Zika virus polypeptide

Abstract

To provide vaccine compositions capable of increasing an immune response against a flavivirus, such as Zika virus, in a mammal (e.g., a human), and to provide methods of increasing the immune response.SOLUTION: This document provides methods and materials related to selected Zika virus polypeptides. For example, vaccine compositions are provided that contain one or more selected Zika virus polypeptides provided herein and have the ability to increase an immune response against a flavivirus, such as Zika virus, in a mammal (e.g., a human).SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 984,520, filed March 3, 2020. The disclosure of the prior application is considered part of (and incorporated by reference into) the disclosure of this application. [Technical Field]

[0002] This document provides methods and materials related to selected Zika virus polypeptides. For example, this document provides vaccine compositions that contain one or more selected Zika virus polypeptides provided herein and that are capable of increasing an immune response to a flavivirus, such as a Zika virus, in a mammal (e.g., a human). [Background technology]

[0003] Zika virus (ZIKV), a previously obscure pathogen, has emerged as a global public health problem. Since 2007, ZIKV has spread throughout the Pacific and South America, resulting in highly publicized outbreaks between 2015 and 2016 (Gatherer et al., J. Gen. Virol. 97:269-273 (2016)). Localized ZIKV transmission has also been reported in the continental United States, with approximately 60% of the U.S. population living in areas that allow seasonal transmission by Aedes mosquito vectors. Compelling evidence links ZIKV infection in pregnant women to a number of fetal malformations and neurological abnormalities, highlighting the need for effective vaccines to combat the spread of this emerging disease (Krauer et al., PLOS Med. 14:e1002203 (2017)). Summary of the Invention

[0004] This document provides methods and materials related to selected Zika virus polypeptides. For example, this document provides isolated polypeptides set forth in Tables 1, 2, and 3. In some cases, the selected Zika virus polypeptides provided herein can be substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17. This document also provides vaccine compositions containing one or more selected Zika virus polypeptides provided herein and capable of increasing an immune response to a flavivirus such as Zika virus in a mammal (e.g., a human), methods and materials for making vaccine compositions containing one or more selected Zika virus polypeptides provided herein and capable of increasing an immune response to a flavivirus such as Zika virus in a mammal (e.g., a human), kits comprising one or more selected Zika virus polypeptides provided herein, methods of using such kits to identify a mammal (e.g., a human) as having been previously or currently infected with Zika virus, and methods of using such kits to identify a mammal (e.g., a human) as having humoral immunity specific to a flavivirus such as Zika virus.

[0005] As described herein, select Zika virus polypeptides have been identified as having the ability to induce a broad recall immune response against Zika virus.

[0006] In general, one embodiment of this document features a substantially pure polypeptide consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17. The polypeptide can be covalently conjugated to a stabilizer selected from the group consisting of sucrose, lactose, monosodium salt of glutamic acid, human serum albumin, and gelatin.

[0007] In another aspect, this document features a composition including at least four polypeptides, each of the at least four polypeptides consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17. Each of the at least four polypeptides can be a polypeptide ...9. The composition can include a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 9. The composition can include an adjuvant. The adjuvant may be selected from the group consisting of CpG oligonucleotide motifs, aluminum sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, monophosphoryl lipid A, aluminum phosphate, MF59, AS03, and AS04.

[0008] In another aspect, this document features a method for increasing an immune response to a flavivirus in a mammal. The method includes administering to the mammal a composition comprising a polypeptide consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-17. The mammal can be a human. The flavivirus can be a Zika virus. The composition can include an adjuvant. The adjuvant can be selected from the group consisting of a CpG oligonucleotide motif, aluminum sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, monophosphoryl lipid A, aluminum phosphate, MF59, AS03, and AS04. The composition can include at least four polypeptides, each of which is a polypeptide consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-17. Each of the at least four polypeptides can be a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-17. Each of the at least four polypeptides can be a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-9. The composition may comprise a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 9.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains.Although the present invention can be carried out using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.In addition, materials, methods, and examples are only illustrative and are not intended to be limiting.

