Recombinant virus expressing alpha-1,3-galactosyltransferase and its use

Abstract

Disclosed are viruses and vaccines composed and prepared by such viruses, which include a heterologous nucleic acid fragment encoding α-1,3-galactosyltransferase (α-1,3-GT), so that the nucleic acid fragment expresses α-1,3-GT when the virus infects a host cell. Such viruses produce proteins with α-1,3-galactose. The presence of α-1,3-galactose on the proteins of infected cells can strongly stimulate the host's immune response to the viral proteins of the virus, thereby enhancing the effect of the virus as a vaccine. Also disclosed are vaccines including such viruses and / or produced by such viruses. Also disclosed are methods for preparing and using such viruses and vaccines, such as administering the disclosed vaccines to subjects in need, and preparing vaccines including one or more viral proteins expressed by the disclosed viruses.

Description

[0001] This international patent application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 959,581, filed on January 10, 2020, the entire contents of which are incorporated by reference for all purposes. Technical Field

[0002] The disclosed invention is generally in the field of vaccines, and in particular in the field of live attenuated vaccines. Background Art

[0003] Influenza is a highly contagious disease with a significant impact on global public health (Krammer et al., Nat. Rev. Dis. Primers, 4:3 (2018)). Sixteen HA subtypes and nine NA subtypes have been identified in bird populations. These HA subtypes can be divided into two groups (Group 1: H1, H2, H5, H6, H8, H9, H11, H12, and H16; Group 2: H3, H4, H7, H14, and H15). Of these subtypes, only H1N1, H2N2, and H3N2 viruses are known to cause epidemics.

[0004] The secondary attack rates of seasonal and pandemic influenza in humans are estimated to be approximately 15% and 33%, respectively (World Health Organization, website who.int / csr / disease / swineflu / assess / disease_swineflu_assess_20090511 / en / (visited in July 2019)). In view of the fact that animal influenza viruses can cause epidemics, there is an urgent need for effective "universal" strategies to control influenza. Among the existing control measures, vaccination is one of the most effective ways to prevent influenza infection. Currently, there are two main types of influenza vaccines available for human use on the market, namely inactivated vaccines and live attenuated vaccines (Rajao and Perez, Front. Microbiol., 9:123 (2018); Paules et al., N. Engl. J. Med., 378:7-9 (2018)).

[0005] However, current commercial vaccines generally have poor vaccine effectiveness against drifted antigenic variants and short-lived vaccine-induced protection. In addition, none of the currently available vaccines are designed to induce broad cross-protective immune responses against different animal influenza subtypes, which is a key feature of a "universal influenza vaccine."

[0006] There remains a need to develop an effective and safe "universal" influenza vaccine. There is also a need for strategies to provide an effective and safe "universal" influenza vaccine.

[0007] It is therefore an object of the present invention to provide improved vaccines for the prevention of viral infections such as influenza, varicella, measles, respiratory syncytial virus, infectious mononucleosis and herpes simplex virus infections.

[0008] Another object of the present invention is to provide viral vaccines that can provide stronger and longer-lasting protection, especially for high-risk groups with poor immune response.

[0009] Another specific object of the present invention is to provide improved influenza vaccines. SUMMARY OF THE INVENTION

[0011] Disclosed are viruses and vaccines composed of and prepared from such viruses, which include a heterologous nucleic acid fragment encoding α-1,3-galactosyltransferase (α-1,3-GT), such that the nucleic acid fragment expresses α-1,3-GT when the virus infects a host cell. Such viruses produce proteins with α-1,3-galactose. The presence of α-1,3-galactose on proteins in infected cells can strongly stimulate the host's immune response to viral proteins of the virus, thereby enhancing the effect of the virus as a vaccine. Also disclosed are vaccines comprising such viruses and / or produced by such viruses. Also disclosed are methods for preparing and using such viruses and vaccines, such as administering the disclosed vaccines to subjects in need, and such as preparing vaccines comprising one or more viral proteins expressed by the disclosed viruses.

[0012] Disclosed are viruses comprising heterologous nucleic acid fragments encoding α-1,3-galactosyltransferase (α-1,3-GT), such that when the virus infects a host cell, the nucleic acid fragment expresses α-1,3-GT. Typically, when such a virus infects a host cell, the expression of α-1,3-GT results in proteins in the infected host cell having α-1,3-galactose. For example, in certain forms, when the virus infects a host cell, at least one protein exposed on the surface of the host cell will have α-1,3-galactose. Also disclosed are vaccines comprising such viruses and / or produced by such viruses. Also disclosed are methods for preparing and using such viruses and vaccines. The heterologous nucleic acid fragment encoding α-1,3-GT can be (or is considered to be) a gene or coding portion of a gene encoding α-1,3-GT. When the virus infects a host cell, the nucleic acid fragment / gene should generally be able to express (configured to express) α-1,3-GT, such that when the virus infects a host cell, the virus expresses α-1,3-GT.

[0013] In some forms, at least one viral protein expressed by the virus when the virus infects a host cell will have alpha-1,3-galactose. In some forms, the virus is live and attenuated. In some forms, the nucleic acid fragment is introduced into the genome of the virus.

[0014] In some forms, the nucleic acid fragment is introduced into the reading frame of the viral coding region of the virus in an in-frame manner. In some forms, the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the viral coding region of the virus. In some forms, the virus further comprises a protease cleavage site encoded between the nucleic acid fragment and the viral coding region of the virus.

[0015] In some forms, the virus is an influenza virus. In some forms, the nucleic acid fragment is introduced into the reading frame of the neuraminidase (NA) coding region of the virus in an in-frame manner. In some forms, the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the viral NA coding region. In some forms, the virus is further included in the protease cleavage site encoded between the nucleic acid fragment and the NA coding region of the virus. In some forms, the protease cleavage site is included in the 2A self-cleaving peptide. In some forms, the 2A self-cleaving peptide is derived from porcine teschovirus (teschovirus) -1. In some forms, the 2A self-cleaving peptide is flanked by a short peptide linker. In some forms, the peptide linker is GSG.

[0016] In some forms, the α-1,3-GT is mammalian α-1,3-GT. In some forms, the α-1,3-GT is mouse α-1,3-GT.

[0017] In some forms, the virus is attenuated by continuous passage in cultured cells, continuous passage in heterologous host animals, gene deletion in the virus, site-directed mutagenesis of the virus, changing the codon usage of the virus, selecting cold-adapted mutants, using related viruses from heterologous host species, using naturally occurring attenuated virus strains, or a combination thereof. In some forms, the virus is attenuated by continuous passage in cultured cells, wherein the cultured cells are Madin-Darby canine kidney (MDCK) cells.

[0018] In some forms, the virus is influenza virus, influenza A virus, influenza B virus, human papillomavirus, measles virus, human immunodeficiency virus, rabies virus, Ebola virus, Hanta virus, Marburg virus, Lassa virus, dengue virus, Eastern equine encephalitis virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, hepatitis E virus, human herpes virus, human SARS coronavirus, mumps virus, polio virus, rubella virus, varicella-zoster virus, Rift Valley fever virus, West Nile virus, yellow fever virus, human T-lymphotropic virus or Merkel cell polyomavirus. In some forms, the virus is influenza virus.

[0019] In some forms, the vaccine includes the disclosed viruses. In some forms, the viruses are live and attenuated. In some forms, the vaccine includes one or more viral proteins expressed by the disclosed viruses, wherein the one or more viral proteins contain alpha-1,3-galactose.

[0020] In some forms, methods of using the disclosed vaccines include administering the disclosed vaccines to a subject in need thereof. In some forms, the vaccine is administered intranasally, intrapulmonary, orally, subcutaneously, intramuscularly, intradermally, or intraperitoneally.

[0021] Also disclosed are methods for preparing vaccines comprising one or more viral proteins expressed by the disclosed viruses, wherein the one or more viral proteins comprise α-1,3-galactose. In some forms, the method comprises infecting cells with the virus, thereby producing one or more viral proteins comprising α-1,3-galactose. In some forms, the method further comprises purifying the one or more viral proteins.

[0022] Other advantages of the disclosed methods and compositions will be set forth in part in the following description and will be understood in part from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained by the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0024] Figure 1 is a schematic diagram showing WT and mutant NA fragments in the sense of cRNA. The mouse α-1,3-GT gene ORF was introduced immediately before the stop codon of the NA ORF (open arrow). As shown in the figure, the autoproteolytic cleavage site of porcine Teschovirus-1 flanked by two short peptide linkers (GSG) was introduced into the junction of NA and α-1,3-GT ORF. The 5' and 3' untranslated regions (UTRs) and the start codon (black arrow) are indicated. The red box represents the 5' end vRNA packaging signal sequence of the NA ORF region.

[0025] Figure 2 is a graph showing the replication kinetics of NAGT mutants in MDCK cells (MOI: 0.001). Progeny virus titers were determined using a standard plaque assay. Mean ± SD; **: p < 0.01, ***: p < 0.001.

[0026] Figure 3 is a bar graph showing that NAGT virus-infected human A549 cells enhance the phagocytic activity of human monocyte-derived macrophages. Mean ± SD; *: p < 0.05.

[0027] Figures 4A-4F is a graph showing an antibody-dependent NK cell assay. Figure 4A , 4C and 4E) or not present ( Figure 4B , 4D and 4F), by PBS ( Figure 4E and 4F ), WT virus ( Figure 4C and 4D ) or NAGT( Figure 4A and 4B ) treated human A549 and activated human NK cells were co-incubated. After incubation under different experimental conditions, the percentage of influenza NP positive cells is shown in the figure. These figures are representative FACS graphs using serum samples from healthy donors.

[0028] Figure 5 is a graph showing the stimulatory effect of all tested human sera on NK cells to kill WT and NAGT infected A549 cells (fold reduction of NP+A549 cells; N=6; paired t-test). Mean±SD; *: p<0.05.

[0029] Figure 6 is a graph showing luciferase reporter assay for ADCC activity. A549 cells infected with WT or NAGT were first treated with serially diluted human serum samples and then tested by ADCC assay. Each data point represents the average reading of 6 different human serum samples. Mean ± SD; *: p < 0.05.

[0030] Figure 7 Schematic diagram showing vaccination and virus challenge. Mice were sensitized and boosted with rabbit RBCs via the intraperitoneal route at 4 and 6 weeks of age. Mice were vaccinated with NAGT mutants at 8 weeks of age and then challenged with a lethal dose of influenza virus via the intranasal route. Relevant samples were collected at 4, 6 and 8 weeks of age and at different post-vaccination (1, 5 and 11 days) / post-challenge (0, 3 and 7 days) time points.

[0031] Figure 8 The splenic CD4 +Figure 1. T cell recall response. T cells studied by ICS. Activated T cells (TNFα + ). Data represent mean ± SD; ***: p < 0.001.

[0032] Fig. 9 The splenic CD8 + Figure 1. T cell recall response. T cells studied by ICS. Activated T cells (TNFα + ). Data represent mean ± SD; ***: p < 0.001.

[0033] Figures 10A-10C Figure 2 is a graph showing professional antigen presenting cells (DCs and macrophages) and neutrophils in the spleen after vaccination. Vaccinated WT mice and KO mice were harvested on days 1, 5, and 21 after vaccination. Unvaccinated mice (-) were used as controls. Representative FACS graphs and DCs ( Fig. 10A ), neutrophils ( Fig. 10B ) and macrophages ( Fig. 10C ). Data from vaccinated mice are highlighted in gray boxes. Dotted lines are used for comparison between KO and WT mice at the same time point (t-test). Data represent mean ± SD; *: p < 0.05, **: p < 0.01.

[0034] Figures 11A-11E Figure 2 is a graph showing professional antigen presenting cells (DCs and alveolar macrophages) and neutrophils in the lung after vaccination. Vaccinated WT mice and KO mice were harvested on days 1, 5, and 21 after vaccination. Unvaccinated mice (-) were used as controls. Representative FACS graphs and DCs ( Fig.11A ), CD11b 低 / - DC( Fig. 11B ) and CD11b 高 DC( Fig. 11C ), neutrophils ( Fig.11D ) and alveolar macrophages ( Fig.11E ). Data from vaccinated mice are highlighted in grey boxes. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0035] Figures 12A-12CFigure 2 is a graph showing the protection of mice by NAGT mutants against lethal homologous challenge. Vaccinated and unvaccinated (-) mice were challenged with a lethal dose of H1N1 (A / PR / 8 / 34; 40 MLD) 3 weeks after vaccination. 50 ) to attack. Fig. 12A Shown are the survival rates of the different groups of mice assessed daily for 14 days after challenge (log-rank test). There were 16 mice in each vaccinated group. Fig. 12B The weight loss of different mouse groups is shown. Fig. 12C Lung virus titers are shown for mice at days 3 and 7 post-challenge. Data from vaccinated mice are highlighted in grey boxes.

[0036] Figures 13A-13B Figure 2 shows PR8-specific CD4 in BAL of infected mice at day 7 after infection. + ( Fig.13A ) and CD8 + ( Fig. 13B ) Graph of T cell recall response. Activity was determined by ICS assay. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0037] Figures 14A-14B The PR8-specific CD4 + ( Fig.14A ) and CD8 + ( Fig. 14B ) Graph of T cell recall response. Activity was determined by ICS assay. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0038] Figures 15A-15D Figure 2 is a graph showing protection of mice by NAGT mutants against lethal heterosubtypic H3N2 challenge. Vaccinated and unvaccinated (-) mice were challenged with a lethal dose of H3N2 virus (HK68; 10 MLD 50 ) to attack. Fig.15A Survival rates assessed daily for 14 days after challenge are shown (log-rank test). Each vaccinated group had > 20 mice. Fig. 15B The weight loss of different mouse groups is shown. Fig. 15C Shown are lung virus titers from mice at days 3 and 7 post-challenge. Data from vaccinated mice are highlighted in grey boxes. Fig.15DHK68-specific neutralizing antibody titers in mice before (3 weeks after vaccination) and after (7th and 14th days after challenge) are shown. Data from vaccinated mice are highlighted in gray boxes. Dashed lines are used for comparisons between KO mice and WT mice at the same time point (t test). Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0039] Figures 16A-16B Figure 2 shows HK68-specific CD4 in BAL of infected mice on day 7 after infection. + ( Fig.16A ) and CD8 + ( Fig. 16B ) Graph of T cell recall response. Data represent mean ± SD; **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0040] Figures 17A-17B The HK68-specific CD4 + ( Fig.17A ) and CD8 + ( Fig. 17B ) Graph of T cell recall response. Dashed line is used for comparison between KO mice and WT mice at the same time point (t-test). Data represent mean ± SD; *: p < 0.05, ****: p < 0.0001.

[0041] Figures 18A-18C Figure 2 shows the total DCs in infected lung tissues on day 7 after challenge. Fig.18A ), CD11b 低 / - DC( Fig.18B ) and CD11b 高 CD( Fig.18C ). The dashed line is used for comparison between KO mice and WT mice at the same time point (t test). Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0042] Figures 19A-19C Figure 2 is a graph showing the protection of mice by NAGT mutants against lethal heterosubtypic H5N1 challenge. Vaccinated and unvaccinated (-) mice were challenged with a lethal dose of H5N1 virus (VN1203; 10 MLD 50 ) to attack. Fig.19A Survival rates assessed daily for 14 days after challenge are shown (log-rank test). There were 10 mice per vaccinated group. Fig.19B Shown are lung virus titers in mice at day 7 post-challenge. Fig.19C The weight loss of different mouse groups is shown. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

[0043] Figures 20A-20B is a graph showing that enhanced protection of NAGT mutants in mice requires anti-α-Gal antibodies. Fig. 20A These mice groups were shown to be 50 There were 5 mice in each group. Fig. 20B The weight loss of different mouse groups is shown. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0044] Figures 21A-21B is a graph showing enhanced polyfunctional T cell responses in BAL by NAGT mutant vaccination after viral challenge. Fig.21A PR8-specific multifunctional CD8 + T cells. Fig. 13B The data presented in reanalysis data. Fig. 21B HK68-specific multifunctional CD4 + T cells. Fig.16A Data presented in were reanalyzed. Data represent mean ± SD; *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0046] The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included therein and by reference to the Figures and their previous and following descriptions.

[0047] Influenza A viruses have multiple antigenically diverse HA subtypes. Classical influenza vaccines are subtype specific, and they cannot produce satisfactory heterosubtypic immunity against multiple influenza subtypes. Disclosed herein are live and attenuated viruses that can produce α-Gal epitopes in infected cells and vaccines containing one or more viruses. The disclosed viruses can increase the immunogenicity and protective effect of vaccines, thereby allowing a broad cross-reactive immune response against different influenza subtypes.

[0048] Anti-α-Gal antibodies are naturally produced by humans. Therefore, cells infected with the disclosed viruses can promote opsonization of infected cells, resulting in enhanced vaccine-induced immune responses. For example, cells infected with a virus carrying the α-1,3-galactosyltransferase (α-1,3-GT) gene can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro, and can induce 100% protection against lethal heterosubtypic group 1 (e.g., H5) or group 2 (e.g., H3) influenza challenge in vivo. In contrast, heterosubtypic protection of the virus in mice without anti-α-Gal antibody expression was only partial, indicating that the enhanced vaccine-induced protection involves anti-α-Gal antibodies after vaccination. In addition, the disclosed vaccines can stimulate CD11b 低 / - Lung dendritic cells, which are known to play a key role in influenza virus clearance.

