz-Riano, BY Cheng, JC de la Torre, L. Martinez-Sobrido. J Gen Virol. 94:1175-88, 2013).

[0275] (a) Replication-defective arenavirus

[0276] These techniques can be used to generate infectious, replication-defective arenaviruses for use in this invention, however, the genome of the rescued virus is modified as described in Section 6.1. These modifications may include: i) the removal or functional inactivation of one or more of the four arenavirus ORFs (glycoprotein (GP); nucleoprotein (NP); matrix protein Z; RNA-dependent RNA polymerase L), for example, two, three, or four, to prevent the formation of infectious particles in normal cells, but still allow gene expression in host cells infected with the arenavirus vector; and ii) the introduction of nucleic acids encoding HBV antigens. The infectious, replication-defective viruses described herein can be produced as described in International Patent Application Publication No. WO 2009 / 083210 (Application No. PCT / EP2008 / 010994) and International Patent Application Publication No. WO 2014 / 140301 (Application No. PCT / EP2014 / 055144), each of which is incorporated herein by full reference.

[0277] Once generated from cDNA, the infectious replication-defective arenavirus provided herein can proliferate in supplementary cells. Supplementary cells are cells that provide functionality that has been eliminated from replication-defective arenaviruses through modifications to their genome (e.g., supplementary cells provide the GP protein if the ORF encoding the GP protein is deleted or its function is inactivated).

[0278] Due to the removal or functional inactivation of one or more viral genes in the arenavirus vector (here, deletion of glycoprotein GP is an example), the arenavirus vector can be generated and amplified in cells that trans-provide the deleted viral genes, for example, GP in the current embodiment. Such supplementary cell lines, hereinafter referred to as C-cells, are generated by transfecting mammalian cell lines such as BHK-21, HEK 293, VERO, or others (here, BHK-21 is an example) with one or more plasmids expressing the target viral gene (supplementary plasmids, referred to as C-plasmids). Under the control of one or more expression cassettes suitable for expression in mammalian cells, such as mammalian polymerase II promoters, such as CMV or EF1alpha promoters with polyadenylation signals, the C-plasmid expresses the viral genes deleted from the arenavirus vector to be generated. In addition, supplemental plasmids may contain mammalian selection markers, such as puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells, such as the aforementioned polymerase II expression cassette, or the viral gene transcript may be followed by an internal ribosome entry site, such as one of the encephalocardiitis viruses, followed by a mammalian resistance marker. For production in E. coli, the plasmid additionally contains bacterial selection markers, such as an ampicillin resistance cassette.

[0279] Usable cells, such as BHK-21, HEK 293, MC57G, or other cell lines, can be maintained in culture and transfected using any commonly used strategy, such as calcium phosphate-based, liposome-based, or electroporation with supplemental plasmids. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and highly expressed C-cell clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry. As an alternative to using stably transfected C-cells, transient transfection of normal cells can supplement missing viral genes in each step below using C-cells. Additionally, helper viruses can be used to trans-provide the missing function.

[0280] The plasmids that can be used can be of two types: i) two plasmids, called TF-platmids, for intracellular expression of minimal trans-acting factors of arenavirus in C-cells, derived in the current embodiment from, for example, the NP and L proteins of LCMV; and ii) plasmids, called GS-platmids, for intracellular expression of arenavirus vector genomic fragments in C-cells, for example, fragments with designed modifications. The TF-platmid expresses the NP and L proteins of the corresponding arenavirus vector under the control of an expression cassette suitable for protein expression in mammalian cells, generally a mammalian polymerase II promoter, such as the CMV or EF1alpha promoter, either of which is preferably combined with a polyadenylation signal. The GS-platmid expresses the small (S) and large (L) genomic fragments of the vector. Generally, polymerase I-driven expression cassettes or T7 phage RNA polymerase (T7-)-driven expression cassettes can be used, the latter preferably having a 3'-terminal ribozyme for processing the initial transcript to produce the correct ends. When using a T7-based system, it is necessary to provide additional T7 expression in a stable manner by constructing other expression plasmids that provide T7, such as those similar to TF-plasmids, during the recovery process, or by constructing C-cells to provide T7 expression in C-cells. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via T7, polI, and polII promoters.

[0281] To recover the arenavirus vector, the following procedure can be used. Day 1: C cells, typically 80% confluent on M6-well plates, are transfected with a mixture of two TF-plasmids and two GS-plasmids. In some implementations, the TF and GS plasmids can be the same, i.e., the genomic sequence and trans-acting factors can be transcribed from a single plasmid via the T7, polI, and polII promoters. For this purpose, any commonly used strategy can be utilized, such as calcium phosphate-based, liposome-based protocols, or electroporation.

[0282] After 3-5 days: Harvest the culture supernatant (arenavirus vector product), aliquot it, and store it at 4°C, -20°C, or -80°C, depending on the storage time required before use. Then, assess the infectious titer of the arenavirus vector product using immunofocal analysis on C-cells.

[0283] This invention further relates to the expression of HBV antigen in cell cultures, wherein the cell cultures are infected with an infectious isovirus expressing HBV antigen. When used to express HBV antigen in cultured cells, the following two processes can be used:

[0284] i) Infect the target cell type with one or more, such as two, three or four, multiples of infection (MOI) using the arenavirus vector product described herein, causing the production of the HBV antigen described herein in all cells shortly after infection.

[0285] ii) Alternatively, a lower MOI can be used, and individual cell clones can be selected based on the level of virus-driven HBV antigen expression. Subsequently, due to the non-cytolytic nature of the arenavirus vector, individual clones can be expanded indefinitely. Regardless of the pathway, HBV antigen can then be collected (and purified) from the culture supernatant or from the cells themselves, depending on the nature of the HBV antigen produced. However, the invention is not limited to these two strategies, and other methods using infectious, replication-defective arenaviruses as vectors to drive HBV antigen expression can also be considered.

[0286] Alternatively, a rescue system consisting of three plasmids can be used: (1) the first plasmid expresses protein NP via transcription by polymerase II and subsequent translation in transfected cells; (2) the second plasmid generates the (negative strand) L fragment of the LCMV genome via transcription by polymerase I and generates the L protein via transcription by polymerase II in the opposite direction to the polymerase I promoter; and (3) the third plasmid generates the S-fraction of the LCMV genome (encoding the antigen-coding sequence rather than the LCMV glycoprotein) via transcription by polymerase I. 3 μg of each plasmid was used for electroporation of C-cells, followed by seeding the cells on 6-well plates and incubation at 37°C. After incubation, cells and supernatant from the transfected material were combined with freshly seeded C-cells, the vector was harvested at a post-infection time point, and the cells / debris was washed away. Once the vector has been generated, nucleic acids encoding antigens of oncogenic viruses and / or immunomodulatory peptides, polypeptides, or proteins (see Section 6.2) can be inserted into a plasmid, from which genomic fragments of the infectious replication-defective vector are transcribed using any technique known to those skilled in the art. Due to the removal or functional inactivation of one or more viral genes in the arenavirus vector (here, deletion of the glycoprotein GP is an example), the arenavirus vector can be generated and amplified in cells that trans-provide the deleted or functionally inactivated viral gene (e.g., GP). The resulting virus is infectious in itself, but due to the lack of the deleted or functionally inactivated viral gene (e.g., GP), it cannot generate further infectious progeny particles in non-supplementary cells. Supplementary cells can provide the missing functionality through stable transfection, transient transfection, or infection with a helper virus expressing the missing functionality.

[0287] In some embodiments, the supplementary cell provides a viral gene that has been deleted or functionally inactivated from the arenavirus vector genome. In a specific embodiment, the supplementary cell provides a viral gene from a viral strain identical to the viral strain used to generate the arenavirus vector genome. In another embodiment, the supplementary cell provides a viral gene from a viral strain different from the viral strain used to generate the arenavirus vector genome. For example, the viral gene provided in the supplementary cell is derived from the MP strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 15, 16, 17, or 18. In another embodiment, the viral gene provided in the supplementary cell is derived from the clone 13 strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 21, 22, 23, or 24. In yet another embodiment, the viral gene provided in the supplementary cell is derived from the WE strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 25.

[0288] In a specific embodiment, the supplementary cell provides the GP of the MP strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cell provides the GP of the MP strain of LCMV, the arenavirus vector is obtained from LCMV clone 13, and contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0289] In a specific embodiment, the supplementary cell provides the GP of LCMV clone 13, and the arenavirus vector contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cell provides the GP of LCMV clone 13, the arenavirus vector is obtained from the LCMV MP strain, and contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0290] In a specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, the arenavirus vector is obtained from LCMV clone 13, and contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0291] In a specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, the arenavirus vector is obtained from the LCMV MP strain, and contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0292] In some implementations, the infectious, replication-defective sand virus is tri-fragmented.

[0293] (b) Replication of competent cells in three-segmented arena virus

[0294] The methods and compositions used herein are methods for producing replicating competent arenavirus vectors. The infectious, replicating competent tri-fragmented virus described herein can be produced as described in U.S. Provisional Patent Application No. 62 / 079,493, the entirety of which is incorporated herein by reference.

[0295] In some embodiments, the method for generating tri-fragmented arenavirus particles includes (i) transfecting a host cell with cDNA of one L fragment and two S fragments, or two L fragments and one S fragment; (ii) transfecting a host cell with a plasmid expressing the minimal trans-acting factors NP and L of arenavirus; (iii) maintaining the host cell under conditions suitable for virus formation; and (vi) harvesting the arenavirus particles.

[0296] Once generated from cDNA, the tri-fragmented arenavirus particles (i.e., infectious and replication-competent) can be proliferated. In some embodiments, the tri-fragmented arenavirus particles can proliferate in any host cell that allows the virus to grow to a titer that permits the use of the virus described herein. In one embodiment, the host cell allows the tri-fragmented arenavirus particles to grow to a titer comparable to that determined for the corresponding wild type.

[0297] In some embodiments, the tri-fragmented arenavirus particles can be propagated in host cells. Specific examples of host cells that can be used include BHK-21, HEK 293, VERO, or others. In specific embodiments, the tri-fragmented arenavirus particles can be propagated in cell lines.

[0298] In some embodiments, the host cell is held in a culture and transfected with one or more plasmids. Under the control of one or more expression cassettes suitable for expression in mammalian cells, the plasmid expresses the desired isovirus genome fragment, for example, the expression cassette consisting of a polymerase I promoter and a terminator.

[0299] In a specific implementation, the host cell is held in a culture and transfected with one or more plasmids. Under the control of one or more expression cassettes suitable for expression in mammalian cells, the plasmid expresses the viral gene to be produced; for example, the expression cassette consists of a polymerase I promoter and a terminator.

[0300] Plasmids that can be used to generate trifragmented arenaviruses containing one L fragment and two S fragments include: i) two plasmids, each encoding an S genome fragment, such as a pol-I driven S fragment expression plasmid; and ii) a plasmid encoding an L genome fragment, such as a pol-I driven L fragment expression plasmid. Plasmids required for trifragmented arenaviruses containing two L fragments and one S fragment are: i) two plasmids, each encoding an L genome fragment, such as pol-L; and ii) a plasmid encoding an S genome fragment, such as pol-I S.

[0301] In some embodiments, plasmids encoding arenavirus polymerases that guide the intracellular synthesis of the L and S fragments of the virus can be incorporated into the transfection mixture. For example, plasmids encoding the L protein and NP (pC-L and pC-NP, respectively). The L protein and NP are minimal trans-acting factors essential for viral RNA transcription and replication. Alternatively, along with the NP and L protein, the intracellular synthesis of the viral L and S fragments can be performed using an expression cassette with pol-I and pol-II promoters that read the L and S fragment cDNAs from two separate plasmids, respectively.

[0302] In addition, the plasmid may contain mammalian selection markers, such as puromycin resistance, under the control of an expression cassette suitable for gene expression in mammalian cells, such as the polymerase II expression cassette described above, or the viral gene transcript may be followed by an internal ribosome entry site, such as that of encephalocardiitis virus, and then a mammalian resistance marker. For production in E. coli, the plasmid may also contain bacterial selection markers, such as an ampicillin resistance cassette.

[0303] Plasmid transfection of BHK-21 cells can be performed using any commonly used strategy, such as calcium phosphate-based, liposome-based, or electroporation protocols. Several days later, a suitable selection reagent, such as puromycin, is added at a titrated concentration. Viable clones are isolated and subcloned according to standard procedures, and high-expressing clones are identified using antibodies against the target viral protein, Western blotting, or flow cytometry.

[0304] Generally, polymerase I-driven expression cassettes, RNA polymerase II-driven cassettes, or T7 phage RNA polymerase-driven cassettes can be used, the latter preferably having a 3' terminal ribozyme for processing the initial transcript to produce the correct ends. In some embodiments, the plasmid encoding a fragment of the arenavirus genome can be the same, i.e., the genome sequence and trans-acting factor can be transcribed from a single plasmid via T7, polI, and polII promoters.

[0305] To recover the tri-fragmented isovirus vector, the following procedure is envisioned. Day 1: Cells, typically 80% confluent on M6-well plates, are transfected with a mixture of plasmids as described above. For this, any commonly used strategy can be employed, such as calcium phosphate-based, liposome-based protocols, or electroporation.

[0306] 3-5 days later: Harvest the culture supernatant (arenavirus vector product), aliquot and store at 4°C, -20°C, or -80°C, depending on the storage time required before use. Assess the infectious titer of the arenavirus vector product using immunofocal analysis. Alternatively, transfected cells and supernatant can be passaged into larger containers (e.g., T75 tissue culture flasks) 3-5 days after transfection, and the culture supernatant can be harvested 5 days after passage.

[0307] This application further relates to the expression of heterologous ORFs (e.g., HBV antigens), wherein plasmids encoding genomic fragments are modified to incorporate heterologous ORFs. Heterologous ORFs can be incorporated into plasmids using restriction enzymes. In some embodiments, the heterologous ORF encodes an HBV antigen. In some embodiments, plasmids encoding genomic fragments are modified to incorporate one or more heterologous ORFs. In some embodiments, the heterologous ORF encodes one or more HBV antigens.

[0308] 6.4 Nucleic Acids, Vector Systems, and Cell Lines

[0309] In one embodiment, the nucleic acid sequence described herein is the cDNA of a large genomic fragment (L fragment) of the infectious arenavirus described herein, wherein one ORF of the genomic fragment is deleted or functionally inactivated, and the genomic fragment contains a nucleotide sequence encoding an HBV antigen. In some embodiments, the viral vector of the infectious arenavirus is replication-defective (see Section 6.1(a)). In some embodiments, the viral vector of the infectious arenavirus is replication-competent (see Section 6.1(b)).