[0010] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0011] [Figure 1] IFN-γ ELISpot analysis focusing on responses to candidate polypeptides (left cluster) and comparison polypeptides (right cluster). ZIKV responses for all subjects are shown for comparison. SFU refers to spot-forming units and represents the number of T cells that recognize each peptide by secreting IFN-γ. DETAILED DESCRIPTION OF THE INVENTION

[0012] This document provides methods and materials related to selected Zika virus polypeptides. For example, this document provides isolated polypeptides set forth in Tables 1, 2, and 3. In some cases, the selected Zika virus polypeptides provided herein can be substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 1-17. The term "substantially pure," as used herein with respect to a polypeptide, means that the polypeptide is substantially free from other polypeptides, lipids, carbohydrates, and nucleic acids with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that has been removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In some cases, the substantially pure polypeptides provided herein can be polypeptides that are synthesized to have a purity of at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent. [Table 1] [Table 2]

[0013] In some cases, a Zika virus polypeptide provided herein consisting essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 1-17 is a polypeptide having zero, one, or two amino acid substitutions within the clearly expressed sequence of a sequence identifier (e.g., SEQ ID NO: 1), zero, one, two, three, four, or five amino acid residues before the clearly expressed sequence of a sequence identifier (e.g., SEQ ID NO: 1), and / or zero, one, two, three, four, or five amino acid residues after the clearly expressed sequence of a sequence identifier (e.g., SEQ ID NO: 1), provided that the Zika virus polypeptide has the ability to increase an immune response to a flavivirus, such as a Zika virus, in a mammal (e.g., a human). Examples of Zika virus polypeptides consisting essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 1-17 are shown in Table 3. [Table 3-1] [Table 3-2]

[0014] The polypeptides provided herein (e.g., substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) can be of any suitable length. For example, the polypeptides provided herein (e.g., substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) can be from 9 amino acid residues to 100 amino acid residues in length (e.g., from 9 amino acid residues to 90 amino acid residues, from 9 amino acid residues to 80 amino acid residues, from 9 amino acid residues to 70 amino acid residues, from 9 amino acid residues to 60 amino acid residues, from 9 amino acid residues to 50 amino acid residues, from 9 amino acid residues to 40 amino acid residues, from 9 amino acid residues to 35 amino acid residues, from 9 amino acid residues to 30 amino acid residues, from 9 amino acid residues to 25 amino acid residues, from 9 amino acid residues to 24 amino acid residues, from 9 amino acid residues to 23 amino acid residues, from 9 amino acid residues to 22 amino acid residues, from 9 amino acid residues to 21 amino acid residues, from 9 amino acid residues to 20 amino acid residues, from 9 amino acid residues to 19 amino acid residues, from 9 amino acid residues to 19 amino acid residues, from 9 amino acid residues to 20 amino acid residues, from 9 amino acid residues to 21 ... 18 amino acid residues, 9 amino acid residues to 17 amino acid residues, 9 amino acid residues to 16 amino acid residues, 9 amino acid residues to 15 amino acid residues, 10 amino acid residues to 100 amino acid residues, 11 amino acid residues to 100 amino acid residues, 12 amino acid residues to 100 amino acid residues, 13 amino acid residues to 100 amino acid residues, 14 amino acid residues to 100 amino acid residues, 15 amino acid residues to 100 amino acid residues, 16 amino acid residues Acid residues to 100 amino acid residues, 17 amino acid residues to 100 amino acid residues, 18 amino acid residues to 100 amino acid residues, 19 amino acid residues to 100 amino acid residues, 20 amino acid residues to 100 amino acid residues, 21 amino acid residues to 100 amino acid residues, 22 amino acid residues to 100 amino acid residues, 23 amino acid residues to 100 amino acid residues, 24 amino acid residues to 100 amino acid residues, 25 amino acid residues to 100 amino acid residues,It can be 10 to 80 amino acid residues, 10 to 50 amino acid residues, 10 to 30 amino acid residues, 10 to 25 amino acid residues, or 10 to 20 amino acid residues).