[0049] Viruses and vaccines composed of and prepared from such viruses have been found, including heterologous nucleic acid fragments encoding α-1,3-galactosyltransferase (α-1,3-GT), such that the nucleic acid fragments express α-1,3-GT, which are useful when the virus infects host cells because of the expression of α-1,3-GT. Such viruses produce proteins with α-1,3-galactose. The presence of α-1,3-galactose on proteins of infected cells can strongly stimulate the host's immune response to viral proteins of the virus (because human hosts will produce anti-α-1,3-galactose antibodies), thereby enhancing the effect of the virus as a vaccine. Vaccines comprising such viruses and / or produced by such viruses are also disclosed. Methods of preparing and using such viruses and vaccines are also disclosed, such as administering the disclosed vaccines to subjects in need, and such as preparing vaccines comprising one or more viral proteins expressed by the disclosed viruses.

[0050] It should be understood that unless otherwise specified, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques or specific reagents, and therefore may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0051] As used herein, the term "robust immune response" refers to an antibody-mediated response, a cell-mediated adaptive immune response, an innate immune response, or a combination thereof, which is generated following administration of a virus and is generated at a level that provides immune protection against subsequent challenge with the same virus.

[0052] As used herein, the terms "immune response", "immune response" or "immune response" refer to the development of a humoral (antibody-mediated) response, a cellular (mediated by antigen-specific T cells or their secretory products) response, or both, directed against an antigen. This response can be an active response induced by the administration of an immunogen or a passive response induced by the administration of antibodies or sensitized T cells. The presentation of polypeptide epitopes associated with class I or class II MHC molecules triggers a cellular immune response to activate antigen-specific CD4 + T helper cells, CD8 + cytotoxic T cells, or both. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other innate immune components. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 + The relative contributions of humoral and cellular responses to the protective or therapeutic effects of an immunogen can be distinguished by isolating antibodies and T cells, respectively, from immunized syngeneic animals and measuring the protective or therapeutic effects in a second subject.

[0053] As used herein, the term "immunoprotection" refers to an immune response established for one or more antigens of an infectious agent, which prevents the progression of the disease or one or more symptoms of the disease, preventing future attacks of the infectious agent for at least one year, at least two years, at least three years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least ten years after the immune response is established, thereby protecting the host from future attacks of the pathogen. Future attacks of the infectious agent can come from, for example, pathogens, pathogen subtypes, pathogen strains, or pathogen particles that are identical, similar, or different from the pathogen used to establish the immune protection. For example, when the infectious agent is a virus, the future attacks of the virus can come from, for example, viruses, virus subtypes, virus strains, or virus particles that are identical, similar, or different from the pathogen used to establish the immune protection.

[0054] As used herein, the term "attenuated live virus" or "live attenuated virus" or "attenuated virus" refers to a virus that has been altered from an original parental virus or wild-type virus in the following manner: its ability to infect a host, replicate in a host, be packaged, re-infect a host, or a combination thereof is weakened. Typically, this attenuation can be generally in several or all hosts of the virus, or only in only one or several hosts of the virus. Therefore, the attenuation of the attenuated live virus (i.e., its ability to infect a host, replicate in a host, be packaged, re-infect a host, or a combination thereof is weakened) is generally associated with one or more viral hosts, wherein the attenuated live virus is not significantly or detectably attenuated in one of the more other hosts of the virus. The attenuated live viruses disclosed herein are generally genetically altered and can be referred to as mutated, mutated, genetically engineered, recombinant, or a combination.

[0055] As used herein, the term "live attenuated vaccine" or "live attenuated vaccine" or "attenuated vaccine" refers to a pharmaceutical composition containing a live attenuated pathogen, such as a virus. The pharmaceutical composition contains at least one immunologically active component that induces an immune response against the virus in a subject, protects the subject from the virus or possible death caused by the virus, or both, and optionally may include one or more additional components that enhance the immunological activity of the active component. The vaccine may also include other components typical of pharmaceutical compositions. At least one immunologically active component is one or more live attenuated viruses described herein.

[0056] As used herein, the term "homologous virus challenge" refers to a second or subsequent virus challenge of the same subject, wherein the virus is genetically substantially identical to the virus, virus subtype, strain, or infectious virus particle used in the first virus challenge. In a homologous virus challenge (wherein the second or subsequent virus challenge of the same subject uses a virus of a virus subtype that is substantially identical or similar to the virus used in the first challenge), homologous immune protection can be obtained.

[0057] As used herein, the term "heterologous viral challenge" refers to a second or subsequent viral challenge of the same subject, wherein the virus is significantly genetically different from the virus, viral subtype, strain, or infectious viral particle used in the first viral challenge. In a heterologous viral challenge (wherein the second or subsequent viral challenge of the same subject uses a virus with a significantly different viral subtype than the virus used in the first challenge), heterosubtypic immune protection can be obtained.

[0058] As used herein, the term "replication" refers to genome replication. When referring to viruses, the term "replication" also means "viral replication", which covers the replication of the viral genome, the production of viral proteins, the packaging of the viral genome, and the formation of new viral particles.

[0059] As used herein, in the context of viruses, the terms "unchanged replication", "substantially identical replication" and "similar replication" refer to replication of a virus that is comparable or substantially identical to replication of a reference virus of the same species. For example, when replication of an attenuated live virus is comparable or substantially identical to replication of a wild-type virus of an attenuated live virus, such replication can be referred to as unchanged or similar replication. Replication can be measured by any technique used in the art for measuring viral replication, such as by viral yield or by the rate at which a virus replicates or is produced. For example, replication can be measured by itself, or by the number of plaque-forming units per unit time, or per unit weight of an organ, or per unit weight of protein or nucleic acid, or per unit length of nucleic acid, the number of virions, the number of viral particles, etc. For example, "substantially identical replication rate" refers to a change in the number of viral particles per unit weight of tissue, wherein the change in the number of one virus is substantially the same as the change in the number of another virus. As used herein, the term "substantially identical" refers to identical or similar values, such as replication values ​​or values ​​of changes in viral numbers. When compared to each other, these values ​​may be the same or may differ from each other within a range of ±10%.

[0060] As used herein, in the context of viruses, the terms "slow replication" and "attenuated replication" refer to viral replication that is slower or less than replication of a reference virus of the same species. For example, when the replication of an attenuated live virus is slower or less than replication of a wild-type virus of the attenuated live virus, such replication can be referred to as slow or attenuated replication. Replication can be measured by any technique used in the art to measure viral replication, such as by viral yield or by the rate at which the virus replicates or is produced. For example, the reduction in replication can be any reduction in the number of plaque forming units, the number of virions, the number of viral particles, etc. The range of reduction relative to the number of plaque forming units, the number of virions, or the number of viral particles of the wild-type virus can be, for example, 10% to 90%.

[0061] As used herein, the terms "master strain" and "master virus" refer to a subtype or strain of a virus that can be allowed to recombine with another virulent subtype or strain of the virus to produce a hybrid virus.

[0062] As used herein, the term "synonymous substitution" refers to a change in a nucleotide located in a coding region in the genome of an organism but does not result in a change in the encoded amino acid sequence.

[0063] As used herein, the term "silent mutation" refers to a change in a nucleotide in the genome of an organism that does not significantly change the phenotype of the organism. Silent mutations can occur in non-coding regions (outside of genes within introns) or in exons. When silent mutations occur in exons or coding regions, they either do not result in a change in the encoded amino acid sequence (i.e., a synonymous substitution) or result in the insertion of an alternative amino acid with similar properties to the original amino acid, and in either case, the phenotype does not change significantly.

[0064] As used herein, the term "mammalian host" in the context of a virus refers to any mammalian organism capable of being infected by and propagating the virus.

[0065] As used herein, the term "nucleic acid region" or "genomic region" refers to any region of a genome. The region can be a fragment of a genome, a segment of two or more nucleotides, or the entire genome.

[0066] As used herein, the term "involved in packaging or splicing" in the context of a virus refers to a genomic region encoding a protein responsible for packaging or splicing of genomic material, or a genomic region having nucleic acid sequences that define packaging or splicing sites (such as splice donor sites and splice acceptor sites).

[0067] As used herein, the term "nucleic acid molecule" refers to any nucleic acid in any possible configuration, such as a single strand, a double strand, or a combination thereof. Nucleic acids include, for example, DNA molecules, RNA molecules, analogs of DNA or RNA generated using nucleotide analogs or using nucleic acid chemistry, locked nucleic acid molecules (LNA), protein nucleic acid molecules (PNA) and tecto-RNA molecules (e.g., Liu, B. et al., J.Am.Chem.Soc., 126: 4076-4077 (2004)). PNA molecules are nucleic acid molecules in which the backbone is a pseudopeptide rather than a sugar. Therefore, compared to, for example, DNA or RNA, PNA generally has a charge-neutral backbone. However, PNA is capable of hybridizing at least complementary and substantially complementary nucleic acid chains, just like, for example, DNA or RNA (PNA is considered a structural mimetic). LNA molecules have a modified RNA backbone with a methylene bridge between C4' and O2' that locks the furanose ring in an N-type configuration, thereby providing higher double-strand stability and nuclease resistance for each molecule. Unlike PNA molecules, LNA molecules have a charged backbone. DNA or RNA can be genomic origin or synthetic origin, and can be single-stranded or double-stranded. Such nucleic acids can be copolymers, oligonucleotides, mixed polymers, sense strands and antisense strands of, for example, mRNA, cRNA, vRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, DNA and RNA, or can contain non-natural or derived nucleotide bases. The corresponding nucleic acid can be a nucleic acid containing non-natural nucleotide analogs, a nucleic acid connected to an affinity tag or a label, or both. When mentioned in this article, the terms "nucleotide sequence", "polynucleotide", "nucleic acid sequence", "nucleic acid" and "nucleic acid molecule" are used interchangeably.

[0068] As used herein, the term "wild-type virus" refers to the virus in its native form as it exists in a host in nature prior to genetic or other types of intentional manipulation by humans.

[0069] As used herein, the term "parental virus" refers to a virus prior to initial or further genetic or other types of intentional manipulation by man.

[0070] As used herein, the terms "genetic engineering" and "genetic modification" refer to using biotechnology to carry out genetic manipulation on nucleotide, codon, gene, fragment, framework or chromosome level to organism. By first using molecular cloning methods to separate and replicate interested genetic material to generate DNA sequence, or by synthetic DNA, then the construct is inserted into the host genome, new nucleotides, bases, base pairs or DNA are inserted into the host genome. Nucleotide, codon, gene, fragment, framework or chromosome can be removed (" knock out "), added (" knock in ") or replaced. Genetically engineered organisms can also be referred to as mutated organisms, mutant organisms, recombinant organisms or their combinations. Usually, all of these terms can be applied to genetically engineered organisms interchangeably.

[0071] As used herein, the term "conserved site at the amino acid level" refers to a site in the genome of an organism that uses the same amino acid more than 90% of the time at the site between different strains of the same organism or between different species of the same genus. As used herein, the term "semi-conserved site at the amino acid level" refers to a site in the genome of an organism that uses the same amino acid more than 80% of the time at the site between different strains of the same organism or between different species of the same genus.

[0072] As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which viral genetic material or recombinant expression vectors can be introduced.

[0073] As used herein, the terms "transformation" and "transfection" refer to the introduction of a nucleic acid (eg, a vector) into a cell by a number of techniques known in the art.

[0074] As used herein, the term "immune cell" refers to a cell of hematopoietic origin that plays a role in an immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0075] The term "T cell" refers to a CD4+T cell or a CD8+T cell. The term T cell includes a TH1 cell, a TH2 cell, and a TH17 cell.

[0076] The term "T cell cytotoxicity" includes any immune response mediated by CD8+ T cell activation. Exemplary immune responses include the production of cytokines, proliferation of CD8+ T cells, production of granzymes or perforins, and clearance of infectious agents.

[0077] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, dosage forms, or combinations thereof, which are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues, organs, body fluids, or combinations thereof, without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0078] The terms "individual," "subject," and "patient" refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a mammal. Thus, the subject can be a human. The subject can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and neonatal subjects, whether male or female, are encompassed. The subject can include a control subject or a test subject.

[0079] As used herein, the term "polypeptide" refers to amino acid chains of any length, regardless of modification (eg, phosphorylation or glycosylation).

[0080] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to a dose sufficient to provide treatment for the disorder, disease or condition being treated, to induce or enhance an immune response, or otherwise provide a desired pharmacological effect, physiological effect, or both. The exact dose will vary depending on a variety of factors, such as subject-related variables (e.g., age, immune system health, etc.), the disease, the stage of the disease, and the treatment being administered.

[0081] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic ingredient, natural or synthetic, inactive ingredient in a formulation, which is combined with one or more active ingredients.

[0082] As used herein, the term "adjuvant" refers to an agent, pharmacological agent, immunological agent, or combination thereof, that modifies the effect of other agents. Adjuvants can be added to vaccines to modify the immune response by enhancing the immune response, such as providing higher amounts of antibodies and longer-lasting protection, thereby minimizing the amount of foreign material injected. Adjuvants can also be used to enhance the efficacy of vaccines by assisting in disrupting the immune response to specific cell types of the immune system, such as activating T cells instead of antibody-secreting B cells, depending on the type of vaccine.

[0083] As used herein, the term "dosage regimen" refers to vaccine administration with respect to formulation, route of administration, vaccine dose, dosing interval, and duration of immune protection.

[0084] "Effective amount" or "therapeutically effective amount" refers to the amount of a composition, virus or vaccine that, when administered to a mammal, preferably a human, is sufficient to achieve treatment of a disease or condition or prevention of a disease or condition in a mammal, preferably a human. The amount of a compound, virus or vaccine that constitutes a "therapeutically effective amount" will vary depending on the compound, virus, or vaccine, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art, taking into account his or her own knowledge and the present disclosure.

[0085] As used herein, "treatment" or "therapy" encompasses the treatment of a disease or condition of interest in a mammal, preferably a human, suffering from the disease or condition of interest, and includes:

[0086] (i) preventing the disease or condition from occurring in a mammal, particularly where the mammal is susceptible to the condition but has not yet been diagnosed as having the condition;

[0087] (ii) inhibiting a disease or condition, i.e. preventing its development;

[0088] (iii) alleviate the disease or condition, i.e. cause regression of the disease or condition; or

[0089] (iv) Relief of symptoms caused by a disease or condition, i.e., relief of pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different, in that a particular disease or condition may have no known causative agent (and thus the cause has not yet been addressed) and therefore is not yet considered a disease but rather an undesirable condition or syndrome in which clinicians have identified a more or less specific set of symptoms.

[0090] Material

[0091] A. Virus

[0092] Disclosed is a virus comprising a heterologous nucleic acid fragment encoding an α-1,3-galactosyltransferase (α-1,3-GT), such that the nucleic acid fragment expresses α-1,3-GT when the virus infects a host cell. Typically, when such a virus infects a host cell, the expression of α-1,3-GT results in a protein in the infected host cell having α-1,3-galactose. For example, in certain forms, when the virus infects a host cell, at least one protein exposed on the surface of the host cell will have α-1,3-galactose. The heterologous nucleic acid fragment encoding α-1,3-GT can be (or is considered to be) a gene or coding portion of a gene encoding α-1,3-GT. When the virus infects a host cell, the nucleic acid fragment / gene should generally be able to express (configured to express) α-1,3-GT, such that the virus expresses α-1,3-GT when the virus infects the host cell.

[0093] In some forms, when the virus infects a host cell, at least one viral protein expressed by the virus will have alpha-1,3-galactose. In some forms, the virus is live and attenuated.

[0094] In some forms, the nucleic acid fragment is introduced into the genome of the virus. In some forms, the nucleic acid fragment is introduced into the reading frame of the viral coding region of the virus in an in-frame manner. In some forms, the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the viral coding region of the virus. In some forms, the virus further comprises a protease cleavage site encoded between the nucleic acid fragment and the viral coding region of the virus.

[0095] In some forms, the virus is an influenza virus. In some forms, the nucleic acid fragment is introduced into the reading frame of the neuraminidase (NA) coding region of the virus in an in-frame manner. In some forms, the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the viral NA coding region. In some forms, the virus is further included in a protease cleavage site encoded between the nucleic acid fragment and the NA coding region of the virus. In some forms, the protease cleavage site is included in the 2A self-cleaving peptide. In some forms, the 2A self-cleaving peptide is derived from porcine Teschovirus-1. In some forms, the 2A self-cleaving peptide is flanked by a short peptide linker. In some forms, the peptide linker is GSG.

[0096] In some forms, the α-1,3-GT is mammalian α-1,3-GT. In some forms, the α-1,3-GT is mouse α-1,3-GT.

[0097] In some forms, the virus is attenuated by continuous passage in cultured cells, continuous passage in heterologous host animals, gene deletion in the virus, site-directed mutagenesis of the virus, changing the codon usage of the virus, selecting cold-adapted mutants, using related viruses from heterologous host species, using naturally occurring attenuated virus strains, or a combination thereof. In some forms, the virus is attenuated by continuous passage in cultured cells, wherein the cultured cells are Madin-Darby canine kidney (MDCK) cells.