[0310] In one embodiment, the present invention describes a nucleic acid sequence encoding a short genomic fragment (S fragment) of the infectious arenavirus described herein, wherein an ORF of the genomic fragment is deleted or functionally inactivated, and wherein the short genomic fragment contains a nucleotide sequence encoding an HBV antigen. In another embodiment, the present invention describes a nucleic acid sequence encoding a short genomic fragment (S fragment) of the infectious arenavirus described herein, wherein an ORF of the glycoprotein gene is deleted or functionally inactivated, and wherein the short genomic fragment contains a nucleotide sequence encoding an HBV antigen. In a more specific embodiment, the HBV antigen is the antigen described in section 6.2.

[0311] In some embodiments, the nucleic acid sequences provided herein may be derived from specific strains of LCMV. LCMV strains include clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, 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 LCMV clone 13. In other specific embodiments, the nucleic acid is derived from the LCMV MP strain.

[0312] In a more specific embodiment, this document provides a nucleic acid comprising a fragment of a sand virus genome containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another embodiment, this document provides a nucleic acid comprising a fragment of a sand virus genome containing (i) a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotides 1639 to 3315 of SEQ ID NO: 11; and (ii) a nucleotide sequence encoding an HBV antigen.

[0313] In another embodiment, this document provides a nucleic acid comprising a fragment of a sand virus genome, the sand virus genome comprising (i) a nucleotide sequence encoding an expression product, the amino acid sequence of which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 11 from 1639 to 3315; and (ii) a nucleotide sequence encoding an HBV antigen.

[0314] In another embodiment, this document provides a nucleic acid comprising a fragment of a sand virus genome comprising (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the sequence of nucleotides 1640 to 3316 of SEQ ID NO: 12; and (ii) a nucleotide sequence encoding an HBV antigen.

[0315] In another embodiment, this document provides a nucleic acid comprising a fragment of a sand virus genome, the sand virus genome comprising (i) a nucleotide sequence encoding an expression product, the amino acid sequence of which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 12 from 1640 to 3316; and (ii) a nucleotide sequence encoding an HBV antigen.

[0316] In one embodiment, this document describes a vector system comprising one or more vectors, said one or more vectors collectively containing the genome of the infectious arenavirus particle described herein. Specifically, this document provides a vector system in which one or more vectors contain two arenavirus genome fragments, namely, the L fragment and the S fragment, of the infectious arenavirus described herein. Such a vector system may comprise (on one or more separate DNA molecules):

[0317] The arenavirus S genome segment, which is modified so that arenavirus particles carrying this modified S genome segment cannot produce infectious progeny virus particles, and the arenavirus L genome segment, which contains nucleotide sequences encoding (positive or negative) HBV antigens.

[0318] The arenavirus L genome segment, which is modified so that arenavirus particles carrying this modified L genome segment cannot produce infectious progeny virus particles, and the arenavirus S genome segment, which contains nucleotide sequences encoding (positive or negative) HBV antigens.

[0319] A isovirus S genome fragment, modified such that isovirus particles carrying this modified S genome fragment cannot produce infectious progeny virus particles, and wherein said isovirus S genome fragment contains a nucleotide sequence encoding (sense or antisense) HBV antigens and contains a wild-type isovirus L genome fragment; or

[0320] The arenavirus L genome fragment is modified such that arenavirus particles carrying this modified L genome fragment cannot produce infectious progeny virus particles, and the arenavirus L genome fragment contains a nucleotide sequence encoding (positive or negative) HBV antigens and contains a wild-type arenavirus S genome fragment.

[0321] In some embodiments, this document describes a nucleic acid sequence comprising a segment of a arenavirus (e.g., LCMV) genome, wherein the ORF encoding the GP segment of the S genome is replaced by a nucleotide sequence comprising:

[0322] Nucleotide sequences encoding hepatitis B pre-S2 / S protein or antigenic fragments thereof;

[0323] The nucleotide sequence encoding the hepatitis B virus HBc protein or its antigenic fragment;

[0324] Nucleotide sequences encoding hepatitis B virus HBs protein or antigenic fragments thereof;

[0325] Nucleotide sequences encoding fusions of hepatitis B virus HBs and HBc proteins or antigenic fragments thereof;

[0326] Nucleotide sequences encoding the hepatitis B virus HBe protein or its antigenic fragments.

[0327] In some embodiments, this document describes a nucleic acid sequence comprising a segment of a arenavirus (e.g., LCMV) genome, wherein the ORF encoding the GP of the S genome segment is replaced by a nucleotide sequence encoding one or more HBV antigens (e.g., one or more of those listed in the paragraphs above).

[0328] In another embodiment, this document provides cells comprising the nucleic acid or vector systems described above in this section. This document also provides cell lines derived from such cells, cultures comprising such cells, and methods for culturing such cells infected with nucleic acid or vector systems. In some embodiments, this document provides cells comprising nucleic acids containing a large genomic fragment (L fragment) of the infectious arenavirus described herein, wherein an ORF of the genomic fragment is deleted or functionally inactivated, and the genomic fragment contains a nucleotide sequence encoding an HBV antigen.

[0329] In other embodiments, this document provides a cell containing a nucleic acid sequence comprising a short genomic fragment (S fragment) of an infectious arenavirus described herein, wherein an ORF of the genomic fragment is deleted or functionally inactivated, and wherein the short genomic fragment contains a nucleotide sequence encoding an HBV pre-S2 / S protein or an antigenic fragment thereof.

[0330] In other embodiments, this document provides a cell, wherein the cell contains a nucleic acid sequence comprising a short genomic fragment (S fragment) of the infectious arenavirus described herein, wherein an ORF of the genomic fragment is deleted or functionally inactivated, and wherein the short genomic fragment contains a nucleotide sequence encoding an HBV HBc protein or an antigenic fragment thereof.

[0331] In other embodiments, this document provides a cell, wherein the cell contains a nucleic acid sequence comprising a short genomic fragment (S fragment) of an infectious arenavirus described herein, wherein an ORF of the genomic fragment is deleted or functionally inactivated, and wherein the short genomic fragment contains a nucleotide sequence encoding an HBV HBs protein or an antigenic fragment thereof.

[0332] In other embodiments, this document provides a cell, wherein the cell comprises a nucleic acid sequence comprising a short genome fragment (S fragment) of an infectious arenavirus described herein, wherein an ORF of the genome fragment is deleted or functionally inactivated, and wherein the short genome fragment comprises a nucleotide sequence encoding a fusion protein comprising at least one domain from HBV HBs protein and HBV HBc protein.

[0333] In other embodiments, this document provides a cell containing a nucleic acid sequence comprising a short genome fragment (S fragment) of an infectious arenavirus described herein, wherein an ORF of the genome fragment is deleted or functionally inactivated, and wherein the short genome fragment contains a nucleotide sequence encoding one or more HBV antigens.

[0334] In another embodiment described herein, cells are provided, wherein the cells comprise the two nucleic acid or vector systems described herein. Cell lines derived from such cells, cultures comprising such cells, and methods for culturing such cells infected with nucleic acid or vector systems are also provided herein.

[0335] In some embodiments, this document provides a nucleic acid comprising a nucleotide sequence that is 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 to SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, this document provides an expression vector comprising a nucleotide sequence that is 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 to SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, this document provides a host cell containing a nucleotide sequence that is 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 to SEQ ID NO: 13 or SEQ ID NO: 14.

[0336] In some embodiments, this document provides a nucleic acid comprising a nucleotide sequence that encodes 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 to the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18. In some embodiments, this document provides an expression vector comprising a nucleotide sequence that encodes 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 to the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18. In some embodiments, this document provides a host cell comprising a nucleotide sequence that encodes 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 to the amino acid sequence of SEQ ID NO: 15, 16, 17, or 18.

[0337] In some embodiments, this document provides an isolated protein comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 15, 16, 17, or 18. In some embodiments, this document provides a host cell expressing a protein comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 15, 16, 17, or 18. In some embodiments, the host cells are cultured in a cell culture medium.

[0338] In some embodiments, this document provides a nucleic acid comprising a nucleotide sequence that is 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 to SEQ ID NO: 12 or SEQ ID NO: 7. In some embodiments, this document provides an expression vector comprising a nucleotide sequence that is 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 to SEQ ID NO: 12 or SEQ ID NO: 7. In some embodiments, this document provides a host cell containing a nucleotide sequence that is 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 to SEQ ID NO: 12 or SEQ ID NO: 7.

[0339] In some embodiments, this document provides a nucleic acid comprising a nucleotide sequence that encodes 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 to the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In some embodiments, this document provides an expression vector comprising a nucleotide sequence encoding 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 to the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24. In some embodiments, this document provides a host cell comprising a nucleotide sequence that encodes 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 to the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24.

[0340] In some embodiments, this document provides an isolated protein comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 21, 22, 23, or 24. In some embodiments, this document provides a host cell expressing a protein comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 21, 22, 23, or 24. In some embodiments, the host cells are cultured in a cell culture medium.

[0341] 6.5 Usage Instructions

[0342] This document provides immunotherapy for hepatitis B virus infection. In one embodiment, this document provides a method for treating an infection in a subject, comprising administering to the subject one or more areonaviruses or combinations thereof expressing HBV antigens as described herein. In some embodiments, the infectious areonavirus is replication-defective. In some embodiments, the infectious areonavirus is replication-competent. In a specific embodiment, the method for treating an infection described herein comprises administering to a subject in need an effective amount of one or more infectious areonaviruses expressing HBV antigens as described herein. The subject may be a mammal, such as, but not limited to, humans, mice, rats, guinea pigs, domesticated animals, such as, but not limited to, cattle, horses, sheep, pigs, goats, cats, dogs, hamsters, and donkeys. In a specific embodiment, the subject is a human.

[0343] In another embodiment, this document provides a method for inducing an immune response against HBV in a subject, comprising administering to the subject a sand virus expressing HBV antigen or a combination thereof.

[0344] In another embodiment, the subject receiving the infectious isovirus or a composition thereof expressing HBV antigen described herein is HBV infected, susceptible to HBV infection, or at risk of HBV infection. In yet another specific embodiment, the subject receiving the infectious isovirus or a composition thereof expressing HBV antigen described herein is HBV infected, susceptible to HBV infection, or at risk of HBV infection.

[0345] In another embodiment, a subject receiving the infectious isovirus or a composition thereof expressing HBV antigen described herein is infected with HBV, is susceptible to HBV infection, or is at risk of HBV infection, for example, in the liver. In a specific embodiment, a subject receiving the infectious isovirus or a composition thereof expressing HBV antigen described herein is infected with HBV, is susceptible to HBV infection, or is at risk of HBV infection in one or more organs of the body, such as the liver.

[0346] In another embodiment, the subject administering the infectious isovirus expressing the HBV antigen or a composition thereof described herein has test results indicating liver damage (e.g., blood test results). In some embodiments, the subject has alanine aminotransferase (ALT) levels in the blood indicating liver damage. In some embodiments, the subject has aspartate aminotransferase (AST) levels in the blood indicating liver damage. In some embodiments, the subject has alkaline phosphatase levels in the blood indicating liver damage. In some embodiments, the subject has lactate dehydrogenase (LDH) levels in the blood indicating liver damage. In some embodiments, the subject has one or more of the ALT, AST, alkaline phosphatase, and LDH levels in the blood indicating liver damage.

[0347] In some embodiments, the subject has a blood level of alpha-fetoprotein (AFP) indicating liver cancer or susceptibility to liver cancer. In some embodiments, the subject has a blood level of bilirubin (e.g., conjugated bilirubin) indicating liver damage. In some embodiments, the subject has a blood level of albumin indicating liver damage.

[0348] In some embodiments, the object has abdominal ultrasound results indicating liver injury. In some embodiments, the object has CAT scan results indicating liver injury. In some embodiments, the object has MRI results indicating liver injury.

[0349] In another embodiment, the subject administering the infectious isovirus expressing HBV antigen or a composition thereof described herein has detectable levels of HBs antigen (HBsAg) in their blood. In some embodiments, the subject has detectable levels of IgM antibody against HBc antigen (HBcAg) in their blood. In some embodiments, the subject has detectable levels of HBe antigen (HBeAg, the extracellular / secreted version of the HBc protein) in their blood. In some embodiments, the subject has detectable levels of antibody against HBsAg in their blood.

[0350] In another embodiment, the subject receiving the infectious isovirus expressing HBV antigen or a composition thereof described herein has a persistent level of HBsAg indicating chronic hepatitis. In some embodiments, the subject has a persistent level of HBeAg indicating chronic hepatitis. In some embodiments, the subject has both HBsAg and HBeAg indicating chronic hepatitis.

[0351] In another embodiment, the subject receiving the infectious isovirus or a composition thereof expressing the HBV antigen described herein has symptoms of HBV infection, including but not limited to loss of appetite, fatigue, nausea, vomiting, itching, abdominal pain, bloating, or jaundice.

[0352] In another embodiment, the subject to which the infectious isonavirus or a composition thereof expressing HBV antigen described herein is administered has HBV manifestations, including but not limited to acute hepatitis B, chronic HBV infection, cirrhosis, and hepatocellular carcinoma (HCC). In another embodiment, the infectious isonavirus or a composition thereof expressing HBV antigen described herein is administered to a subject suffering from asymptomatic HBV.

[0353] In another embodiment, the infectious arenavirus or its composition expressing HBV antigen described herein is administered to subjects of any age group who are infected with HBV, susceptible to HBV infection, or at risk of HBV infection. In a specific embodiment, the infectious arenavirus or its composition expressing HBV antigen described herein is administered to subjects with compromised immune systems, pregnant subjects, subjects undergoing organ or bone marrow transplantation, subjects taking immunosuppressive drugs, subjects undergoing hemodialysis, subjects with cancer, or subjects who are infected with, susceptible to, or at risk of HBV infection. In a more specific embodiment, the infectious arenavirus or its composition expressing HBV antigen described herein is administered to subjects with compromised immune systems due to HBV infection, who are infected with HBV, susceptible to HBV infection, or at risk of HBV infection. In yet another specific embodiment, the infectious arenavirus or its composition expressing HBV antigen described herein is administered to children aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age who are infected with, susceptible to, or at risk of HBV infection. In yet another specific embodiment, the infectious arenavirus or a composition thereof expressing HBV antigen described herein is administered to an infant who is infected with, susceptible to, or at risk of HBV infection. In yet another specific embodiment, the infectious arenavirus or a composition thereof expressing HBV antigen described herein is administered to an infant aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months who is infected with, susceptible to, or at risk of HBV infection. In yet another specific embodiment, the infectious arenavirus or a composition thereof expressing HBV antigen described herein is administered to an elderly subject who is infected with, susceptible to, or at risk of HBV infection.