[0015] A polypeptide provided herein (e.g., a substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) can be capable of increasing an immune response to a flavivirus, such as Zika virus, in a mammal (e.g., a human). For example, after administering a polypeptide provided herein (e.g., a substantially pure polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) or a nucleic acid encoding a polypeptide provided herein (e.g., a nucleic acid encoding a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) to a mammal (e.g., a human), the mammal can generate an increased immune response (e.g., an increased antibody response and / or an increased T cell response) to a flavivirus, such as Zika virus. Any suitable method can be used to identify the development of an increased immune response to a flavivirus, such as Zika virus. For example, a kit provided herein comprising one or more polypeptides provided herein can be used to evaluate a sample obtained from the mammal being tested for the presence, absence, or level of antibodies capable of binding to the polypeptides.

[0016] Any suitable method can be used to obtain the polypeptides provided herein (e.g., substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17). For example, the polypeptides provided herein (e.g., substantially pure polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) can be obtained by isolating the polypeptide of interest from a cell that expresses the polypeptide (e.g., a cell engineered to express the polypeptide of interest from an exogenous nucleic acid encoding the polypeptide, or a cell infected with a virus (e.g., Zika virus) that expresses the polypeptide of interest from the virus), or by synthesizing the polypeptide of interest using suitable solid-phase peptide synthesis techniques, such as those described elsewhere (Introduction to Peptide Synthesis. Gregg B. Fields. Current Protocols in Protein Science. Vol. 26, Issue 1: pp. 18.1.1-18.1.9 (2001)).

[0017] This document also provides nucleic acids encoding the polypeptides provided herein (e.g., polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17). For example, this document provides plasmids and viral vectors containing nucleic acids encoding the polypeptides provided herein (e.g., polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) in a manner such that the polypeptides can be expressed intracellularly. In some cases, nucleic acids encoding the polypeptides provided herein (e.g., polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17) can include a regulatory nucleic acid sequence (e.g., a promoter sequence) operably linked to the sequence encoding the polypeptide such that the polypeptide is expressed intracellularly. Examples of promoter sequences that can be used as described herein include, but are not limited to, a CMV promoter, an EF1a promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a CAG promoter, a tetracycline response element promoter, and an H1 promoter.

[0018] This document also provides compositions comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of the polypeptides provided herein and / or nucleic acids encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of the polypeptides provided herein. For example, the compositions provided herein can include at least four (e.g., 4, 5, 6, 7, or 8) of the polypeptides (or nucleic acids encoding those polypeptides) set forth in Table 1. In some cases, the compositions provided herein can include each of the polypeptides (or nucleic acids encoding those polypeptides) set forth in Table 1. In some cases, the compositions provided herein can include at least four (e.g., four, five, six, or seven) of the polypeptides (or nucleic acids encoding those polypeptides) set forth in Table 2. In some cases, the compositions provided herein can include each of the polypeptides (or nucleic acids encoding those polypeptides) set forth in Table 2. Examples of other specific combinations of polypeptides that can be used to make the compositions provided herein non-specifically include those set forth in Table 4. [Table 4]

[0019] Any suitable method can be used to formulate the compositions provided herein (e.g., compositions comprising one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the polypeptides provided herein and / or nucleic acids encoding one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more of the provided polypeptides). For example, one or more polypeptides provided herein (and / or nucleic acids encoding such one or more polypeptides) can be combined with a pharmaceutically acceptable carrier and / or pharmaceutical excipient. The term "pharmaceutically acceptable" generally refers to a compound that is non-toxic, inert, and / or physiologically compatible. The term "pharmaceutical excipient" includes materials such as carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, coloring agents, and preservatives.

