Mammalian-specific replication-deficient arbovirus

Abstract

It provides a mutant arbovirus and its use as an immunogen. [Solution] Arboviruses have modified furin cleavage sites that result in enhanced cleavage of precursor polyproteins such as prE2 or prM. Dengue virus particles may have amino acid modifications within amino acids 80-130 of prM. Zika virus particles may have modifications in and / or around the furin cleavage site. The virus can be produced in insect cells. The virus does not form progeny viruses in mammalian cells.

Description

[Technical Field]

[0001]

[0001] The present invention generally relates to arboviruses, which are a small group of viruses having unique and unparalleled properties that replicate in insect cells and human cells. The present invention relates to mutant arboviruses and their use as immunogens. In view of this, the present invention relates to a modified arbovirus that no longer regenerates in mammalian host cells but continues to replicate in insect cells and can be harvested therefrom, and continues to express and present arbovirus antigens in and to the mammalian immune system.

[0002]

[0002] The present invention relates to the arbovirus group of viruses. The basic characteristic of arboviruses is their replication in mammalian and insect cells. Many arboviruses are transmitted to mammals by ticks or mosquitoes. Under one classification scheme, arboviruses include the genera Flavivirus, Alphavirus, and Orthobunyavirus. Under another classification scheme, arboviruses include the families Bunyaviridae, Flaviviridae, Reoviridae, and Togaviridae. Examples of arboviruses include African swine fever virus, tick-borne encephalitis virus, Rift Valley fever virus, Colorado tick-borne fever virus, equine encephalopathy virus, Chikungunya virus, dengue fever virus (DV), Zika virus (ZV), and West Nile virus. [Background technology]

[0003]

[0003] Flaviviruses have been studied in part due to their connection to human pathology. See, for example, Gubler & Kuno (eds.): Dengue and Dengue Hemorrhagic Fever. Wallingford, CAB International, 1997; and Porterfield: Exotic Viral Infections. Chapman and Hall Medical, London, 1995.

[0004]

[0004] The flavivirus genome consists of a single linear, single-stranded +sense RNA. The +strand RNA infects suitable host cells. The entire genome can range from 10 to 11 kb. There is no 3' polyadenylation. The 5' end has a methylated cap.

[0005]

[0005] The flavivirus genome does not contain an internal ribosome entry site (IRES) that provides a translation initiation site to the host ribosome. Instead, the flavivirus uses ribosome scanning to initiate protein synthesis.

[0006]

[0006] Flavivirus virions can be spheres with a diameter of 40-65 nm. Beneath the lipid envelope is an icosahedral capsid coat with a diameter of approximately 25-30 nm.

[0007] Dengue virus (types 1-5), yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and West Nile virus are significant causative factors of morbidity and mortality in humans.

[0007]

[0008] For example, the yellow fever virus can cause an epidemic. In the first cycle, the virus is transmitted by Aedes africanus and other Aedes mosquitoes (Africa) or Hemogogus mosquitoes (America); monkeys act as carriers, and generally, human infected individuals are those who venture deep into forests and jungles and are exposed to these vectors. In the second cycle, Aedes aegypti, an inland mosquito that lives in close contact with humans, can directly transmit the virus to humans, the sole host during the cycle.

[0008]

[0009] Flaviviruses cause other diseases, such as Murray Valley encephalitis, Rocio and Powassan encephalitis, as well as West Nile fever and Zika fever, which have been observed more recently in the United States.

[0009]

[0010] Several flaviviruses are commercially important veterinary pathogens, including the jumping disease virus that causes neurological disorders in sheep, the West Nile virus that causes encephalitis in horses, and the Japanese encephalitis virus, which also causes encephalitis in horses and stillbirths in pigs.

[0010]

[0011] Given the urgent need for arbovirus treatment, a robust method is required to produce safe and effective arbovirus treatment compositions. Theoretically, attenuated live vaccines induce the most effective long-term specific immunity, while inactivated viral vaccines, including recombinant subunit vaccines, offer a higher level of safety. An ideal vaccine would be one that combines the efficacy of live vaccines with the safety of subunit vaccines.

[0011]

[0012] These goals were achieved in developing a scalable method for producing mammalian-specific replication-restricted arboviruses. The target replication-restricted viruses are present in and express arbovirus antigens, inducing an immune response to arbovirus antigens (1 or more) in infected mammals, but do not produce progeny viruses in mammalian cells. Therefore, infected cells do not contribute to radially spreading cell infection and lesions in the mammalian host. The replication of the target replication-restricted viruses is not inhibited in insect cells. Thus, the target dysfunctional viruses can be economically produced in insect cell lines. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Dengue and Dengue Hemorrhagic Fever.Wallingford,CAB International,1997 [Non-Patent Document 2] Exotic Viral Infections.Chapman and Hall Medical,London,1995 [Overview of the project]

[0013]

[0013] The present invention relates to materials and methods for producing replication-restricted arbovirus particles that continue to replicate in insect cells but no longer replicate (or only replicate at a low level) in mammalian cells (e.g., human cells), and therefore infected mammalian cells do not produce progeny viruses. The target arbovirus infects host mammalian cells, replicates in those host mammalian cells, and can produce arbovirus proteins recognized by the mammalian host immune system, for example, in cellular and / or humoral responses, but does not produce or release progeny arbovirus particles. The target deficient virus contains all the genes necessary to ensure viral genome replication and arbovirus protein production, which can be released from infected cells or expressed on the surface of host cells, but does not produce progeny viruses from infected host mammalian cells. Therefore, the target arbovirus particles can be as immunogenic as wild-type viruses, but they should contribute, if any, to disease or lesions in the infected host, if not to a significant extent. This is because the target modified arbovirus exhibits reduced replication, if any, in mammalian cells.

[0014]

[0014] The target replication-restricted arbovirus does not readily reproduce in mammalian or human cells, and releases a reduction in viral particles of 4 log or less, 5 log or less, 6 log or less, 7 log or less, 8 log or less, or 9 log or less, compared to the level or amount of progeny viruses produced in cells of the same type infected with the wild-type arbovirus of the same species or in the wild-type arbovirus of the sample before the replication-restricted treatment, or a state of no progeny arbovirus at all.

[0015]

[0015] On the other hand, the growth-restricted or growth-deficient arbovirus of interest readily propagates in insect cells such as arthropod cells to produce similar progeny arboviruses. The level of progeny arbovirus produced by the insect cells can be approximately equal to or exceed the level or amount of progeny virus produced by an appropriate control wild-type arbovirus from a sample used for processing to confer growth restriction in the same species or the same insect cells.

[0016]

[0016] In an embodiment, the defective arbovirus of interest comprises enhanced furin activity (that is, can be cleaved at a faster rate, can be cleaved more efficiently, or both, as compared to a wild-type arbovirus of the same species or a wild-type arbovirus from a sample before processing to confer growth restriction). Furin cleavage of the precursor polyprotein can be essential for the maturation of arbovirus progeny particles. In some arboviruses, furin cleavage occurs at the junction of pr and M. Accordingly, the defective arbovirus of interest comprises enhanced furin activity in, at or around the prM protein or polypeptide. In some arboviruses, furin cleavage occurs at the junction of E3 and E2. Accordingly, the defective arbovirus of interest comprises enhanced furin activity in, at or around the precursor E2 protein or polypeptide.

[0017]

[0017] The growth-restricted arbovirus of interest comprises: a modification in a furin consensus tetrapeptide, which can be, for example, at the junction of pr and M; a modification upstream of the consensus tetrapeptide (for example, toward the amino terminus in the pr polypeptide); a modification downstream of the consensus tetrapeptide (for example, toward the carboxy terminus in the M protein); or a combination thereof.

[0018]

[0018] In an embodiment, the arbovirus is dengue virus (DV) comprising a prM polyprotein.

[0019] In one embodiment, one or more amino acid substitutions at and / or around the furin cleavage site between pr and M result in a virus with enhanced furin cleavage of prM; it reproduces in insect cells; and does not reproduce in mammalian or human cells.

[0019]

[0020] In one embodiment, enhanced furin activity in DV is obtained by one or more of the following: 1) modification upstream (amino-terminal direction) of the furin recognition and cleavage site of the prM polypeptide of DV, i.e., in an 8-amino acid stretch of amino acids 80-87 of prM; 2) modification at the tetrapeptide furin recognition site; 3) modification downstream (carboxy-terminal direction) of the furin recognition and cleavage site, i.e., in a 39-amino acid stretch of amino acids 92-130 of prM; 4) modification upstream of amino acid 80; and 5) modification downstream of amino acid 130. Any combination of the above may exist, for example, 1), 2), and 3). Modifications 1), 2), and 3) may exist.

[0020]

[0021] In one embodiment, the DV fulin cleavage site in the prM protein, NH2-(88)Arg-Glu-Lys-Arg-COOH / (SEQ ID NO: 1) [in this respect, cleavage occurs after the Lys-Arg residue (indicated by " / " in the above cleavage recognition site)], is modified at the Glu site to enhance fulin cleavage.

[0021]

[0022] In one embodiment, Glu is substituted with any amino acid.

[0023] In one embodiment, the amino acid substituting Glu is a non-acidic, non-neutral amino acid such as Gln, Asn, Gly, Lys, Arg, His, Thr, Ser, Tyr, Met, or Cys. In one embodiment, the substituted amino acid does not contain sulfur. In one embodiment, the substituted amino acid does not contain a hydroxyl group in the R-group side chain. In one embodiment, the substituted amino acid is a basic amino acid. In one embodiment, the substituted amino acid is Lys, Arg, or His. In one embodiment, the substituted amino acid is Arg.

[0022]

[0024] In one embodiment, the target deletion DV includes one or more modifications to the polypeptide sequence in the eight amino acids upstream of SEQ ID NO: 1, for example, upstream of the Arg-Glu residue. In another embodiment, the target deletion DV includes one or more modifications to the polypeptide sequence in the 39 amino acids downstream of SEQ ID NO: 1, for example, downstream from the Lys-Arg residue. The modifications may be at or within a furin recognition or cleavage site. The modifications may be present in the M protein. The modifications may be located within the first 39 amino-terminal amino acids of a membrane protein. The modifications may be present in the pr polypeptide. The modifications may be located within the last eight carboxy-terminal amino acids of the pr polypeptide.