[0354] In another embodiment, the infectious isonavirus or its composition expressing HBV antigen described herein is administered to a subject at increased risk of HBV infection transmission. In a specific embodiment, the infectious isonavirus or its composition expressing HBV antigen described herein is administered to a neonatal subject with an immature neonatal immune system. In another embodiment, the infectious isonavirus or its composition expressing HBV antigen described herein is administered to a subject at increased risk of HBV infection receiving intravenous medication.

[0355] In another embodiment, the infectious arenavirus expressing HBV antigen, or a composition thereof, described herein, is administered to a subject infected with one or more genotypes or subtypes of HBV. In some embodiments, the genotype is one or more genotypes AZ, or another genotype. In some embodiments, the subtype is one or more subtypes A1-A6, B1-B4, C1-C6, D1-D7, F1-F4, or another subtype.

[0356] In another embodiment, administration of the infectious isavirus expressing HBV antigen described herein to a subject confers cell-mediated immunity (CMI) against HBV infection. Without being limited by theory, in another embodiment, the infectious isavirus expressing HBV antigen described herein, or a composition thereof, infects and expresses the target antigen in antigen-presenting cells (APCs) of the host (e.g., macrophages) to guide antigen presentation on major histocompatibility complexes (MHCs) of classes I and II. In another embodiment, administration of the infectious isavirus expressing HBV antigen described herein, or a composition thereof, to a subject induces high-level, multifunctional, co-producing HBV-specific CD4+ and CD8+ T cell responses (IFN-γ produced by CD4+ and CD8+ T cells, and TNF-α produced by CD4+ T cells) to treat or prevent HBV infection.

[0357] In another embodiment, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or higher, compared to the risk of HBV infection without such treatment.

[0358] In another embodiment, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces the symptoms of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the symptom presentation of HBV infection without such treatment.

[0359] In another embodiment, administration of an infectious isavirus expressing HBV antigen or a composition thereof to a subject with an immature neonatal immune system induces a cell-mediated immune (CMI) response against HBV infection by at least about 10%, at least about 20%, at least 25%, at least 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 higher, compared to a cell-mediated immune (CMI) response against HBV infection without such treatment.

[0360] In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces ALT levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces AST levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces alkaline phosphatase levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces LDH levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces one or more of the levels of ALT, AST, alkaline phosphatase, and LDH in the blood.

[0361] In some embodiments, administration of an infectious isoflavone virus expressing HBV antigen or a composition thereof reduces AFP levels in the blood. In some embodiments, administration of an infectious isoflavone virus expressing HBV antigen or a composition thereof reduces bilirubin (e.g., conjugated bilirubin) levels in the blood. In some embodiments, administration of an infectious isoflavone virus expressing HBV antigen or a composition thereof increases albumin levels in the blood.

[0362] In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces HBsAg levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces IgM antibody levels against HBcAg in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces HBeAg levels in the blood. In some embodiments, administration of an infectious isovirus expressing HBV antigen or a composition thereof reduces antibody levels against HBsAg in the blood.

[0363] In some embodiments, administration of infectious isavirus expressing HBV antigen or a composition thereof reduces the number of inclusion bodies detected in salivary glands or another histological sample. In some embodiments, administration of infectious isavirus expressing HBV antigen or a composition thereof reduces the number of anti-HBV antibodies detected in patient blood samples. In some embodiments, administration of infectious isavirus expressing HBV antigen or a composition thereof reduces the number of HBV detected in urine, saliva, blood, tears, semen, or breast milk. In some embodiments, administration of infectious isavirus expressing HBV antigen or a composition thereof reduces the level of virus cultured from urine, throat swabs, bronchoalveolar lavage fluid, or tissue samples. In some embodiments, administration of infectious isavirus expressing HBV antigen or a composition thereof reduces the level of virus detected by quantitative or qualitative PCR testing.

[0364] The alteration of cell-mediated immune (CMI) response to HBV infection induced by administration of infectious isovirus expressing HBV antigen or a combination thereof in subjects can be measured by any analysis known to those skilled in the art, including but not limited to flow cytometry (see, e.g., Perfetto SP et al, Nat Rev Immun. 2004; 4(8):648-55), lymphocyte proliferation analysis (see, 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), analysis measuring lymphocyte activation, including measuring changes in surface marker expression after measurement of T lymphocyte cytokine activation (see, e.g., Caruso A. et al, Cytometry. 1997; 27:71-6), ELISPOT analysis (see, e.g., Czerkinsky CC et al., J Immunol. Methods. 1983; 65: 109-121; and Hutchings P.R. et al, J Immunol Methods. 1989; 120: 1-8) or natural killer cell cytotoxicity assay (see, for example, Bonilla F. et al, Ann Allergy Asthma Immunol. 2005 May; 94(5Suppl 1): S1-63).

[0365] In another embodiment, this document describes a method using an infectious arena virus (e.g., LCMV) expressing HBV antigens, wherein the ORF encoding the S genome segment is replaced by a nucleotide sequence comprising the following:

[0366] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0367] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0368] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0369] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0370] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0371] In another embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering an infectious isovirus expressing HBV antigens as described herein to a person of childbearing age. See section 6.2. In a specific embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering an infectious isovirus expressing HBV antigens as described herein to a seronegative person of childbearing age. In yet another embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering an infectious isovirus expressing HBV antigens as described herein to a person of childbearing age intending to conceive.

[0372] In another embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering to a person of childbearing age one or more infectious arenaviruses expressing HBV antigens as described herein. See Section 6.2. In a specific embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering to a seronegative person of childbearing age one or more infectious arenaviruses expressing HBV antigens as described herein. In yet another embodiment, this document provides a method for preventing HBV transmission from mother to unborn infant, comprising administering to a person of childbearing age intending to conceive one or more infectious arenaviruses expressing HBV antigens as described herein.

[0373] In another embodiment, this document provides a method for preventing HBV transmission from the mother and / or infection of the unborn infant, comprising administering to a pregnant subject an infectious isovirus expressing HBV antigens as described herein. In a specific embodiment, this document provides a method for preventing HBV transmission from the mother and / or infection of the unborn infant, comprising administering to a pregnant subject an effective amount of an infectious isovirus expressing HBV antigens as described herein.

[0374] In another embodiment, this document provides a method for preventing HBV transmission from the mother and / or infection of the unborn infant, comprising administering to a pregnant subject an infectious isovirus expressing HBV antigens as described herein. In a specific embodiment, this document provides a method for preventing HBV transmission from the mother and / or infection of the unborn infant, comprising administering to a pregnant subject an effective amount of an infectious isovirus expressing HBV antigens as described herein.

[0375] In another embodiment, administration of infectious arenavirus expressing HBV antigen reduces congenital HBV infection. In yet another embodiment, administration of one or more infectious arenaviruses expressing HBV antigen reduces congenital HBV infection.

[0376] In another embodiment, administration of infectious isavirus expressing HBV antigen reduces the incidence of congenital HBV infection by at least about 10%, at least about 20%, 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 higher. In yet another specific embodiment, administration of infectious isavirus expressing HBV antigen reduces the mortality rate of newborns with congenital HBV infection.

[0377] In another embodiment, administration of one or more infectious arenaviruses expressing HBV antigens reduces the incidence of congenital HBV infection by at least about 10%, at least about 20%, 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 higher. In another specific embodiment, administration of one or more infectious arenaviruses expressing HBV antigens reduces the mortality rate of newborns with congenital HBV infection.

[0378] This manifestation of congenital HBV includes, but is not limited to, acute hepatitis B, chronic HBV infection, cirrhosis, and hepatocellular carcinoma (HCC).

[0379] 6.6 Composition, Application and Dosage

[0380] The present invention also relates to vaccines, immunogenic compositions, and pharmaceutical compositions comprising the genetically engineered isoviruses described herein. Such vaccine and pharmaceutical compositions can be formulated according to standard procedures in the art.

[0381] In another embodiment, this document provides a composition comprising the infectious isovirus described herein. Such a composition can be used in methods of treating and preventing disease. In a specific embodiment, the composition described herein is used to treat subjects infected with HBV or susceptible to HBV infection. In another specific embodiment, the immunogenic composition provided herein can be used to induce an immune response in a host to which the composition has been administered. The immunogenic composition described herein can be used as a vaccine and thus can be formulated as a pharmaceutical composition. In a specific embodiment, the immunogenic composition described herein is used to prevent HBV infection in subjects (e.g., human subjects). In some embodiments, the viral vector of the infectious isovirus is replication-defective (see section 6.1(a)). In some embodiments, the viral vector of the infectious isovirus is replication-competent (see section 6.1(b)).

[0382] In some embodiments, this document provides an immunogenic composition comprising the arenavirus vectors described herein (or combinations of different arenavirus vectors). In some embodiments, such an immunogenic composition further comprises a pharmaceutically acceptable excipient. In some embodiments, such an immunogenic composition further comprises an adjuvant. The adjuvant for co-administration with the compositions described herein may be administered before, concurrently with, or after administration of the compositions. In some embodiments, the term "adjuvant" refers to a compound that, when administered with or as part of the compositions described herein, increases, enhances, and / or strengthens the immune response to infectious arenavirus particles, but does not produce an immune response against infectious arenavirus particles when the compound is administered alone. In some embodiments, the adjuvant produces an immune response against the infectious arenavirus particles without causing allergic reactions or other adverse reactions. Adjuvants can enhance the immune response through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages. When the vaccine or immunogenic composition of the present invention contains an adjuvant, or is administered with one or more adjuvants, the adjuvants that may be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticle adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Application WO2007 / 109813), and saponins, such as QS21 (see Kensil et al, in Vaccine Design: The Subunit and Adjuvant). Approach (eds. Powell & Newman, Plenum Press, NY, 1995; U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (e.g., squalene or peanut oil), optionally combined with an immunostimulant, such as monophosphoryl lipid A (see Stoute et al, N. Engl. J. Med. 336, 86-91 (1997)).

[0383] The composition comprises either the infectious isovirus described herein alone or with a pharmaceutically acceptable vector. A suspension or dispersion of genetically engineered isoviruses, particularly isotonic aqueous suspensions or dispersions, can be used. The pharmaceutical composition may be sterilized and / or may contain excipients, such as preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, osmotic-adjusting salts and / or buffers, prepared in a manner known in the art, for example, by conventional dispersion and suspension processing. In some embodiments, such dispersions or suspensions may contain viscosity modifiers. The suspensions or dispersions are maintained at a temperature of about 2-8°C, or preferably frozen for longer storage and then thawed immediately before use. For injectable formulations, the vaccine or immunogenic product may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks's solution, Ringer's solution, or physiological saline buffer. This solution may contain formulation reagents such as suspending agents, stabilizers, and / or dispersants.

[0384] In some embodiments, the compositions described herein additionally contain a preservative, such as the mercury derivative thimerosal. In specific embodiments, the pharmaceutical compositions described herein contain 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not contain a preservative.

[0385] The pharmaceutical composition contains about 10 3 To about 10 11 Genetically engineered sand-like viruses forming lesion units. The unit dosage form for parenteral administration is, for example, an ampoule or vial containing approximately 10... 3 Up to 10 10 One lesion forming unit or 10 5 Up to 10 15 A genetically engineered sand virus consisting of physical particles.

[0386] In another embodiment, the vaccine or immunogenic composition provided herein is administered to the subject via routes including but not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, local, subcutaneous, transdermal, intranasal, and inhalation, as well as through skin penetration (scraping away the top layer of skin, e.g., using a bifurcated needle). Specifically, subcutaneous, intramuscular, or intravenous routes may be used.

[0387] For intranasal or inhalation administration, a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide, or other suitable gas, is used to conveniently deliver the article of use according to the invention in the form of an aerosol spray from a pressurized package or nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver the measured amount. For example, gelatin capsules and cartridges used in inhalers or blowpipes can be formulated as a powder mixture containing a compound and a suitable powder base, such as lactose or starch.

[0388] The dosage of the active ingredient depends on the type and recipient of the vaccine, their age, weight, individual condition, individual pharmacokinetic data, and method of administration.

[0389] This document also provides a process and use for manufacturing vaccines in the form of pharmaceutical articles containing genetically engineered isoviruses as active ingredients. The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example, by conventional mixing and / or dispersion processes.

[0390] 6.7 Optimization of LCMV Carriers

[0391] By removing or functionally inactivating one or more viral genes in the arenavirus vector (deletion of glycoprotein GP as an example), the arenavirus vector can be generated and amplified in cells that "trans-" provide the deleted or functionally inactivated viral gene (e.g., GP). The generated virus is infectious in itself, but cannot produce further infectious progeny particles in non-supplementary cells due to the lack of the deleted or functionally inactivated viral gene (e.g., GP). Supplementary cells can provide the missing functionality through stable transfection, transient transfection, or infection with a helper virus expressing the missing functionality.

[0392] In some embodiments, the supplementary cell provides a viral gene that has been deleted or functionally inactivated from the arenavirus vector genome. In a specific embodiment, the supplementary cell provides a viral gene from a viral strain identical to the viral strain used to generate the arenavirus vector genome. In another embodiment, the supplementary cell provides a viral gene from a viral strain different from the viral strain used to generate the arenavirus vector genome. For example, the viral gene provided in the supplementary cell is derived from the MP strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 15, 16, 17, or 18. In another embodiment, the viral gene provided in the supplementary cell is derived from the clone 13 strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 21, 22, 23, or 24. In yet another embodiment, the viral gene provided in the supplementary cell is derived from the WE strain of LCMV and encodes a protein having the amino acid sequence SEQ ID NO: 25.

[0393] In a specific embodiment, the supplementary cell provides the GP of the MP strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cell provides the GP of the MP strain of LCMV, the arenavirus vector is obtained from LCMV clone 13, and contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.

[0394] In a specific embodiment, the supplementary cell provides the GP of LCMV clone 13, and the arenavirus vector contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cell provides the GP of LCMV clone 13, the arenavirus vector is obtained from the LCMV MP strain, and contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 22.