[0020] In some cases, the compositions provided herein (e.g., compositions comprising one or more of the polypeptides provided herein and / or nucleic acids encoding one or more of the polypeptides provided herein) can be vaccine compositions. For example, a composition containing four or more of the polypeptides set forth in SEQ ID NOS: 1-9 can be formulated into a polypeptide-based vaccine for use in mammals (e.g., humans). Any suitable method can be used to produce polypeptide-based vaccines, such as those described elsewhere (Belyakov et al., Proc. Natl. Acad. Sci. USA, 95:1709-1714 (1998); Jackson et al., Proc. Natl. Acad. Sci. USA, 101:15440-15445 (2004); Makarkov et al., NPJ Vaccines, 15(4):17 (2019)), Hekele et al., Emerg. Microbes Infect., 2(8):e52 (2013); Parlane et al., Biochem. Biophys. Res. Commun., pii:S0006-291X(20)30264-3 (2020), and Bounds et al. al., Hum. Vaccin. Immunother., 13(12):2824-2836 (2017)). In some cases, the vaccine compositions provided herein can include one or more polypeptides provided herein (e.g., four or more different polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any of SEQ ID NOS: 1-17) in combination with one or more adjuvants. Examples of adjuvants that can be included in the vaccine compositions provided herein include CpG oligonucleotide motifs, aluminum (e.g., aluminum salts such as aluminum sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), monophosphoryl lipid A, aluminum hydroxyphosphate sulfate, MF59, AS03, AS04, CpG1018, and ASO1. BAdjuvants included in the vaccine compositions provided herein include, but are not limited to, non-natural (e.g., artificial) adjuvants. In some cases, the vaccine compositions provided herein can include one or more polypeptides provided herein (e.g., four or more different polypeptides comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1-17), one or more adjuvants, and one or more pharmaceutically acceptable carriers and / or pharmaceutical excipients.

[0021] In some cases, the polypeptides of the vaccine compositions provided herein can be conjugated to, for example, polysaccharides (e.g., sucrose or lactose), amino acids (e.g., glycine or the monosodium salt of glutamic acid), and / or proteins (e.g., human serum albumin or gelatin) to improve the stability or immunogenicity of the vaccine composition. In some cases, the polypeptides provided herein can be formulated into the vaccine composition in combination with a delivery vehicle such as a nanoparticle. For example, four or more polypeptides provided herein can be included within a nanoparticle (e.g., embedded or displayed on its surface).

[0022] In some cases, the vaccine compositions provided herein can be multivalent vaccine compositions capable of increasing immune responses in mammals (e.g., humans) against multiple members of the Flavivirus family. For example, the vaccine compositions provided herein can be capable of increasing immune responses against Zika virus, Dengue virus, West Nile virus, Yellow fever virus, or any combination thereof. In some cases, the vaccine compositions provided herein can be used as multivalent vaccine compositions capable of increasing immune responses against one or more lineages, clades, or strains of Zika virus. For example, the vaccine compositions provided herein can be capable of increasing immune responses against East African Zika virus, West African Zika virus, Asian Zika virus, South American Zika virus, or any combination thereof.

[0023] This document also provides methods for increasing the immune response to a flavivirus, such as Zika virus, in a mammal (e.g., a human). For example, the vaccine compositions provided herein can be administered to a mammal (e.g., a human) to increase the immune response to a flavivirus, such as Zika virus (e.g., increasing an antibody response and / or increasing a T cell response). Any suitable mammal can be administered a vaccine composition provided herein to increase the immune response to a flavivirus, such as Zika virus, in the mammal. For example, humans, non-human primates (e.g., monkeys or apes), horses, dogs, cats, bovine species, pigs, sheep, mice, rats, goats, ducks, water buffalo, and bats can be administered a vaccine composition provided herein to increase the immune response to a flavivirus, such as Zika virus. In some cases, the vaccine compositions provided herein can be administered to a mammal identified as needing an increased immune response to a flavivirus, such as Zika virus. For example, a person identified as having recent contact (e.g., within one to two weeks) with one or more people who have or are suspected of having a flavivirus infection (e.g., Zika virus infection) can be identified as needing an increased immune response to a flavivirus, such as Zika virus, and can be administered a vaccine composition provided herein. In some cases, a person traveling or planning to travel to a location where flavivirus infection (e.g., Zika virus infection) is widespread or where a flavivirus infection (e.g., Zika virus infection) is suspected to have previously occurred as an outbreak can be identified as needing an increased immune response to a flavivirus, such as Zika virus, and can be administered a vaccine composition provided herein. In some cases, a pregnant mammal (e.g., a pregnant human) can be administered a vaccine composition provided herein to increase the immune response to a flavivirus, such as Zika virus.