[0098] Any virus can be used and prepared for the disclosed viruses. Viruses that cause human diseases are preferred. For example, the virus can be Aichi virus, Australian bat lyssavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus La Crosse, Bunyavirus snowshoe hare, Cercopithecine herpesvirus, Chandipura virus, virus), Chikungunya virus, Coxsackie virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhori virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, Hantavirus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis D virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, 70,Human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus, human papillomavirus 16, human papillomavirus 18, human parainfluenza virus, human parvovirus, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human T-lymphotropic virus, human torovirus, influenza virus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus virus), Lagos bat virus, Lake Victoria Marburg virus, Langate virus, Lassa virus, Lozdare virus, jumping disease virus, lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, molluscum contagiosum virus, monkeypox virus, mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, polio virus , Punta Toro sandfly virus, Puumala virus, rabies virus, Rift Valley fever virus, Ross River virus, rotavirus, rotavirus A, rotavirus B, rotavirus C, rubella virus, Lushan virus, salivirus A, sandfly fever Sicilian virus, Sapporo virus, Semliki Forest virus, Seoul virus, Sindbis virus, Southampton virus, St. Louis encephalitis virus, tick-borne Powassan virus, Tuscany virus, Ukuniemi virus, varicella-zoster virus, smallpox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, Western equine encephalitis virus, West Nile virus, yellow fever virus, or Zika virus. Characteristics of some of the human viruses that cause disease are shown in Table 1. ,

[0099] Preferred viruses include influenza virus, influenza A virus, influenza B virus, human papillomavirus, measles virus, human immunodeficiency virus, rabies virus, Ebola virus, Hanta virus, Marburg virus, Lassa virus, dengue virus, Eastern equine encephalitis virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, hepatitis E virus, human herpes virus, human SARS coronavirus, mumps virus, polio virus, rubella virus, varicella-zoster virus, Rift Valley fever virus, West Nile virus, yellow fever virus, human T-lymphotropic virus and Merkel cell polyomavirus.

[0100] Table 1. Human pathogenic viruses

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] The example of the virus that is suitable for generating attenuated live virus includes the virus of following virus family: Adenoviridae, Papillomaviridae, Parvoviridae, Herpesviridae, Poxviridae, Hepaciviridae, Polyomaviridae, Anelloviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae, Astroviridae, Bornaviridae, Arteriviridae, Hepaciviridae. Virus must generate mRNA from their genome to produce protein and self-replicate, but in each virus family, different mechanisms are used to realize this. Viral genome can be single-stranded (ss) or double-stranded (ds) RNA or DNA, and can use or can not use reverse transcriptase (RT). In addition, ssRNA virus can be sense (+) or antisense (-).

[0109] This classification divides viruses into seven groups:

[0110] I: dsDNA viruses (e.g. adenovirus, herpes virus, pox virus)

[0111] II: ssDNA viruses (+ strand or "positive sense") DNA (e.g., parvoviruses)

[0112] III: dsRNA viruses (e.g. reovirus)

[0113] IV: (+)ssRNA viruses (+ strand or positive sense) RNA (e.g., picornaviruses, togaviruses)

[0114] V: (-)ssRNA virus (-strand or antisense) RNA (e.g., orthomyxovirus, rhabdovirus)

[0115] VI: ssRNA-RT viruses with a DNA intermediate in their life cycle (positive strand or positive sense) RNA (e.g. retroviruses)

[0116] VII: dsDNA-RT viruses (e.g. hepatitis viruses)

[0117] Examples of DNA and RNA viruses are listed in Tables 2 and 3.

[0118] Table 2. Examples of DNA viruses that can be used to produce live attenuated viruses.

[0119]

[0120] Table 3. Examples of RNA viruses that can be used to produce live attenuated viruses.

[0121]

[0122]

[0123] Viruses with segmented genomes are also suitable for forming the attenuated live viruses described herein. Viruses with segmented genomes include viruses of the families Orthomyxoviridae, Bunyaviridae, and Arenaviridae. Orthomyxoviridae include influenza A, influenza B, and influenza C. Bunyaviridae include Bunyanvira, La Crosse, California encephalitis, Rift Valley fever, and Hamtan virus. Arenaviridae include lymphocytic choriomeningitis virus (LCMV), Lassa, Junin (Argentine hemorrhagic fever).

[0124] 1. Influenza virus

[0125] In some forms, the virus described herein is an influenza A virus or an influenza B virus. The influenza A virus or influenza B virus disclosed herein can be classified according to the influenza virus naming system revised by the World Health Organization (Bulletin of the World Health Organization, 58 (4): 585-591 (1980)). The revised naming system is similar to the 1971 system and consists of two parts: (a) type and strain name, and (b) for influenza A virus, a description of the antigenic specificity of the surface antigens (H and N).

[0126] The strain names of influenza A, B, and C viruses contain the following information:

[0127] 1. Description of the viral antigenic type based on the antigenic specificity of the NP antigen (type A, B or C).

[0128] 2. Host of origin. This does not apply to strains isolated from human sources, but applies to all strains isolated from non-human hosts (such as pigs, horses, chickens and turkeys). For viruses from non-human species, the Latin binomial nomenclature and common name of the host of origin are used, for example, Anas acuta (pintail duck). Thereafter, the common name of the species is used for the strain, such as A / duck / USSR / 695 / 76(H2N3). When the virus is isolated from non-biological material, the nature of the material is specified, such as A / lake water / Wisconsin / 1 / 79.

[0129] 3. Geographical origin.

[0130] 4. Strain number.

[0131] 5. Year of separation.

[0132] For influenza A viruses, the antigen description in parentheses follows the strain name and includes the following information.

[0133] (a) An index describing the antigenic properties of the hemagglutinin, i.e., H1, H2, H3, H4, etc. The subtype numbering is a simple sequential system uniformly applied to influenza viruses from all sources.

[0134] (b) An index describing the antigenic characteristics of the neuraminidase, i.e., N1, N2, N3, N4, etc. As with the H antigen subtype, this is a simple sequential numbering system that is uniformly applied to all influenza A viruses.

[0135] Exemplary nomenclature for influenza A viruses isolated from humans is listed in Table 4.

[0136] Table 4. Examples of reference strains and subtypes of hemagglutinin antigens and neuraminidase antigens of influenza A viruses isolated from humans.

[0137] H and N subtypes Reference strains HkDJ A / PR / 8 / 34(H1N1) A / Weiss / 43(H1N1) A / FM1 / 47(H1N1) A / England / 1 / 51(H1N1) A / Denver / l / 57(H1N1) A / New Jersey / 8 / 76(H1N1) A / USSR / 90 / 77(H1N1) H2N2 A / Singapore / 1 / 57(H2N2) A / Japan / 305 / 57(H2N2) A / England / 12 / 64(H2N2) A / Tokyo / 3 / 67(H2N2) H3N2 A / Hong Kong / 1 / 68(H3N2) A / England / 42 / 72(H3N2) A / Port Chalmers / 1 / 73(H3N2) A / Victoria / 3 / 75(H3N2) A / Texas / 1 / 77(H3N2)

[0138] i. Influenza A

[0139] Attenuated live influenza A virus described herein, including, for example, corresponding viruses in pharmaceutical compositions, can be based on any influenza A virus, such as avian influenza, human influenza, swine influenza, equine influenza or canine influenza. There are various influenza A virus subtypes, with different properties of HA and NA glycoproteins on their surfaces. Therefore, influenza A viruses are generally divided into subtypes according to the combination of protein forms of the hemagglutinin and neuraminidase (two proteins located on the surface of the viral envelope) present. 18 hemagglutinin forms (H1 to H18) and 11 neuraminidase forms (N1 to N11) have been identified.

[0140] Suitable virus strains include, but are not limited to, H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H2N1, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8, H3N9, H4N1, H4N2 , H4N3, H4N5, H4N6, H4N7, H4N8, H5N1, H5N2, H5N3, H5N4, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6 N3, H6N4, H6N5, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H8N1, H 8N2, H8N3, H8N4, H8N5, H8N6, H8N7, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N 1. H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, H10N9, H11N1, H11N2, H11N3, H11N4, H11N5 , H11N6, H11N7, H11N8, H11N9, H12N1, H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H13N1, H13N2, H13N3, H13N4, H13N5, H13N6, H13N8, H14N3, H14N5, H14N6, H15N8, H15N9, H16N3. In some forms, the influenza virus is one of the H1N1, H1N2, H2N2, H3N1, H3N2, H5N1 and H7N7 strains.

[0141] Examples of H1N1 virus strains are influenza A virus strains A / Brisbane / 59 / 2007 (H1N1), A / Puerto Rico / 8 / 1934 (H1N1), with Genbank accession numbers NC 002016, NC 002017, NC 002018, NC 002019, NC 002020, NC 002021, NC 002022, NC 002023. Other examples of H1N1 strains are influenza A strain A / Brevig Mission / 1 / 1918 H1N1) (influenza A virus (strain A / South Carolina / 1 / 1918 H1N1), influenza A virus strain A / Russia: St. Petersburg / 8 / 2006 H1N1, influenza A virus strain A / United States: Texas / UR06-0195 / 2007 H1N1-strain A / Brevig Mission / 1 / 1918 H1N1), influenza A virus strain A / Russia: St. Petersburg / 8 / 2006 H1N1, influenza A virus strain A / United States: Texas / UR06-0195 / 2007 H1N1-strain A / Brevig Mission / 1 / 1918H1N1, influenza A virus strain A / South Carolina / 1 / 1918H1N1, influenza A virus strain A / swine / Lowa / 15 / 1930H1N1, influenza A virus strain A / Wilson-Smith / 1933H1N1, influenza A virus strain A / WS / 1933H1N1, and strain A / United States: Philadelphia / 1935H1N1. Other examples of H1N1 strains are influenza A virus strain A / New Zealand: South Canterbury / 35 / 2000H1N1. An example of an H1N2 strain is influenza A virus strain A / Xianfeng / 3 / 1989H1N2. Two examples of H1N3 strains are influenza A / duck / NZL / 160 / 1976H1N3 and strain A / whale / Pacific / 19 / 1976 H1N3. An example of an H1N4 strain is influenza A virus strain A / wild duck / Netherlands / 30 / 2006H1N4. An example of an H1N5 strain is influenza A virus strain A / pintail duck / ALB / 631 / 1981H1N5. An example of an H1N6 strain is influenza A virus strain A / sea murre / Alaska / 305 / 1976H1N6. An example of an H1N7 strain is influenza A virus strain A / swine / England / 191973 / 92H1N7. An example of an H1N8 strain is strain A / Egyptian goose / South Africa / AI1448 / 2007. An example of an H2N1 strain is influenza A virus strain A / Japan / Bellamy / 57H2N1. An example of an H2N2 strain is influenza A virus strain A / Korea / 426 / 68H2N2, whose Genebank accession number is NC 007366, NC 007367, NC 007368, NC 007369, NC007370, NC 007374, NC 007375, NC 007376, NC 00737, NC 007378, NC 007380, NC 007381 and NC 007382.Three other examples of H2N2 strains are influenza A strain A / Japan / 305 / 1957H2N2, A / Czech Republic / 1 / 1966H2N2 and strain A / Singapore / Singapore / 1 / 1957H2N2. An example of an H2N3 strain is influenza A strain A / Wild Duck / Minnesota / -00692 / 2008H2N3. An example of an H2N4 strain is A / Wild Duck / Alberta / 149 / 2002H2N4. An example of an H2N5 strain is influenza A strain A / Tern / Australia / 1 / 04H2N5. An example of an H2N6 strain is influenza A strain A / Thick-billed Murre / Alaska / 44145-199 / 2006H2N6. The example of H2N7 strain is influenza A virus strain A / northern shoveler / California / HKWF1128 / 2007H2N7. The example of H2N8 strain is influenza A virus strain A / turkey / California / 1797 / 2008H2N8. The example of H2N9 strain is influenza A virus strain A / duck / Germany / 1972H2N9. The example of H3N1 strain is influenza A virus strain A / wild duck / ALB / 26 / 1976H3N1. The example of H3N2 strain is influenza A virus strain A / New York / 392 / 2004H3N2, whose Genebank accession numbers are NC 007371, NC 007372 and NC 007373. The example of H3N2 strain is influenza virus strain NX-31H3N2, strain A / Hong Kong / 5 / 1983H3N2, A / Rio / 6 / 69H3N2, A / Hong Kong / MA / 1968H3N2 and influenza virus strain A / Shanghai / N12 / 2007H3N2. The example of H3N3 strain is influenza virus strain A / duck / Hong Kong / 22A / 1976H3N3. The example of H3N4 strain is influenza virus strain A / wild duck / ALB / 1012 / 1979H3N4. The example of H3N5 strain is influenza virus strain A / northern shoveler / California / HKWF1046 / 2007H3N5. The example of H3N6 strain is influenza virus strain A / chicken / Nanchang / 9-220 / 2000H3N6. Examples of H3N8 strains are influenza A strain A / horse / Miami / 1 / 1963H3N8 and strain A / duck / Ukraine / 1 / 1963H3N8. An example of an H3N9 strain is influenza A strain A / swan / Shimane / 227 / 01H3N9.

[0142] The example of H4N1 strain is influenza virus strain A / chicken / Singapore / 1992 (H4N1). The example of H4N2 strain is influenza virus strain A / duck / Hong Kong / 24 / 1976 (H4N2). The example of H4N3 strain is influenza virus strain A / wild duck / Sweden / 65 / 2005 (H4N3). The example of H4N4 strain is influenza virus strain A / grey teal / Australia / 2 / 1979H4N4. The example of H4N5 strain is influenza virus strain A / duck / Hokkaido / 1058 / 2001 (H4N5). Two examples of H4N6 strain are influenza virus strain A / duck / Czechoslovakia / 1956H4N6 and influenza virus strain A / duck / Alberta / 28 / 1976H4N6. The example of H4N7 strain is influenza A virus strain A / duck / Mongolia / 583 / 02H4N7. The example of H4N8 strain is influenza A virus strain A / chicken / Alabama / 1 / 1975H4N8. The example of H4N9 strain is influenza A virus strain A / WDk / ST / 988 / 2000 (H4N9). The example of H5N1 strain is influenza A virus (A / goose / Guangdong / 1 / 96 (H5N1)), and its Genebank accession number is NC 007357, NC 007358, NC 007359, NC 007360, NC 007362, NC 007363 and NC 007364. Other examples of H5N1 strains are influenza A strain A / duck / Hong Kong / 2986.1 / 2000H5N1, influenza A strain / silkie / Hong Kong / SF189 / 2001H5N1, influenza A strain A / chicken / Hong Kong NU562 / 2001H5N1, influenza A strain A / chicken / Hong Kong / FY150 / 2001H5N1, influenza A strain A / chicken / Hong Kong / 715.5 / 2001H5N1, influenza A strain A / guinea fowl / Hong Kong / 38 / 2002H5N1, influenza A strain A / chicken / Hong Kong / 31.2 / 2002H5N1, influenza A strain A / chicken / Hong Kong / 32.3 / 2002H5N1. Strain A / chicken / Hong Kong / 37.4 / 2002H5N1, influenza A strain A / skinny chicken / Hong Kong NU100 / 2002H5N1, influenza A strain A / chicken / Hong Kong / 96.1 / 2002H5N1, influenza A strain A / chicken / Hong Kong NU22 / 2002H5N1, influenza A strain A / teal / China / 2978.1 / 2002H5N1, influenza A strain A / Hong Kong / 212 / 2003H5N1, influenza A strain A / chicken / Shantou / 4231 / 2003H5N1, influenza A strain A / goose / Guangxi / 345 / 2005H5N1. The example of H5N2 strain is influenza A strain A / chicken / Pennsylvania / 1370 / 1983H5N2. An example of an H5N3 strain is influenza A strain A / Duck / Malaysia / F119-3 / 97 H5N3.The example of H5N4 strain is influenza A strain A / environment / New York / 200269-18 / 2002H5N4. The example of H5N5 strain is influenza A strain A / duck / Massachusetts / Sg-00440 / 2005H5N5. The example of H5N6 strain is A / duck / Potsdam / 2216-4 / 1984H5N6. The example of H5N7 strain is A / wild duck / Denmark / 64650 / 03H5N7. The example of H5N8 strain is strain A / duck / Ireland / 113 / 1983H5N8. Two examples of H5N9 strain are influenza A strain A / turkey / Ontario / 7732 / 1966H5N9 and strain A / chicken / Italy / 22AM 998H5N9.

[0143] The example of H6N1 strain is A / chicken / Taiwan / PF1 / 02 (H6N1). The example of H6N2 strain is influenza A strain A / chicken / California / 1316 / 2001 (H6N2). The example of H6N5 strain is influenza A strain A / seagull / Australia / 1972H6N5. The example of H6N8 strain is influenza A strain A / turkey / Minnesota / 501 / 1978H6N8. The example of H7N1 strain is influenza A strain A / fowl plague virus / Rostock / 8 / 1934H7N1. The example of H7N2 strain is influenza A strain A / duck / Hong Kong / 293 / 1978 (H7N2). The example of H7N3 strain is influenza A strain A / turkey / Oregon / 1971H7N3. The example of H7N7 strain is influenza A strain A / horse / Detroit / 1 / 1964H7N7, influenza A strain A / horse / Cambridge / 1 / 1973H7N7 and influenza A strain A / horse / Saint Paul / 1 / 1976H7N7, influenza A strain A / horse / Prague / 1 / 1956H7N7 and influenza A strain A / chicken / Weybridge H7N7. The example of H8N2 strain is influenza A strain A / duck / Alaska / 702 / 1991 (H8N2). The example of H8N4 strain is influenza A strain A / turkey / Ontario / 6118 / 1968H8N4. The example of H8N4 strain is influenza A strain A / duck / Tsukuba / 255 / 2005 (H8N5). An example of an H8N7 strain is influenza A strain A / Duck / Alaska / 702 / 1991 (H8N7).