[0395] In a specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, the arenavirus vector is obtained from LCMV clone 13, and contains the ORF of the human HBV antigen described herein instead of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0396] In a specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, and the arenavirus vector contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the supplementary cells provide the GP of the WE strain of LCMV, the arenavirus vector is obtained from the LCMV MP strain, and contains the ORF of the human HBV antigen described herein in place of the ORF encoding the GP protein. In a more specific embodiment, the GP protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25.

[0397] 6.8 Combination Therapy

[0398] 6.8(a) Method

[0399] In one embodiment, this document provides a method for treating and / or preventing HBV infection in a subject, comprising administering to the subject two or more infectious arenaviruses expressing HBV antigens as described herein. See, for example, section 6.2. In a specific embodiment, the method for treating and / or preventing HBV infection comprises administering a first infectious arenavirus expressing HBV antigens as described herein, for example, wherein the ORF of the GP encoding an S genome segment is replaced by a nucleotide sequence encoding an HBV antigen, wherein the HBV antigen may be, but is not limited to:

[0400] a) The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0401] b) The nucleotide sequence encoding the HBV HBc protein or its antigenic fragment;

[0402] c) The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0403] d) The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or their antigenic fragments; and

[0404] e) The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment;

[0405] The second type isovirus expressing HBV antigens as described herein, for example, wherein the ORF encoding the S genome segment of the GP is replaced by a nucleotide sequence encoding the HBV antigen, wherein the HBV antigen may be, but is not limited to:

[0406] a) The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0407] b) The nucleotide sequence encoding the HBV HBc protein or its antigenic fragment;

[0408] c) The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0409] d) The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or their antigenic fragments; and

[0410] e) The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0411] In some embodiments, the first and second infectious arenaviruses are replication-defective. In some embodiments, the first and second infectious arenaviruses are replication-competent. In some embodiments, either the first or second infectious arenavirus is replication-defective. In some embodiments, the first and second infectious arenaviruses are bifragmented. In some embodiments, the first and second infectious arenaviruses are trifragmented. In some embodiments, either the first or second infectious arenavirus is bifragmented, and the other is trifragmented.

[0412] In specific embodiments, this document provides a method for treating and / or preventing HBV infection, comprising administering a first infectious isovirus expressing a first HBV antigen as described herein, said antigen being selected from: HBV pre-S2 / S protein or an antigenic fragment thereof; HBV HBc protein or an antigenic fragment thereof, HBV HBs protein or an antigenic fragment thereof, or HBV HBe protein or an antigenic fragment thereof; and a second infectious isovirus expressing a second HBV antigen, said antigen being selected from: a nucleotide sequence encoding HBV pre-S2 / S protein or an antigenic fragment thereof; HBV HBc protein or an antigenic fragment thereof, HBV HBs protein or an antigenic fragment thereof, or HBV HBe protein or an antigenic fragment thereof.

[0413] In some embodiments, this document provides a method for treating and / or preventing infection, comprising administering two arenavirus vector constructs expressing HBV antigens as described herein. In specific embodiments, the two arenavirus vector constructs express different HBV antigens.

[0414] In some embodiments, this document provides methods for treating and / or preventing infection, including administering two or more arenavirus vector constructs expressing HBV antigens as described herein. In specific embodiments, this document provides methods for treating and / or preventing infection, including administering three or more arenavirus vector constructs expressing HBV antigens as described herein. In some embodiments, the arenavirus vector construct may be LCMV-based.

[0415] In some embodiments, this document provides methods for treating and / or preventing infection, including administering two or more arenavirus vector constructs, each expressing a different HBV antigen, as described herein. In specific embodiments, this document provides methods for treating and / or preventing infection, including administering three or more arenavirus vector constructs, each expressing a different HBV antigen, as described herein. In some embodiments, the arenavirus vector construct may be LCMV-based.

[0416] In a specific implementation, the antigen is the HBV pre-S2 / S protein or a fragment thereof (see, for example, section 6.2(a)).

[0417] In some embodiments, the antigen is HBV HBc protein or a fragment thereof (see, for example, section 6.2(b)).

[0418] In some embodiments, the antigen is HBV HBs protein or a fragment thereof (see, for example, section 6.2(c)).

[0419] In some embodiments, the antigen is a fusion of HBV HBs and HBc proteins or antigenic fragments thereof (see, for example, section 6.2(d)).

[0420] In some embodiments, the antigen is HBV HBe protein or a fragment thereof (see, for example, section 6.2(e)).

[0421] In some embodiments, the vectors encoding one or more HBV antigens produced as described herein comprise one or more nucleic acids or combinations thereof encoding HBV antigens as described herein. In specific embodiments, the HBV antigens described herein are separated by various linkers, spacers, and cleavage sites as described herein.

[0422] In another embodiment, the vector encoding one or more HBV antigens generated by the first infectious isovirus as described herein can be based on LCMV clone 13 or LCMV MP strain (see, for example, section 7.1).

[0423] In another embodiment, the vector encoding one or more HBV antigens generated from the second infectious arenavirus as described herein may be based on LCMV clone 13 or LCMV MP strain (see, for example, section 7.1). In another embodiment, the vector encoding one or more HBV antigens generated from the first infectious arenavirus as described herein may be based on Junin virus.

[0424] In another embodiment, the vector encoding one or more HBV antigens generated by the second infectious isovirus as described herein can be based on Junin virus.

[0425] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof.

[0426] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0427] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0428] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0429] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc protein or an antigenic fragment thereof.

[0430] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBVpre-S2 / S protein or an antigenic fragment thereof.

[0431] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV pre-S2 / S protein or antigenic fragments thereof.

[0432] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBe protein or antigenic fragments thereof.

[0433] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBc proteins or antigenic fragments thereof.

[0434] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus expressing HBVHBe protein or an antigenic fragment thereof.

[0435] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0436] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0437] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0438] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBVpre-S2 / S protein or an antigenic fragment thereof.

[0439] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBs protein or antigenic fragments thereof.

[0440] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBVpre-S2 / S protein or an antigenic fragment thereof.

[0441] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof.

[0442] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof.

[0443] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof.

[0444] In a specific implementation, this document provides a method for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof.

[0445] In another embodiment, the first infectious isovirus expressing HBV antigen is the primary vaccine antigen, and the second infectious isovirus expressing another HBV antigen is the second vaccine antigen.

[0446] In some embodiments, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or HBV HBc protein, provides better immunoprotective effect against HBV after vaccination compared to administration of an infectious arenavirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBc protein. In other embodiments, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, elicits a greater immune response compared to administration of an infectious arenavirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBc protein. In another embodiment, compared to administration of a single infectious arenavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBc protein, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, elicits a greater CD8+ T cell response. In other embodiments, compared to administration of a single infectious arenavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBc protein, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBc protein, elicits higher titers of neutralizing antibodies.

[0447] In some embodiments, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or HBV HBs protein, provides better immunoprotective effect against HBV after vaccination compared to administration of an infectious arenavirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBs protein. In other embodiments, administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, and a second infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, elicits a greater immune response compared to administration of an infectious arenavirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBs protein. In another embodiment, administration of a first infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, and a second infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, evokes a greater CD8+ T cell response compared to administration of a single infectious isavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only HBs protein. In other embodiments, administration of a first infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, and a second infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBs protein, evokes higher titers of neutralizing antibodies compared to administration of a single infectious isavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only HBs protein.

[0448] In some embodiments, administration of a first infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins, and a second infectious isovirus expressing HBV pre-S2 / S protein or a fusion of HBV HBs and HBc proteins, provides better immunoprotective effect against HBV after vaccination compared to administration of a single infectious isovirus expressing HBV antigen, such as a fusion expressing only the pre-S2 / S protein (or a fragment thereof) or only HBV HBs and HBc proteins. In other embodiments, administration of a first infectious isovirus expressing HBV pre-S2 / S protein or a fusion of HBV HBs and HBc proteins, and a second infectious isovirus expressing HBV pre-S2 / S protein or a fusion of HBV HBs and HBc proteins, elicits a greater immune response compared to administration of a single infectious isovirus expressing HBV antigen, such as a fusion expressing only the pre-S2 / S protein (or a fragment thereof) or only HBV HBs and HBc proteins. In another embodiment, compared with the administration of a single infectious arenavirus expressing HBV antigen, such as a fusion expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBV HBs and HBc proteins, the administration of a first infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins, and a second infectious arenavirus expressing HBV pre-S2 / S protein or a fragment thereof, or a fusion of HBV HBs and HBc proteins, elicited a greater CD8+ T cell response. In other embodiments, administration of a first infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins, or administration of a second infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins, elicited higher titers of neutralizing antibodies compared to administration of a single infectious isovirus expressing HBV antigen, such as a fusion of HBV pre-S2 / S protein or a fragment thereof or a fusion of HBV HBs and HBc proteins.

[0449] In some embodiments, administration of a first infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, and a second infectious isovirus expressing HBV pre-S2 / S protein or HBV HBe protein, provides better immunoprotection against HBV after vaccination compared to administration of an infectious isovirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBe protein. In other embodiments, administration of a first infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, and a second infectious isovirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, elicits a greater immune response compared to administration of an infectious isovirus expressing only HBV antigen, such as expressing only pre-S2 / S protein (or a fragment thereof) or only HBe protein. In another embodiment, administration of a first infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, and a second infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, elicits a greater CD8+ T cell response compared to administration of a single infectious isavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBe protein. In other embodiments, administration of a first infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, and a second infectious isavirus expressing HBV pre-S2 / S protein or a fragment thereof or HBV HBe protein, elicits higher titers of neutralizing antibodies compared to administration of a single infectious isavirus expressing HBV antigen, such as expressing only the pre-S2 / S protein (or a fragment thereof) or only the HBe protein.

[0450] In yet another embodiment, this document provides a combination of the replication-defective arenavirus expressing HBV antigen described herein with one or more replication-defective viral vectors. In a more specific embodiment, the replication-defective viral vector is selected from the group consisting of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rod-shaped viruses, polioviruses, adenovirus-associated viruses, and Sendai viruses, and mixtures thereof. In a specific embodiment, the poxvirus is the modified vaccine Ankara.

[0451] In another embodiment, this document provides a combination of the replication-defective isovirus expressing HBV antigen described herein with one or more replication-defective viral vectors expressing HBV antigen. In a more specific embodiment, the replication-defective viral vector is selected from the group consisting of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rod-shaped viruses, polioviruses, adenovirus-associated viruses, and Sendai viruses, and mixtures thereof. In a specific embodiment, the poxvirus is the modified vaccine Ankara.

[0452] In another embodiment, a first infectious isovirus expressing HBV antigen, as described herein, is administered before or after a second infectious isovirus expressing HBV antigen, as described herein. For example, the first infectious isovirus expressing HBV antigen is administered 30-60 minutes before or after the first administration of the second infectious isovirus.

[0453] In another embodiment, the first infectious isovirus expressing the vaccine antigen is administered prior to the second infectious isovirus expressing the vaccine antigen. In some embodiments, there are time periods of 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, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year between the administration of the first and second infectious isoviruses.

[0454] In another embodiment, the two infectious arenaviruses are administered in a treatment regimen of approximately 1:1 to 1:1000, particularly including: 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000 molar ratios.

[0455] In another embodiment, the subject to be administered two or more infectious arenaviruses expressing HBV antigens as described herein is infected with HBV, susceptible to HBV infection, or at risk of HBV infection.

[0456] In another embodiment, the subject to be simultaneously administered two or more infectious arenaviruses expressing HBV antigens as described herein is HBV infected, susceptible to HBV infection, or at risk of HBV infection.

[0457] In another embodiment, the subject to sequential administration of two or more infectious arenaviruses expressing HBV antigens as described herein is HBV infected, susceptible to HBV infection, or at risk of HBV infection.

[0458] In another embodiment, the two or more infectious arenaviruses expressing HBV antigens described herein are further administered in combination with at least one other drug for the treatment and / or prevention of HBV. Therapeutic drugs for the treatment and / or prevention of HBV include, but are not limited to, entecavir (…). Bristol-Myers Squibb, Lamivudine (EPIVIR) GlaxoSmithKline, Adefovir Dipivoxil ( Gilead Sciences), Interferon α2b ( Schering), pegylated interferon ( Roche, Telbivudine Novartis and tenofovir ( Gilead Sciences.

[0459] In another embodiment, the two or more infectious arenaviruses expressing HBV antigens described herein are further administered in combination with at least one other immunomodulator. In a more specific embodiment, the two or more infectious arenaviruses expressing HBV antigens described herein are further administered in combination with at least one Th1-specific adjuvant. In a more specific embodiment, the Th1-specific adjuvant is BCG.

[0460] In another embodiment, the administration regimen may involve administering a second infectious isovirus expressing the HBV antigen, as described herein, to a symptomatic subject. In yet another embodiment, the administration regimen may involve administering a second infectious isovirus expressing the HBV antigen, as described herein, to a subject with an compromised immune system, particularly a transplant recipient, an HIV-infected person, a pregnant person, or a person with cancer. In yet another embodiment, two or more infectious isoviruses expressing the HBV antigen, as described herein, are administered to a child aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age who has, is susceptible to, or is at risk of HBV infection.

[0461] In another embodiment, the administration protocol may involve administering a first isovirus expressing HIV antigen to a child subject and a second isovirus expressing HBV antigen to the same subject who is an adolescent. In a specific embodiment, the administration protocol may involve administering the first isovirus expressing HBV antigen described herein to a subject aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age, and administering a second infectious isovirus expressing HBV antigen to the same subject aged 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 years of age.

[0462] In another embodiment, the administration regimen may involve administering a second infectious arenavirus expressing HBV antigen to a pre-pubescent subject. In another embodiment, the administration method may involve administering a second infectious arenavirus expressing HBV antigen as described herein to adolescent males aged 12 to 18 years. In another embodiment, the administration method may involve administering a second infectious arenavirus expressing HBV antigen to females aged 12 to 18 years.

[0463] In another implementation, administration of two or more infectious arena viruses expressing HBV antigens reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the risk of HBV infection without such treatment.

[0464] In another implementation, isolated administration of two or more infectious arena viruses expressing HBV antigens reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the risk of HBV infection without such treatment.

[0465] In another implementation, consecutive administration of two or more infectious arena viruses expressing HBV antigens reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the risk of HBV infection without such treatment.

[0466] Unrestricted by theory, administration of the first infectious isovirus and subsequent administration of the second infectious isovirus vector produced an initial immune-enhancing effect.