[0024] This document also provides methods for treating a mammal (e.g., a human) infected with a flavivirus, such as Zika virus. For example, the vaccine compositions provided herein can be administered to a mammal (e.g., a human) with a Zika virus infection to reduce the severity of the Zika virus infection. Any suitable mammal can be administered the vaccine compositions provided herein to treat a flavivirus infection, such as a Zika virus infection. For example, humans, non-human primates (e.g., monkeys or apes), horses, dogs, cats, bovine species, pigs, sheep, mice, rats, goats, ducks, water buffalo, and bats can be administered the vaccine compositions provided herein to treat a flavivirus infection, such as a Zika virus infection. In some cases, a pregnant mammal (e.g., a pregnant human) can be administered the vaccine compositions provided herein to treat a flavivirus infection, such as a Zika virus infection. In some cases, a mammal identified as having a flavivirus infection, such as a Zika virus infection, can be administered the vaccine compositions provided herein to treat the infection. Any suitable method can be used to identify a mammal as having a flavivirus infection, such as a Zika virus infection. For example, an immunoassay can be used to identify a mammal (e.g., a human) as having antibodies specific to a flavivirus (e.g., Zika virus). In some cases, the presence of flavivirus nucleic acid (e.g., Zika virus nucleic acid) in a sample obtained from the mammal is detected, thereby indicating that the mammal has a flavivirus infection. Any suitable sample can be obtained from the mammal to be tested and evaluated as described herein. For example, a biological sample, such as a fluid sample (e.g., blood (e.g., whole blood, plasma, and serum), urine, breast milk, saliva, amniotic fluid, cerebrospinal fluid, or semen) or a tissue sample (e.g., a placental tissue sample), can be obtained from the mammal and evaluated as described herein.

[0025] In some cases, the vaccine compositions provided herein can be administered to a mammal (e.g., a human) with a flavivirus infection (e.g., Zika virus infection) under conditions effective to reduce the duration and / or severity of one or more symptoms or disease complications of the infection. Symptoms of flavivirus infection include, but are not limited to, fever, skin rash (e.g., papular rash), muscle pain, joint pain, back pain, conjunctivitis, vomiting, headache, fatigue, weakness, swollen extremities, diarrhea, loss of appetite, dizziness, and more severe symptoms such as jaundice, renal failure, systemic shock, and death.

[0026] When administering the compositions provided herein (e.g., vaccine compositions) to a mammal (e.g., a human), any suitable route of administration can be used. For example, the compositions provided herein (e.g., vaccine compositions) can be administered to a mammal (e.g., a human) intravenously (e.g., via intravenous injection or infusion), subcutaneously (e.g., via subcutaneous injection), intraperitoneally (e.g., via intraperitoneal injection), orally, via inhalation, or intramuscularly (e.g., via intramuscular injection). In some cases, the route and / or mode of administration of the compositions provided herein (e.g., vaccine compositions) can be tailored to the mammal being treated.

[0027] An effective dose of a composition (e.g., a vaccine composition) provided herein can vary depending on the route of administration, the age and general health of the subject, the use of excipients, the possibility of coadministration with other therapeutic treatments, such as the use of other drugs, and the judgment of the treating physician. In some cases, an effective amount of a composition (e.g., a vaccine composition) provided herein can be an amount that increases the immune response in a mammal (e.g., a human) to a flavivirus, such as Zika virus, without causing significant toxicity to the mammal. For example, an effective amount of a composition (e.g., a vaccine composition) provided herein can be about 3 μg / dose to about 150 μg / dose (e.g., 3 μg / dose to 150 μg / dose, 5 μg / dose to 150 μg / dose, 10 μg / dose to 150 μg / dose, 15 μg / dose to 150 μg / dose, 20 μg / dose to 150 μg / dose, 25 μg / dose to 150 μg / dose). The amount may be 30 μg / dose to 150 μg / dose, 3 μg / dose to 125 μg / dose, 3 μg / dose to 100 μg / dose, 3 μg / dose to 90 μg / dose, 3 μg / dose to 75 μg / dose, 10 μg / dose to 125 μg / dose, 15 μg / dose to 100 μg / dose, 15 μg / dose to 90 μg / dose, or 20 μg / dose to 75 μg / dose). In some cases, an effective amount of a composition (e.g., a vaccine composition) provided herein can range from about 3 μg of total Zika virus polypeptide content of the composition to about 150 μg of total Zika virus polypeptide content of the composition (e.g., 3 μg to 150 μg, 5 μg to 150 μg, 10 μg to 150 μg, 15 μg to 150 μg, 20 μg to 150 μg, 25 μg to 150 μg, 30 μg to 150 μg, 3 μg to 125 μg, 3 μg to 150 ... The total Zika virus polypeptide content may be between 1 and 20 μg (a total Zika virus polypeptide content of between 1 and 20 μg, ...