[0023]

[0025] Such modifications, or combinations of modifications, reduce or eliminate the replication, maturation, and release of DV from infected mammalian cells. Thus, infected mammalian cells contain and present DV antigens to the mammalian host immune system, but do not release infectious progeny DV particles.

[0024]

[0026] However, such modifications to the furin cleavage site or its surrounding area do not negate the replication of the target DV in insect cells, and consequently provide methods and means for producing DV particles containing the modified furin site disclosed herein for use in infecting but not replicating in mammalian cells. Reproduction in insect cells is robust and provides a high viral yield, and the growth or production of DV in insect cells can be scaled to produce viral particles in greater volume, quantity, and response.

[0025]

[0027] In one embodiment, the arbovirus is a Zika virus (ZV) containing the prM polyprotein.

[0028] The furin cleavage site of ZV is located after amino acid 93 of prM. In one embodiment, the prM sequence His-His-Lys-Lys-Gly-Glu-Ala-Arg-Arg-Ser-Arg-Arg / (SEQ ID NO: 2), which is upstream of and encompasses the ZV furin recognition site, is modified at the serine residue of the tetrapeptide furin recognition site (cleavage occurs after the fourth Arg in the above sequence), resulting in a sequence of five arginine residues to enhance furin cleavage. In one embodiment, the glutamic acid residue in the region can be modified to histidine to enhance furin cleavage. Other modifications can be made to the above sequence of residues. Modifications can be made upstream of amino acid 82 and / or downstream of amino acid 93.

[0026]

[0029] Enhancement of furin activity in ZV can be achieved by one or more of the following: (1) modification of one or more amino acids upstream of the ZV furin cleavage site, for example, in an eight-residue sequence starting from the 82nd amino acid residue from the N-terminus of prM (i.e., amino acid residues 82-89 of prM), but also upstream of amino acid 82; (2) modification of the ZV furin tetrapeptide cleavage site; and (3) modification of one or more amino acid residues downstream of the furin cleavage site, i.e., modification of one or more amino acid residues downstream of amino acid 94, for example, in a 26-residue stretch of amino acids downstream of the tetrapeptide. Any combination of (1), (2), and (3) is possible.

[0027]

[0030] The proliferation of the target ZV in mammalian cells is minimized or virtually absent, while the proliferation of its dysfunctional ZV in insect cells remains virtually unaffected.

[0031] Further features and advantages of the present invention will be described in the following detailed description of the invention and in the drawings, and will become apparent therefrom. [Brief explanation of the drawing]

[0028] [Figure 1]

[0032] Figure 1 shows a survival graph including data obtained from mice given either phosphate-buffered saline (PBS) as a control or the target DV mutant (D2-89R) prior to attack with wild-type DV. [Figure 2]

[0033] Figure 2 shows a survival graph including data obtained from mice given either phosphate-buffered saline (PBS) as a control or the target SV mutant (M2) prior to attack with wild-type SV. [Figure 3]

[0034] Figure 3 shows a safety graph including data obtained from mice given either the wild-type SV or the SV M2 mutant. [Modes for carrying out the invention]

[0029]

[0035] As used herein, “dysfunction” or its grammatical form indicates an enhancement of a characteristic or function resulting from a change or modification, compared to the presence of a level or characteristic of function or function observed before or without the change or modification. For example, a modified and dysfunctional enzyme site may be an enzyme site that is always recognized by the enzyme or cleaved by a cognate enzyme at a faster rate, more efficiently, etc., resulting in an increase in the level or amount of the enzyme reaction product. For example, in one embodiment, a dysfunctional furin cleavage site is cleaved at an enhanced rate or value, or at a rate or value exceeding the rate or value observed for a non-dysfunctional furin cleavage site. Thus, a decrease in the amount or level of prM in infected cells is the result. A synonym for dysfunction is “deficiency” or its grammatical form.

[0030]

[0036] "Enhanced" or its grammatical variation is a metric or level that exceeds the baseline or reference level or metric. The baseline level can be determined by sampling several units and taking the mean, or by obtaining a population mean derived from the literature. In some embodiments, the enhanced level is any level higher than the level found in the control sample. Thus, in bioassays, chemical assays, etc., two samples can be tested in parallel, for example, an experimental sample suspected of having an enhanced metric and a control which may be a sample from a known wild type representative of the population, such as a standard, unmodified, and unmutated sample, and a higher metric, e.g., a larger quantity of product, faster kinetics, larger product, etc., will be revealed in the experimental sample. In some embodiments, the baseline level is that found in the wild-type unit. In some embodiments, enhancement includes an increase in the level of enzyme activity. In some embodiments, enhancement includes an increase in the level of catalytic activity. In some embodiments, enhancement includes an increase in the level of solubility. In some embodiments, enhancement includes an increase in the level of polypeptide cleavage by furin. In one embodiment, enhanced furin activity can be identified by comparing the amount of prM in samples from cells infected with the target mutant and samples from cells infected with the wild-type virus. In another embodiment, enhanced furin activity can be identified by a decrease in the level of prM or an increase in the level of pr or M in a comparison between the mutant and the wild-type.

[0031]

[0037] "Modification," or its grammatical form, is a change or denaturation of the wild-type amino acid sequence at one or more residues. Therefore, a modified polypeptide may contain alleles. The change can be an amino acid substitution, deletion, or insertion that includes two or more amino acids at a site in the wild-type sequence. Modification results in a dysfunctional virus of the target.

[0032]

[0038] The term "stretch," or its grammatical form, refers to a sequence of nucleotides in nucleic acids or a sequence of amino acids in proteins. A sequence of nucleotides in nucleic acids (nucleotide stretches) is polymerized as linear oligonucleotides. A sequence of amino acids in proteins (amino acid stretches) is polymerized as linear oligopeptides.

[0033]

[0039] "Reproduction" or its grammatical form includes the process by which a virus infects a compatible host cell; replicates within the infected cell to produce progeny viruses; and the infected cell releases the progeny viruses into the extracellular space. Synonyms include "regeneration," "replication," and "proliferation."

[0034]

[0040] "Immunogenicity" or its grammatical form includes generating or inducing an immune response in a host. Therefore, immunogenicity, in embodiments, includes having an epitope or determinant. In embodiments, for example, an immunogenic composition is a composition having an epitope or determinant that, when introduced into a host, induces an immune response by the host immune system to that epitope or determinant. The immune response may, to some extent, “remove” the immunogenic composition from the host, and in this regard, “remove” may include actually destroying the composition, i.e., altering or digesting the composition; isolating the composition so that it is contained within a structure and becomes inactive; detoxifying the composition; neutralizing the composition; biologically inactivating the composition; making the composition safe for and in the host; and so on. Immunogenicity does not mean or guarantee immunodefence in any one exposed host. The immune response may be humoral, cellular, etc., or a combination thereof.

[0035]

[0041] The terms “does not replicate in human or mammalian cells,” “does not reproduce in human or mammalian cells,” “does not grow in human or mammalian cells,” “does not proliferate in human or mammalian cells,” “does not regenerate in human or mammalian cells,” “reproduction is restricted in human or mammalian cells,” “reproduction is restricted,” “reproduction is defective,” and their equivalents, grammatical forms, etc., are synonymous terms or phrases as described herein, and include human or mammalian cells infected with the mutant virus of the subject which produce or do not produce progeny arboviruses of 4 log or less, 5 log or less, 6 log or less, 7 log or less, 8 log or less, or 9 log or less, compared to the level or amount of progeny virus produced by the same type of cells infected with the same species, strain, lineage, etc., of wild-type arbovirus; or by similar cells infected with the dysfunctional virus as described herein; or by appropriate and acceptable negative controls.

[0036]

[0042] The terms “replicate in insect cells,” “reproduce in insect cells,” “grow in insect cells,” “proliferate in insect cells,” “regenerate in insect cells,” “unrestricted growth in insect cells,” “not growth-deficient,” “unrestricted growth,” and their equivalents, grammatical forms, etc., are synonyms or synonymous terms or phrases as described herein, and include insect cells infected with the mutant virus of the subject that produce approximately the same amount or more of progeny arboviruses compared to the level or quantity of progeny viruses produced by insect cells of the same type infected with wild-type arbovirus of the same species, strain, lineage, etc.; by similar cells infected with a dysfunctional virus as described herein; or by a suitable and acceptable negative control.

[0037]

[0043] "Approximately" is an approximation to a value such that, for example, with respect to a certain value, there exists a variable reflected in the error or deviation that provides a range where the limits of the range are 10% smaller than the value, encompass the value, and are 10% larger than the value. Thus, by citing approximately 50 as used herein, it is understood that the value may be in the range of 45 to 55. Synonymous terms include "essentially" and "substantially."

[0038]

[0044] "Scalable" or its grammatical form refers to a method that is carried out on a bench or laboratory scale and can be translated to a larger scale or presentation, such as when carried out for the commercial manufacture of food, beverages, consumer goods, industrial chemicals, etc., where the reaction vessel can have a capacity of several hundred or several thousand liters. Therefore, in the context of the present invention, insect cell culture can be carried out in volumes, quantities, or reactions of 10 liters, 100 liters, 1000 liters, or more.