[0467] In some embodiments, this document provides a method for treating and / or preventing HBV infection, comprising the sequential administration of two or more arenavirus vector constructs, each expressing the same or different HBV antigens. The time interval between each administration may be approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 3 months, approximately 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, approximately 18 months, or approximately 24 months.

[0468] In some embodiments, the first infectious isovirus and the second infectious isovirus are homologous. In some embodiments, the first infectious isovirus and the second infectious isovirus are heterologous.

[0469] In some specific embodiments, the first infectious isovirus is an Old World isovirus, and the second infectious isovirus is an Old World isovirus. In some specific embodiments, the first infectious isovirus is an Old World isovirus, and the second infectious isovirus is a New World isovirus. In some specific embodiments, the first infectious isovirus is a New World isovirus, and the second infectious isovirus is a New World isovirus. In some specific embodiments, the first infectious isovirus is a New World isovirus, and the second infectious isovirus is an Old World isovirus.

[0470] In some specific embodiments, the first infectious areca virus is derived from LCMV, and the second infectious areca virus is derived from LCMV. In some specific embodiments, the first infectious areca virus is derived from LCMV, and the second infectious areca virus is derived from Junin virus. In some specific embodiments, the first infectious areca virus is derived from Junin virus, and the second infectious areca virus is derived from Junin virus. In some specific embodiments, the first infectious areca virus is derived from Junin virus, and the second infectious areca virus is derived from LCMV.

[0471] In some embodiments, this document provides a method for treating and / or preventing HBV infection, wherein a first infectious isovirus is administered as a “primary immunization” and a second infectious isovirus is administered as a “boost.” The first and second infectious isovirus vectors may express the same or different HBV antigens. In some specific embodiments, the “primary immunization” is performed using an infectious isovirus derived from LCMV, and the “boost” is performed using an infectious isovirus derived from Junin virus.

[0472] In some embodiments, administration of a first infectious isovirus expressing HBV antigen or a fragment thereof, followed by administration of a second infectious isovirus expressing HBV antigen or a fragment thereof, produces a greater antigen-specific CD8+ T cell response compared to administration of a single infectious isovirus expressing HBV antigen or a fragment thereof. In some embodiments, the antigen-specific CD8+ T cell count increases by 50%, 100%, 150%, or 200% after the second administration compared to the first administration. In some embodiments, administration of a third infectious isovirus expressing HBV antigen produces a greater antigen-specific CD8+ T cell response compared to administration of two consecutive infectious isoviruses expressing HBV antigen. In some embodiments, the antigen-specific CD8+ T cell count increases by approximately 50%, approximately 100%, approximately 150%, approximately 200%, or approximately 250% after the third administration compared to the first administration.

[0473] In some embodiments, this document provides a method for treating and / or preventing infection, comprising administering two or more isotropic viral vector constructs, wherein the two or more isotropic viral vector constructs are homologous, and wherein the time interval between each administration is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 3 months, approximately 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, approximately 18 months, or approximately 24 months.

[0474] In some implementations, administration of a first infectious arena virus expressing HBV antigen or fragments thereof and a heterologous second infectious arena virus expressing HBV antigen or fragments thereof produces a greater CD8+ T cell response compared to administration of a first infectious arena virus expressing HBV antigen or fragments thereof and a second infectious arena virus expressing HBV antigen or fragments thereof.

[0475] In some specific embodiments, the first infectious arenavirus expressing the HBV pre-S2 / S protein is LCMV, and the heterologous second infectious arenavirus expressing the HBV pre-S2 / S protein is Junin virus.

[0476] In some specific embodiments, the primary infectious isavirus expressing HBV HBc protein is Junin virus, and the heterologous secondary infectious isavirus expressing HBV HBc protein is LCMV.

[0477] In some specific embodiments, the first infectious arenavirus expressing the HBV HBs and HBc fusion protein is LCMV, and the heterologous second infectious arenavirus expressing the HBV HBs and HBc fusion protein is Junin virus.

[0478] In some specific embodiments, the primary infectious isanone virus expressing the HBV HBe protein is LCMV, and the heterologous secondary infectious isanone virus expressing the HBV HBe protein is Junin virus.

[0479] In certain specific embodiments, administration of first-infectious arenavirus expressing HBV pre-S2 / S protein and heterologous second-infectious arenavirus expressing HBV pre-S2 / S protein elicits a greater CD8+ T cell response compared to administration of first-infectious arenavirus expressing HBV pre-S2 / S protein and homologous second-infectious arenavirus expressing HBV pre-S2 / S protein. In certain specific embodiments, administration of first-infectious arenavirus expressing HBV pre-S2 / S protein and heterologous second-infectious arenavirus expressing HBV pre-S2 / S protein elicits a CD8+ T cell response that is approximately 20%, approximately 40%, approximately 60%, approximately 80%, approximately 100%, approximately 120%, approximately 140%, approximately 160%, approximately 180%, or approximately 200% greater than administration of first-infectious arenavirus expressing HBV pre-S2 / S protein and homologous second-infectious arenavirus expressing HBV pre-S2 / S protein.

[0480] In certain specific embodiments, administration of first-infectious arenavirus expressing HBV HBc protein and heterologous second-infectious arenavirus expressing HBV HBc protein elicits a greater CD8+ T cell response compared to administration of first-infectious arenavirus expressing HBV HBc protein and homologous second-infectious arenavirus expressing HBV HBc protein. In certain specific embodiments, administration of first-infectious arenavirus expressing HBV HBc protein and heterologous second-infectious arenavirus expressing HBV HBc protein elicits a CD8+ T cell response that is approximately 20%, approximately 40%, approximately 60%, approximately 80%, approximately 100%, approximately 120%, approximately 140%, approximately 160%, approximately 180%, or approximately 200% greater than administration of first-infectious arenavirus expressing HBV HBc protein and homologous second-infectious arenavirus expressing HBV HBc protein.

[0481] In certain specific implementations, administration of first-infectious arenavirus expressing HBV HBs and HBc fusion proteins and heterologous second-infectious arenavirus expressing HBV HBs and HBc fusion proteins elicits a greater CD8+ T cell response compared to administration of first-infectious arenavirus expressing HBV HBs and HBc fusion proteins and homologous second-infectious arenavirus expressing HBV HBs and HBc fusion proteins. In certain specific implementations, the CD8+ T cell responses induced by administration of first-infectious arenavirus expressing HBV HBs and HBc fusion proteins and homologous second-infectious arenavirus expressing HBV HBs and HBc fusion proteins are approximately 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% greater than those induced by administration of first-infectious arenavirus expressing HBV HBs and HBc fusion proteins and heterologous second-infectious arenavirus expressing HBV HBs and HBc fusion proteins.

[0482] In certain specific embodiments, administration of first-infectious arenavirus expressing HBV HBe protein and heterologous second-infectious arenavirus expressing HBV HBe protein elicits a greater CD8+ T cell response compared to administration of first-infectious arenavirus expressing HBV HBe protein and homologous second-infectious arenavirus expressing HBV HBe protein. In certain specific embodiments, administration of first-infectious arenavirus expressing HBV HBe protein and heterologous second-infectious arenavirus expressing HBV HBe protein elicits a CD8+ T cell response that is approximately 20%, approximately 40%, approximately 60%, approximately 80%, approximately 100%, approximately 120%, approximately 140%, approximately 160%, approximately 180%, or approximately 200% greater than administration of first-infectious arenavirus expressing HBV HBe protein and homologous second-infectious arenavirus expressing HBV HBe protein.

[0483] In some embodiments, this document provides a method for treating and / or preventing infection, comprising administering two or more isotropic viral vector constructs, wherein the two or more isotropic viral vector constructs are heterologous, and wherein the time interval between each administration is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 3 months, approximately 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, approximately 18 months, or approximately 24 months.

[0484] In yet another embodiment, this document provides a combination of the replication-defective arenavirus expressing HBV antigen described herein with one or more replication-defective viral vectors. In a more specific embodiment, the replication-defective viral vector is selected from the group consisting of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rod-shaped viruses, polioviruses, adenovirus-associated viruses, and Sendai viruses, and mixtures thereof. In a specific embodiment, the poxvirus is the modified vaccine Ankara.

[0485] In another embodiment, this document provides a combination of the replication-defective isovirus expressing HBV antigen described herein with one or more replication-defective viral vectors expressing HBV antigen. In a more specific embodiment, the replication-defective viral vector is selected from the group consisting of poxviruses, adenoviruses, alphaviruses, herpes simplex viruses, paramyxoviruses, rod-shaped viruses, polioviruses, adenovirus-associated viruses, and Sendai viruses, and mixtures thereof. In a specific embodiment, the poxvirus is the modified vaccine Ankara.

[0486] In another embodiment, a first infectious isovirus expressing HBV antigen, as described herein, is administered before or after a second infectious isovirus expressing HBV antigen, as described herein. For example, the first infectious isovirus expressing HBV antigen is administered 30-60 minutes before or after the first administration of the second infectious isovirus.

[0487] In another embodiment, the first infectious isovirus expressing the vaccine antigen is administered prior to the second infectious isovirus expressing the vaccine antigen. In some embodiments, there are time periods of 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, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year between the administration of the first and second infectious isoviruses.

[0488] In another embodiment, the two infectious arenaviruses are administered in a treatment regimen of approximately 1:1 to 1:1000, particularly including: 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000 molar ratios.

[0489] In another embodiment, the subject receiving two or more infectious arenaviruses expressing HBV antigens as described herein is HBV infected, susceptible to HBV infection, or at risk of HBV infection.

[0490] The methods described in this article can be used to treat individuals who are susceptible to HBV infection or at risk of HBV infection.

[0491] In another embodiment, the two or more infectious arenaviruses expressing HBV antigens described herein further express at least one other immunostimulatory peptide, polypeptide, or protein. In some embodiments, the immunostimulatory peptide, polypeptide, or protein is calreticulin (CRT) or a fragment thereof; pervasive protein or a fragment thereof; granulocyte-macrophage colony-stimulating factor (GM-CSF) or a fragment thereof; invariant chain (CD74) or an antigenic fragment thereof; Mycobacterium tuberculosis heat shock protein 70 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-associated tyrosine kinase 3 (Flt3) ligand or an antigenic fragment thereof.

[0492] A heterologous primary immunization-enhancement method using infectious replication-defective arenavirus vectors, wherein the two infectious replication-defective arenavirus vectors are derived from different arenaviruses (e.g., LCMV and Junin virus), is also provided. These infectious replication-defective arenavirus vectors can express antigens, such as HBV antigens.

[0493] A heterologous primary immunization-enhancement method using infectious, replicating, competent arenavirus vectors derived from different arenaviruses (e.g., LCMV and Junin virus) is also provided. These infectious, replicating, competent arenavirus vectors can express antigens, such as HBV antigens.

[0494] 6.8(b) Composition

[0495] The present invention also relates to vaccines, immunogenic compositions, and pharmaceutical compositions comprising the genetically engineered isoviruses described herein. Such vaccine and pharmaceutical compositions can be formulated according to standard procedures in the art.

[0496] In one embodiment, this document provides a composition comprising two or more infectious arenaviruses expressing HBV antigens as described herein. See, for example, section 6.2. In a specific embodiment, the composition described herein comprises administering to a subject a first infectious arenavirus expressing HBV antigens as described herein, for example, wherein the ORF of the GP encoding an S genome segment is replaced by a nucleotide sequence encoding an HBV antigen. The HBV antigen may be, but is not limited to:

[0497] a) The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0498] b) The nucleotide sequence encoding the HBV HBc protein or its antigenic fragment;

[0499] c) The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0500] d) The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or their antigenic fragments; and

[0501] e) The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment;

[0502] And the second infectious arenavirus expressing HBV antigens described herein, for example, wherein the ORF encoding the S genome segment of the GP is replaced by a nucleotide sequence encoding the HBV antigen. The HBV antigen may be, but is not limited to:

[0503] a) The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0504] b) The nucleotide sequence encoding the HBV HBc protein or its antigenic fragment;

[0505] c) The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0506] d) The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or their antigenic fragments; and

[0507] e) A nucleotide sequence encoding the HBV HBe protein or an antigenic fragment thereof. In some embodiments, the first and second infectious arenaviruses are replication-defective. In some embodiments, the first and second infectious arenaviruses are replication-competent. In some embodiments, either the first or the second infectious arenavirus is replication-defective.

[0508] In specific embodiments, this document provides a method for treating and / or preventing HBV infection, comprising administering a first infectious isovirus expressing a first HBV antigen selected from: HBV pre-S2 / S protein or an antigenic fragment thereof; HBV HBc protein or an antigenic fragment thereof; HBV HBs protein or an antigenic fragment thereof; a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; or HBV HBe protein or an antigenic fragment thereof, as described herein; and a second infectious isovirus expressing a second HBV antigen selected from: HBV pre-S2 / S protein or an antigenic fragment thereof; HBV HBc protein or an antigenic fragment thereof; or HBV HBs protein or an antigenic fragment thereof, a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; or HBV HBe protein or an antigenic fragment thereof.

[0509] In some embodiments, this document provides compositions suitable for methods of treating and / or preventing HBV infection, the methods comprising administering two arenavirus constructs expressing HBV antigens as described herein. In a specific embodiment, the two arenavirus vector constructs express HBV antigens.

[0510] In some embodiments, this document provides compositions comprising two or more arenavirus vector constructs expressing HBV antigens as described herein. In specific embodiments, this document provides compositions comprising three or more arenavirus vector constructs expressing HBV antigens as described herein. In some embodiments, the arenavirus may be LCMV.

[0511] In a specific implementation, the antigen is the HBV pre-S2 / S protein or a fragment thereof. (See, for example, section 6.2(a)).

[0512] In some embodiments, the antigen is HBV HBc protein or a fragment thereof. (See, for example, section 6.2(b)).

[0513] In some embodiments, the antigen is HBV HBs protein or a fragment thereof. (See, for example, section 6.2(c)).

[0514] In some embodiments, the antigen is a fusion of HBV HBs and HBc proteins or antigenic fragments thereof (see, for example, section 6.2(d)).

[0515] In some embodiments, the antigen is HBV HBe protein or a fragment thereof. (See, for example, section 6.2(e)).

[0516] In some embodiments, the vectors encoding one or more HBV antigens produced as described herein comprise one or more nucleic acids or combinations thereof encoding HBV antigens as described herein. In specific embodiments, the HBV antigens described herein are separated by various linkers, spacers, and cleavage sites as described herein.