[0028] This document also provides kits that include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) polypeptides provided herein (e.g., one or more substantially pure polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 1-17). For example, the kits provided herein may include at least four (e.g., 4, 5, 6, 7, or 8) of the polypeptides set forth in Table 1. In some cases, the kits provided herein may include each of the polypeptides set forth in Table 1. In some cases, the kits provided herein may include at least four (e.g., 4, 5, 6, or 7) of the polypeptides set forth in Table 2. In some cases, the kits provided herein may include each of the polypeptides set forth in Table 2.

[0029] In some cases, the kits provided herein can be used to detect an immune response (e.g., a humoral antibody response or a cellular immune response) in a mammal (e.g., a human). For example, cells obtained from a mammal (e.g., a human) can be incubated with a kit provided herein containing antigen-presenting cells that present one or more of the polypeptides provided herein to detect the presence or absence of antigen-specific T cells capable of recognizing one or more of the polypeptides included in the kit. In some cases, the kits provided herein can be used to detect antigen-specific T cells after vaccination of a mammal (e.g., a mammal administered a vaccine composition provided herein) to determine the effectiveness of immunization. In some cases, the kits provided herein can be used to detect HLA class II-restricted T helper cells capable of recognizing one or more of the polypeptides included in the kit. Any suitable technique can be used to determine the presence or absence of cells (e.g., T cells such as HLA class II-restricted T helper cells) capable of recognizing one or more of the polypeptides included in the kit provided herein. For example, flow cytometry, enzyme-linked immunospot (ELISPOT), cytokine secretion, direct cytotoxicity assay, and lymphoproliferation assay can be used to detect antigen-specific T cells. In some cases, cytokine production and / or degranulation can be used as markers to determine the presence or absence of cells (e.g., T cells, such as HLA class II-restricted T helper cells) capable of recognizing one or more of the polypeptides included in the kits provided herein. Examples of cytokines that can be assessed include, but are not limited to, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 10 (IL-10), interferon alpha (IFN-alpha), transforming growth factor beta (TGF-beta), interleukin (IL-12), and interleukin 17 (IL-17).Examples of degranulation markers that can be evaluated include, but are not limited to, intracellular expression of perforin, intracellular expression of granzyme B, or cell surface expression of CD107a. In some cases, the kits provided herein may include one or more of the polypeptides provided herein in the form of an MHC-polypeptide tetramer that is labeled (e.g., covalently labeled) with a fluorescent dye. In such cases, the labeled MHC-polypeptide tetramers of the kits provided herein can be used to bind to antigen-specific T cells in a sample, and the bound cells can be counted by flow cytometry.

[0030] As described herein, the methods and materials provided herein can be used to increase an immune response to a flavivirus in a mammal (e.g., a human), can be used to treat a mammal (e.g., a human) infected with a flavivirus, can be used to identify a mammal (e.g., a human) as having a flavivirus infection, and / or can be used to identify a mammal (e.g., a human) as having an immune response (e.g., a humoral antibody response or a cellular immune response) to a flavivirus. Examples of such flaviviruses include, but are not limited to, Zika virus, dengue virus, West Nile virus, yellow fever virus, Spondweni virus, Japanese encephalitis virus, St. Louis encephalitis virus, Powassan virus, tick-borne encephalitis virus, Kyasanur Forest disease virus, deer tick virus, Omsk hemorrhagic fever virus, Entebbe virus, Modoc virus, and Rio Bravo virus. When the flavivirus is a Zika virus, the Zika virus can be any appropriate lineage, clade, or strain of Zika virus. Examples of such Zika viruses include, but are not limited to, East African Zika virus, West African Zika virus, Asian / Pacific Zika virus, and Asian / American Zika virus.