[0144] The example of H9N1 strain is influenza A virus A / duck / Shantou / 2030 / 00 (H9N1). The example of H9N2 strain is influenza A virus A / Hong Kong / 1073 / 99 (H9N2), and its Genebank accession number is NC 004905, NC 004906, NC 004907, NC 004908, NC 004909, NC 004910, NC 004911 and NC 004912. The example of H9N3 strain is influenza A virus A / duck / Vietnam / 340 / 2001H9N3. The example of H9N4 strain is influenza A virus A / shorebird / Germany / 231 / 2003H9N4. The example of H9N5 strain is influenza A virus A / duck / Hong Kong / 702 / 79H9N5. The example of H9N7 strain is A / turkey / Scotland / 70 (H9N7). The example of H9N8 strain is A / chicken / Korea / 04164 / 2004 (H9N8). The example of H9N9 strain is A / turkey / France / 03295 / 2003H9N9. The example of H10N1 strain is influenza A virus A / duck / Hong Kong / 938 / 80H10N1. The example of H10N2 strain is influenza A virus A / duck / Alaska / 658 / 1991H10N2. The example of H10N5 strain is influenza A virus A / duck / Hong Kong / 15 / 1976H10N5. Examples of H10N7 strains are influenza A strain A / chicken / Germany / n / 1949H10N7, strain A / duck / Germany / 1949H10N7 and strain A / duck / Manitoba / 1 / 1953H10N7. Examples of H10N7 strains are influenza A strain A / duck / Germany / 1949H10N7. Examples of H11N1 strains are influenza A strain A / duck / Miyagi / 47 / 1977H11N1. Examples of H11N2 strains are A / duck / Yangzhou / 906 / 2002H11N2. Examples of H11N3 strains are A / chicken / Thailand / CU5388 / 2009H11N3. Examples of H11N6 strains are influenza A strain A / duck / England / 1 / 1956H11N6. The example of H11N8 strain is strain A / duck / Ukraine / 2 / 1960H11N8. Two examples of H11N9 strains are influenza A strain A / duck / Ukraine / 1 / 1960H11N9 and influenza A strain A / tern / Australia / G70C / 1975H11N9. The example of H12N1 strain is A / wild duck / Alberta / 342 / 1983 (H12N1). The example of H12N2 strain is A / duck / Primorie / 3691 / 02H12N2. The example of H12N3 strain is A / whooper swan / Mongolia / 232 / 2005H12N3. The example of H12N5 strain is influenza A strain A / duck / Alberta / 60 / 1976H12N5.An example of an H12N6 strain is A / Wild Duck / Alberta / 221 / 2006H12N6. An example of an H12N7 strain is A / Duck / Victoria / 30a / 1981H12N7. An example of an H12N8 strain is A / Wild Duck / Netherlands / 20 / 2005H12N8. An example of an H12N9 strain is A / Red-breasted Knot / Australia / 5745 / 1981H12N9.

[0145] An example of an H13N1 strain is A / Guano / Illinois / 185997-30 / 2007H13N1. An example of an H13N2 strain is influenza A strain A / Whale / Maine / 328 / 1984H13N2. An example of an H13N3 strain is A / Shorebird / New Zealand / 840 / 1986H13N3. Two examples of H13N6 strains are influenza A strain A / Gull / Maryland / 704 / 1977H13N6 and strain A / Gull / Minnesota / 945 / 1980H13N6. An example of an H13N8 strain is A / Black-headed Gull / Sweden / 1 / 2005H13N8. An example of an H14N3 strain is A / Wild Duck / Gur / 263 / 82H14N3. Three examples of H14N5 strains are A / Wild Duck / Gurjev / 263 / 1982H14N5, A / Wild Duck / Astrakhan / 266 / 1982H14N5 and A / Herring Gull / Astrakhan / 267 / 1982H14N5. An example of an H14N6 strain is strain A / Wild Duck / Gurjev / 244 / 1982H14N6. An example of an H15N8 strain is A / Duck / Australia / 341 / 1983H15N8. An example of an H15N9 strain is A / Seagull / Western Australia / 2576 / 79H15N9. An example of an H16N3 strain is A / Black-headed Gull / Sweden / 2 / 99H16N3.

[0146] Such virus subtypes are serologically distinguishable, meaning that antibodies specific for one subtype will not bind to another subtype with comparable high affinity.However, the nucleic acid positions characterizing the influenza A virus genes according to the present invention are applicable to any influenza A virus strain.

[0147] ii. Influenza B

[0148] The attenuated live virus described herein may also be an influenza B virus. The attenuated live influenza B virus, including the attenuated live influenza B virus in the pharmaceutical composition, may be based on any influenza B virus strain. Suitable virus strains include, but are not limited to, influenza B virus strain B / Maryland / 1959, strain B / Yamagata / 1 / 1973, strain B / Victoria / 3 / 1985, strain B / USSR / 100 / 1983, strain B / Tokyo / 942 / 1996, strain B / Texas / 4 / 1990, strain B / Singapore / 222 / 1979, strain B / South Dakota / 5 / 1989, strain B / Paris / 329 / 1990, strain B / Leningrad / 179 / 1986, strain B / Hong Kong / 8 / 1973, strain B / Fukuoka / 80 / 1981, strain B / Bangkok / 163 / 1990, strain B / Beijing / 1987, strain B / Switzerland / 9359 / 99, strain B / Wisconsin / 6 / 2006, strain B / West Virginia / 01 / 2009, strain B / Washington / 08 / 2009, strain B / Uruguay / NG / 02, strain B / Texas / 18 / 2001, strain B / Taiwan / 3799 / 2006, strain B / Spain / WV45 / 2002, strain B / Seoul / 232 / 2004, strain B / Rio Grande do Sul / 57 / 2008, strain B / Quebec / 517 / 98, strain B / Philippines / 5072 / 2001, strain B / Oslo / 1871 / 2002, strain B / Osaka / 983 / 1997, strain B / Milan / 05 / 2006, strain B / Johannesburg / 116 / 01 or strain B / Arizona / 12 / 2003.

[0149] B. Attenuation

[0150] When the disclosed virus is used as a vaccine or for a vaccine, attenuated viruses are useful. This is because the virus will be designed and expected to infect host cells, replicate and produce viral proteins, but not cause disease. Attenuated viruses are viruses produced by reducing the virulence of the virus but still maintaining its vitality (or "survival") (Badgett et al., J. Virol. 76 (20): 10524–10529 (2002)). Attenuation takes the virus and changes it to make it harmless or less toxic. These viruses are in contrast to viruses produced by "killing" the virus (inactivated vaccines).

[0151] There are many methods for weakening viruses. Each kind has useful elements, but most methods have some limitations in use or usefulness. The example of attenuated form includes continuous passage in cultured cells, continuous passage in heterologous host animals, gene deletion in viruses, site-directed mutagenesis of viruses, codon usage of change viruses, selection of cold-adapted mutants, use of related viruses from heterologous host species, use of naturally occurring attenuated virus strains or its combination. In some forms, viruses are attenuated by continuous passage in cultured cells, and the cells of wherein culture are Madin-Darby canine kidney (MDCK) cells. Attenuated categories include empirical attenuated viruses (empirically attenuated virus), replication-defective viruses, single-cycle viruses, replication-fidelity attenuated viruses, codon deoptimization viruses, viruses controlled by miRNA, and viruses controlled by zinc finger nucleases (ZFNs).

[0152] Empirically attenuated viruses are usually generated by blind passage through different cell types. By adapting to the new environment, the virus accumulates mutations that mediate attenuation. Host immunity can limit the virulence and spread of the attenuated virus. Empirically attenuated viruses have excellent immunogenicity and require very low doses. Only some viruses can be empirically attenuated, and empirically attenuated viruses are prone to reverting to wild type and breakthrough disease.

[0153] Replication-deficient viruses are usually produced by deleting one or several genes required for genome replication from the virus. Viral vaccines can then be produced in helper cell lines that express the deleted proteins in trans (Dudek and Knipe, Virology 344: 230–239 (2006)). The administered virus is unable to replicate its genome. Replication-deficient viruses generally provide good immunogenicity and have known attenuation mechanisms. The effectiveness of replication-deficient viruses may be limited because replication in the host is limited to the inoculation site.

[0154] Single-cycle viruses are usually produced by deleting one or several genes required for viral assembly and propagation. Single-cycle viruses are distinguished from replication-deficient viruses by their ability to replicate their genome (Loudon et al., J Gene Med. 3: 458–467 (2001)). Single-cycle viruses are able to replicate their genomes but are defective in assembly or propagation. Single-cycle viruses generally provide good immunogenicity and have known attenuation mechanisms. The effectiveness of replication-deficient viruses may be limited because replication in the host is limited to the inoculation site.

[0155] Replication fidelity attenuated viruses are usually produced by engineering or selecting viruses that are resistant to nucleoside analogs. Resistant viruses have higher replication fidelity, which results in less diversity in viral offspring, resulting in attenuation (e.g., the host is more effective in attacking the virus) (Vignuzzi et al., Nature 439:344-348 (2006); Pfeiffer and Kirkegaard, PLoS Pathog.1:e11 (2005)). Replication fidelity attenuated viruses generally provide strong immunogenicity, have known attenuation mechanisms, and are insensitive to antigenic shift / drift. Replication fidelity attenuated viruses are limited to RNA viruses. It is possible to revert to wild type. The safety of viruses with higher replication fidelity has not yet been clearly determined.

[0156] Codon deoptimization viruses are typically produced by changing codons in the virus to synonymous codons that are less biased in the host (Baker et al., Future Virology 10(6):715-730(2015)). The loss of expression efficiency of codon deoptimization viruses results in reduced virus yield. Codon deoptimization viruses generally provide strong immunogenicity, do not revert to wild type, and are suitable for many viruses. Possible safety issues. The safety of codon deoptimization viruses has not yet been clearly determined.

[0157] miRNA-controlled viruses are typically created by adding miRNA sequences to a virus (Fay and Langlois, Non-Coding RNA 4(4):25 (2018)). The miRNA sequence is chosen to restrict which host cells the virus can replicate in (e.g., altered tropism). miRNA-controlled viruses can provide strong immunogenicity, have known attenuation mechanisms, and can prevent latent infection. miRNA-controlled viruses are limited to a few RNA viruses. The safety of miRNA-controlled viruses has not been clearly established.

[0158] Zinc finger nuclease (ZFN)-controlled viruses are typically generated by introducing a sequence encoding a ZFN that targets a sequence in the virus for viral replication and other important viral processes. Following inoculation, both the immunogenic viral gene and the virus-specific ZFN are expressed. While viral proteins stimulate an innate immune response, the ZFN cleaves the viral DNA and restricts replication. ZFN-controlled viruses are strongly immunogenic, have known attenuation mechanisms, and can protect against latent infection. ZFN-controlled viruses are limited to non-integrating DNA viruses. The safety of ZFN-controlled viruses has not been clearly established.

[0159] C. Vaccine

[0160] A composition disclosed herein is a vaccine. The vaccine may contain nucleic acids, amino acids, or a combination thereof. The vaccine (or immunogenic composition) includes a composition of an immunogenic amount (preferably an effective amount or a protective amount) in a pharmaceutically acceptable excipient and an optional adjuvant, such as an outer membrane protein (isolated or purified, or present in an outer membrane vesicle, ghost or dead, live or attenuated whole cell preparation). In this case, the immunogenic amount can be defined as the amount of protein sufficient to elicit an antibody response in a host. A preferred vaccine is a vaccine comprising the disclosed virus and / or produced by the disclosed virus.

[0161] An immunogenic amount of one of the disclosed compositions can be formulated in a pharmaceutically acceptable excipient and an optional adjuvant to prevent or treat infectious diseases. The vaccine can be used to induce an immune response in a mammal susceptible to infection by administering an effective amount of the vaccine to the mammal (an effective amount is an amount that can protect the host from infection to a certain extent). The vaccine can also be used to prevent infection by administering an effective amount to a mammal.

[0162] Vaccines are able to induce cross-protective immune responses against multiple viruses.

[0163] Vaccines described herein are generally generated based on basic information about pathogens, such as how viruses infect cells and how the immune system responds to them, as well as practical considerations, such as where in the world the vaccine will be used. Vaccines described herein can be, for example, live attenuated vaccines; inactivated vaccines; subunit vaccines; toxoid vaccines; conjugate vaccines; DNA vaccines; or recombinant vector vaccines.

[0164] Typically, the attenuated live viruses described herein can be used in attenuated live vaccines. Exemplary vaccines include attenuated live influenza virus vaccines, which can effectively protect hosts from influenza by immunizing with a single effective dose.

[0165] Vaccines can elicit humoral responses, cell-mediated immune responses, or a combination thereof. Ideally, the immune response provides protection when subsequently challenged with an unattenuated virus of the same or different subtype or strain. When the attenuated live virus described herein is used as a vaccine, it can provide homosubtype immune protection, heterosubtype immune protection, or both.

[0166] i. Live attenuated vaccines

[0167] Live attenuated vaccines contain versions of live viruses that have been weakened in the laboratory so they cannot cause disease. Because live attenuated vaccines are the closest to natural infection, these vaccines are great teachers for the immune system: They elicit strong cellular and antibody responses, often providing lifelong immunity with just one or two doses.

[0168] Despite the advantages of live attenuated vaccines, there are also some disadvantages. It is the nature of living things to change or mutate, and the organisms used in live attenuated vaccines are no exception. There is a small possibility that the attenuated virus in the vaccine may revert to a virulent form and cause disease. However, by introducing a large number of mutations into the viral genome, this possibility is significantly reduced. In addition, not everyone can safely receive a live attenuated vaccine. For their own protection, people with impaired or weakened immune systems (for example because they have undergone chemotherapy or are infected with HIV) cannot receive live vaccines.

[0169] Another limitation is that live, attenuated vaccines generally need to be refrigerated to maintain their potency. If the vaccine needs to be shipped overseas and stored by health care workers in developing countries that lack widespread refrigeration, live vaccines may not be the best choice.

[0170] ii. Inactivated vaccines

[0171] Inactivated vaccines are produced by killing disease-causing pathogens with chemicals, heat or radiation. Such vaccines are more stable and safer than live vaccines: dead pathogens cannot mutate back to their pathogenic states. Inactivated vaccines generally do not require refrigeration, and they can be easily stored and transported in freeze-dried form, making them accessible to people in developing countries.

[0172] However, most inactivated vaccines stimulate a weaker immune system response than live vaccines. Therefore, multiple additional doses, or booster shots, may be needed to maintain an individual's immunity. This can be a disadvantage in areas where people do not have regular access to medical care and cannot get booster shots on time.

[0173] iii. Subunit vaccines

[0174] Subunit vaccines include only the antigens that are most likely to stimulate the immune system, as opposed to the entire pathogen. In some cases, these vaccines use epitopes, which are very specific parts of an antigen that antibodies or T cells recognize and bind to. Because subunit vaccines contain only the essential antigens and not all the other molecules that make up the microbe, the chances of having an adverse reaction to the vaccine are lower.

[0175] Subunit vaccines can contain 1 antigen, 20 antigens, any number between 1 and 20 antigens, or more than 20 antigens. Once the antigens that best stimulate the immune system are identified, they can be prepared into a subunit vaccine in one of two ways: 1) the pathogen can be grown in a lab and then chemicals can be used to break it down and collect the important antigens; or 2) the antigens can be prepared using recombinant DNA technology. Vaccines produced this way are called "recombinant subunit vaccines."

[0176] iv. Toxoid vaccines

[0177] Toxoid vaccines can be used against pathogens that secrete toxins. These vaccines are used when the pathogen's toxins are the main cause of disease. The toxins are usually inactivated by treatment with formalin (a solution of formaldehyde and sterile water). This "detoxified" toxin, called a toxoid, is safe for use in vaccines.

[0178] When the immune system receives a vaccine containing a harmless toxoid, it learns how to fight the natural toxin. The immune system produces antibodies that latch onto and block the toxin. Vaccines against diphtheria and tetanus are examples of toxoid vaccines.

[0179] v. Conjugate vaccines

[0180] Conjugate vaccines are usually prepared when the pathogen has a polysaccharide outer coating, such as many harmful bacteria. The polysaccharide coating masks the bacteria's antigens, making it impossible for the immature immune systems of infants and young children to recognize or respond to them. Conjugate vaccines are special subunit vaccines that can solve this problem.

[0181] In making conjugate vaccines, an antigen or toxoid from a pathogen that the infant's immune system can recognize is often attached to a polysaccharide. This link helps the immature immune system respond to the polysaccharide coating and fight off the disease-causing bacteria.

[0182] vi. DNA vaccines

[0183] DNA vaccines use pathogen genes that encode immunogenic antigens. Studies have found that when genes for pathogen antigens are introduced into the body, some cells take up that DNA. The DNA then instructs these cells to make antigen molecules. The cells secrete the antigens and display them on the cell surface. DNA vaccines against pathogens cause strong antibody responses to free-floating antigens secreted by cells, and the vaccine also stimulates strong cellular responses against microbial antigens displayed on the cell surface. In addition, DNA vaccines are relatively easy and inexpensive to design and produce.