[0039]

[0045] The term “wild-type” or its grammatical form generally refers to the most common or dominant form, trait, gene, protein, phenotype, etc., in a population of spontaneously occurring arboviruses. In aspects, synonyms are “standard” or “reference” arboviruses. Population traits can be determined by more than one allele at a single locus or by polygenes. Therefore, sets of alleles or polygenes are considered equivalent herein, as they produce the same or substantially the same trait. Wild-type arboviruses do not contain dysfunctional furin sites and produce enhanced furin cleavage, as is present in the modified arbovirus of interest. In aspects herein, comparisons are made to demonstrate the characteristics of the modified arbovirus of interest to normal or wild-type arboviruses that do not contain dysfunctional furin cleavage sites. In aspects, the arbovirus of interest has substantially the same characteristics as normal or reference arboviruses, such as proliferation in insect cells. For example, in one embodiment, the reference DV is serotype 2, New Guinea strain available from ATCC (accession number VR-1584). Therefore, the DV prM and its amino acid numbering as described herein can be compared with the prM of VR-1584; or with the prM and its amino acid numbering as the wild-type form of mutant DV. In another embodiment, the reference ZV is available from ATCC under accession number VR-1838. Therefore, the ZV prM and its amino acid numbering as described herein can be compared with the prM of VR-1838; or with those of the wild-type form of mutant ZV. Similar to wild-type arboviruses, the arboviruses in question reproduce in insect cells. However, with respect to amino acid numbering as described herein, due to inherent population variation, polymorphism, or variability, the numbering in one strain, lineage, or species may not directly correspond to the numbering in another strain, lineage, or species. Therefore, the numbering as described herein is not absolute with respect to the position of any single polypeptide, cleavage site, etc. This is because numbering can vary even within the range of lineages, strains, and species due to naturally occurring variations.Therefore, the numbering used herein is specific to particular cells, viruses, etc., and should not be interpreted as absolutely representative of all arboviruses that have the same numbering as presented herein. Instead, the various features provide markers to identify sites and enable those skilled in the art to carry out the claimed subject matter using any arbovirus as a design choice. For example, the frin consensus tetrapeptide at the pr-M junction, downstream or carboxyl terminus of pr, and upstream or amino terminus of M are markers that can be used to adapt protein manipulation and modification. Thus, in one embodiment, the numbering takes into account the number of amino acids in the tetrapeptide to determine whether or not there is variability in the size of pr and M, and if so, to provide a suitable reference frame by making adjustments or correlations between those taught herein and those used by those skilled in the art in different arboviruses.

[0040]

[0046] The “prM” protein or polypeptide of DV is 166 amino acids long in DV serotypes 1-4 and is cleaved by furin after amino acid 91 to produce the pr polypeptide and M protein. The wild-type prM amino acid sequence of the DV2 NGC strain is FHLTTRNGEP HMIVSRQEKG KSLLFKTEDG VNMCTLMAMD LGELCEDTIT YNCPLLRQNE PEDIDCWCNS TSTWVTYGTC TTTGEHRREK RSVALVPHVG MGLETRTETW MSSEGAWKHA QRIETWILRH PGFTIMAAIL AYTIGTTYFQ RVLIFILLTA VAPSMT (SEQ ID NO: 8). As is known, in natural mutations, the wild-type strain can be found to have one or more amino acid changes from the sequence provided in SEQ ID NO: 8 without disruption or enhancement of furin-mediated prM cleavage and without reduced reproduction in mammalian cells. The modified sequence number 8 in question shows enhanced Furin cleavage and viral replication in insect cells, but little to no viral replication in mammalian cells.

[0041]

[0047] The ZV "prM" protein or polypeptide is 168 amino acids long and is cleaved by furin after amino acid 93 to produce the pr polypeptide and M protein. The wild-type prM amino acid sequence is AEITRRGSAY YMYLDRSDAG KAISFATTLG VNKCHVQIMD LGHMCDATMS YECPMLDEGV EPDDVDCWCN TTSTWVVYGT CHHKKGEARR SRRAVTLPSH STRKLQTRSQ TWLESREYTK HLIKVENWIF RNPGFALVAV AIAWLLGSST SQKVIYLVMI LLIAPAYS (SEQ ID NO: 9). As is known, in natural mutations, the wild-type strain can be found to have one or more amino acid changes from the sequence provided in SEQ ID NO: 9 without disruption or enhancement of furin-mediated prM cleavage and without reduced reproduction in mammalian cells. The modified sequence number 9 in question shows enhanced Furin cleavage and viral replication in insect cells, but not in mammalian cells.

[0042]

[0048] As used herein, “furin cleavage site,” “furin recognition site,” and “furin recognition and cleavage site” are generally interchangeable and equivalent, as furin is known to recognize the consensus tetrapeptide Arg-X-Lys / Arg-Arg (SEQ ID NO: 16) [wherein X is any amino acid (and the upstream and / or downstream sequences of the tetrapeptide may include a furin cleavage site)] and cleave the peptide bond either downstream or at the carboxyl terminal of the tetrapeptide. Generally, in arboviruses, the furin cleavage site is located at the junction of two polypeptides in a precursor polyprotein such as prM and precursor E2 (prE2). In some arboviruses, the furin cleavage site is located at the pr-M junction. Other arboviruses with different genomic compositions do not contain prM; for example, in some alphaviruses, the furin cleavage site is located at the E3-E2 junction.

[0043]

[0049] Where used herein, “Xlog or less,” “Xlog or less,” or “Xlog or less,” where X is a rational number, refers to a logarithmic scale for describing the number of virus particles. As is well known, this scale is nonlinear and based on a base-10 number of digits. The logarithmic scale is used when the range of parameter values ​​is wide. Thus, 4log is 10 4 It reflects the difference between the two values, that is, one value is 10,000 units smaller or larger than the other value, and 5log = 10 5 This reflects the difference between two values, i.e., one value is 100,000 units smaller or larger than the other. By “lower,” “less than,” or “fewer,” phrases containing these words relate to quantities such as a lower value, a smaller number, or, in the context herein, to a smaller number of virus particles. Thus, “4log or less” means that the difference between two values ​​is 10 4 , 10 4.5 , 10 5 This means that the number of virus particles produced in one sample is 10,000 fewer, approximately 31,600 fewer, or approximately 100,000 fewer than in other samples, and in the context of this specification, this means that one sample contains 10,000 fewer virus particles than in other samples.

[0044]

[0050] The focus of this invention is to manipulate the arbovirus genome to produce arboviruses with mammalian cell-specific replication defects. In this regard, mammalian cells infected with such arboviruses do not release mature progeny viruses but continue to express arbovirus epitopes and determinants that can be recognized by the mammalian host, such as determinants expressed by M and E proteins. A cellular and / or humoral response to the arbovirus may occur in the mammalian host. The target arbovirus is essentially similar to the wild-type virus and therefore presents the mammalian host immune system with a region (universe) of arbovirus epitopes that can be found in arboviruses within the wild-type arbovirus. The target arbovirus is simply unable to replicate in host mammalian cells and therefore cannot contribute to infection of other host mammalian cells and to lesions or diseases in the host.

[0045]

[0051] Accordingly, the present invention relates to deficient or dysfunctional arboviruses that express, but not all, of the structural proteins, or at least the majority of polypeptides containing determinants or epitopes of antibodies or immune cells generated by an infected host against the arbovirus. For example, in the case of flaviviruses, the majority of the pr, M, and / or E proteins are preferably expressed because they contain the immunogenic sites of the wild-type virus. The C protein may be present, but less than the target of the host immune system.

[0046]

[0052] All known arboviruses require fulin cleavage of a precursor polyprotein for proper viral maturation and release from host cells, for example, cleavage of prE2 to E3 and E2 in some alphaviruses, and cleavage of prM to pr and M in some flaviviruses, as well as for proper viral maturation and final release of progeny viruses from infected cells.

[0047]

[0053] The target mutant arboviruses include, for example, modified or dysfunctional furin cleavage sites in prE2 or prM, and optionally, modifications upstream and / or downstream of, for example, furin consensus cleavage sites in prE2 or prM, resulting in, for example, enhanced furin cleavage in prE2 or prM, producing viruses that continue to replicate in insect cells but no longer replicate or only very poorly in mammalian cells (not to mention humans). Thus, for example, modifications in and / or around the prE2 or prM furin cleavage site in arboviruses result in replication-limited viruses that continue to replicate in insect cells but no longer replicate or hardly replicate in human or mammalian cells. In this regard, replication indicates that progeny viruses are released by infected cells.

[0048]

[0054] Generally, during the maturation of early flavivirus particles in the Golgi apparatus, immature particles present spikes containing trimers of prM and E proteins, which are rearranged to form a smooth surface on the particle during passage through the Golgi apparatus, possibly due to changes in pH. This rearrangement or transformation of the spikes exposes the furin cleavage sites of prM. prM is cleaved by furin, and some of the pr fragments remain attached to the particle, likely based on the pH in the Golgi apparatus. These pr fragments are thought to prevent membrane fusion. Once the early particles are released from the Golgi apparatus into the extracellular environment, pr is released from the early particles, forming mature particles that can fuse with the cell membrane for release from the cell. Some of the growing particles may not mature. Nevertheless, the viral proteins may bind to the intracellular membrane and be incorporated into the cell membrane, where the viral proteins are expressed on the cell surface. Viral particles and components can be released into the environment upon cell death and lysis.

[0049]

[0055] While not attempting to establish a theory, a mutant viral genome in question containing a modified frin cleavage site in prM, which enables enhanced prM cleavage, could potentially induce prM cleavage before growing viral particles enter the Golgi apparatus under different pH conditions. As a result, pr would not bind to the particle surface when the particle enters the Golgi. Therefore, without pr, which prevents membrane fusion, the viral particle would be prevented from properly maturing during passage through the Golgi and would be trapped within the Golgi. By prematurely cleaving prM before entering the Golgi, the arbovirus particles in question would not contain pr and therefore could not be properly processed in the Golgi. Consequently, the viral particles would not bind to and fuse with the Golgi membrane and would not be released from the Golgi membrane, and therefore could not be released from the cell.

[0050]

[0056] However, this process is not modified in insect cells, and the timing and / or potency of, for example, prE2 or prM cleavage are not related to, or do not eliminate, the maturation of appropriate arbovirus particles and their release from the host insect cell. Therefore, infected insect cells produce progeny arboviruses at levels equivalent to, the same as, or exceeding those found in insect cells infected with wild-type virus.