[0031] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0032] Example 1: Peptide-based vaccine development against Zika virus In this example, immortalized human B cells expressing HLA-A*0201 and HLA-DRB1*0401 were infected with ZIKV, and polypeptides were isolated by denaturing HLA molecules on the cell surface using an acidic buffer. The polypeptides were identified using two-dimensional (2D) liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). Subsequently, a series of computational methods were used to analyze the polypeptides and evaluate their antigenic properties and binding to different HLA molecules. The synthetic polypeptides were used to screen IFN-γ T cell responses in immune cells from convalescent subjects.

[0033] Identification of polypeptides by mass spectrometry Immortalized B cells homozygous for HLA-A*02:01 (A2 supertype) and HLA-DRB1*04:01 (DR4 supertype) were infected with ZIKV (PRVABC59, MOI = 0.1) and cultured for 48 hours to allow for sufficient processing and presentation of viral proteins. Cells were treated with acidic citrate-phosphate buffer (pH = 3.0) to denature HLA molecules, and polypeptides were isolated from protein contaminants by centrifugal filtration. The polypeptides were further purified by fractionation using strong cation exchange (SCX) chromatography and analyzed by nanoscale liquid chromatography-tandem mass spectrometry (nLC-MS / MS). Deconvolution analysis using UniProt identified 90 polypeptides derived from viral proteins: 59 from nonstructural (NS) protein 1 (NS1), 2 from NS2A, 7 from NS3, 4 from NS4B, 8 from NS5, 8 from capsid (C), and 2 from envelope (E). Redundant sequences or polypeptides with a high degree of sequence similarity (e.g., sequences nested within larger sequences) were excluded from further analysis, narrowing the list to 34 unique polypeptides for testing. Polypeptides were individually synthesized in large batches (5 mg) for functional testing.

[0034] Target for recovery period Peripheral blood mononuclear cells (PBMCs) from seven healthy human donors previously confirmed to have ZIKV infection were used. The subjects are represented herein by their unique numerical study identifiers: 591, 596, 602, 625, 626, 627, and 629. Subject 591 provided samples on two separate days, approximately 21 and 138 days post-infection. These samples are subsequently designated 591-1 and 591-3, respectively.

[0035] IFN-γ T cell ELISpot recall response Polypeptide pool stimulation Viral polypeptides were randomly sorted into pools of 8–9 polypeptides with overlap between adjacent pools. PBMCs (2 × 10 5 Cells (1000 cells / well) were seeded into 96-well polyvinylidene fluoride (PVDF)-lined microtiter plates coated with anti-human IFN-γ antibody and treated with one of the following conditions: culture medium (unstimulated), 20 μg of pooled ZIKV polypeptides, ZIKV (MOI = 1), or 20 μg of pooled actin polypeptides as a negative control. Cells were incubated for 18 hours, and immune responses were quantified using a human IFN-γ ELISpot kit. Additionally, data were grouped by subject to better visualize the unique recall response profile of each individual. Samples stimulated with ZIKV polypeptides or live virus were tested in triplicate, while unstimulated samples and negative controls were tested in quadruplicate. Responses were highly variable between polypeptide pools and individual subjects; pools that stimulated a positive IFN-γ ELISpot response in at least one subject were subsequently analyzed at the individual polypeptide level for responding subjects.

[0036] Individual polypeptide stimulation PBMCs (2 × 10 5Cells (1000 cells / well) were seeded into 96-well PVDF-backed microtiter plates coated with anti-human IFN-γ antibody and treated with one of the following conditions: culture medium (unstimulated), 10 μg of individual ZIKV polypeptides, ZIKV (MOI = 1), or 20 μg of pooled actin polypeptides as a negative control. Cells were incubated for 18 hours, and immune responses were quantified using a human IFN-γ ELISpot kit. Samples stimulated with ZIKV polypeptides or live virus were tested in triplicate, while unstimulated samples and the negative control were tested in quadruplicate. Nine polypeptides (Table 1) that stimulated positive recall immune responses in four of seven subjects were selected as major ZIKV-derived polypeptides for detailed informatics analysis. Eight polypeptides that stimulated limited recall responses (one of seven subjects, Table 2) were selected for comparison.