[0184] So-called naked DNA vaccines consist of DNA administered directly into the body. These vaccines can be administered with a needle and syringe or with a needle-free device that uses high-pressure gas to shoot tiny gold particles coated with DNA directly into cells. Sometimes the DNA is mixed with molecules that promote cellular uptake.

[0185] vii. Recombinant vector vaccines

[0186] Recombinant vector vaccines are similar to DNA vaccines, but they use attenuated viruses or bacteria to introduce microbial DNA into the body's cells. "Vector" refers to the virus or bacteria used as a vehicle. The vehicle virus carries the pathogen's DNA to the cells. Recombinant vector vaccines are very similar to natural infections, so they stimulate the immune system.

[0187] Attenuated bacteria can also be used as vectors. In this case, the inserted genetic material causes the bacteria to display antigens of other microorganisms on their surface. In effect, the harmless bacteria mimic the harmful microorganism, thus triggering an immune response.

[0188] 1. Carrier

[0189] Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the active compound into a pharmaceutically acceptable formulation. The appropriate formulation depends on the selected route of administration. In one form, administration is by inhalation into the upper or lower respiratory tract mucosa. Typical formulations include carriers such as sterile saline or phosphate buffered saline. Viscosity modifiers and preservatives are also often added.

[0190] Optional pharmaceutically acceptable excipients particularly for enteral, topical and mucosal administration include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers and surfactants. Diluents, also known as "fillers," are typically necessary to increase the volume of solid dosage forms to provide a practical size for tablet compression or the formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium dioxide, magnesium aluminum silicate and powdered sugar.

[0191] Binders are used to impart binding properties to solid dosage forms, thereby ensuring that tablets or beads or particles remain intact after the dosage form is formed. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as gum arabic, tragacanth, sodium alginate, cellulose including hydroxypropyl methylcellulose, hydroxypropyl cellulose, ethyl cellulose and magnesium aluminum silicate, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, methacrylic acid aminoalkyl ester copolymers, polyacrylic acid / polymethacrylic acid and polyvinyl pyrrolidone.

[0192] Disintegrants are used to promote disintegration or "breakdown" of the dosage form after administration and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginic acid, gums, or cross-linked polymers such as cross-linked PVP (available from GAF Chemical Corp. XL).

[0193] Stabilizers are used to inhibit or retard decomposition reactions, including, for example, oxidation reactions.

[0194] Surfactants can be anionic, cationic, amphoteric or nonionic surfactants. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long-chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates such as sodium dodecylbenzene sulfonate; sodium dialkyl sulfosuccinates such as bis-(2-ethylthioxy)-sodium sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethyl benzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of the nonionic surfactant include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan esters, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearyl monoisopropanolamide and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-b-alanine, sodium N-lauryl-b-iminodipropionate, myristic amphoacetic acid, lauryl betaine and lauryl sulfobetaine.

[0195] If desired, the composition may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents or preservatives.

[0196] 2. Adjuvant

[0197] In one form, the adjuvant is the synthetic glycolipid alpha-galactosylceramide (αGalCer). Dendritic cells presenting antigens in the context of CD1d can lead to rapid innate and prolonged production of cytokines such as interferon and IL-4 by natural killer T cells (NKT cells). CD1d is a major histocompatibility complex class I molecule that presents glycolipid antigens to a subset of NKT cells. Advantageously, αGalCer is not toxic to humans and has been shown to act as an adjuvant, eliciting antigen-specific CD4+ and CD8+ T cell responses. For example, it has been shown that αGalCer combined with a malaria vaccine can result in a cytotoxic response against infected cells, an ideal approach for vaccines against infectious diseases. In addition to αGalCer, other glycolipids can be used as adjuvants to activate NKT cell-mediated immune responses.

[0198] In another form, the adjuvant can be, but is not limited to, one or more of the following: oil emulsions (e.g., Freund's adjuvant); saponin preparations; virosomes and virus-like particles; bacterial and microbial derivatives, including but not limited to carbohydrates such as lipopolysaccharide (LPS); immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives, mucoadhesives, or both; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolones; and surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0199] Adjuvants can also include immunomodulators, such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-.gamma.), macrophage colony stimulating factor and tumor necrosis factor; and costimulatory molecules, such as molecules of the B7 family. Such protein adjuvants can be provided as full-length polypeptides or active fragments thereof, or in the form of DNA, such as plasmid DNA.

[0200] D. Test kit

[0201] Also provided is one or more kits for immunizing a subject with a live attenuated virus as described herein. The kit may include, for example, a live attenuated virus, a pharmaceutically acceptable carrier, an adjuvant, a dispenser, and instructional materials for using them. In a further form, the live attenuated virus may be one or more polioviruses, one or more rhinoviruses, one or more influenza viruses, etc. In the case of a plurality of different isolates of a particular virus being desired to immunize a host, more than one virus may be preferred. The instructions may provide any information useful for directing the administration of the live attenuated virus.

[0202] method

[0203] Methods of making and using the disclosed viruses and vaccines are disclosed.

[0204] A. Methods of preparing viruses

[0205] Methods for preparing recombinant viruses with segmented or single genomes are generally known in the art. These include methods using standard reverse genetics techniques and genome manipulation in molecular biology (Hoffmann et al., Proc. Natl. Acad. Sci. USA, 97 (11): 6108-6113 (2000); Zhou et al., J. Virol., 72 (4): 3241-3247 (1998)). All synthetic genes are usually subcloned into expression constructs. The constructs are then contacted in vitro, i.e., infecting the same host cells. The host cells then produce packaged viruses containing the manipulated genome. The packaged viruses are then harvested and their titers are determined using standard virological techniques and used for further characterization.

[0206] Methods for quantifying viral particles are known in the art. Examples include plaque-based assays, which are used to determine viral concentration based on infectious dose. Viral plaque assays determine the number of plaque forming units (pfu) in a viral sample, which is a measure of the amount of virus. The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis to detect plaque formation, the FFA employs an immunostaining technique using fluorescently labeled antibodies specific for viral antigens to detect infected host cells and infectious viral particles prior to actual plaque formation. The FFA is particularly useful for quantifying viral species that do not lyse the cell membrane, as these viruses are not amenable to plaque assays. Another assay is the endpoint dilution assay (50% tissue culture infectious dose (TCID 50 )). TCID 50 Is a measure of infectious virus titer. This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or produce a cytopathic effect in 50% of inoculated tissue culture cells.

[0207] Viral proteins of a virus can be isolated or purified from the virus produced as described herein. Methods for doing so are generally known.

[0208] B. Administration of viruses and vaccines

[0209] The disclosed viruses can be used for the preventive treatment of viral infection, the therapeutic treatment of viral infection, or both; that is, they can be used to treat viral infection, prevent viral infection, or both. The virus can be administered as a pharmaceutical composition by any route known in the art. Typically, the pharmaceutical composition can be administered, for example, intravenously, subcutaneously, intramuscularly, or intranasally. For this purpose, the virus of the pharmaceutical composition can be provided in a suitable injectable or inhalable form. The attenuated live virus of the present disclosure can be included in some forms in a device for applying the virus in an inhalable or injectable form to a subject.

[0210] Pharmaceutical compositions comprising the viruses of the present disclosure can be prepared in a manner known per se, for example by conventional mixing, dissolving, granulating, dragee making, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. The composition can be an immunogenic composition such as a vaccine. The corresponding vaccine forming the main component of the vaccine composition of the present disclosure can include a single antigen, a combination of antigens, a single virus or a combination of viruses, for example at least two or three viruses of the same species, including one or more reassortants.

[0211] Some forms of any of the immunization and treatment methods of the present invention further include administering at least one adjuvant to the subject. A variety of adjuvants (including particulate adjuvants) suitable for vaccines based on proteins and nucleic acids, as well as methods of combining adjuvants with antigens, are well known to those skilled in the art. Suitable adjuvants for nucleic acid-based vaccines include, but are not limited to, Quil A, imiquimod, resiquimod, and interleukin-12 delivered in the form of purified protein or nucleic acid. Adjuvants suitable for protein immunization include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), saponin, Quil A, and QS-21.

[0212] Exemplary routes of administration of the pharmaceutical compositions of the present disclosure include oral, transdermal and parenteral delivery. Suitable routes of administration may, for example, include implantation, oral, rectal, transmucosal or intestinal administration; parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, and intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection.

[0213] As an illustrative example, for injection, the pharmaceutical compositions according to the present disclosure can be formulated into aqueous solutions, for example in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For oral administration, the corresponding pharmaceutical compositions can be easily formulated by combining the virus with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the virus of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated.

[0214] Pharmaceutical preparations for oral use can be obtained by adding solid excipients, optionally grinding the resulting mixture and processing the granular mixture, if desired, after adding suitable auxiliaries, to obtain tablets or dragee cores. Suitable excipients are especially fillers, such as sugars, including lactose, glucose, sucrose, mannitol or sorbitol; starch and its derivatives, such as corn starch, dextrin and wheat starch, rice starch, potato starch, hydroxypropyl starch, wheat starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP) or a combination thereof; cellulose preparations, such as methylcellulose, carboxymethylcellulose and hydroxypropylcellulose; inorganic compounds, such as sodium chloride, boric acid, calcium sulfate, calcium phosphate and precipitated calcium carbonate. If desired, disintegrants, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salts such as sodium alginate can be added.

[0215] The dragee core is provided with a suitable coating. For this purpose, a concentrated sugar solution can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol, titanium dioxide or a combination thereof; a lacquer solution; and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablet or dragee coating for identification or characterization of different combinations of viral doses. Suitable fluidizing agents include, but are not limited to, magnesium oxide, synthetic aluminum silicate, metasilicic acid, magnesium aluminum oxide, hydrated silicic acid, anhydrous silicic acid, talc, magnesium stearate and kaolin. Suitable binders include, but are not limited to, polyethylene glycol, polyvinyl pyrrolidine, polyvinyl alcohol, gum arabic, tragacanth, sodium alginate, gelatin and gluten. Suitable stabilizers include, but are not limited to, proteins such as albumin, protamine, gelatin and globulin; and amino acids and their salts. Suitable thickeners include, but are not limited to, sucrose, glycerol, methylcellulose and carboxymethylcellulose. Suitable pH regulators include, but are not limited to, hydrochloric acid, sodium hydroxide, phosphates, citrates and carbonates.

[0216] Pharmaceutical compositions that can be used orally include, but are not limited to, push-in capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-in capsules may contain attenuated live viruses and may be mixed with fillers such as lactose, binding agents such as starch, lubricants (such as talc or magnesium stearate), or both, and optionally stabilizers. In the soft capsules, one or more viruses may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All formulations for oral administration should be in a dosage suitable for this administration.

[0217] For buccal administration, corresponding pharmaceutical compositions may take the form of tablets or lozenges formulated in conventional manner.

[0218] For administration by inhalation, pharmaceutical compositions for use according to the present disclosure may be conveniently delivered as an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, may be formulated for use in an inhaler or insufflator containing a powder mix of the virus and a suitable powder base such as lactose or starch.

[0219] The corresponding pharmaceutical composition can be formulated for parenteral administration by injection, for example by rapid bolus injection or continuous infusion. The injection preparation can be present in unit dosage form, for example, in ampoules or multidose containers and with the addition of preservatives. The composition can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle and can contain a formulating agent, such as a suspending agent, a stabilizer, a dispersant or a combination thereof.

[0220] Pharmaceutical preparations for parenteral administration include aqueous solutions of the virus in water-soluble form. In addition, the viral suspension can be prepared into a suitable oily injection suspension. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

[0221] In some forms, the active ingredient, such as a virus described herein, can be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free (SPF) water, before use.

[0222] Examples of suitable mammals include, but are not limited to, mice, rats, cows, goats, sheep, pigs, dogs, cats, horses, guinea pigs, canines, hamsters, minks, seals, whales, camels, chimpanzees, rhesus monkeys, and humans.

[0223] The dosage and desired concentration of the attenuated virus disclosed herein can vary depending on the specific use envisioned. The determination of an appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for determining effective doses for human prophylactic and therapeutic use. Interspecies scaling of effective doses can be performed according to Mordenti, J. and Chappell, W. "The use of interspecies scaling intoxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., eds., Pergamon Press, New York 1989, pp. 42-96.

[0224] The amount or dose of the material administered should be sufficient to affect a therapeutic or prophylactic response in the subject within a reasonable time frame. For example, the dose of the material should be sufficient to prevent symptomatic infection caused by an unattenuated wild-type virus. The dose should be sufficient to stimulate an immune response, treat or prevent wild-type virus infection, or both.

[0225] Many assays for determining the dosage to be administered are known in the art. For the purposes of the present method, assays including comparisons of antiviral antibodies and immune cell types present in response to several different doses of attenuated virus administered to a group of mammals may be performed. The dosage may also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration. Various factors may be considered in determining the dosage, such as age, weight, general health, diet, sex, administration material, route of administration, and severity of the disease being treated.

[0226] The dose of the attenuated live virus vaccine for a subject, such as a mammalian adult organism, may be about 10 2 to about 10 15 , for example about 10 3 to about 10 12 , about 10 3 to about 10 10 , about 10 3 to about 10 8 , about 10 5 to about 10 8 , about 10 3 to about 10 6 , about 10 4 to about 10 8 , about 10 4 to about 10 7 , about 10 4 to about 10 6 or about 10 4 to about 10 5 Plaque forming units (PFU) / kg, or any range or value therein. However, the dosage should be a safe and effective amount determined by conventional methods using existing vaccines as a starting point.

[0227] The pharmaceutical composition used according to the present disclosure can be prepared in a conventional manner using one or more pharmaceutically acceptable carriers, including excipients and adjuvants, which help process the virus into a pharmaceutically acceptable preparation. The appropriate formulation depends on the selected route of administration. If the administration of the composition (including its components) can be tolerated by the recipient subject, it is considered to be "pharmacologically acceptable". If the amount applied is physiologically significant, the agent is said to be administered in a "therapeutically effective amount". If the presence of the composition of the present disclosure causes a detectable change in the physiology of the recipient subject, such as enhancing at least one primary or secondary humoral or cellular immune response to at least one infectious virus strain, the composition of the present disclosure has physiological significance.

[0228] In some forms, effective doses of the attenuated live viruses and pharmaceutical compositions described herein may generally include about 10 2 pfu, 10 3 pfu, 10 4 pfu, 10 5 pfu, 10 6 pfu, 10 7 pfu, 10 8 pfu, 10 9 pfu or more. The administration rate can be different. Typically, the administration rate can be, for example, once a month, once every three months, once every six months, once a year, or booster vaccinations are performed as needed.

[0229] As used herein, "immunogen" or "immunogenic amount" refers to the ability of a substance (antigen) to induce an immune response. An immune response is a reactive change in the immune system of an organism in response to an antigen, which in vertebrates may involve antibody production, induction of cell-mediated immunity, complement activation, or the development of immune tolerance.

[0230] As used herein, an "adjuvant" is a substance that increases the ability of an antigen to stimulate the immune system.

[0231] As used herein, "attenuation" refers to a procedure that weakens a disease agent (pathogen). In the context of such disease agents, the term "attenuation" refers to such disease agents that have been weakened. For example, an attenuated virus is a weakened, less viable virus. Vaccines for viral diseases can be prepared from attenuated, less toxic strains of the virus that can stimulate an immune response and produce immunity, but will not cause disease or cause less severe disease. Attenuation can be achieved by chemical treatment, radiation or genetic modification of the pathogen using methods known to those skilled in the art. Attenuation may result in reduced proliferation, attachment to host cells or reduced production or intensity of toxins.

[0232] As used herein, the term "in need of treatment" refers to the judgment made by a caregiver (e.g., a physician, nurse, nurse practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that a subject requires or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's expertise, but includes the knowledge that the subject is or will become ill as a result of a condition treatable by the disclosed compounds.

[0233] As used herein, "subject" includes, but is not limited to, animals, plants, bacteria, viruses, parasites, and any other organism or entity. The subject can be a vertebrate, more specifically, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cattle, cat, guinea pig, or rodent), fish, bird, or reptile or amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses, whether male or female, are covered. A patient refers to a subject suffering from a disease or condition. The term "patient" includes both human and veterinary subjects.

[0234] "Therapy" and "treatment" refer to the medical management of a subject with the purpose of curing, improving, stabilizing or preventing a disease, pathological condition or condition. The term includes active treatment, i.e., treatment specifically directed to improving a disease, pathological condition or condition, and also includes etiological treatment, i.e., treatment directed to eliminating the cause of the relevant disease, pathological condition or condition. In addition, the term also includes palliative treatment, i.e., treatment aimed at relieving symptoms rather than curing a disease, pathological condition or condition; preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological condition or condition; and supportive treatment, i.e., treatment used to supplement another specific therapy for the improvement of the relevant disease, pathological condition or condition. It should be understood that treatment, while intended to cure, improve, stabilize or prevent a disease, pathological condition or condition, does not actually need to result in cure, improvement, stabilization or prevention. The effect of treatment can be measured or evaluated in a manner applicable to the disease, pathological condition or condition involved as described herein and known in the art. Such measurement and evaluation can be performed in a qualitative and / or quantitative manner. Thus, for example, the characteristics or features of a disease, pathological condition or condition and / or the symptoms of a disease, pathological condition or condition can be reduced to any effect or to any amount.