[0051]

[0057] The target arbovirus's furin consensus tetrapeptide (RXK / RR (SEQ ID NO: 16)) is located, for example, at the E3-E2 junction and the pr polypeptide-M protein junction. The amino acids of the tetrapeptide are modified by known methods, such as amino acid substitution or site-directed mutagenesis of the nucleic acid encoding those amino acids, for example, by constructing the tetrapeptide to contain Arg as the second or X site. The modified furin site is positioned, for example, in the full-length nucleic acid by homologous recombination using known methods. Nucleic acid manipulation can be performed on cDNA, which is then transcribed using methods known in the art to form viral RNA containing a modified furin cleavage coding sequence as taught herein. The viral nucleic acid can be packed into particles, for example, by electroporation of the viral RNA into a suitable host cell. Transfected cells are cultured, and viral particle production is monitored to obtain viral particles having RNA containing the modified furin cleavage site coding region. DNA, RNA, vectors, and host cells are well known, and several reagents are commercially available, for example, Polo et al., Zeng et al., Khromykh et al. J Virol 75(10)4633-4640,2001; Gehrke et al.,J Virol 77(6)8924-8933, 2003; and Shustov et al. See also al., J Virol 81(21)11737-11748, 2007. Subsequently, appropriate cells are infected with viral particles and cultured as known in the art. The production of progeny viruses is monitored using the methods taught herein to determine whether the modification affects viral replication in mammalian cells but not in insect cells, and to what extent reproduction is modified in the two types of cells.

[0052]

[0058] This invention relates in part to flaviviruses, some of which include prM polyproteins.

[0059] In this embodiment, the present invention relates to DV (for example, any one of serotypes 1 to 5).

[0053]

[0060] The DV genome consists of a single linear, single-stranded +sense RNA. The entire genome can range from 10 to 11 kb. There is no 3' polyadenylation. The 5' end has a methylation cap. The DV genome does not contain an internal ribosome entry site (IRES) that provides a translation initiation site to the host ribosome. Instead, DV uses ribosome scanning to initiate protein synthesis.

[0054]

[0061] Dengue fever virions are spheres with a diameter of 40–65 nm. Beneath the lipid envelope is an icosahedral capsid coat with a diameter of approximately 25–30 nm.

[0062] Dengue fever is an acute infectious disease characterized by biphasic fever, headache, pain in various parts of the body, collapse, rash, lymphadenopathy, and leukopenia (Holstead, 1980, Immunological parameters of togavirus disease syndromes, pp. 107-173, Schlesinger (ed.), “The Togaviruses”, Academic Press, Inc., New York; and Sabin, 1959, Dengue, pp. 361-373, Rivers & Horsfall (eds.), “Viral and Rickettsial Infections of Man”, JB Lippincott Co., Philadelphia). DV is transmitted by mosquitoes. Infection with one dengue serotype can provide lifelong immunity to that subtype but not cross-protective immunity to other serotypes.

[0055]

[0063] Dengue hemorrhagic fever (DHF) is a severe febrile illness characterized by impaired hemostasis and increased vascular permeability, and in some cases, can lead to hypovolemic shock syndrome or dengue shock syndrome (DSS) (WHO: 1975. Technical Guides for Diagnosis, Treatment Surveillance, Prevention and Control of Dengue Hemorrhagic Fever, Geneva, Switzerland). The mechanism of DHF / DSS can vary from case to case. Major contributing factors to DHF / DSS include viral toxicity, the patient's health status, and secondary infection with different serotypes of DV.

[0056]

[0064] Currently, there are five serotypes of DV: 1, 2, 3, 4, and 5. Immunity to one serotype generally does not confer immunity to another.

[0065] Inexplicably, humans do not always respond equally to individual serotypes. Therefore, individuals infected with at least two serotypes of DV appear to initiate an immune response to only one serotype. Furthermore, subsequent infections, generally with a different serotype than the primary or previous infection, but also when the initial or previous infection was benign, mild, or asymptomatic, can result in more severe illness and lesions.

[0057]

[0066] The causes of serotype dominance, selectivity, or interference are unknown. In situ, viral replication, proliferation, maturation, and release from host cells, or antigen presentation, can induce a host immune response to a particular serotype.

[0058]

[0067] In any case, unpredictable host responses, serotype interference, and asymptomatic infections present many obstacles to the development of effective multivalent vaccines.

[0068] Current polyvalent live vaccines lack the ability to provide equivalent immunity against all serotypes, even when the relative amounts of each serotype in the vaccine fluctuate in an attempt to compensate for a distorted immune response.

[0059]

[0069] In carrying out the subject of this study, various DV genomes were constructed using different amino acid substitutions at the Glu residues of the prM furin cleavage site; upstream of the consensus tetrapeptide, for example, the 8-amino acid upstream stretch adjacent to the consensus, i.e., amino acids 88-91; downstream of the consensus tetrapeptide, for example, the 39-amino acid downstream stretch adjacent to the consensus of amino acids 88-91, i.e., amino acids 92-130 (i.e., within amino acids 80-130 of prM), or in combination thereof. Amino acid changes (greater than 1) enhance the furin cleavage of prM. Upon introduction into mammalian host cells, replication and expression occurred with the production of viral particles. However, mature particles intended for extracellular migration were not obtained. Therefore, although the target particles infect mammalian cells and the mutant viral genome is replicated and expressed in the mammalian host cells, mature progeny viral particles are not released by the infected host mammalian or human cells.

[0060]

[0070] In this embodiment, the DV frintetrapeptide cleavage site in the prM protein, NH 2-(88)Arg-Glu-Lys-Arg-COOH(Sequence ID: 1)[Regarding this] The cleavage occurs downstream of the Lys-Arg residue, and this is modified at the Glu site to enhance furin cleavage.

[0061]

[0071] In one embodiment, Glu is substituted with any amino acid. The substituted amino acid can be a non-acidic, non-neutral amino acid such as Gln, Asn, Gly, Lys, Arg, His, Thr, Ser, Tyr, Met, or Cys. In one embodiment, the substituted amino acid does not contain sulfur. Therefore, the substituted amino acid is a non-acidic, non-neutral amino acid other than Met or Cys. In one embodiment, the substituted amino acid does not contain an R hydroxyl group. Therefore, in one embodiment, the substituted amino acid is not Tyr, Ser, or Thr. In one embodiment, the substituted amino acid is a basic amino acid. In one embodiment, the substituted amino acid is Lys, Arg, or His. In one embodiment, the substituted amino acid is Arg.

[0062]

[0072] In one embodiment, the amino acids upstream of the tetrapeptide consensus are modified. For example, the amino acids in the 8-amino acid stretch immediately upstream (or towards the amino terminus) of the consensus of the fulin cleavage site are modified to enhance the fulin cleavage of prM ("modified upstream site"). Thus, the modification can occur at amino acids 80-87 of the prM polypeptide.

[0063]

[0073] In one embodiment, the amino acids downstream of the tetrapeptide consensus are modified. For example, the amino acids in the 39-amino acid stretch immediately downstream (or carboxyl-terminal) of the consensus of the fulin cleavage site are modified to enhance the fulin cleavage of prM ("modified downstream site"). This modification can occur at amino acids 92-130 of the M polypeptide.

[0064]

[0074] The target DV includes at least one of the following: (1) a modified frintetrapeptide cleavage site; (2) a modified upstream site; and (3) a modified downstream site. The target DV may include any two combinations of (1), (2), and (3). The target DV may include all three modifications.

[0065]

[0075] In this embodiment, the arbovirus is a ZV containing the prM polyprotein.

[0076] The ZV genome consists of a single linear, single-stranded positive-sense RNA of 10,794 nucleotides (however, some isolates exhibit different sizes [e.g., Baronti et al., Genome Announc 2(3)1-2, 2014]). In addition to structural and non-structural genes, 3' and 5' non-coding ends are present.

[0066]

[0077] ZV exhibits a small amount of polymorphism. For example, at least two subtypes have been recognized: African lineage viruses and Asian / South American lineage viruses. The two lineages can be distinguished serologically, suggesting diversity in envelope proteins.

[0067]

[0078] Symptoms of Zika fever include fever, conjunctivitis, joint pain, headache, and maculopapular rash, and generally last less than 7 days. While initial infection is not considered fatal for normal adults, infection during pregnancy can cause malformations and abnormalities such as microcephaly in the developing embryo and / or fetus. Infection in adults is associated with Guillain-Barré syndrome.

[0068]

[0079] ZV is primarily transmitted by mosquitoes. Following a large-scale Zika outbreak in Brazil in 2015, ZV has spread through Latin America and the Caribbean, and is now even reaching the United States.

[0080] The life cycle of ZV begins with virion attachment to the host cell surface, followed by entry into the cell via receptor-mediated endocytosis. As is currently understood, acidification of the endosomal vesicle triggers conformational changes in the virion, fusion of the virus with the cell membrane, and degradation of the particle. Once the genome is released into the cytoplasm, the positive sense RNA is translated into a single polyprotein, which is then processed by viral and host proteases to produce 10 gene products: three structural proteins—core protein (C), premembrane protein (prM), and envelope protein (E)—as well as seven non-structural (NS) proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

[0069]

[0081] The furin cleavage site of ZV is located after amino acid 93 of prM. In one embodiment, the furin tetrapeptide cleavage site, His-His-Lys-Lys-Gly-Glu-Ala-Arg-Arg-Ser-Arg-Arg / (SEQ ID NO: 2) upstream and including the prM protein sequence, with respect to this, the cleavage occurs after the terminal Arg, indicated by a slash, and the serine residue is substituted with Arg (tetrapeptide site, Arg-Ser-Arg-Arg(SEQ ID NO: 10)) to provide a sequence of five arginine residues to enhance the furin cleavage. Furthermore, the glutamate residue in the upstream stretch can be modified, for example, to histidine to enhance the furin cleavage. Other modifications of residues in the upstream stretch enhance the furin cleavage. Substitutions downstream of the tetrapeptide enhance the furin cleavage. For example, the modification can be for any or all of the downstream 26 amino acid stretches of the tetrapeptide to enhance the furin cleavage.

[0070]

[0082] Enhanced furin activity is obtained by one or more of the following: (1) modification of one or more amino acids upstream of the furin cleavage site, e.g., an 8-residue sequence starting at amino acid residue 82 from the N-terminus of the ZV prM polypeptide (i.e., amino acid residues 82-89 of prM); (2) modification at the furin cleavage site; and (3) modification of one or more amino acid residues downstream of the furin cleavage site (downward from amino acid 94), e.g., a 26-residue sequence downstream of the furin cleavage site. Any combination of (1), (2), and (3) may exist.