[0037] Modeling polypeptide structure and properties The structures of all ZIKV-derived and comparative polypeptides shown in SEQ ID NOS: 1-9 were modeled using PEP-FOLD 3.5, an online server that predicts polypeptide structure based on the properties of each amino acid in the sequence. The properties of individual polypeptides in both groups were determined in silico using the Protparam tool hosted on the Expasy server. The polypeptides shown in SEQ ID NOS: 1-9 were predicted to have a largely ordered structure with some degree of helical conformation when modeled under physiological conditions. In contrast, the majority of the comparative polypeptides (5 out of 8) adopted a largely disordered structure exhibiting a high degree of coiled and extended structural features under identical modeling parameters. The ZIKV-derived polypeptides shown in SEQ ID NOS: 1-9 were predicted to have a longer theoretical half-life than the comparative group (16.21 h vs. 8.99 h), which correlated with the average instability index of the two groups (24.72 vs. 43.75). A larger instability index (>40) indicates an unstable polypeptide structure. The ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9 were also predicted to have a greater mean aliphatic index than the comparator (125.41 vs. 103.67), which is a positive metric for thermal stability. The overall mean hydropathicity (GRAVY) index differed between the ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9 and the comparator (0.27 vs. -0.24), indicating that the comparator polypeptides were slightly more hydrophilic. Polypeptide stability can be used in the design of polypeptide-based vaccines and therapeutics, and collectively, these modeling data suggest that the ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9 are stable for formulation and use as components of a ZIKV vaccine.

[0038] Optimized IFN-γ ELISpot recall response The analysis of IFN-γ ELISpot recall responses was modified to focus only on the cluster of ZIKV-derived and comparison polypeptides set forth in SEQ ID NOS: 1-9. While a tight cluster of polypeptides eliciting recall responses was observed among convalescent subjects, as expected, for the ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9, the comparison cluster exhibited a very narrow response profile across subjects. Subjects 602 and 625 were the strongest responders in the comparison group, with subject 625 responding to four of the eight epitopes (Figure 1). Three subjects (626, 596, and 629) did not respond to any of the comparison group polypeptides. In contrast, all subjects exhibited recall responses to at least three of the ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9, illustrating the breadth of coverage provided by the ZIKV-derived polypeptides set forth in SEQ ID NOS: 1-9.

[0039] Other embodiments While the present invention has been described in conjunction with its detailed description, the above description is intended to be illustrative, not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A substantially pure polypeptide consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17.

2. 2. The substantially pure polypeptide of claim 1, wherein the polypeptide is covalently conjugated to a stabilizer selected from the group consisting of sucrose, lactose, monosodium salt of glutamic acid, human serum albumin, and gelatin.

3. A composition comprising at least four polypeptides, each of the at least four polypeptides consisting essentially of or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1-17.

4. The composition according to claim 3, wherein each of the at least four polypeptides is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17.

5. The composition according to claim 3, wherein each of the at least four polypeptides is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

9.

7. The composition of any one of claims 1 to 6, wherein the composition comprises an adjuvant.

8. 8. The composition of claim 7, wherein the adjuvant is selected from the group consisting of CpG oligonucleotide motifs, aluminum sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, monophosphoryl lipid A, aluminum phosphate, MF59, AS03, and AS04.

9. A method for increasing an immune response to a flavivirus in a mammal, the method comprising administering to the mammal a composition comprising a polypeptide consisting essentially of or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1-17.

10. 10. The method of claim 9, wherein the mammal is a human.

11. The method of any one of claims 9 to 10, wherein the flavivirus is Zika virus.

12. The method of any one of claims 9 to 11, wherein the composition comprises an adjuvant.

13. 13. The method of claim 12, wherein the adjuvant is selected from the group consisting of CpG oligonucleotide motifs, aluminum sulfate, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, monophosphoryl lipid A, aluminum phosphate, MF59, AS03, and AS04.

14. 14. The method of any one of claims 9 to 13, wherein the composition comprises at least four polypeptides, and each of the at least four polypeptides is a polypeptide consisting essentially of or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17.

15. The method according to claim 14, wherein each of the at least four polypeptides is a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 17.

16. The method according to claim 14, wherein each of the at least four polypeptides is a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 9.

17. The method of any one of claims 9 to 16, wherein the composition comprises a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

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