[0235] The cell can be in vitro. Alternatively, the cell can be in vivo and can be present in a subject. A "cell" can be a cell from any organism, including but not limited to bacteria.

[0236] In one aspect, the compounds described herein can be administered to a subject in need of relief or improvement from a recognized medical condition, including humans or animals, including but not limited to mice, dogs, cats, horses, cows, sheep, etc.

[0237] As provided herein, the term "effective amount" of a compound refers to an amount of a compound that is non-toxic but sufficient to provide the desired result. As will be noted below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, the specific compound used, its mode of administration, etc. Therefore, it is not possible to specify an exact "effective amount". However, a suitable effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

[0238] The dose or amount of the compounds described herein is large enough to produce the desired effect in the method of delivery. The dose should not be so large as to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Generally, the dose will vary with the age, condition, sex, and degree of disease of the subject, and can be determined by those skilled in the art. The individual physician can adjust the dose according to the clinical condition of the subject involved. The dose, dosing regimen, and route of administration can be varied.

[0239] The efficacy of a particular dose of a compound or composition administered according to the methods described herein can be determined by evaluating particular aspects of the medical history, signs, symptoms, and objective laboratory tests known to be useful in evaluating the subject's status. These signs, symptoms, and objective laboratory tests will vary depending on the particular disease or condition being treated or prevented, as is known to any clinician treating such patients or researcher conducting experiments in the field. For example, a particular treatment regimen would be considered effective if, based on comparison with an appropriate control group and / or knowledge of the normal progression of the disease in the general population or a particular individual: (1) the subject's physical condition appears to be improved (e.g., a tumor has partially or completely regressed), (2) the progression of the disease or condition appears to be stabilized, or slowed, or reversed, or (3) the need for other drugs to treat the disease or condition is reduced or eliminated.

[0240] "Pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effect or interacting in a deleterious manner with any other components of the pharmaceutical composition in which it is contained.

[0241] Any compound of formula I can be used in combination with a pharmaceutically acceptable carrier for therapeutic use. The compounds described herein can be conveniently formulated into a pharmaceutical composition consisting of one or more compounds and a pharmaceutically acceptable carrier. See, for example, Remington's Pharmaceutical Sciences, latest edition, from EWMartin MackPub.Co., Easton, PA, which discloses typical carriers and conventional methods for preparing pharmaceutical compositions, which can be used in conjunction with the preparation of the preparations of the compounds described herein, and are incorporated herein by reference. These are most typically standard carriers for administering the composition to humans. On the one hand, standard carriers for administration to humans and non-humans include solutions such as sterile water, saline, and buffered solutions of physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0242] In addition to the selected molecule, the pharmaceutical compositions described herein may include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. The pharmaceutical compositions may also include one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, etc.

[0243] Compounds and pharmaceutical compositions described herein can be applied to subjects in a variety of ways, depending on whether local or systemic treatment and the area to be treated are desired. Therefore, for example, compounds or pharmaceutical compositions described herein can be applied to the surface of the eye as ophthalmic solutions and / or ointments. In addition, compounds or pharmaceutical compositions can be applied to subjects by vaginal, rectal, intranasal, oral, by inhalation or parenteral routes, such as by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. If parenteral administration is used, it is usually characterized by injection. Injections can be prepared into conventional forms, either as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. The parenteral administration method recently revised involves the use of sustained release or sustained release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0244] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, which may also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc.

[0245] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0246] Compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binding agents may be desirable.

[0247] The terms "high", "higher", "increase", "elevate" or "elevate" refer to an increase above a basal level, e.g., compared to a control. The terms "low", "lower", "decrease" or "decrease" refer to a decrease below a basal level, e.g., compared to a control.

[0248] As used herein, the term "modulate" refers to the ability of a compound to change an activity in some measurable way compared to an appropriate control. Due to the presence of the compound in the assay, the activity can be increased or decreased compared to a control in which these compounds are not present. Preferably, the activity is increased by at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, the activity is reduced by preferably at least 25%, more preferably at least 50%, and most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an "agonist." A compound that decreases or prevents a known activity is an "antagonist."

[0249] The term "inhibit" means to reduce or decrease an activity or expression. This can be complete inhibition or partial inhibition of activity or expression. The inhibition can be compared to a control or standard level. The inhibition can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0250] As used herein, the term "monitoring" refers to any method in the art by which an activity can be measured.

[0251] As used herein, the term "providing" refers to any means of adding a compound or molecule to a thing known in the art. Examples of providing may include using a pipette, a pipette, a syringe, a needle, a catheter, a gun, etc. It may be manual or automatic. It may include transfection of providing nucleic acids to a culture dish, a cell, a tissue, a cell-free system by any means or any other means, and may be performed in vitro or in vivo.

[0252] As used herein, the term "preventing" refers to the administration of a compound prior to the onset of clinical symptoms of a disease or condition to prevent abnormal physical manifestations associated with the disease or condition.

[0253] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0254] 1. A virus comprising a heterologous nucleic acid segment encoding α-1,3-galactosyltransferase (α-1,3-GT), wherein when the virus infects a host cell, the nucleic acid segment expresses α-1,3-GT, wherein when the virus infects the host cell, at least one protein exposed on the surface of the host cell comprises α-1,3-galactose.

[0255] 2. The virus of paragraph 1, wherein when the virus infects the host cell, at least one viral protein expressed by the virus comprises α-1,3-galactose.

[0256] 3. The virus of paragraph 1 or 2, wherein the virus is live and attenuated.

[0257] 4. The virus of any one of paragraphs 1-3, wherein the nucleic acid fragment is introduced into the genome of the virus.

[0258] 5. The virus of any one of paragraphs 1-4, wherein the nucleic acid fragment is introduced in-frame into the reading frame of the viral coding region of the virus.

[0259] 6. The virus of paragraph 5, wherein the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the viral coding region of the virus.

[0260] 7. The virus of paragraph 6, wherein the virus further comprises a protease cleavage site encoded between the nucleic acid fragment and the viral coding region of the virus.

[0261] 8. The virus of any of paragraphs 1-7, wherein the virus is an influenza virus.

[0262] 9. The virus of paragraph 8, wherein the nucleic acid fragment is introduced in-frame into the reading frame of the neuraminidase (NA) coding region of the virus.

[0263] 10. The virus of paragraph 9, wherein the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the NA coding region of the virus.

[0264] 11. The virus of paragraph 10, wherein the virus further comprises a protease cleavage site encoded between the nucleic acid fragment and the NA coding region of the virus.

[0265] 12. The virus of any one of paragraphs 7-11, wherein the protease cleavage site is contained in the 2A self-cleaving peptide.

[0266] 13. The virus of paragraph 12, wherein the 2A self-cleaving peptide is derived from porcine Teshchinavirus-1.

[0267] 14. The virus of paragraph 12 or 13, wherein the 2A self-cleaving peptide is flanked by short peptide linkers.

[0268] 15. The virus of paragraph 14, wherein the peptide linker is GSG.

[0269] 16. The virus of any of paragraphs 1-15, wherein the α-1,3-GT is a mammalian α-1,3-GT.

[0270] 17. The virus of any of paragraphs 1-16, wherein the α-1,3-GT is mouse α-1,3-GT.

[0271] 18. The virus of any one of paragraphs 1-17, wherein the virus is attenuated by serial passaging in cultured cells, serial passaging in a heterologous host animal, gene deletion in the virus, site-directed mutagenesis of the virus, altering the codon usage of the virus, selecting cold-adapted mutants, using related viruses from heterologous host species, using naturally occurring attenuated virus strains, or a combination thereof.

[0272] 19. The virus of paragraph 18, wherein the virus is attenuated by serial passaging in cultured cells, wherein the cultured cells are Madin-Darby canine kidney (MDCK) cells.

[0273] 20. The virus of any of paragraphs 1-19, wherein the virus is influenza virus, influenza A virus, influenza B virus, human papillomavirus, measles virus, human immunodeficiency virus, rabies virus, Ebola virus, Hantaan virus, Marburg virus, Lassa virus, dengue virus, Eastern equine encephalitis virus, hepatitis C virus, hepatitis A virus, hepatitis B virus, hepatitis E virus, human herpes virus, human SARS coronavirus, mumps virus, polio virus, rubella virus, varicella-zoster virus, Rift Valley fever virus, West Nile virus, yellow fever virus, human T-lymphotropic virus or Merkel cell polyomavirus.

[0274] 21. The virus of paragraph 20, wherein the virus is an influenza virus.

[0275] 22. A vaccine comprising the virus of any of paragraphs 1-21, wherein the virus is live and attenuated.

[0276] 23. A vaccine comprising one or more viral proteins expressed by the virus of any of paragraphs 1-21, wherein the one or more viral proteins comprise alpha-1,3-galactose.

[0277] 24. A method of vaccination, the method comprising administering the vaccine of paragraph 22 or 23 to a subject in need thereof.

[0278] 25. The method of paragraph 24, wherein the vaccine is administered intranasally, pulmonary, orally, subcutaneously, intramuscularly, intradermally or intraperitoneally.

[0279] 26. A method of preparing the vaccine of paragraph 23, the method comprising infecting a cell with the virus, thereby producing one or more viral proteins comprising α-1,3-galactose.

[0280] 27. The method of paragraph 26 further comprises purifying the one or more viral proteins.

[0281] The recombinant influenza virus PR8 / NAGT described herein was deposited at the American Type Culture Collection (ATCC) at PO Box 1549, Manassas, VA 20110, USA, on August 22, 2019, in accordance with the Budapest Treaty, with the following deposit number: PTA-125887. As of August 22, 2019, the viability of the deposited material has been verified. Example

[0282] Example 1. Characterization of NA mutants expressing α-1,3-galactosyltransferase in vitro.

[0283] Materials and methods

[0284] Generation of NAGT mutants

[0285] The mouse α-1,3-GT gene amplified by RT-PCR from spleen tissue of Balb / c mice was introduced into a plasmid expressing PR8 NA vRNA (pHW2000-PR8-NA) (34) as Figure 1 As shown. Recombinant PR8 WT virus and NAGT virus were generated by standard reverse genetics techniques (Hoffmann et al., PNAS, 97: 6108-6113 (2000)). The rescued viruses were cultured in 10-day-old embryonic eggs for further amplification. The viral sequence of the rescued viruses was confirmed by standard Sanger sequencing.

[0286] Western blot analysis

[0287] Infected A549 cells were harvested 24 hours after infection (MOI = 1). Cell lysates were separated in 12% separation PAGE gel. In the assay, 1000-fold diluted rabbit anti-neuraminidase antibody (ab21304; Abcam) and 2000-fold diluted horseradish peroxidase (HRP) goat anti-rabbit IgG (ab205718; Abcam) were used as primary and secondary antibodies, respectively.

[0288] Immunofluorescence staining assay

[0289] As described in Leung et al., J. Virol., 86: 10704-13 (2012), infected cells (MOI = 1) were fixed in 4% paraformaldehyde solution in PBS 24 hours after infection. In the assay, 5-fold diluted mouse anti-α-Gal monoclonal IgM (M86, Enzo life sciences) and 2000-fold diluted fluorescent dye-conjugated goat anti-mouse IgG / IgA / IgM antibody (10667, Invitrogen) were used as primary and secondary antibodies, respectively. DAPI was used as a nuclear counterstain (Invitrogen). Human A549 cells infected with WT virus or NAGT virus 24 hours after infection were stained for α-Gal epitopes. In order to detect the α-Gal epitope on non-permeabilized cells, human A549 cells treated with NAGT mutants or PBS (control) were stained. The staining scheme was as described above, except that Triton X-100 was not used in the initial cell fixation.

[0290] Virus replication kinetics assay

[0291] MDCK cells were infected with influenza virus (MOI = 0.001) in triplicate. After a 1-hour virus adsorption period at 37°C, infected cells were briefly washed once and twice with acidified PBS (pH 2.0) and PBS (pH 7.0), respectively, and then supplemented with virus culture medium as described in Leung et al., J. Virol., 86: 10704-13 (2012). The titer of progeny virus was determined by standard plaque assay.

[0292] Antibody-dependent NK cell assay

[0293] As described in Jegaskanda et al., J. Immunol., 190: 1837-1848 (2013), human A549 cells were infected with NAGT or WT PR8 virus at an MOI of 10 for 5 hours. Infected cells were stained with PE-Cy7 anti-human HLA-A, B, C antibodies (clone W6 / 32, Biolegend). PBS-treated cells were used as negative controls. In the presence of heat-inactivated human serum (1:20 dilution), the treated A549 cells were incubated with CD16.NK92 cells (FoxChase Cancer Centre) treated with anti-human CD107a APC (clone LAMP-1, Biolegend) for 5 hours. After incubation, the cell mixture was stained with anti-human CD56-PE antibody (clone MEM188, Biolegend) and fixed. The treated cell mixture was then stained with anti-NP-FITC (Abcam) antibody. NK cell activation (CD56) was obtained by BD LSR Fortessa. +CD107a + ) and infected A549 cells (NP + HLA + ). Signals from infected cells were generated without preincubation with human serum, while signals from mock infected cells with / without preincubation with human serum were used as controls. All data were analyzed using FlowJo. The fold reduction (%) of infected cells due to α-Gal expression is equal to:

[0294]

[0295] ADCC

[0296] A commercial luciferase reporter gene assay kit was used to measure ADCC activity (ADCC receptor bioassay kit, Promega). Briefly, A549 cells were infected with WT or NAGT mutants (10 MOI). Five hours after infection, cells were incubated with diluted human serum and recombinant Jurkat effector cells for 6 hours. The cell mixture without human serum was used as a control. The luciferase activity of effector cells induced by infected cells was measured by a standard luciferase assay. The induction fold of the ADCC reaction was equal to:

[0297]

[0298] Phagocytosis assay

[0299] Human monocyte-derived macrophages were prepared as described in Manches et al., Haematologica, 90: 625-34 (2005). One day before the experiment, A549 cells were infected with WT virus or NAGT virus (MOI = 1) and then cultured overnight. On the day of the experiment, the infected cells were incubated with human serum at 4°C for 30 minutes. Serum-treated cells were added to macrophages in a ratio of 3:1, and the cell mixture was incubated at 37°C for 2 hours. The incubated cell mixture was washed with PBS, fixed in 4% paraformaldehyde, and then stained with Giemsa. The stained macrophages were examined under a standard light microscope. The work of collecting primary human monocytes and human serum samples was approved by the local IRB.

[0300] Statistical analysis

[0301] Unless otherwise stated, one-way ANOVA was used to determine effects on the amplitude of cellular immune responses and cell population profiles.

[0302] result

[0303] The uptake of antigens by professional antigen presenting cells (APCs) such as dendritic cells (DCs) plays a key role in acquired immunity. The antigen uptake process can be enhanced by conditioning the antigen, and high levels of pre-existing antibodies specific for the antigen can promote this process (Boudreau and Alter, Front. Immunol., 10: 440 (2019)). The expression of galactose-α-1,3-galactose (α-Gal) epitopes on the membrane surface can enhance their conditioning, resulting in phagocytosis of APCs mediated by anti-Gal antibodies naturally expressed in healthy individuals. This leads to enhanced phagocytosis of infected cells by professional APCs (Galili, Immunology, 140: 1-11 (2013)). Human polyclonal anti-α-Gal antibodies exist in different forms (IgA, IgG and IgM), which are expressed in large quantities in healthy individuals (about 1% of circulating immunoglobulins). α-Gal epitopes are expressed by a variety of living organisms. This epitope is presented by surface glycoproteins or glycolipids after an enzymatic reaction of α-1,3-galactosyltransferase (α-1,3-GT) in the endoplasmic reticulum (Galili, Immunology, 140:1-11 (2013)). However, humans, apes, and Old World monkeys have defective α-1,3-GT genes and are unable to express this epitope (Galili, Immunology, 140:1-11 (2013)). Due to the continuous stimulation of the α-Gal epitope in food and normal flora, these primates naturally produce high levels of anti-α-Gal antibodies throughout their lives.

[0304] The use of α-Gal epitopes to stimulate T cell and B cell responses by enhancing antigen uptake has been demonstrated in cancer therapy and experimental vaccines (Albertini et al., Cancer Immunol. Immunother., 65:897-907 (2016); Abdel-Motal et al., Vaccine, 28:1758-65 (2010); Henion et al., Vaccine, 15:1174-82 (1997)). Previous viral vaccine studies were based on inactivated viral antigens artificially coated with α-Gal epitopes, which have been shown to enhance adaptive immune responses against homologous viral infections. However, this strategy requires additional enzymatic treatment to generate α-Gal epitopes on these antigens, thereby reducing antigen yield and cost-effectiveness. In this paper, an attenuated influenza virus containing an α-1,3-GT gene was generated to express the α-Gal epitope in infected cells.