[0517] In another embodiment, the vector encoding one or more HBV antigens generated by the first infectious isovirus as described herein can be based on LCMV clone 13 or LCMV MP strain. (See, for example, section 7.1).

[0518] In another embodiment, the vector encoding one or more HBV antigens generated by the second infectious isovirus as described herein can be based on LCMV clone 13 or LCMV MP strain. (See, for example, section 7.1).

[0519] In a specific embodiment, this document provides a composition suitable for treating and / or preventing HBV infection in a subject, the method comprising administering to the subject a first infectious isovirus composition expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus composition expressing HBV HBc protein or an antigenic fragment thereof.

[0520] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0521] In a specific embodiment, this document provides a composition suitable for infecting a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0522] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0523] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0524] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof.

[0525] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV pre-S2 / S protein or antigenic fragments thereof.

[0526] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBe protein or antigenic fragments thereof.

[0527] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBc proteins or antigenic fragments thereof.

[0528] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising administering to the subject a first infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof.

[0529] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof.

[0530] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0531] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof, and a second infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof.

[0532] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof.

[0533] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing a fusion of HBV HBs and HBc proteins or antigenic fragments thereof, and a second infectious isovirus expressing HBV HBs protein or antigenic fragments thereof.

[0534] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising sequentially administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV pre-S2 / S protein or an antigenic fragment thereof.

[0535] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof.

[0536] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof.

[0537] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBs protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof.

[0538] In a specific embodiment, this document provides a composition suitable for treating and / or preventing infection in a subject, comprising simultaneously administering to the subject a first infectious isovirus expressing HBV HBe protein or an antigenic fragment thereof and a second infectious isovirus expressing HBV HBc protein or an antigenic fragment thereof.

[0539] In another embodiment, the first infectious isovirus composition expressing HBV antigen is a primary vaccine antigen, and the second infectious isovirus expressing another HBV antigen is a second vaccine antigen.

[0540] In yet another embodiment, this document provides a combination of the replication-defective arenavirus composition expressing HBV antigen described herein with one or more replication-defective viral vector compositions. In a more specific embodiment, the replication-defective viral vector composition may be, but is not limited to: poxvirus, adenovirus, alphavirus, herpes simplex virus, paramyxovirus, rod-shaped virus, poliovirus, adenovirus-associated virus, and Sendai virus, and mixtures thereof. In a specific embodiment, the poxvirus is the modified vaccine Ankara.

[0541] In another embodiment, the two infectious sand virus compositions have a molar ratio of about 1:1 to 1:1000, particularly including: 1:1, 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000.

[0542] In another embodiment, the two or more infectious arenavirus compositions expressing HBV antigens described herein are suitable for administration to subjects who are infected with HBV, susceptible to HBV infection, or at risk of HBV infection. In another embodiment, the subject receiving the two or more infectious arenavirus compositions expressing HBV antigens or compositions thereof described herein is infected with HBV, susceptible to HBV infection, or at risk of HBV infection.

[0543] In another embodiment, the two or more infectious arenavirus compositions further comprise at least one other drug for treating and / or preventing HBV infection. Therapeutic drugs for treating and / or preventing HBV include, but are not limited to, entecavir (…). Bristol-Myers Squibb), Lamivudine GlaxoSmithKline, Adefovir Dipivoxil ( Gilead Sciences), Interferon α2b (INTRON) Schering), pegylated interferon ( Roche, Telbivudine Novartis and tenofovir ( Gilead Sciences.

[0544] In another embodiment, the composition is suitable for administering to symptomatic subjects a second infectious isovirus composition expressing HBV antigen or fragments thereof as described herein. In yet another embodiment, the composition is suitable for administering to subjects with compromised immune systems, particularly transplant recipients, HBV-infected individuals, pregnant subjects, or subjects with cancer, a second infectious isovirus composition expressing HBV antigen or fragments thereof as described herein. In another embodiment, two or more infectious isovirus compositions expressing HBV antigen or fragments thereof as described herein are suitable for administration to subjects who are children aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age who have, are susceptible to, or are at risk of HBV infection.

[0545] In another embodiment, the composition is suitable for administering a first isovirus expressing HIV antigen to a child subject and a second isovirus expressing HBV antigen to the same subject who is an adolescent. In a specific embodiment, the administration regimen may involve administering the first isovirus expressing HBV antigen described herein to a subject aged 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age, and administering a second infectious isovirus expressing HBV antigen to the same subject aged 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 years of age.

[0546] In another embodiment, the composition is suitable for administering a second infectious arenavirus expressing HBV antigen to a pre-pubescent subject. In another embodiment, the administration may involve administering a second infectious arenavirus expressing HBV antigen as described herein to adolescent males aged 12 to 18 years. In another embodiment, the administration may involve administering a second infectious arenavirus expressing HBV antigen to females aged 12 to 18 years.

[0547] In another embodiment, compared with the risk of HBV infection without such treatment, an infectious isovirus composition expressing two or more HBV antigens or fragments thereof, as described herein, reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more.

[0548] In another embodiment, the separate administration of two or more infectious isovirus compositions expressing HBV antigens or fragments thereof, as described herein, reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the risk of HBV infection without such treatment.

[0549] In another embodiment, sequential administration of two or more infectious isovirus compositions expressing HBV antigens or fragments thereof, as described herein, reduces an individual's risk of HBV infection by 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 about 70%, at least about 80%, at least about 90%, or more, compared to the risk of HBV infection without such treatment.

[0550] In another embodiment, the invention provided herein provides a vaccine composition comprising a synergistic combination of two or more infectious replication-defective arena viruses expressing HBV antigens.

[0551] In another embodiment, the invention provided herein provides a vaccine composition comprising a synergistic combination of two or more infectious, replicating competent arenaviruses expressing HBV antigens.

[0552] 6.9 Analysis

[0553] Any analysis known to a skilled technician can be used to measure the infectivity of isovirus vectors. For example, virus / vector titers can be determined by focal forming unit (FFU) analysis. In short, supplementary cells, such as HEK 293 cells expressing the LCMV GP protein, are inoculated with virus / vector samples at different dilutions. After a latent period, cells are allowed to form a monolayer and the virus is allowed to attach to the cells; the monolayer is covered with methylcellulose. When the plates are further incubated, the originally infected cells release viral progeny. Due to the methylcellulose covering, the spread of new virus is confined to neighboring cells. Thus, each infectious particle produces a circular area of ​​infected cells called a lesion. Using an antibody against LCMV-NP and an HRP-based colorimetric reaction, such lesions can become visible and countable. The virus / vector titer can be calculated in lesion forming units per milliliter (FFU / mL).

[0554] To determine the infectious titer (FFU / mL) of a transgenic vector, this analysis was modified by using the corresponding transgenic-specific antibody instead of the anti-LCMV-NP antibody.

[0555] The determination of humoral immune responses during vaccination in animals (e.g., mice, guinea pigs) can be performed using antigen-specific serum ELISA (enzyme-linked immunosorbent assay). In short, plates are coated with an antigen (e.g., recombinant protein) to block and prevent non-specific antibody binding, and incubated with serial dilutions of serum. After incubation, bound serum-antibodies can be detected, for example, using enzyme-linked anti-species (e.g., mouse, guinea pig) specific antibodies (detecting total IgG or IgG subclasses) and a subsequent color reaction. Antibody titers can be determined, for example, as an endpoint geometric mean titer.

[0556] Immunocapture ELISA (IC-ELISA) can also be performed (see Shanmugham et al, 2010, Clin. Vaccine Immunol. 17(8): 1252-1260), in which the capture reagent is cross-linked to the beads.

[0557] Neutralization assay in ARPE-19 cells to determine the neutralizing activity of induced antibodies in serum was performed using the following cell assays with ARPE-19 cells from ATCC and GFP-labeled virus. Supplemented guinea pig serum was used as a source of exogenous complement. The assays began one or two days prior to neutralization in 6.5 × 10⁶ wells seeded in 384-well plates. 3Cells / well (50 μl / well). Neutralization was performed for 1 hour at 37°C in 96-well sterile tissue culture plates for Key Cells. After the neutralization incubation step, the mixture was added to the cells and incubated for an additional 4 days for GFP detection using a plate reader. Positive neutralized human serum was used as an analytical positive control on each plate to check the reliability of all results. Titers (EC50) were determined using 4-parameter logistic curve fitting. As an additional test, the reaction wells were examined using a fluorescence microscope.

[0558] In short, plaque reduction analysis of hepatitis B virus (HBV) can be performed using HBV isolates labeled with green fluorescent protein (GFP), with 5% rabbit serum used as a source of exogenous complement. Spots are counted using a fluorescence microscope. The neutralization titer is defined as the highest serum dilution that causes a 50% reduction in plaques compared to a control (pre-immunization) serum sample.

[0559] Neutralization assay in guinea pig lung fibroblasts (GPL) cells: In summary, serial dilutions of test serum and control (pre-inoculation) serum were prepared in GPL complete medium supplemented with rabbit serum (1%) as an exogenous complement source. The dilution series ranged from 1:40 to 1:5120. Serum dilutions were incubated with eGFP-labeled virus (100–200 pfu per well) at 37°C for 30 min, then transferred to 12-well plates containing confluent GPL cells. Samples were performed in triplicate. After 2 h of incubation at 37°C, cells were washed with PBS, re-fed in GPL complete medium, and incubated at 37°C / 5% CO2 for 5 days. Plaques were visualized by fluorescence microscopy, counted, and compared with control wells. Serum dilutions with a 50% reduction in plaque number compared to controls were determined as the neutralizing titer.

[0560] LCMVRNA genomes were isolated using the QIAamp Viral RNA mini kit (QIAGEN) according to the manufacturer's protocol. III Using a one-step qRT-PCR kit (Invitrogen) and primers and probes specific to certain LCMV NP coding regions (FAM reporter and NFQ-MGB quencher), quantitative PCR was performed on the StepOnePlus Real-Time PCR system (Applied Biosystems) to detect LCMV RNA genomic equivalents. The reaction temperature distribution was: 60°C for 30 min, 95°C for 2 min, followed by 45 cycles of 95°C for 15 s and 56°C for 30 s. RNA was quantified by comparing the sample results with a standard curve prepared from a log10 dilution series of spectrophotometrically quantified, in vitro transcribed RNA fragments corresponding to LCMV NP coding sequences containing primer and probe binding sites.

[0561] Western blots were performed on infected cells grown in tissue culture flasks or suspensions, lysed at indicated time points post-infection using RIPA buffer (Thermo Scientific), or used directly without cell lysis. Samples were heated to 99°C for 10 minutes with reducing agent and NuPage LDS sample buffer (NOVEX), cooled to room temperature, and then loaded onto 4–12% SDS-PAGE gels for electrophoresis. Protein spots were blotted onto the membrane using an Invitrogens iBlot Gel transfer device and visualized by Ponceau staining. Finally, the blots were probed with a primary antibody against the target protein and a secondary antibody that binds to alkaline phosphatase, followed by staining with 1-Step NBT / BCIP solution (INVITROGEN).

[0562] MHC-peptide multimer staining analysis for detecting antigen-specific CD8+ T cell proliferation: Any analysis known to a skilled technician can be used to test for antigen-specific CD8+ T cell responses. For example, MHC-peptide tetramer staining analysis can be used (see, e.g., Altman JD et al., Science. 1996; 274:94-96; and Murali-Krishna K. et al., Immunity. 1998; 8:177-187). Briefly, this analysis includes the following steps: tetramer analysis is used to detect the presence of antigen-specific T cells. To detect T cell-specific peptides, it is necessary to identify the peptide and a tetramer of MHC molecules (typically fluorescently labeled) tailored for specific antigen-specific T cells. The tetramer is then detected by flow cytometry using fluorescent labeling.

[0563] The ELISPOT assay for detecting antigen-specific CD4+ T cell proliferation is any assay known to a skilled technician that can be used to test for antigen-specific CD4+ T cell responses. For example, the ELISPOT assay can be used (see, for example, Czerkinsky CC et al, J Immunol Methods. 1983; 65:109-121; and Hutchings PREtal, J Immunol Methods. 1989; 120:1-8). Briefly, the assay comprises the following steps: an immunospot plate is coated with an anti-cytokine antibody. Cells are incubated on the immunospot plate. Cells secrete cytokines, which are then washed away. The plate is then coated with a second biotinylated anti-cytokine antibody and visualized using the avidin-HRP system.

[0564] Intracellular cytokine analysis for assessing the functionality of CD8+ and CD4+ T cell responses: Any analysis known to a skilled technician can be used to test the functionality of CD8+ and CD4+ T cell responses. For example, intracellular cytokine analysis in combination 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, Clinical and Diagnostic Laboratory Immunology. 2001; 8:628-63). Briefly, the analysis includes the following steps: activating cells with a specific peptide or protein, and retaining cytokines intracellularly by adding an inhibitor of protein transport (e.g., brefidobacterium A). After washing, antibodies against other cellular markers can be added to the cells. The cells are then fixed and permeabilized. Anti-cytokine antibodies are added, and the cells can be analyzed by flow cytometry.

[0565] Any analysis known to a skilled craftsman to determine the concentration of infectious and replication-competent viral particles can be used to measure replication-defective viral particles in a sample. For example, FFU analysis using non-supplementary cells (as described in

[00408] ) can be used for this purpose.

[0566] Furthermore, plaque-based analysis is a standard method for determining the concentration of virus in a viral sample according to plaque-forming units (PFU). Specifically, a confluent monolayer of non-supplementary host cells is infected with varying dilutions of virus and covered with a semi-solid medium, such as agar, to prevent uncontrolled spread of viral infection. Viral plaques are formed when the virus successfully infects and replicates itself within the cells of the fixed cell monolayer (see, e.g., Kaufmann, SH; Kabelitz, D. (2002) Methods in Microbiology Vol. 32: Immunology of Infection. Academic Press. ISBN 0-12-521532-0). Plaque formation can take 3–14 days, depending on the virus being analyzed. Plaques are generally counted manually, and this result, along with the dilution factor used to prepare the plate, is used to calculate the number of plaque-forming units per unit volume of sample (PFU / mL). The PFU / mL result represents the number of infectious, replicating competent particles within the sample.

[0567] Measuring viral load in blood or liver: Any analysis known to a skilled technician can be used to detect the number of HBV particles per volume of blood or liver (see, for example, Mendy et al, 2010, J. Viral Hepat. 17(2): 115-122). Non-limiting examples of such analyses include nucleic acid-based tests, such as PCR, as well as non-nucleic acid-based tests.