[0071]

[0083] With respect to yellow fever virus, California encephalitis virus, Rift Valley fever virus, tick-borne encephalitis virus, West Nile virus, equine encephalopathy virus, Colorado tick-borne fever virus, chikungunya virus, African swine fever virus, and other arboviruses, the furin consensus tetrapeptide recognition site is located based on known consensus furin recognition sequences, and amino acid substitutions are performed to identify variants that no longer reproduce or reproduce very little in mammalian cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications can also be performed upstream and / or downstream of the furin consensus cleavage site.

[0072]

[0084] The creation, propagation, and maintenance of viruses in cell lines and animals are known. The creation of arbovirus variants by modifying the coding sequence using molecular techniques is also known. Various tests for determining viral propagation in cells, the production of progeny viruses from cells, and other assays used in carrying out the present invention are known, and their selection (one or more) is a design choice.

[0073]

[0085] Another object of the present invention is to develop a scalable system for producing a target immunogenic arbovirus composition. This objective was achieved by producing a target arbovirus that expresses a dysfunctional furin cleavage site that is cleaved at an enhanced level by furin in an arthropod cell line, such as an insect cell line, e.g., a mite or mosquito cell line. In this regard, materials and methods for large-scale production of viruses using insect cells are known and available. Arthropod cell lines such as silkworm and mosquito cell lines are commercially available.

[0074]

[0086] The target arbovirus replicates without impairment in insect cells. Therefore, the target arbovirus with dysfunctional furin cleavage sites can replicate, reproduce, mature, and be released into the environment by host insect cells at levels approximately the same as or the same as wild-type arboviruses. Consequently, the target arbovirus can produce progeny viruses at levels comparable to or higher than those of arboviruses without the target dysfunctional furin cleavage sites in insect cells.

[0075]

[0087] The degree of progeny virus production (or reproduction) can be assessed, for example, by plaque assays or hemagglutinin assays, as are known in the art and taught herein. A simple and highly sensitive method for assessing virus production is to count fluorescently labeled cells using a flow cytometer, as described in Drayman & Oppenheim, “Rapid Titration of Viruses by Flow Cytometry”, Curr Prot Cell Biol 26.11.1-26.11.7, 2011. Thus, sequentially diluted infected cells are collected, separated as necessary, recovered, fixed, and exposed to one or more antibody reagents having at least one antibody directed against the viral protein and at least one fluorescently labeled antibody, and then, for example, at least about 10,000 cells are read by a flow cytometer to determine the number of fluorescently labeled cells expressing the viral protein. Therefore, the target arbovirus can produce progeny viruses at approximately the same level, or in greater quantities, than the amount produced by cells infected with a reference arbovirus (e.g., DV, serotype 2, New Guinea strain, ATCC No. VR-1584) in C6 / 36 cells (ATCC accession number CRL-1660), or by wild-type viruses of the same strain or lineage that do not contain the target mutant virus and contain enhanced furin activity or exhibit enhanced furin cleavage or prM.

[0076]

[0088] The target arbovirus does not mature properly in mammalian cells, and the particles are not released (or reproduce) by the host mammalian cell. Instead, immature particles are captured and accumulate within the host mammalian cell. Therefore, the target arbovirus produces little to no progeny viruses in human or mammalian cells. The target arbovirus produces fewer progeny viruses compared to wild-type arboviruses, for example, producing fewer than 4log, 5log, 6log, 7log, 8log, or 9log of progeny, or no progeny at all, compared to the amount of progeny produced when using HT1080 cells (ATCC accession number CCL-121).

[0077]

[0089] Target mutant viruses that do not reproduce in mammalian cells are desirable because they minimize or avoid disease or lesions. However, very little or no release of progeny viruses is not essential or necessary. Dysfunctional viruses that, when infecting mammalian cells, produce, for example, fewer than 4log, 5log, 6log, or fewer progeny viruses compared to similar cells infected with wild-type viruses, can be used as immunogens because, despite the release of low or small amounts of progeny viruses, the host immune system ultimately eliminates the virus and the cells expressing viral antigens.

[0078]

[0090] Viruses containing the modified, dysfunctional furin region are infectious but cannot, or do not, produce infectious progeny virions from infection of mammalian cells. Therefore, non-replicating particles are particles that infect host mammalian cells, such as human cells, but the host cell does not produce or release progeny viral particles resulting from the infection. However, the infected mammalian host cell expresses an arboviral epitope within and on the infected cell.

[0079]

[0091] The unique characteristics of the target deficiency virus provide a source of effective and safe arbovirus immunogenic compositions, allowing for cost-effective acquisition of large quantities of virus by propagating the target virus in insect cells. The target deficiency virus, when infecting the same type of insect cells, can produce approximately the same amount of progeny virus as the reference virus. In one embodiment, the target deficiency virus produces more virus from insect cells compared to the reference virus infecting the same insect cell type. In another embodiment, the target deficiency virus produces less virus than the reference virus. Highly immunogenic deficiency virus strains mimic the natural arbovirus infection process, enabling mammalian hosts to produce extensive arbovirus immunity over long periods, with or without minimal lesions. Therefore, the modified arbovirus of the target contains genetic material to express many or all of the epitopes expressed by the wild-type prM, M, and E proteins, and possibly C, of ​​the wild-type arbovirus.

[0080]

[0092] The target arbovirus can infect many, if not all, susceptible cells after appropriate titration. The degree of infectivity can be assessed using standard and known assays. For example, Vero cells in a 6-well dish are infected with the arbovirus in serial dilutions, and the cells are agitated in a rocker platform at 37°C. The cell culture is collected after 48 hours. The number of viruses released into the culture medium can be determined by performing known methods such as those taught herein.

[0081]

[0093] For example, to prepare partially purified arbovirus particles, the culture medium is clarified by centrifugation at 16,000 × g in a microcentrifuge for 15 minutes at 4°C, and the particles are pelletized from the supernatant by ultracentrifugation at 40,000 rpm for 2 hours at 4°C in an AH650 rotor of a Sorvall OTD55B centrifuge. The pellet is resuspended in 50 μL of phosphate-buffered saline (PBS) and left to dissolve overnight at 4°C.

[0082]

[0094] To determine the titer of arboviruses, for example, baby hamster kidney (BHK)-21 cells on an 8-well chamber slide are infected with 50 μL of serially 10-fold diluted cell culture medium or resuspended pelletized material at 37°C for 2 hours. This fluid is then replaced with 1 mL of Dulbecco's Minimum Essential Medium supplemented with 2% fetal bovine serum. After incubating the cells in a CO2 incubator at 37°C for 24 hours, they are subjected to immunofluorescence (IF) analysis using an arbovirus-specific Ab or mAb (commercially available or prepared as known in the art), HMAF as described below, or a polyclonal antiserum with the required specificity, a virus-specific reagent with a detectable reporter to form a sandwich, and an appropriate control.

[0083]

[0095] The viral titer (infectious units (IU) / milliliter) present in the collected culture medium (CF) was also determined, for example, by infection of rhesus monkey renal epithelial cells (e.g., LLC-MK2 (ATCC) cells), followed by indirect immunofluorescence (IF) analysis in hyperimmunized mouse ascites (HMAF) containing arbovirus-specific and labeling reagents, such as mAbs.

[0084]

[0096] Both humoral antibodies and cellular immune responses are involved in defense against and recovery from DV infection. The target arbovirus induces both arms of the immune response. The particles are composed of prM, M, and / or E proteins, and therefore the target particles are broad immunogens that simulate infection by wild-type virus. However, the target particles infect mammalian host cells, and in these cells, further prM, M, and / or E proteins are expressed in or by the host cells. The prM, M, and / or E proteins can be released from these cells to provide further antigenic stimulation to the host, or they can be expressed on the surface of infected host cells to provide the host with further other forms of antigenic stimulation. The target arbovirus can optionally express non-structural proteins such as NS1. Previous reports have demonstrated that these NS viral proteins can induce a protective immune response (Heinz & Roehrig, 1990, “Immunochemistry of Virus, Vol II,” Amsterdam-NY-Oxford, Elsevier, pp. 289-305; Heinz, 1986, Adv Virus Res 31:103-168; Bray & Lai, 1991, Virol, 185:505-508; Henchal et al., 1988, J Gen Virol 69:2101-2107; and Schlesinger et al., 1986, J Virol 60:1153-1155).

[0085]

[0097] A suitable model for testing the efficacy of a pharmaceutical composition in question is an animal model that simulates arbovirus infection. For example, protocols used for immunizing mice and subsequent arbovirus attack are described (Bray et al.). (al., 1989, J Virol 63:2853-2856). Briefly, in each group of 10 mice, female BALB / c mice were immunized with viruses such as DV by intraperitoneal (ip) inoculation at 3 weeks of age (day 1) and again at 14 days. Control animals were given phosphate-buffered saline (PBS). Blood samples were taken from all animals on day 0 and day 21. In the case of DV, the 50% lethal dose (LD50) was administered on day 22. 50 Mice are attacked by intracerebral injection of 100 times the normal dose of the virus. After the attack, the mice are observed for pathological signs for 21 days, and mice exhibiting symptoms (encephalitis, paralysis, or death) are recorded daily. In addition, serum is collected from surviving individuals and compared to serum from before the attack.