[0305] In order to express the α-gal epitope in infected cells, a recombinant A / PR / 8 / 34 H1N1 (PR8) virus carrying the mouse α-1,3-GT gene in its neuraminidase (NA) vRNA segment (hereinafter referred to as the NAGT mutant) was prepared ( Figure 1 ). The α-1,3-GT gene was inserted in the same manner immediately before the stop codon of the NA open reading frame (ORF). The 2A peptide derived from porcine Teschovirus-1 flanked by a short peptide linker (GSG) was inserted into the junction of NA and α-1,3-GT ORF to allow efficient cleavage of the NA / α-1,3-GT polypeptide into NA and α-1,3-GT proteins (Kim et al., PLoS One, 6: e18556 (2011)). The cleavage of the NA / α-1,3-GT polypeptide into NA and α-1,3-GT proteins was confirmed in the detection of influenza NA protein infection by Western blotting, where human A549 cells infected with WT virus and NAGT virus (MOI of 1) were harvested 48 hours after infection (data not shown). The packaging signal (157 nt in the UTR and ORF regions) at the 5' end of the NAvRNA was inserted near the stop codon of the NA / GT ORF sequence. The NAGT mutant was attenuated in MDCK cells (virus titer decreased by approximately 2.5 log), but it was still able to achieve relatively robust replication in infected cells with a maximum titer of approximately 5.8e5 pfu / ml ( Figure 2 ). Preliminary mouse infection studies showed that the NAGT mutant was attenuated in mice (MLD50 of WT = 178 pfu; MLD50 of NAGT = 2700 pfu). The NAGT mutant was approximately 15 times less toxic in mice than its WT control. Although this viral background (PR8) is lethal in mice, it is the standard master strain for preparing seasonal influenza vaccines. Further attenuation of the mutant may be required, for example by fine-tuning the level of viral attenuation using codon bias (Fan et al., J. Virol., 89: 10762-73 (2015)). Therefore, the level of attenuation of the NAGT mutant can be adjusted by using this strategy or other methods.

[0306] Human cells infected by NAGT mutants but not WT virus can express α-Gal epitopes in the cytoplasm and on the cell surface, as demonstrated by immunofluorescence staining of α-Gal epitopes 24 hours after infection in human A549 cells infected with WT virus or NAGT virus (data not shown).

[0307] Expression of the α-Gal epitope by the NAGT mutant can lead to binding of human anti-α-Gal antibodies to infected cells, enhancing opsonization of infected cells by APCs and thus enhancing vaccine-induced adaptive responses. To demonstrate this, cells infected with WT and NAGT viruses were treated with normal human serum, and the ability of primary human monocyte-derived macrophages to phagocytose these cells was obtained. Figure 3 As shown, human A549 cells infected with NAGT mutants can significantly enhance the phagocytic activity of human macrophages (p<0.05). Cells infected with NAGT mutants are more easily killed by NK cells after incubation with human serum ( Figure 4A and Figure 4B This enhanced killing effect was much smaller in cells infected with WT virus ( Figure 4C and Figure 4D The lower killing effect stimulated by cells infected with WT virus may be due to the presence of influenza virus antibodies commonly found in normal individuals. In 5 of the 6 human sera tested, cells infected with NAGT mutants were more likely to be killed by NK cells than cells infected with WT virus ( Figure 5 ; p<0.05). The stimulatory effect of NAGT mutants on immune effector cells was confirmed by using an antibody-dependent cellular cytotoxicity (ADCC) reporter gene assay ( Figure 6 ).

[0308] In summary, the above results indicate that α-Gal epitope expression on NAGT-infected cells can stimulate APC phagocytosis, NK cell-mediated cytotoxicity, and ADCC. These enhanced effects suggest that opsonization of infected cells with anti-α-Gal antibodies can enhance vaccine-induced responses.

[0309] Example 2. Characterization of α-1,3-GT knockout mice after vaccination with NAGT mutants.

[0310] Materials and methods

[0311] α-1,3-GT KO mouse model and immune studies

[0312] WT C57BL / 6 and α-1,3-GT KO mice (Tearle et al., Transplantation, 61:13-9 (1996)) were purchased from Shanghai Model Organisms Center Inc., China. To induce anti-α-Gal antibodies in KO mice, rabbit RBC membranes (3×10 in 100 μl PBS) were injected intraperitoneally into 4- and 6-week-old mice. 8) (La Temple and Galili, Xenotransplantation, 5: 191-6 (1998)). Sera from treated mice were collected at 4, 6 and 8 weeks of age. Serum anti-α-Gal IgG1 antibody levels in treated mice were measured by ELISA as described below. For vaccination, NAGT virus (150 pfu in 25 μl PBS) was administered intranasally to KO mice and WT mice at 8 weeks of age. Mock-vaccinated (PBS) WT mice and KO mice were used as controls.

[0313] ELISA

[0314] Galα1-3Galβ1-4GlcNAc-BSA (3-atom spacer, Dextra), recombinant NP or HA (SinoBiological) were coated overnight at 4°C on a 96-ELISA plate (Nunc MaciSorp). Pooled serum samples (N≥4 per group) were serially diluted 2-fold starting from 1:40. Anti-mouse IgG1 HRP-conjugated antibody (31430, Invitrogen) was used as a secondary antibody. The absorbance at 450 nm was measured. Each sample was tested in duplicate. A difference of ≥4-fold in the endpoint titer was considered significant.

[0315] Cell Atlas in Infected Samples

[0316] Mouse lung and spleen tissue (n≥5) were harvested at 1, 3, 5 and 21 days after vaccination and 3 and 7 days after infection using the scheme described in Valkenburg et al., PNAS, 111:5676-81 (2014). After hemolysis with RBC lysis buffer (eBioscience), cells were stained with Zombie Aqua (Biolegend), and then different cell markers were stained (by Fc blocking as follows: anti-CD16 / CD32, BD biosciences; anti-CD11b-FITC, CD11c-PerCP / Cy5.5, Ly6G-APC, F4 / 80-PE, MHC II-PB, Biolegend). As Valkenburg et al., PNAS, 111:5676-81 (2014) described in fixed stained cells (BD Cytofix / cytoperm buffer). DC markers in the lung (CD11c+MHCII high Ly6G-F4 / 80-), neutrophil markers (Ly6G high CD11b high), alveolar macrophage markers (CD11c+F4 / 80+) and macrophage markers in the spleen (CD11b+F4 / 80+) were studied. Signal acquisition was performed on a BD LSRFortessa and data were analyzed by FlowJo. The gating strategy for these cells is shown in the corresponding figures.

[0317] ICS assay

[0318] CD4 + and CD8 + T cell recall response was determined as described in Horton et al., J. Immunol. Methods, 323: 39-54 (2007) and Yan et al., Virology, 525: 73-82 (2018). The production of type 1 (IFN-γ, TNF-α, IL-2) and type 2 (IL-4) cytokines of T cells was detected. Splenocytes and cells from BAL were harvested on day 7 after infection (n ≥ 3 per group). After RBC lysis, isolated lymphocytes were stimulated with PR8 or HK68 virus and incubated with anti-CD28, anti-CD49d (BDbiosciences), IL-2 (Roche) for 6 hours, and then incubated with Golgi plug (Brefeldin A, BDbiosciences) overnight. For spleen cells 3 weeks after vaccination, cells were stimulated with PR8, HK68, H1N1 / Brisbane / 07(H1N1), and HK / MPF461 / 07(H5N2) viruses. Treated cells were first treated with Zombie Aqua TM(Biolegend) staining, followed by T cell markers (Fc blocking by: anti-CD16 / CD32, BDbiosciences; anti-CD4 APC / Cy7, CD8 PerCP / Cy5.5, Biolegend). The stained cells were then fixed with Cytofix / cytoperm buffer (BD) and then intracellular cytokine staining (IFN-γFITC, TNF-αAPC, IL-2PE, IL-4PE / Cy7, Biolegend). Signal acquisition was performed on BD LSR Fortessa and data were analyzed by FlowJo.

[0319] Statistical analysis

[0320] Unless otherwise stated, one-way ANOVA was used to determine effects on the amplitude of cellular immune responses and distribution of cell populations.

[0321] result

[0322] WT laboratory mice have a functional α-1,3-GT gene and they do not produce anti-α-Gal antibodies to avoid autoimmunity. Therefore, the vaccine potential of NAGT mutants was evaluated using the α-1,3-GT knockout (KO) mouse strain ( Figure 7 ) (Tearle et al., Transplantation, 61: 13-9 (1996)). These KO mice were first injected intraperitoneally with rabbit RBCs twice to stimulate anti-α-Gal antibody production (LaTemple and Galili, Xenotransplantation, 5: 191-6 (1998)). RBC-treated KO mice (weeks 8-11) can stably produce and maintain high levels of anti-α-Gal antibodies (Table 5). In contrast, no anti-α-Gal antibodies were detected in RBC-treated WT mice.

[0323] Table 5. Anti-α-Gal antibody titers in different experimental groups before virus challenge at week 11.

[0324]

[0325] At the predetermined intranasal vaccination dose (150 pfu), both vaccinated WT and KO mice produced high levels of neutralizing antibody titers against H1N1 but not H3N2 viruses 3 weeks after vaccination (Table 6), demonstrating that the viral vaccine itself does not stimulate the production of heterosubtypic neutralizing antibodies. Influenza virus-specific CD4 T cells against different influenza virus subtypes were measured in vitro in the spleen tissue of vaccinated mice using the intracellular cytokine staining (ICS) assay. + or CD8 +T recall response (TNF-α + ). Compared with the results of mock-vaccinated KO mice, splenic CD4 + T cells can be stimulated by different virus subtypes (H1N1, H3N2, and H5N2; p<0.05) ( Figure 8 ), demonstrating the presence of cross-reactive CD4 + T cell responses. No cross-reactive CD8 + T cell responses, but vaccinated WT and KO mice had some but not significant CD8 + Recall reaction ( Fig. 9 ). Thus, at day 21 after vaccination, antibody and T cell responses were similar between vaccinated WT and KO mice.

[0326] Table 6. H1N1 and H3N2 virus-specific neutralizing antibody titers in different experimental groups before virus challenge at week 11.

[0327]

[0328] To determine whether the NAGT mutant could induce a stronger response in other immune cells, the spleens of vaccinated mice were studied 1, 5, and 21 days after vaccination. Figures 10A-10C ) and lungs ( Figures 11A-11E ) in the spleen tissue of KO mice. On day 1 after vaccination, DC populations (CD11c + MHCII 高 6G - F4 / 80 - ) Fig. 10A ), indicating that the mutant can stimulate DC migration to the spleen. In contrast, the neutrophil or macrophage profiles in the spleen of these WT and KO mice at these time points were similar ( Fig. 10B and Fig. 10C ).

[0329] Lung DC (CD11c + MHCII 高 6G - F4 / 80 - ) frequencies increased, reaching peaks on days 5 and 21 in vaccinated WT and KO mice, respectively ( Fig.11A In addition to the kinetic differences, the DC populations studied in WT and KO mice at these time points were slightly different (CD11b 高 CD11b低 / - )( Fig. 11B and Fig. 11C The DC population increased in vaccinated KO mice was mainly CD11b 低 / - ( Fig. 11B ). In contrast, CD11b was detectable in vaccinated WT mice at day 5 after vaccination. 高 A significant increase in the DC population ( Fig. 11C ) and CD11b 低 / - A slight increase (not significant relative to unvaccinated controls) Fig. 11B On day 1 after vaccination, an increase in neutrophil frequency was observed in vaccinated WT mice ( Fig.11D ), suggesting that the NAGT mutant caused a relatively more severe inflammatory response in WT mice because these mice lack anti-α-Gal antibodies that control infection (Ueki et al., PNAS, 115: E6622-E6629 (2018)). Both vaccinated WT and KO mice had reduced levels of alveolar macrophages after vaccination ( Fig.11E ), but the reduction is indistinguishable.

[0330] DC is essential for immune surveillance. This cell population can help eliminate pathogens through adaptive immune responses. Conditioned immune complexes can be effectively internalized by monocyte-derived DCs through the interaction between the Fc portion of the antibody and the Fcγ receptor of the DC (Guilliams et al., Nat. Rev. Immunol., 14: 94-108 (2014)). DCs loaded with antigens can migrate to draining lymph nodes to activate T cells. Increasing this DC-T cell interaction in the lymphatic system is expected to enhance the immune memory response to influenza virus (Eisenbarth, Nat. Rev. Immunol., 19: 89-103 (2019); Lehmann et al., J. Exp. Med., 214: 1509-1528 (2017)). Lung mouse lung DC (CD11c + MHCII 高 ) can be divided into two major subsets: CD11b 低 / - DC and CD11b 高 DC (Condon et al., J. Leukoc. Biol., 90:883-95 (2011)). In this study, both vaccine groups had increased total DC populations in lung tissue after vaccination, but the increased DC subsets in these vaccinated mice were different. The increased DC population in the vaccinated KO mice was entirely CD11b 低 / -, while the increased DC population in vaccinated WT mice was mainly CD11b 高( Fig. 11B ). This suggests that NAGT mutants can modulate protective DC responses in these groups of mice.

[0331] Overall, these results suggest that NAGT mutants can direct DC and other APC responses in both WT and KO mice following vaccination.

[0332] Healthy individuals are frequently exposed to α-Gal epitopes through external sources (e.g., food). These regular exposures also explain why different forms of α-Gal antibodies (including serum IgG) remain at high levels in normal individuals. Further testing of the safety of this vaccine regimen is necessary, such as any side effects in healthy individuals repeatedly immunized with NAGT mutants.

[0333] Example 3. NAGT mutants can protect mice from lethal viral challenge.

[0334] Materials and methods

[0335] Virus attack research

[0336] At 8 weeks of age, NAGT virus (150 pfu in 25 μl PBS) was administered intranasally to KO and WT mice. Mock-vaccinated (PBS) WT and KO mice were used as controls. Three weeks after vaccination, treated mice were anesthetized and treated with a lethal dose (H1: 40 MLD) as described in Valkenburg et al., PNAS, 111: 5676-81 (2014). 50 ; H3 or H5: 10MLD 50 ) influenza virus for intranasal challenge. The influenza viruses used in this study were: PR8 (A / Puerto Rico / 8 / 1934; H1N1), mouse-adapted HK68 (A / Hong Kong / 1 / 68; H3N2), or highly pathogenic avian H5N1 (A / Vietnam / 1203 / 2004; H5N1) viruses. Morbidity and weight loss were monitored for 14 days, and survival curves were recorded. Thirty percent weight loss was set as the humane endpoint for euthanasia. Tissue samples from representative mice were collected at different time points as specified. All H5 studies were performed in a biosafety level 3 facility. All animal experiments were approved by the Committee on Live Animal Teaching and Research (CULATR) of the University of Hong Kong.

[0337] Viral load determination

[0338] Lungs of challenged mice were harvested on days 3 and 7 post-infection (N ≥ 3) and then mechanically homogenized. The expression of TCID in MDCK cells was determined using the Reed-Muench formula. 50 Determine titrated viral load.

[0339] Serological assays

[0340] Heat-treated sera before and after virus challenge were studied by ELISA and microneutralization (MN) assay to measure influenza virus-specific antibody titers as previously described (Valkenburg et al., Proc. Natl. Acad. Sci. USA 111:5676-81 (2014); Valkenburg et al., Vaccine 36:4198-4206 (2018)).

[0341] ELISA was performed as described above. For MN assay, pooled serum samples treated with RDE (n≥3 per group) were serially diluted 2-fold starting from 1:10 and tested against PR8 or HK68 viruses. Neutralizing antibody titers were expressed as geometric mean titer values.

[0342] Statistical analysis

[0343] Unless otherwise stated, one-way ANOVA was used to determine the effects on the amplitude of cellular immune responses, cell population profiles, and viral load within the WT or KO groups, while t-tests were used for the same purpose between WT and KO mice at the same time points. Survival curves based on weight loss were analyzed by GraphPad Prism. Log-rank tests were used to compare survival rates between groups.

[0344] result

[0345] NAGT mutants protect mice from lethal homologous challenge

[0346] The vaccine potential of NAGT mutants was first tested by homologous challenge. Rabbit RBC-sensitized WT and KO mice were challenged with a lethal dose of WT PR8 virus (10 MLD 50 ) challenge. All mock-vaccinated WT mice and KO mice succumbed to infection as expected. All vaccinated KO mice and 94% of vaccinated WT mice survived the challenge ( Fig. 12A ), there was no significant weight loss in the vaccinated KO mice group ( Fig. 12B Both vaccinated groups developed a good antibody response to influenza NP protein after challenge (Table 7). Although both vaccinated groups had greatly reduced viral lung titers compared to the unvaccinated control group, the vaccinated KO mice had a faster viral clearance rate than the vaccinated WT mice ( Fig. 12C ).

[0347] Vaccine-induced influenza-specific T cell responses were measured in respiratory tract (bronchoalveolar lavage, BAL) and spleen samples of treated mice on day 7 post-challenge. Influenza-specific CD4 T cells were measured by IFN-γ ICS assay after ex vivo stimulation with PR8. + and CD8 + T cell response. In BAL, CD8 + T cell responses were much higher than those in WT mice (e.g., IFN-γ + and TNF-α + )( Fig. 13B ). In the spleen, IFN-γ from WT mice + CD4 + T cells and IFN-γ + CD8 + The T cell response was only slightly better than that of KO mice, but significant enough ( Fig.14A and Fig. 14B ). The DC, neutrophil, and macrophage profiles in lung and spleen tissues were also determined at days 0, 3, and 7 post-challenge, but no significant differences between these mice were detected.

[0348] Table 7. NP-specific antibody titers in mice on days 0, 7, and 14 after viral challenge a .

[0349]

[0350] a Antibody levels were investigated by ELISA assay.

[0351] In short, the rapid viral clearance and highly robust CD8+ T cell responses observed in vaccinated KO mice suggest that, in the presence of anti-Gal antibodies, NAGT mutants can induce enhanced immune responses in this mouse model, which expresses high levels of anti-α-Gal antibodies in vivo.