[0568] Liver biopsy: Any procedure known to a skilled technician performing a liver biopsy can be used to determine the extent of liver damage, for example, to test for chronic HBV infection or liver cancer in a patient. Non-limiting examples of liver biopsy types include percutaneous needle biopsy, laparoscopic biopsy, and transvenous biopsy. In some embodiments, liver biopsy is used to determine the presence of ground-glass hepatocytes when examining cells under a light microscope. The observation of ground-glass hepatocytes indicates the presence of HBsAg in the hepatocytes.

[0569] Analysis of viral antigen expression: Any analysis known to a skilled craftsman can be used to measure viral antigen expression. For example, FFU analysis (as described in

[00408] ) can be performed. For detection, a monoclonal or monoclonal antibody preparation (transgenic-specific FFU) against the corresponding viral antigen is used.

[0570] In addition, Western blotting can be performed (as described in

[00415] ).

[0571] Microparticle enzyme immunoassay HBsAg (Abbott) is a microparticle enzyme immunoassay (MEIA) used to detect HBsAg in the serum or plasma of adults, children, and newborns, including pregnant women. This analysis can be used as an adjunct to the diagnosis of acute or chronic HBV. It can also be used to confirm the presence of HBV infection.

[0572] To perform the analysis, a sample of the patient's blood is placed in a reaction well containing a detector antibody and microparticles coated with an antibody against HBV (e.g., against HBV antigen). If the blood sample contains HBV proteins (e.g., HBsAg), they will bind to the microparticles in the reaction well. This reaction is detected by another light-emitting substance, which is then measured to determine the presence of HBV (e.g., HBV antigen) in the blood. If the initial test is positive, the patient's blood is retested to confirm the presence of HBV (e.g., HBV antigen). Any microparticle enzyme immunoassay known to a skilled craftsman can be used to measure the presence of HBsAg or other HBV antigens.

[0573] Other HBV analyses involve placing a patient's blood sample in contact with HBV antibodies or HBV antigens. Antibodies and / or antigens include HBsAg, antibodies against HBeAg, antibodies against HBsAg, HBeAg, antibodies against HBcAg, and antibodies against HBcAg. If the patient is infected with HBV, the antigens and / or antibodies present in the blood will trigger a chemical reaction during test execution. Depending on which HBV antigens and / or antibodies are present in the patient's blood, this analysis allows for the detection of HBV at different stages.

[0574] Any analysis known to those skilled in the art can be used to assess levels of HBV, HBV antigen, or HBV antibody. Non-limiting examples of such analyses can be found, for example, Mayer et al, 2012, BMC Clin. Pathol. 12:8, Van Helden et al, 2004, Clin. Lab. 50(l-2):63-73, and Villar et al, 2011, J. Med. Virol. 83(9):1522-1529.

[0575] The tolerability and immunogenicity of vaccines or compositions thereof expressing HBV antigens described herein can be tested in animal models. In some embodiments, animal models used herein for testing the safety, tolerability, and immunogenicity of vaccines and compositions thereof include mice, guinea pigs, rats, monkeys, and chimpanzees. In a preferred embodiment, animal models used herein for testing the safety, tolerability, and immunogenicity of vaccines and compositions thereof include mice.

[0576] In specific instances, transgenic mouse models can be used to evaluate the antiviral potential of pharmacological agents such as immunotherapies or vaccines, as well as to assess physiological processes, including immune responses (see, for example, Guidotti et al, 1995, J.Virol. 69(10): 6158-69). Such transgenic mouse models can express human molecules, such as human class I and II HLA molecules and / or hepatitis B surface antigen (HBsAg) (see, for example, Bourgine et al, 2012, Virology 430(1): 10-9).

[0577] In another specific instance, the marmot (Marmota monax) can be used as an animal model for the development and testing of treatments and prevention for chronic hepatitis virus infections, such as chronic hepatitis B (see, e.g., Kosinska et al., Hepat. Res. Treat. 2010:817580). Marmot models are well-suited for evaluating the immunogenicity and other immune responses to potential immunotherapies such as vaccines (see, e.g., Vaccine 27(25-26):3271-3275).

[0578] 6.10 sequence

[0579] The sequences in Table 3 are illustrative amino acid and nucleotide sequences that can be used in the methods and compositions described herein. In some cases, DNA sequences are used to describe RNA sequences of viral genome fragments. RNA sequences can be readily deduced from DNA sequences. Table 3. Illustrative amino acid sequences.

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624] The following are some specific implementation schemes of this application.

[0625] 1. An infectious arenavirus vector, wherein the arenavirus open reading frame is removed and replaced with a nucleotide sequence selected from:

[0626] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0627] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0628] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0629] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0630] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0631] 2. The viral vector of embodiment 1, wherein the pre-S1 / S protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

[0632] 3. The viral vector of embodiment 1, wherein the HBc protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2.

[0633] 4. The viral vector of embodiment 1, wherein the fusion of HBV HBs and HBc proteins or their antigenic fragments comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.

[0634] 5. The viral vector of embodiment 1, wherein the HBe protein or its antigenic fragment comprises an amino acid sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26.

[0635] 6. The viral vector of implementation scheme 1 contains at least two of the following:

[0636] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0637] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0638] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0639] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0640] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0641] 7. The viral vector of implementation scheme 1 contains at least three of the following:

[0642] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0643] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0644] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0645] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0646] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0647] 8. The viral vector of embodiment 6 or 7, wherein expression of the nucleotide sequence generates an antigenic protein complex, the antigenic protein complex triggering a higher titer of neutralizing antibody compared to the expression of each component of the protein complex.

[0648] 9. The viral vector of any of the foregoing embodiments, wherein the sand virus is a lymphocytic choroid plexus meningitis virus.

[0649] 10. The viral vector of any of the aforementioned embodiments, wherein the open reading frame encoding the glycoprotein of the sand virus is deleted or functionally inactivated.

[0650] 11. The viral vector of any of the aforementioned embodiments, wherein the genomic information encoding the infectious isovirus viral vector is derived from the clonal strain 13 of lymphocytic choroid plexus meningitis virus.

[0651] 12. The viral vector of any of the foregoing embodiments, wherein the genomic information encoding the infectious isovirus viral vector is derived from the lymphocytic choroid plexus meningitis virus (MP) strain.

[0652] 13. A viral vector of any of the foregoing embodiments, wherein the viral vector comprises a genomic fragment, wherein the genomic fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical nucleotide sequences to nucleotides 1639 to 3315 of SEQ ID NO: 11 or 1640 to 3316 of SEQ ID NO: 12.

[0653] 14. A viral vector of any of the foregoing embodiments, wherein the viral vector comprises a genomic fragment, the genomic fragment comprising a nucleotide sequence encoding an expression product, and the amino acid sequence of the expression product being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 11 1639 to 3315 or SEQ ID NO: 12 1640 to 3316.

[0654] 15. The viral vector of any of the aforementioned embodiments, wherein the sand-like virus is a hump virus.

[0655] 16. The viral vector of embodiment 15, wherein the genomic information encoding the infectious isovirus viral vector is derived from the Candid#1 strain of Juninvirus.

[0656] 17. A viral vector according to any one of embodiments 1 to 16, wherein the growth or infectivity of the sand-like virus is not affected by the heterologous nucleic acid.

[0657] 18. A pharmaceutical composition comprising a viral vector and a pharmaceutically acceptable vector of any of the foregoing embodiments.

[0658] 19. An immunogenic composition comprising a viral vector and a pharmaceutically acceptable vector of any one of embodiments 1 to 17.

[0659] 20. A vaccine comprising a viral vector and a pharmaceutically acceptable vector according to any one of embodiments 1 to 17.

[0660] 21. A method for treating or preventing hepatitis B virus infection in a patient, wherein the method comprises administering to the patient a viral vector of any one of embodiments 1 to 17, a pharmaceutical composition of embodiment 18, an immunogenic composition of embodiment 19, or a vaccine of embodiment 20.

[0661] 22. Use of the viral vector of any one of embodiments 1 to 17, the pharmaceutical composition of embodiment 18, the immunogenic composition of embodiment 19, or the vaccine of embodiment 20 for the treatment or prevention of hepatitis B virus infection in a patient.

[0662] 23. The use of embodiment 22, wherein the viral vector of any one of embodiments 1 to 17, the pharmaceutical composition of embodiment 18, the immunogenic composition of embodiment 19, or the vaccine of embodiment 20 is suitable for intramuscular injection.

[0663] 24. The use of embodiment 22, wherein the viral vector of any one of embodiments 1 to 17, the pharmaceutical composition of embodiment 18, the immunogenic composition of embodiment 19, or the vaccine of embodiment 20 is suitable for intravenous injection.

[0664] 25. An isolated nucleic acid, wherein the nucleic acid comprises a sand virus genome fragment, wherein an open reading frame of the genome fragment is deleted or functionally inactivated, and wherein the genome fragment comprises one or more of the following:

[0665] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0666] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0667] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0668] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0669] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0670] 26. The isolated nucleic acid of implementation scheme 25, wherein the genomic fragment is a short fragment in which the open reading frame encoding GP is deleted.

[0671] 27. A method for generating an infectious, replication-defective arenavirus vector, comprising:

[0672] a. Transfect the nucleic acid of implementation plan 25 or 26 into the host cell;

[0673] b. Maintain the host cell under conditions suitable for virus formation; and

[0674] c. Harvest the infectious, replication-defective sand-like virus vector;

[0675] The host cell expresses a deleted or functionally inactivated open reading frame of the genomic fragment.

[0676] 28. The sand-like virus vector of embodiment 1, wherein the sand-like virus open reading frame is a glycoprotein (GP) open reading frame.

[0677] 29. An infectious, replication-defective arenavirus viral vector engineered to contain a genome, capable of amplifying and expressing its genetic information in infected cells, but unable to produce further infectious progeny particles in normal, unengineered cells, wherein an arenavirus open reading frame is removed and replaced with a nucleotide sequence encoding an HBV antigen or an antigenic fragment thereof, wherein administration of the arenavirus viral vector to a subject induces a durable immune response against the HBV antigen or an antigenic fragment thereof.

[0678] 30. The isovirus viral vector of embodiment 29, wherein the durable immune response induces a detectable antibody titer against HBV antigen or an antigenic fragment thereof.

[0679] 31. The isovirus vector of embodiment 29, wherein the persistent immunization induces a detectable antibody titer against HBV antigen or antigenic fragments thereof for at least 4 weeks.

[0680] 32. The isovirus vector of embodiment 30 or 31, wherein the durable immune response increases the antibody titer against the HBV antigen or an antigenic fragment thereof by at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0681] 33. A pharmaceutical composition comprising a first infectious, replication-defective arenavirus viral vector engineered to contain a genome, capable of amplifying and expressing its genetic information in infected cells, but unable to produce further infectious progeny particles in normal, unengineered cells, wherein an arenavirus open reading frame is removed and replaced with a first nucleotide sequence selected from:

[0682] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0683] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0684] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0685] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0686] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment;

[0687] And a second infectious, replication-defective arenavirus vector, engineered to contain a genome, capable of amplifying and expressing its genetic information in infected cells, but unable to produce further infectious progeny particles in normal, unengineered cells, wherein one of the arenavirus open reading frames is removed and replaced with a second nucleotide sequence selected from the following:

[0688] a. The nucleotide sequence encoding the HBV pre-S2 / S protein or its antigenic fragment;

[0689] b. The nucleotide sequence encoding HBV HBc protein or its antigenic fragment;

[0690] c. The nucleotide sequence encoding HBV HBs protein or its antigenic fragment;

[0691] d. The nucleotide sequence encoding a fusion of HBV HBs and HBc proteins or antigenic fragments thereof; and

[0692] e. The nucleotide sequence encoding the HBV HBe protein or its antigenic fragment.

[0693] 34. The pharmaceutical composition of embodiment 33, wherein the first and second nucleic acid sequences are different.

[0694] 35. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV pre-S2 / S protein or a fragment thereof, and wherein the second nucleic acid sequence encodes HBV HBc protein or a fragment thereof.

[0695] 36. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV pre-S2 / S protein or a fragment thereof, and wherein the second nucleic acid sequence encodes a fusion of HBV HBs and HBc proteins or fragments thereof.

[0696] 37. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV HBc protein or a fragment thereof, and wherein the second nucleic acid sequence encodes a fusion of HBV HBs and HBc protein or a fragment thereof.

[0697] 38. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV pre-S2 / S protein or a fragment thereof, and wherein the second nucleic acid sequence encodes HBV HBe protein or a fragment thereof.

[0698] 39. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV HBe protein or a fragment thereof, and wherein the second nucleic acid sequence encodes a fusion of HBV HBs and HBc proteins or fragments thereof.

[0699] 40. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV HBs protein or a fragment thereof, and wherein the second nucleic acid sequence encodes HBV HBe protein or a fragment thereof.

[0700] 41. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV pre-S2 / S protein or a fragment thereof, and wherein the second nucleic acid sequence encodes HBV HBs protein or a fragment thereof.

[0701] 42. The pharmaceutical composition of embodiment 33 or 34, wherein the first nucleic acid sequence encodes HBV HBs protein or a fragment thereof, and wherein the second nucleic acid sequence encodes HBV HBc protein or a fragment thereof.

[0702] 43. A pharmaceutical composition according to any one of embodiments 33 to 42, wherein the composition is suitable for intramuscular administration.

[0703] 44. A pharmaceutical composition according to any one of embodiments 33 to 42, wherein the composition is suitable for intravenous administration.

[0704] 45. The method of embodiment 27, further comprising in step a. transfecting the host cell with: cDNA of a second isovirus genome fragment, nucleic acid containing the L protein ORF and / or nucleic acid containing the NP protein ORF.

[0705] 46. ​​The method of implementation scheme 21, wherein the administration causes a reduction in liver damage in the patient.

[0706] 47. The method of embodiment 21, wherein the administration causes a decrease in one or more of the levels of HBsAg, HBeAg and HBcAg in the patient's blood.

[0707] 48. The method of embodiment 21, wherein the administration causes a decrease in the level of antibodies against HBV antigen in the patient's blood.

[0708] 49. The viral vector of embodiment 1, wherein the pre-S2 / S protein or its antigenic fragment comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1.

[0709] 50. The viral vector of embodiment 1, wherein the HBc protein or an antigenic fragment thereof comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2.

[0710] 51. The viral vector of embodiment 1, wherein the fusion of HBV HBs and HBc proteins or antigenic fragments thereof comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3.