[0086]

[0098] The dosage of each virus is determined. The serological response of immunized mice to individual arbovirus proteins is analyzed, for example, by ELISA or radioimmunoprecipitation of labeled arbovirus antigens using commercially available labeling kits and antibodies. The titer of arbovirus-specific neutralizing antibodies in mouse serum can be measured using a plaque reduction assay. For example, first, approximately 0.5 mL of serum sample to be used in the assay is thermally inactivated by incubation at 56°C for 30 minutes. Using medium M199 containing 2% thermally inactivated fetal bovine serum (FBS) as a diluent, a 4-fold dilution of serum is prepared, starting with a 1:10 dilution, to a final volume of 0.3 mL. Equivolute medium containing 150-180 plaque-forming units (PFUs) of virus is added to each 0.3 mL aliquot of diluted serum. The virus and serum are mixed and incubated at 37°C for 30 minutes. Each assay also includes a serum-free control and a control consisting of, for example, two dilutions of each arbovirus type-specific Ab. The virus / serum and control mixture is plated at 0.2 mL / well onto a fusion monolayer of LLC-MK2 cells in, for example, a Costar 6-well plate (Corning Inc., Corning, New York). Two wells are infected with each sample. Virus adsorption is performed at room temperature for 1 hour with manual agitation every 15 minutes. The wells are then covered with a medium containing 1% agarose in Earl's equilibrium salt solution (SeaKem LE; BioWhittaker, Rockland, ME) and 10% FBS supplemented with 6 mL / well of essential vitamins and amino acids (Invitrogen). The plates are incubated at 37°C in 5% CO2 for 7 days. The wells are then covered with a 4% neutral red solution containing 1% agarose (4 mL of neutral red solution added to 96 mL of PBS). The plates are incubated at 37°C for 24 hours. The 50% reduction in plaque count levels is calculated using the average plaque count.

[0087]

[0099] In some embodiments, the target viral composition is polyvalent, i.e., bivalent, trivalent, tetravalent, etc., and generally immunizes the host against multiple arbovirus serotypes of a single virus, but may also contain antigens from multiple viral species, such as those containing multiple target mutant viruses. No interference or dominance of one serotype at the expense of one or more other serotypes is observed. This may be because immunogenicity does not depend on viral production and sustained replication of novel viruses.

[0088]

[0100] The target arbovirus is incorporated into a pharmaceutical composition suitable for administration to act as an immunogen, as is known in the field of immunology. Such compositions typically contain an active ingredient and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc., that are suitable for pharmaceutically active administration. The use of such media and agents for pharmaceutically active substances is known in the art as design choices. Unless any conventional media or agent is incompatible with the active compound, their use in the composition is intended. Supplementary pharmacologically active compounds, such as adjuvants, may also be incorporated into the composition.

[0089]

[0101] The pharmaceutical compositions of the present invention for use as disclosed herein are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, intravenous, intradermal, subcutaneous, transdermal (including topical), transmucosal, and rectal administration.

[0090]

[0102] Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents such as water for injection, physiological saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with an acid or base, such as HCl or NaOH. Parenteral preparations may be sealed in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0091]

[0103] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF; Parsippany, New Jersey), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be injectable. The composition must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and ascorbic acid. It may be preferable to include isotonic agents, such as sugars, polyhydric alcohols like mannitol and sorbitol, or sodium chloride in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0092]

[0104] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above as needed, into a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components derived from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying to obtain powders of the active ingredient and any additional desired components from a pre-sterilized filtered solution.

[0093]

[0105] Oral compositions generally contain an inert diluent or edible carrier. The composition can be encapsulated in gelatin capsules or compressed into tablets, which can be coated or treated to provide, for example, enteric-coated compositions, delayed-release formulations, etc. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier to obtain syrups, elixirs, or liquid formulations, or for use as a mouthwash. In the latter case, the compound in the fluid carrier is administered orally, either swished and spat out, or swallowed.

[0094]

[0106] pharmaceutically compatible binders and / or adjuvant materials may be incorporated as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. The particles of interest may be encapsulated in a form that survives through the gastric environment. Such forms are commonly known as enteric-coated formulations.

[0095]

[0107] When administered by inhalation, the compound can be delivered, for example, in the form of a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or in the form of a nebulizer or aerosol spray from a mist. The formulation can be a liquid, dry, or, for example, a finely ground powder.

[0096]

[0108] Systemic administration can also be carried out by mucosal or percutaneous means. In the case of mucosal or percutaneous administration, a suitable penetrating agent is used in the formulation to create a barrier for penetration. Such penetrating agents are generally known in the art and, for example, in the case of mucosal administration, include detergents, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved by the use of nasal sprays or enemas. In the case of percutaneous administration, the active compound can be formulated into ointments, plasters, foams, gels, or creams, as is generally known in the art. The composition may also be delivered using patches applied to the skin.

[0097]

[0109] When the composition in question is in the form of a suppository, it may include conventional suppository bases such as cocoa butter and other glycerides.

[0110] In this embodiment, the active compound is prepared in the form of a controlled-release formulation, for example, an implant and a microencapsulation delivery system, using a carrier that protects the compound from rapid elimination from the body. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.

[0098]

[0111] The method for preparing such formulations will be obvious to those skilled in the art. The materials can be obtained from commercial sources such as Johnson & Johnson and Encapsula Nano Sciences (Brentwood, Tennessee).

[0099]

[0112] Liposome suspensions (containing liposomes targeted with monoclonal antibodies and other such targeting molecules) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent Publication No. 4522811.

[0100]

[0113] For ease of administration and uniformity of dosage, it may be advantageous to formulate oral or parenteral compositions in unit dosage forms. As used herein, "unit dosage form" refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0101]

[0114] The pharmaceutical composition can be included in a container, pack, kit or dispenser together with instructions for administration.

[0115] Other administration methods include adding the compound of interest in or together with food or beverages, as a food supplement or additive, or as a dosage form taken on a prophylactic basis similarly to vitamins.

[0102]

[0116] Dosages, for example, preferred routes and amounts of administration, can be obtained based on empirical data derived from preclinical and clinical studies carrying out methods known in the art. In the case of repeated administration over several days, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may also be useful. The progress of the treatment is monitored by conventional techniques and assays. An exemplary dosage regimen is disclosed in WO94 / 04188. The specifications for the unit dosage forms of the present invention are determined by, and can directly depend on, the unique characteristics of the active compound, the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulating such active compounds for the treatment of individuals. Therefore, the number of viral particles administered to an adult can be extrapolated from the therapeutic doses delivered to model animals such as mice, rats, and monkeys. For example, based on studies in mice, about 10 6 particles (or IU) to about 10 7 particles to about 10 8 particles to about 10 9The amount or dose can be administered from particles, or more, in dose-based or divided doses. The amount or dose can be adjusted, as empirical data are available, as is known in the art.

[0103]

[0117] The target virus can be used to induce an effective immune response in an individual, where effective includes minimizing, reducing, or preventing symptoms or lesions in the individual. Since the host immune response is critical to obtaining a therapeutic response, which can be scored in any single individual, the target virus may or may not be therapeutic, and may or may not elicit a desired response from the virus recipient, as is the case with any drug. The use of administration, viral presentation routes and means, adjuvants, and other immunological factors can resolve a lack or incomplete host immune response in an individual.

[0104]

[0118] This phenomenon is evident at the population level, and not all recipients in a population of the target virus initiate a therapeutic immune response, and the number of responders in any one population may differ from that of other populations.

[0105]

[0119] The usefulness of the target dysfunctional virus depends on factors such as a larger proportion of responders in the population, a higher level of immune defense in the population, commercial viability, and commercial success, but in a single individual, the target dysfunctional virus elicits a therapeutic immune response that reduces or prevents symptoms or disease.

[0106]

[0120] Herein, the present invention is illustrated in the following non-limiting embodiments. [Examples]

[0107] Example 1

[0121] DV virions are composed of 6% RNA, 66% protein, 9% carbohydrates, and 17% lipids (Russell et al., Chemical and Antigenic Structure of Flaviviruses, Schlesinger ed., “The Togaviruses: Biology, Structure, Replication,” New York, Academic, 1980, pp. 503-529; and Trent & Naeve, Biochemistry). (and Replication, Monath, ed., “St. Louis Encephalitis,” Washington, DC, American Public Health Association, 1980, pp. 159-199). Electron-dense nucleocapsids are composed of C (capsid) protein and genomic RNA. Envelope protein E and membrane (M) protein are embedded in the lipid bilayer by a C-terminal hydrophobic anchor. However, immature particles found within intracellular vesicles contain only untreated M precursor (prM) and are less infectious than released virions (Morens, Clin Infect Dis, 1994, 19:500-512).

[0108]

[0122] The DV genome is homogeneous, consisting of a single-stranded positive-sense RNA molecule of approximately 10-11 kb containing a single ORF, which makes up about 95% of the genome (Chambers et al., Ann Rev Microbiol, 1990, 44:649-688). The full-length genomic RNA appears to be the only virus-specific messenger RNA (mRNA) molecule in DV-infected cells. Upon infection, viral RNA is translated into polyproteins of approximately 3400 amino acids, which are processed into 10 gene products: three structural proteins, namely C, prM, and E; and seven non-structural (NS) proteins, namely 1, 2A, 2B, 3, 4A, 4B, and 5 (Bhamarapravati & Yokan, Live attenuated tetravalent vaccine, Gubler & Kuno eds., “Dengue and Dengue Hemorrhagic Fever,” Wallingford, CAB International, 1997, pp. 367–377; and Falgout & Markoff, 1995, The family flaviviridae and its diseases, pp. 47–66, Porterfield ed., “Exotic Viral Infections,” Chapman and Hall Medical, London, UK). Example 2

[0123] The mutant DV was generated from a full-length infectious cDNA clone of the DV2 New Guinea C strain (ATCC number VR-1584). The mutant DNA fragment was produced by PCR and introduced into the full-length clone by homologous recombination as described (Zeng et al., J Virol. September 1998; 72(9): 7510-22). The mutation was confirmed by DNA sequencing. The RNA of the mutant DV was synthesized by in vitro transcription as described by Polo et al., J Virol. 1997; 71(7): 5366-74.

[0109]

[0124] The proliferation dynamics of mutant DV showed differences in viral replication between infected Vero cells (ATCC CCL-81) (human) and infected C6 / 36 cells (CRL-1660) (mosquito). Wild-type DV2 (DENV2)prM (SEQ ID NO: 3) and four mutants in which the furin recognition site Glu (E in DENV2) is replaced by Arg(R) (D2-89R, SEQ ID NO: 4), Val(V) (D2-89V, SEQ ID NO: 5), Ser(S) (D2-89S, SEQ ID NO: 6), or Gly(G) (D2-89G, SEQ ID NO: 7) were tested for proliferation in human Vero cells and mosquito C6 / 36 cells.