[0352] NAGT mutants protect mice from lethal heterosubtypic H3N2 challenge

[0353] To determine the protective effect of NAGT mutants against heterosubtypic infection, mouse-adapted A / HK / 1 / 68H3N2 (HK68; 10 10 MLD 50 ) virus. All vaccinated KO mice survived, whereas 38% of vaccinated WT mice and all unvaccinated mice died from infection ( Fig.15A In addition, vaccinated KO mice lost less weight compared to controls ( Fig. 15B ), the virus is cleared faster in the lungs ( Fig. 15C). At day 7 after challenge, strong H3N2 virus-specific neutralizing antibody and NP-specific antibody responses could be detected in vaccinated KO mice but not in vaccinated WT mice ( Fig.15D and Table 8). In addition, at day 14 post-challenge, surviving vaccinated WT mice had much less NP-specific antibodies than vaccinated KO mice. These results suggest that the NAGT mutant can generate an effective heterosubtypic antibody response against H3N2 virus in KO mice. This enhanced heterosubtypic protection was confirmed to require the presence of anti-α-Gal antibodies in KO mice (see Example 4 below).

[0354] To determine the mechanism of protection in vaccinated KO mice, CD4 + and CD8 + T cell responses. The H3N2 influenza virus-specific CD4+ T cell responses in the lung and spleen tissues of vaccinated KO mice were found to be much stronger than those of vaccinated WT mice ( Fig.16A and Fig.17A In contrast, no differences were detected between the CD8+ T cell responses of the two vaccinated groups ( Fig. 16B and Fig. 17B ).

[0355] DC, neutrophil, and macrophage populations were investigated in infected lung and spleen tissues. Lung tissues from both vaccinated groups had increased DC frequencies after challenge ( Fig.18A ) and these are mainly CD11b 高 DC response ( Fig.18B and Fig.18C Interestingly, although there was a significant difference in CD11b between vaccinated WT and KO mice, 高 DC levels were similar, but at day 7 after challenge, CD11b 低 / - The DC level was significantly higher than that of the vaccinated WT group ( Fig.18B and Fig.18C In the spleen, no significant increase in DC populations after challenge was observed in any of the vaccinated groups. Neutrophil or macrophage profiles were also similar in infected WT and KO mice.

[0356] Table 8. NP-specific antibody titers in mice on days 0, 7, and 14 after viral challenge a .

[0357]

[0358] a Antibody levels were investigated by ELISA assay.

[0359] NAGT mutants protect mice from lethal, highly pathogenic H5N1 virus challenge

[0360] In mouse models (A / VN / 1203 / 04; 10MLD 50 ) studied the protective effects of NAGT mutants against infection with the highly pathogenic H5N1 avian influenza virus. All vaccinated KO mice survived the lethal virus challenge ( Fig.19A ), while the survival rate of vaccinated WT mice was significantly reduced (70%; p < 0.05). There was no significant difference in lung virus titers between the two vaccinated groups on day 7 after challenge ( Fig.19B ). However, faster recovery was observed in the vaccinated KO group at week 2 post-challenge ( Fig.19C ).

[0361] Thus, these results, together with those from the H3N2 studies described above, suggest that NAGT mutants can elicit strong, broadly cross-reactive immune responses against both group 1 (H1 and H5) and group 2 (H3) influenza virus infection.

[0362] With just one intranasal vaccination, vaccinated KO mice were able to mount enhanced humoral and / or cell-mediated responses following viral challenge. Vaccinated mice had no detectable pre-existing neutralizing antibodies against the H3N2 virus prior to challenge. Results from the H3 virus challenge model indicate that vaccinated KO mice can mount rapid and robust antibody responses against both viral surface and internal proteins following challenge ( Fig.15D and Table 8). These results suggest that NAGT-sensitized KO mice have potent cross-reactive memory responses to heterosubtypic influenza viruses, activating early humoral responses to control infection. As enhanced T cell responses were observed in the mouse model, the enhanced protection may involve interactions between the T cell compartment and the B cell compartment.

[0363] Homologous challenge elicited robust CD8 + T cell recall response ( Fig.13A and Fig. 13B ), while heterotypic attack can induce strong CD4 + T cell recall response ( Fig.16A and Fig. 16B Many of the T cells in the BAL of these mice were multifunctional cytokine-producing T cells ( Fig.21A and Fig. 21B ), indicating that vaccinated KO mice had better T cell responses to control viral infection than vaccinated WT mice.+ and CD8 + The responses also suggest that the protective mechanisms of NAGT mutants in the H1 and H3 models studied are different. Specifically, after H3 virus challenge, CD4 + Enhanced T cell recall response. Memory CD4 + T cells are essential for heterosubtypic influenza protection (Valkenburg et al., PNAS, 111:5676-81 (2014); Richards et al., J. Virol., 83:6566-77 (2009)). Depletion of memory CD4 in mice vaccinated with vaccinia-vectored universal influenza vaccine + T cells increase mortality, delay antibody production, and reduce CD8 + T cell recall response (Valkenburg et al., Vaccine, 36:4198-4206 (2018)). The results of the H3 model confirmed these findings.

[0364] Increased CD11b was observed in vaccinated KO mice after heterosubtypic challenge 低 / -DC frequency( Fig. 15C The gating strategy for DCs also showed that these were classical DCs (cDCs). Non-lymphoid cDCs have two major subsets: CD103 + CD11b - cDC and CD103 - CD11b + Therefore, in this study, CD11b 低 / - DCs are considered to be CD103 + cDC (Condon et al., J. Leukoc. Biol., 90:883-95 (2011); Desch et al., Immunologic Research, 55:178-186 (2013); Heath and Carbone, Nat. Immunol., 10:1237 (2009); Merad et al., Annual Rev. Immunol., 31:563-604 (2013)). CD11b 高 cDCs and CD11b 低 / - cDCs have different functions. In short, lung CD11b 高 cDCs are potent inducers of Th2 and Th17 responses to extracellular pathogens (Mesnil et al., PLoS One, 7: e53242 (2012); Plantinga et al., Immunity, 38: 322-335 (2013)). +cDCs sensitized CD4 + T cells cross-present soluble and apoptotic cell-associated antigens to CD8 + T cells to promote Th1 response (Desch et al., J. Exp. Med., 208: 1789-1797 (2011); Furuhashi et al., Am. J. Respir. Cell Mol. Biol., 46: 165-72 (2012)). In addition, CD11b 低 / - cDC plays a key role in influenza virus clearance (GeurtsvanKessel et al., J.Exp.Med., 205: 1621-34 (2008)). This cell population acquires influenza antigens by phagocytosis of infected cells rather than through infection, and it can promote the lung homing ability of differentiated T cells to lymph nodes (GeurtsvanKessel et al., J.Exp.Med., 205: 1621-34 (2008); Kim et al., Immunity, 40: 400-13 (2014); Helft et al., J.Clin.Invest., 122: 4037-47 (2012)). NAGT enhances CD11b 低 / - The ability of cDCs to respond may therefore be beneficial in resisting influenza virus infection. 低 / Further characterization of the -cDC population may help explain the heterosubtypic protection induced by NAGT mutants.

[0365] Example 4. Enhanced heterosubtypic protection involves anti-α-Gal antibodies in KO mice.

[0366] Materials and methods

[0367] To demonstrate that the enhanced vaccine-induced protection in KO mice was due to recognition of the α-Gal epitope by anti-α-Gal antibodies, KO mice with or without prior injection of rabbit RBCs were vaccinated and challenged using the same protocol as used for the H3 model described above.

[0368] result

[0369] Mice not treated with RBC had no detectable levels of anti-α-Gal antibodies prior to vaccination and viral challenge (Table 9). In the absence of anti-α-Gal antibodies in vivo, the enhanced heterosubtypic protection of NAGT mutants was abolished ( Fig. 20A , KO NR group). The survival rate of this vaccinated group (40%) was similar to that observed in vaccinated WT mice (38%) ( Fig.15A In addition, this group of mice had more severe weight loss than the fully protected group ( Fig. 20B). These results indicate that α-1,3-GT KO mice and WT mice are similarly susceptible to influenza virus infection in the absence of prior injection of rabbit RBCs. These results also confirm that the enhanced protection of NAGT mutants depends on the presence of anti-α-Gal antibodies in vivo.

[0370] Table 9. Anti-α-Gal antibody titers in mice before vaccination and virus challenge a .

[0371]

[0372] a The serum anti-α-Gal antibody titers of these mouse groups were determined by ELISA.

[0373] b KO mice were vaccinated at 8 weeks of age with (KO) or without (KO NR) rabbit RBC stimulation.

[0374] c Mock-treated KO mice (KO(-)NR) were used as controls.

[0375] Attenuated influenza viruses containing the α-1,3-GT gene to express the α-Gal epitope in infected cells were generated. Expression of viral RNA and viral proteins in infected cells after vaccination can further enhance vaccine-induced immune responses. The results suggest that this approach is a promising vaccine strategy to induce heterosubtypic protection against influenza.

[0376] The use of NAGT mutants as LAIVs has several advantages over enzymatic labeling of antigens with α-Gal epitopes in vitro to prepare experimental inactivated vaccines. First, expression of α-Gal epitopes by infected cells can avoid the above-mentioned enzymatic labeling and its downstream purification steps. Second, administration of NAGT mutants via the respiratory route can induce mucosal immune responses. Third, naturally expressed anti-α-Gal antibodies can safely control the extent of NAGT virus replication in humans. Fourth, infected cells labeled with anti-α-Gal antibodies can trigger antibody-mediated cellular immune responses. Finally, the diverse viral antigens expressed by infected cells are more easily taken up by professional APCs for antigen presentation, thereby enhancing downstream vaccine-induced responses against a broad spectrum of viral proteins.

[0377] Other references:

[0378] Wu et al., Clin. Infect. Dis., 51: 1184-91 (2010).

[0379] Tam et al., Paediatr. Child Health, 23:31-34 (2018).

[0380] Misharin et al., Am. J. Respir. Cell Mol. Biol., 49:503-10 (2013).

[0381] It should be understood that the disclosed methods and compositions are not limited to the specific methods, protocols and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0382] Disclosed are materials, compositions and components that can be used, can be used in combination, can be used to prepare, or are products of disclosed methods and compositions. These materials and other materials are disclosed herein, and it should be understood that when the combination, subset, interaction, group, etc. of these materials are disclosed, although there may be no specific reference to each different individual and collective combination and arrangement of these compounds that is explicitly disclosed, each is specifically considered and described herein. For example, if a virus is disclosed and discussed, and a variety of modifications that can be made to a variety of viruses including the virus are discussed, then unless otherwise specifically indicated to the contrary, each combination and arrangement of the virus and possible modifications are specifically considered. Therefore, if an example of a class of molecules A, B and C and a class of molecules D, E and F and a combination molecule AD are disclosed, then each molecule is considered individually and collectively even if each molecule is not listed separately. Therefore, in this example, each of the combinations of AE, AF, BD, BE, BF, CD, CE and CF is specifically considered and should be considered to be disclosed from the disclosure of A, B and C; D, E and F; and the example combination AD. Similarly, any subset or combination of these is also specifically considered and disclosed. Thus, for example, the subgroups of AE, BF, and CE are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the example combination AD. Furthermore, each of the materials, compositions, components, etc. contemplated and disclosed above may also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed by any specific embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically contemplated and should be considered disclosed.

[0383] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of such viruses and reference to "the virus" is a reference to one or more viruses and equivalents thereof known to those skilled in the art, and so forth.

[0384] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "include" and "comprising", mean "including but not limited to", and are not intended to exclude, for example, other additives, ingredients, integers or steps.

[0385] “Optional” or “optionally” means that the subsequently described event, circumstance or material may or may not occur or exist, and that the description includes instances where the event, circumstance or material occurs or exists and instances where it does not.

[0386] Unless the context clearly indicates otherwise, use of the word "may" indicates an option or capability for the object or condition being referred to. Generally, use of "may" in this manner means stating an option or capability affirmatively, while also leaving open the possibility that the option or capability may not exist in other forms or embodiments of the object or condition being referred to. Unless the context clearly indicates otherwise, use of the word "might" indicates an option or capability for the object or condition being referred to. Generally, use of "might" in this manner means stating an option or capability affirmatively, while also leaving open the possibility that the option or capability may not exist in other forms or embodiments of the object or condition being referred to. Unless the context clearly indicates otherwise, "might" as used herein does not refer to an unknown or questionable characteristic of the object or condition.

[0387] Range can be expressed as from "about" a specific value, and / or to "about" another specific value in this article.When expressing such a range, unless the context clearly states otherwise, it is also specifically considered and considered to disclose a range from a specific value and / or to another specific value.Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment specifically considered, unless the context clearly states otherwise, the embodiment should be considered to be disclosed.It will be further understood that the endpoints of each range are significantly related to another endpoint and independent of another endpoint, unless the context clearly states otherwise.It should be understood that all single values ​​and sub-ranges of values ​​included in the clearly disclosed range are also specifically considered and should be considered to be disclosed, unless the context clearly states otherwise.Finally, it should be understood that all ranges refer to the ranges listed as ranges and from and including the first endpoint to and including the set of each number of the second endpoint.In the latter case, it should be understood that any single number can be selected as a form of the quantity, value or feature referred to by the range. In this way, a range describes a set of numbers or values ​​from (including) a first endpoint to (including) a second endpoint, from which a single member of the set (i.e., a single number) can be selected as the quantity, value or characteristic referred to by the range. The above applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular case.

[0388] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present methods and compositions, particularly useful methods, devices and materials are as described. The publications cited herein and the materials cited therein are specifically incorporated by reference herein. Nothing herein should be construed as admitting that the present invention is not entitled to such disclosures due to prior inventions. It is not admitted that any reference constitutes prior art. The discussion of the references illustrates the author's claims, and the applicant reserves the right to question the accuracy and relevance of the cited documents. It will be clearly understood that although many publications are mentioned herein, such reference does not constitute an admission that any of these documents constitute part of the common general knowledge in the art.

[0389] Although the descriptions of materials, compositions, ingredients, steps, techniques, etc. may include many options and alternatives, this should not be interpreted as nor is it an admission that such options and alternatives are equivalent to one another, especially obvious choices. Thus, for example, a list of different viruses does not indicate that the listed viruses are obvious from one another, nor is it an admission of equivalence or obviousness.

[0390] Each virus disclosed herein is intended and should be deemed to be specifically disclosed herein. In addition, each subgroup that can be identified in the present disclosure is intended and should be deemed to be specifically disclosed herein. Therefore, it is specifically contemplated that any virus or subgroup of viruses can be specifically included or excluded from use, or included or excluded from a virus list.

[0391] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A virus comprising a heterologous nucleic acid fragment encoding α-1,3-galactosyltransferase (α-1,3-GT), wherein when the virus infects a host cell, the nucleic acid fragment expresses α-1,3-GT, and at least one protein exposed on the surface of the host cell comprises α-1,3-galactose; wherein the virus is a PR8 influenza virus, and the nucleic acid fragment is introduced in-frame into the reading frame of the neuraminidase (NA) coding region of the virus; the nucleic acid fragment is introduced immediately before the stop codon of the reading frame of the NA coding region of the virus; the virus further comprises a protease cleavage site encoded between the nucleic acid fragment and the NA coding region of the virus; the protease cleavage site is a 2A self-cleaving peptide.

2. The virus of claim 1, wherein at least one viral protein expressed by the virus when the virus infects the host cell comprises α-1,3-galactose.

3. The virus of claim 1 or 2, wherein the virus is live and attenuated.

4. The virus of claim 1 or 2, wherein the 2A self-cleaving peptide is derived from porcine Teshchinavirus-1.

5. The virus of claim 1 or 2, wherein the 2A self-cleaving peptide is flanked by short peptide linkers.

6. The virus of claim 5, wherein the short peptide linker is GSG.

7. The virus of claim 1 or 2, wherein the α-1,3-GT is mammalian α-1,3-GT.

8. The virus of claim 1 or 2, wherein the α-1,3-GT is mouse α-1,3-GT.

9. The virus of claim 1 or 2, wherein the virus is attenuated by serial passage in cultured cells, serial passage in a heterologous host animal, gene deletion in the virus, site-directed mutagenesis of the virus, altering the codon usage of the virus, selecting cold-adapted mutants, using a related virus from a heterologous host species, using a naturally occurring attenuated virus strain, or a combination thereof.

10. The virus of claim 9, wherein the virus is attenuated by serial passaging in cultured cells, wherein the cultured cells are Madin-Darby canine kidney (MDCK) cells.

11. A vaccine comprising the virus of any one of claims 1 to 10, wherein the virus is live and attenuated.

12. The viral vaccine of claim 11, wherein the virus expresses one or more viral proteins, the one or more viral proteins comprising α-1,3-galactose.

13. Use of the virus according to any one of claims 1 to 10 in the preparation of a vaccine.

14. The use of claim 13, wherein the vaccine is administered intranasally, intrapulmonary, orally, subcutaneously, intramuscularly, intradermally or intraperitoneally.

15. A method of preparing the vaccine of claim 12, comprising infecting cells with the virus, thereby producing the one or more viral proteins comprising alpha-1,3-galactose.

16. The method of claim 15, further comprising purifying the one or more viral proteins.

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