[0711] 52. The pharmaceutical composition of embodiment 33, wherein the first nucleic acid sequence is derived from LCMV and the second nucleic acid sequence is derived from Junin virus.

[0712] 53. The pharmaceutical composition of embodiment 33, wherein the first nucleic acid sequence is derived from Junin virus and the second nucleic acid sequence is derived from LCMV.

[0713] 54. A viral vector according to any one of embodiments 1 to 17, wherein the sand virus is replication-defective and engineered to contain a genome, having the ability to amplify and express its genetic information in infected cells, but not to produce further infectious progeny particles in normal, unengineered cells.

[0714] 55. A viral vector according to any one of embodiments 1 to 9, wherein the sand-like virus is a replicating competent state.

[0715] 56. The viral vector of implementation scheme 1, wherein the sand-particle virus is bifragmented.

[0716] 57. A viral vector according to any one of embodiments 1 to 17, wherein the sand-like virus is tri-fragmented.

[0717] 58. An infectious arenavirus vector, wherein the arenavirus open reading frame is removed and replaced with a nucleotide sequence encoding a fusion of HBVHBs and HBc proteins or antigenic fragments thereof.

[0718] 59. The viral vector of embodiment 58, wherein the sand-like virus is a lymphocytic choroid plexus meningitis virus.

[0719] 60. The viral vector of implementation scheme 58 or 59, wherein the open reading frame encoding the glycoprotein of the sand virus is deleted or functionally inactivated.

[0720] 61. A viral vector according to any one of embodiments 58 to 60, wherein the viral vector is replication-defective.

[0721] 62. The viral vector of embodiment 58 or 59, wherein the viral vector is a replication competent state.

[0722] 63. A viral vector according to any one of embodiments 58, 59 or 62, wherein the viral vector is tri-fragmented.

[0723] 64. A method for treating or preventing hepatitis B virus infection in a patient, wherein the method comprises administering to the patient a viral vector of any one of embodiments 58 to 63.

[0724] 7. Example

[0725] 7.1 Design of the genome / vector construct of the arenavirus vector

[0726] Based on established methods (US Patent Application Publication No. US 2010 / 0297172A1; and), vaccine vectors based on LCMV and Junin virus (JUNV) expressing corresponding HBV antigens or certain domains thereof were designed (see appendix). Figure 1 ).

[0727] 7.2 Vaccines against Hepatitis B virus

[0728] Candidate vaccines against hepatitis B virus (HBV) include vectors based on rLCMV and rJUNV (Junin vaccine strain Candid#l) that express pre-S2 / S (rLCMV / pre-S2 / S, rJUNV / Pre-S2 / S), HBc (rLCMV / HBc, rJUNV / HBc), fusion proteins consisting of full-length HBs and HBc ORFs (rLCMV / HBsHBc), and HBe (rLCMV / HBe, rJUNV / HBe). The vectors will be replication-deficient (r2LCMV, also known as rLCMV, r2JUNV, also known as rJUNV) and replication-competent tri-fragmented constructs (r3LCMV, r3JUNV; see, e.g., Emone et al., 2009, PNAS, 106(9):3473-3478), in which the transgenes are arranged in a so-called “artificial” manner (r3LCMV). art r3JUNV art In primary immunization-boost vaccination with either homologous or heterologous conjugates, mice (e.g., C57BL / 6 mice) are immunized with one or a combination of these constructs. Administration is via intraperitoneal, intramuscular, or intravenous routes. The dose will be 10... 4 Up to 10 7 Within the range of foci-forming units (FFU). HBV-specific CD8+ T cells in the blood and / or spleen are measured at time points from 7 to 100 days post-immunization. For example, T cells can be measured using a combination of MHC class I tetramer and anti-CD8 antibody to identify the magnitude of the CD8+ T cell response against HBV-derived epitopes.

[0729] In a supplemental approach, synthetic peptides were used to selectively and directly stimulate blood and / or spleen-derived CD8+ T cells in vitro via intracellular cytokine analysis. Intracellular cytokine analysis measured the frequency of CD8+ T cells producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and / or interleukin (IL)-2. Surface expression of CD107a served as a marker of cell lysis and degranulation in flow cytometry (FACS). Peptide specificity was analyzed, including: HBs-derived epitopes VWLSVIWM (SEQ ID NO: 8), IPQSLDSWWTSL (SEQ ID NO: 9), and HBc-derived epitopes MGLKFRQL (SEQ ID NO: 10).

[0730] 7.3 Immunogenicity of vectors expressing HBV antigen based on replication-defective arenavirus

[0731] C57BL / 6 mice (n=5 per group) were administered 10 via intravenous route. 5 FFU rLCMV / HBs-HBc (group 1), rLCMV / HBc (group 3), rLCMV / Pre-S2 (group 4), or use 10 4 FFU-mediated rLCMV / HBs-HBc (Group 2) immunization was administered once. The control group received no treatment. CD8+ T cells in the blood were measured 10 days post-immunization using MHC class I multimers. H-2K complexed with the HBs-derived epitope VWLSVIWM. b dextramers, and H-2K complexed with the HBc-derived epitope MGLKFRQL. b Dextramers are used in combination with anti-CD8a antibodies to identify hepatitis B virus-specific CD8+ T cells. The counted cells are represented as total CD8+ T cells in peripheral blood. + B220 - Percentage of T cell repertoire.

[0732] As attached Figure 3 As shown, the results indicate that inoculation with rLCMV / HBsHBc, rLCMV / HBc, and rLCMV / Pre-S2 induced substantial antigen-specific CD8+ T cell responses against the antigens expressed by their respective vectors. The anti-HBs and anti-HBc CD8+ T cell responses induced by rLCMV / HBs-HBc inoculation showed a clear dose-dependent relationship. The higher frequency of anti-HBc CD8+ T cells during rLCMV / HBs-HBc immunization compared to rLCMV / HBc immunization suggests that fusion with HBs resulted in amplified immunogenicity of HBc.

[0733] Compared with immunization with rLCMV / HBs-HBc, the frequency of anti-HBs CD8+ T cells was slightly higher after immunization with rLCMV / Pre-S2, suggesting the possibility that anti-HBc CD8+ T cell responses compete with anti-HBs responses for antigen availability.

[0734] 7.4 Immunogenicity of vectors expressing HBV antigen based on attenuated replicating competent arenavirus

[0735] C57BL / 6 mice (n=5 per group) were administered 10 via intravenous route. 5 FFU r3LCMV / HBs-HBc (group 1), r3LCMV / HBc (group 2), r3LCMV / Pre-S2 (group 3), or with 10 5 FFU-enzymed mice were immunized once with rLCMV / HBs-HBc (group 4). Control mice were not immunized. Eight days post-immunization, HBs and HBc epitope-specific CD8+ T cells in the blood were measured using MHC class I multimers. H-2K complexed with the HBs-derived epitope VWLSVIWM b dextramers, and H-2K complexed with the HBc-derived epitope MGLKFRQL. b Dextramers are used in combination with anti-CD8a antibodies to identify hepatitis B virus-specific CD8+ T cells. Cell counts are represented in two different ways: total CD8+ T cells in peripheral blood. + B220 - The percentage of T cell pool (Figure 4A), or the percentage of circulating lymphocytes in the blood (Figure 4B).

[0736] As shown in Figure 4, the results indicate that all r3LCMV-based constructs and the replication-defective rLCMV / HBs-HBc reference vector were immunogenic, evoking epitope-specific CD8+ T cells targeting their respective vectorized antigens. Furthermore, when used as a percentage of circulating lymphocytes to count epitope-specific CD8+ T cells, the replicating r3LCMV / HBs-HBc showed greater immunogenicity than its replication-defective counterpart, rLCMV / HBs-HBc.

[0737] Equivalents and Combination by Reference: The embodiments described herein are intended to be merely exemplary, and those skilled in the art will recognize, or be able to identify, numerous equivalents of the specific processes described herein without departing from conventional experimentation. All such equivalents are considered to be within the scope of the invention and covered by the following embodiments. All references cited herein (including patent applications, patents, and publications) are incorporated herein by full reference for all purposes, to the same extent as specifically and individually indicated to be incorporated herein by full reference for all purposes.

Claims

1. An infectious, replicating three-segment isonin virus vector, wherein the isonin virus vector comprises an L segment and two S segments, wherein the two S segments are a first S segment and a second S segment, wherein... a. The open reading frame (ORF) encoding the first HBV antigen or its antigenic fragment in the first S segment is under the control of the 5' UTR of the arenavirus genome, and the ORF encoding the arenavirus glycoprotein (GP) is under the control of the 3' UTR of the arenavirus genome; b. The ORF encoding the second HBV antigen or an antigenic fragment thereof in the second S segment is controlled by the 5' UTR of the arenavirus genome, and the ORF encoding the arenavirus nucleoprotein (NP) is controlled by the 3' UTR of the arenavirus genome; and c. The ORF encoding the arenavirus matrix protein Z (Z protein) in the L segment is controlled by the 5' UTR of the arenavirus genome, and the ORF encoding the arenavirus RNA-dependent RNA polymerase L (L protein) is controlled by the 3' UTR of the arenavirus genome. and, a. The first HBV antigen is the HBV pre-S2 / S protein or its antigenic fragment, the HBV HBc protein or its antigenic fragment, the HBV HBs protein or its antigenic fragment, a fusion protein of HBV HBs and HBc proteins, and b. The second HBV antigen is HBV pre-S2 / S protein or its antigenic fragment, HBV HBc protein or its antigenic fragment, HBV HBs protein or its antigenic fragment, or a fusion protein of HBV HBs and HBc proteins. The sand-like virus mentioned above is a lymphocytic choriomeningitis virus.

2. The three-segment sand-particle virus vector according to claim 1, wherein: The first HBV antigen is different from the second HBV antigen.

3. The three-segment sand virus vector according to claim 1, wherein the first HBV antigen and the second HBV antigen are the same HBV antigen.

4. The three-segment sand virus vector according to claim 1, wherein the pre-S2 / S protein or its antigenic fragment comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:

1.

5. The three-segment sand virus vector according to claim 1, wherein the HBc protein or its antigenic fragment comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:

2.

6. The three-segment isovirus viral vector according to claim 1, wherein the fusion of HBV HBs and HBc proteins or their antigenic fragments comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:

3.

7. The three-segment isovirus vector according to claim 2, wherein the expression of the first and second HBV antigens or fragments thereof generates an antigenic protein complex, the antigenic protein complex triggering a higher titer of neutralizing antibody compared to the expression of each component of the protein complex.

8. The three-segment isovirus vector according to claim 1, wherein the lymphocytic choroid plexus meningitis virus is the lymphocytic choroid plexus meningitis virus clone 13 strain or the lymphocytic choroid plexus meningitis virus MP strain.

9. The three-segment arenavirus vector according to claim 1, wherein the arenavirus comprises a genome fragment, and wherein the nucleotide sequence comprising the genome fragment is... a. The sequence of nucleotides 1639 to 3315 of SEQ ID NO: 11 or nucleotides 1640 to 3316 of SEQ ID NO: 12; or b. An expression product, wherein the amino acid sequence of the expression product is the amino acid sequence encoded by SEQ ID NO: 11, 1639 to 3315 or SEQ ID NO: 12, 1640 to 3316.

10. The three-segment isovirus vector of claim 1, wherein administration of the isovirus vector to a subject induces a durable immune response against the first and / or second HBV antigen or an antigenic fragment thereof.

11. A pharmaceutical composition, immunogenic composition, or vaccine comprising the three-segment sand virus vector of claim 1 and a pharmaceutically acceptable vector.

12. Use of the three-segment isovirus vector of claim 1 in the preparation of a medicament or kit for the treatment or prevention of HBV infection or HBV infection symptoms in patients.

13. A method for generating an infectious, replicating three-segment isonin virus vector, the method comprising: a. Transfect the following cDNA into the host cell: i. The first S segment, wherein the ORF encoding the first HBV antigen or an antigenic fragment thereof in the first S segment is under the control of the 5' UTR of the arenavirus genome and the ORF encoding GP is under the control of the 3' UTR of the arenavirus genome; ii. The second S segment, wherein the ORF encoding the second HBV antigen or an antigenic fragment thereof in the second S segment is under the control of the 5' UTR of the arenavirus genome and the ORF encoding the NP is under the control of the 3' UTR of the arenavirus genome; and iii. The L segment, wherein the ORF encoding the Z protein in the L segment is controlled by the 5' UTR of the arenavirus genome and the ORF encoding the L protein is controlled by the 3' UTR of the arenavirus genome; b. Transfect host cells with plasmids expressing the minimal trans-acting factors NP and L proteins of arenavirus; c. Maintain the host cells under conditions suitable for virus formation; and d. Harvest the infectious, replicating three-segment sand virus particles. The sand-like virus mentioned above is a lymphocytic choriomeningitis virus.

14. The pharmaceutical composition, immunogenic composition, or vaccine according to claim 11, wherein the pharmaceutical composition, immunogenic composition, or vaccine is suitable for intramuscular or intravenous administration.

15. A group of nucleic acids, which includes: (a) A first nucleic acid, wherein the first nucleic acid comprises cDNA of the first S segment of the three-segment sand virus viral vector of claim 1; (b) a second nucleic acid, wherein the second nucleic acid comprises cDNA of the second S segment of the three-segment arenavirus viral vector of claim 1; and (c) A third nucleic acid, wherein the third nucleic acid comprises cDNA of the L segment of the three-segment sand virus viral vector of claim 1.

16. A method for identifying hepatitis B virus-specific CD8+ T cells, wherein the method includes (1) C57BL / 6 mice were administered 10 via intravenous route. 5 FFU rLCMV / HBs-HBc, rLCMV / HBc, rLCMV / Pre-S2, or using 10 4 One FFU-immunized r3LCMV / HBs-HBc group; control group received no treatment. (2) Ten days after immunization, CD8+ T cells in the blood were measured by using MHC class I multimers; (3) H-2K complexed with HBs-derived epitope VWLSVIWM b dextramers, and H-2K complexed with the HBc-derived epitope MGLKFRQL. b Dextramers are used in combination with anti-CD8a antibodies to identify hepatitis B virus-specific CD8+ T cells.

17. Candidate vaccines against hepatitis B virus (HBV) include r3LCMV-based vectors that express pre-S2 / S (r3LCMV / pre-S2 / S), HBc (r3LCMV / HBc), and a fusion protein consisting of full-length HBs and HBc ORF (r3LCMV / HBsHBc).

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