[0110]

[0125] The D2-89R mutant, in which Glu is replaced by R, reproduces in insect cells but does not replicate in human cells. Example 3

[0126] The mutant was generated from a full-length infectious cDNA clone of DV1, Western Pacific, 74 strains (Genbank number U88536), using the second serotype of DV. Mutant DNA fragments were produced by PCR and introduced into full-length clones by homologous recombination as described (Markoff et al., J Virol. 2002;76(7):3318-28). The mutation was confirmed by DNA sequencing. RNA of the mutant DV was synthesized by in vitro transcription as described by Polo et al.

[0111]

[0127] The replication dynamics of the mutant DV showed differences in viral replication between infected Vero cells (ATCC CCL-81) (human) and infected C6 / 36 cells (CRL-1660) (mosquito). Example 4

[0128] DV1 mutants containing the following sequence were prepared as described in Examples 2 and 3: CSQTGEHRRRKRSVALAPHVGLGLETRTETWMSSEGAWKHAQRIETWILRH(Sequence ID: 17).

[0129] The sequence begins at amino acid 80 of the DV1 prM polypeptide, and in the consensus frin tetrapeptide, D is changed to R. Cleavage occurs between R and S in the above sequence.

[0112]

[0130] The replication dynamics of mutant DV1 showed differences in viral replication between infected Vero cells (ATCC CCL-81) (human) and infected C6 / 36 cells (CRL-1660) (mosquito). Example 5

[0131] Western blot analysis, which involves comparing band intensity and band size to estimate molecular weight using commercially available DV prM mAbs and employing known methods, demonstrated that the furin cleavage efficiency of mutant DV was increased in both Vero cells (CCL-81) and C6 / 36 cells (CRL-1660). Example 6

[0132] The proliferation of mutant DV in mammalian cells was investigated by indirect immunofluorescence staining. mAb 4G2 from ATCC(HB-112) was used in the method described by Polo et al.

[0113]

[0133] Positive fluorescence was observed from day 1 to day 3 post-infection, with increased brightness and a greater number of positive cells compared to positive controls using the parent DV2 virus. Conversely, when infected with the mutant D2-89R, the intensity and number of fluorescent cells gradually decreased over that period. No new colonies formed during days 1-3 post-infection.

[0114]

[0134] Therefore, the D2-89R mutation initiated only one infection in human cells and did not produce infectious progeny virus particles. Example 7

[0135] To verify that the D2-89R mutant is restricted in proliferation in human cells, dermal fibroblasts (CRL-2522, ATCC) were infected with either DV2 or D2-89R in vitro, and the presence of viral envelope antigens was assessed by immunofluorescence at different time intervals (hpi) after infection, as described herein. DV2 proliferated and produced progeny viruses in CRL-2522 cells, but this did not occur with the D2-89R mutant.

[0115]

[0136] In cells infected with wild-type DV2 virus, a gradual increase in viral particle production over time was observed, indicating active viral replication in infected cells.

[0137] On the other hand, cells infected with the mutated D2-89R did not produce any viral offspring at all. Example 8

[0138] To evaluate the protective effect of mammalian cell-specific proliferation restriction of target mutant DV against DV attacks, a group of 6-week-old AG129 mice (N=6 mice / group) were subjected to 10 5 A single intraperitoneal immunization with the infectious unit (IU) mutation D2-89R was administered to the mice. Control mice were intraperitoneally injected with 50 μL of phosphate-buffered saline (PBS).

[0116]

[0139] Neither group of mice exhibited any visually distinct motility or behavior after immunization.

[0140] Ten days after immunization, D2-89R injected mice and PBS-controlled AG129 mice were given 10 5 Plaque-forming units (PFUs) (LD 50 DV2 (100 times the normal dose) was administered by intraperitoneal injection.

[0117]

[0141] PBS-injected control mice inoculated with DV2 developed viremia. In contrast, monoimmunization with mutant D2-89R provided complete protection against DV2 attack, and no detectable viremia (<100 replications / mL) was observed at any time point (N=10).

[0118]

[0142] Furthermore, all D2-89R exposed mice remained healthy throughout the experiment, while all mice exposed to PBS alone died after the DV2 attack. See Figure 1. Example 9

[0143] To evaluate the safety of the DV variant, AG129 mice were used. This is because AG129 mice can lethally infect with DV2 with as little as 1 PFU of virus.

[0119]

[0144] In a group of 6 adult AG129s, 10 6 The IU mutant D2-89R was administered intraperitoneally. For the control group, the same number of mice per group received 10 1 , 10 2 and 10 3 PFU DV2 was administered by intraperitoneal injection. Viral load after DV2 and D2-89R injection was quantified by RT-PCR.

[0120]

[0145] The mice in the DV2-infected group developed detectable viremia and all died after infection.

[0146] On the other hand, mice exposed to the D2-89R mutation did not exhibit detectable viremia and all survived the entire experimental period (15 days) without any signs of disease or weight changes. Example 10

[0147] The electron-dense nucleocapsid of ZV is composed of the C (capsid) protein and genomic RNA. The envelope protein E and membrane (M) proteins are embedded in the lipid bilayer (Russell et al., Chemical and Antigenic Structure of Flaviviruses, Schlesinger (ed.), The Togaviruses: Biology, Structure, Replication. New York: Academic; 1980: 503-529; and Trent & Naeve, Biochemistry and Replication, Monath (ed.), St. Louis Encephalitis. Washington, DC: American Public Health Association; 1980 (pp. 159-199).

[0121]

[0148] The ZV genome is a single-stranded positive-sense RNA molecule of approximately 10-11 kb containing a single ORF that makes up about 95% of the genome (Chambers et al., Ann Rev Microbiol 1990, 44:649-688). Upon infection, the infectious viral RNA is translated and processed into structural and non-structural proteins (Bhamarapravati & Yokan: Live attenuated). tetravalent vaccine, Gubler & Kuno (eds.), Dengue and Dengue Hemorrhagic Fever. Wallingford, CAB International, 1997, pp. 367-377; and Falgout & Markoff, 1995, The family flaviviridae and its diseases, pp. 47-66. (JS Porterfield (ed.), Exotic Viral Infections. Chapman and Hall Medical, London, UK). Example 11

[0149] A full-length infectious cDNA clone of ZV MR766 from ATCC was modified with point mutations to induce restricted proliferation in mammalian cells. The mutations were confirmed by DNA sequencing. Mutant ZV genomic RNA was synthesized by in vitro transcription, and C6 / 36 mosquito cells were transfected with the mutant viral RNA using TransIT®-mRNA (Mirus Bio, Madison, Wisconsin) according to the manufacturer's instructions. Mutant ZV was collected 7 days after transfection. Viral proliferation dynamics were determined in both mammalian (Vero) cells and C6 / 36 cells.

[0122]

[0150] Point mutations were induced in the ZV genome, and the sequence near the flurin cleavage site was modified as follows: Wild type: HHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHIKVENWIFRN (Sequence ID: 11) Mutant: HHKKGEARRRRRAVTLPSHSTRKLQTRSQTWLESREYTKHIKVENWIFRN (Sequence ID: 12)

[0151] A single modification from serine to arginine resulted in reduced reproduction in Vero cells, while reproduction in mosquito cells remained relatively unchanged from the control. Example 12

[0152] The procedure of Example 11 was followed. In addition to using the same Ser-to-Arg substitution, Glu(E) (between G and A) was substituted with His(H) to obtain mutants with two point mutations.

[0123]

[0153] Reproduction in human cells was reduced to a lower level than observed in the mutant in Example 11, and there was no impact on replication in insect cells. Example 13

[0154] The procedure of Example 11 was followed. The first 12 amino acids of the sequence shown in Example 11 were replaced with TTTGEHRRRKRS (Sequence ID: 13).

[0124]

[0155] Reproduction in human cells was reduced to a lower level than observed in the mutant in Example 11, and there was no impact on replication in insect cells. Example 14

[0156] The procedure of Example 11 was followed. The first 22 amino acids of the sequence shown in Example 11 were replaced with TTTGEHRRRKRSVALVPHVGMG (Sequence ID: 14).

[0125]

[0157] Reproduction in human cells was reduced to a lower level than observed in the mutant in Example 11, and there was no impact on replication in insect cells. Example 15

[0158] The procedure of Example 11 was followed. The first 37 amino acids of the sequence shown in Example 11 were substituted with TTTGEHRRRKRSVALVPHVGMGLETRTETWMSSEGAW (SEQ ID NO: 15) to form the ZV M2 mutant.

[0126]

[0159] Reproduction in human cells was reduced to a lower level than observed in the mutant in Example 11, and there was no impact on replication in insect cells. Example 16

[0160] To evaluate the protective effect of mammalian cell-specific proliferation restriction of target mutant SVs against SV attack, a group of 6-week-old AG129 mice (N=6 mice / group) were subjected to 10 5 A single intraperitoneal immunization with the M2 variant of Example 15 (infectious unit (IU)) was administered to the mice in the control group. 50 μL of PBS was injected intraperitoneally.

[0127]

[0161] Neither group of mice exhibited any visually distinct motility or behavior after immunization.

[0162] Fourteen days after immunization, M2 injection mice and PBS control AG129 mice were given 10 5 Plaque-forming units (PFUs) (LD 50 The wild-type SV (100 times the normal dose) was administered by intraperitoneal injection.

[0128]

[0163] PBS-injected control mice inoculated with SV developed viremia. In contrast, monoimmunization with mutant M2 provided complete protection against SV attack, and no detectable viremia (<100 replications / mL) was observed at any time point (N=10).

[0129]

[0164] Furthermore, all M2-exposed mice remained healthy throughout the experiment, while all mice exposed to PBS alone died after SV attack. See Figure 2. Example 17

[0165] To evaluate the safety of mammalian cell-specific proliferation restriction of the target mutant SV, a group of 6-week-old AG129 mice (N=6 mice / group) were subjected to 10 5 A single intraperitoneal immunization with the infectious unit (IU) variant M2 or wild-type ZV was administered.

[0130]

[0166] Mice exposed to M2 remained healthy throughout the experiment, while all mice exposed to PBS alone died after SV attack. See Figure 3. Example 18

[0167] The materials and methods of Examples 2 to 9 are carried out using standard strains of yellow fever virus.

[0131]

[0168] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are made to it. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also made upstream and / or downstream of the fulin cleavage site.

[0132]

[0169] A yellow fever virus variant has been identified that replicates successfully in insect cells but does not replicate well in human cells. Example 19

[0170] The materials and methods of Examples 2-9 are carried out using a standard for California encephalitis virus.

[0133]

[0171] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0134]

[0172] A variant of the California encephalitis virus has been identified that replicates successfully in insect cells but does not replicate well in human cells. Example 20

[0173] The materials and methods of Examples 2-9 are carried out using standard strains of Rift Valley virus.

[0135]

[0174] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0136]

[0175] A variant of the Rift Valley virus that replicates successfully in insect cells but does not replicate well in human cells has been identified. Example 21

[0176] The materials and methods of Examples 2-9 are carried out using standard strains of tick-borne encephalitis virus.

[0137]

[0177] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0138]

[0178] A variant of the tick-borne encephalitis virus has been identified that replicates successfully in insect cells but does not replicate well in human cells. Example 22

[0179] The materials and methods of Examples 2-9 are carried out using standard strains of West Nile virus.

[0139]

[0180] As described above and as is well known in the art, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in mammalian cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0140]

[0181] A West Nile virus variant was identified that replicates successfully in insect cells but does not replicate well in mammalian cells. Example 23

[0182] The materials and methods of Examples 2 to 9 are carried out using standard strains of equine encephalopathy virus.

[0141]

[0183] As described above and as is well known in the art, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in mammalian cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0142]

[0184] A variant of the equine encephalopathy virus has been identified that replicates successfully in insect cells but does not replicate well in mammalian cells. Example 24

[0185] The materials and methods of Examples 2-9 are carried out using standard strains of Colorado tick virus.

[0143]

[0186] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0144]

[0187] A Colorado tick virus was identified that replicates well in insect cells but does not replicate very well in human cells. Example 25

[0188] The materials and methods of Examples 2-9 are carried out using standard strains of chikungunyavirus.

[0145]

[0189] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0146]

[0190] A type of chikungunyavirus was identified that replicates well in insect cells but does not replicate very well in human cells. Example 26

[0191] The materials and methods of Examples 2-9 are carried out using a standard strain of African swine fever virus.

[0147]

[0192] As described above and as is well known in the field, the prM fulin recognition site is positioned and amino acid substitutions are performed. As described herein above, the target mutant is identified as a mutant that no longer reproduces or reproduces only at a low level in human cells. Amino acid modifications such as substitutions, insertions, deletions, and chemical modifications are also performed upstream and / or downstream of the fulin cleavage site.

[0148]

[0193] An African swine fever virus was identified that replicates well in insect cells but does not replicate very well in human cells. Example 27

[0194] Adult rhesus macaques were assigned to experimental and control groups of 4-6 animals per group. The animals in the experimental groups were given the mutant D2-89R from Example 2 at various doses (10 5 ~10 10 The drug was administered in IU. The control group of animals was given an equal volume of PBS.

[0149]

[0195] The animals were retained for four weeks, during which time they were tested for circulating anti-dengue antibodies, viral load, and cellular immunofluorescence to detect the presence, extent, and amount of the virus, as well as the host response to it. Any symptoms of dengue infection, such as fever and weight loss, were also monitored.

[0150]

[0196] Subsequently, the animals were attacked with wild-type DV2, and symptoms such as fever and weight loss, as well as serological parameters such as viral load and dengue antibodies, were monitored.

[0151]

[0197] While control animals exhibit symptoms of dengue fever infection, monkeys given the target mutant do not show any symptoms. Example 28

[0198] Adult rhesus monkeys were assigned to experimental and control groups of 4-6 animals per group. The animals in the experimental groups were given the DV1 mutant from Example 4 at various doses (10 5 ~10 10 The drug was administered in IU. The control group of animals was given an equal volume of PBS.

[0152]

[0199] The animals were retained for four weeks, during which time they were tested for circulating anti-dengue antibodies, viral load, and cellular immunofluorescence to detect the presence, extent, and amount of the virus, as well as the host response to it. Any symptoms of dengue infection, such as fever and weight loss, were also monitored.

[0153]

[0200] Subsequently, the animals were attacked with wild-type DV1, and symptoms such as fever and weight loss, as well as serological parameters such as viral load and dengue antibodies, were monitored.

[0154]

[0201] While control animals exhibit symptoms of dengue fever infection, monkeys given the target mutant do not show any symptoms. Example 29

[0202] Adult rhesus monkeys were assigned to two experimental groups and two control groups, each consisting of 4-6 animals. The animals in the experimental groups were given the mutant Zika virus of Example 14 at various doses (10 5 ~10 10 The drug was administered in IU. The control group of animals was given an equal volume of PBS.

[0155]

[0203] The animals were retained for four weeks, during which time they were tested for circulating anti-Zika antibodies, viral load, and cellular immunofluorescence to detect the presence, extent, and amount of the virus, as well as the host response to it. They were also monitored for any symptoms of Zika infection, such as fever, viremia, rash, and weight loss.

[0156]

[0204] Subsequently, the animals were attacked with wild-type ZV. One experimental group and control group were attacked with the wild-type ZV African strain, and the other experimental group and control group were attacked with the wild-type ZV Asian strain. The animals were monitored for symptoms such as fever, rash, and weight loss, as well as serological parameters such as viral load and Zika antibodies.

[0157]

[0205] While control animals exhibit symptoms of Zika infection, monkeys given the target mutant do not show any symptoms. Example 30

[0206] Adult rhesus monkeys were assigned to two experimental groups and two control groups, each consisting of 4-6 animals. The animals in the experimental groups were given the mutant Zika virus of Example 15 at various doses (10 5 ~10 10 The drug was administered in IU. The control group of animals was given an equal volume of PBS.

[0158]

[0207] The animals were retained for four weeks, during which time they were tested for circulating anti-Zika antibodies, viral load, and cellular immunofluorescence to detect the presence, extent, and amount of the virus, as well as the host response to it. They were also monitored for any symptoms of Zika infection, such as fever, viremia, rash, and weight loss.

[0159]

[0208] Subsequently, the animals were attacked with wild-type ZV. One experimental group and control group were attacked with the wild-type ZV African strain, and the other experimental group and control group were attacked with the wild-type ZV Asian strain. The animals were monitored for symptoms such as fever, rash, and weight loss, as well as serological parameters such as viral load and Zika antibodies.

[0160]

[0209] While control animals exhibit symptoms of Zika infection, monkeys given the target mutant do not show any symptoms.

[0210] All references cited herein are incorporated herein by reference in their entirety.

[0161]

[0211] It should be understood that various changes and modifications to the current preferred embodiments described herein will be obvious to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the invention and without impairing its intended advantages. Accordingly, such changes and modifications are intended to be covered by the scope of the appended claims. This specification includes the disclosure of the following inventions. [1] Arbovirus particles comprising a replication-restricted arbovirus, wherein the precursor polyprotein contains a dysfunctional frintetrapeptide cleavage site, and may also contain modifications in a first stretch of amino acids upstream of the site and / or modifications in a second stretch of amino acids downstream of the site. [2] The particle according to [1], wherein the aforementioned site comprises a consensus sequence of Arg-X-Lys / Arg-Arg (SEQ ID NO: 16). [3] A particle according to [2], containing Arg. [4] The particle according to [1], wherein the first stretch comprises eight amino acids adjacent to the site. [5] The particle according to [1], including modifications in the first stretch. [6] The particle according to [1], including the modification in the second stretch. [7] The particle according to [1], comprising the polyprotein prM. [8] The particle according to [1], comprising the polyprotein prE2. [9] Particles as described in [1], containing the dengue virus (DV).

[10] Particles containing the Zika virus (ZV) as described in [1]. A composition comprising the particles described in

[11] [1] and a pharmaceutically acceptable carrier, diluent, or excipient. Insect cells containing the particles described in

[12] [1].

[13] The cell according to

[12] , wherein the insect includes arthropods.

[14] The cell according to

[12] , wherein the insect includes a tick or a mosquito. Mammalian cells containing the particles described in

[15] [1].

[16] The cells described in

[15] , wherein the cells express the DV antigen.

[17] The cell according to

[15] , wherein the cell expresses the DV antigen on the surface of the cell.

[18] The cells described in

[15] , wherein the cells express the ZV antigen.

[19] The cell according to

[15] , wherein the cell expresses the ZV antigen on the surface of the cell.

[20] Cells as described in

[15] , including human cells.

Claims

1. Arbovirus particles comprising a growth-restricted arbovirus, wherein the precursor polyprotein contains a dysfunctional frintetrapeptide cleavage site, and may also contain modifications in a first stretch of amino acids upstream of the site and / or modifications in a second stretch of amino acids downstream of the site.

2. The particle according to claim 1, wherein the aforementioned portion includes a consensus sequence of Arg-X-Lys / Arg-Arg (sequence number: 16).

3. The particle according to claim 2, wherein X contains Arg.

4. The particle according to claim 1, wherein the first stretch comprises eight amino acids adjacent to the site.

5. The particle according to claim 1, comprising the modification in the first stretch.

6. The particle according to claim 1, comprising the modification in the second stretch.

7. The particle according to claim 1, wherein the polyprotein contains prM.

8. The particle according to claim 1, wherein the polyprotein contains prE2.

9. The particle according to claim 1, comprising the dengue virus (DV).

10. The particle according to claim 1, comprising the Zika virus (ZV).

11. A composition comprising the particles described in claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

12. An insect cell containing the particles described in claim 1.

13. The cell according to claim 12, wherein the insect includes arthropods.

14. The cell according to claim 12, wherein the insect includes a tick or a mosquito.

15. A mammalian cell containing the particles described in claim 1.

16. The cell according to claim 15, wherein the cell expresses the DV antigen.

17. The cell according to claim 15, wherein the cell expresses a DV antigen on the surface of the cell.

18. The cell according to claim 15, wherein the cell expresses the ZV antigen.

19. The cell according to claim 15, wherein the cell expresses the ZV antigen on the surface of the cell.

20. The cell according to claim 15, which includes human cells.

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