CN112521453A

CN112521453A - Zika virus dominant T cell epitope peptide and application thereof in vaccines and diagnosis

Info

Publication number: CN112521453A
Application number: CN201910888604.7A
Authority: CN
Inventors: 金侠; 孙瑾; 郑智航
Original assignee: Institut Pasteur of Shanghai of CAS
Current assignee: Institut Pasteur of Shanghai of CAS
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2021-03-19

Abstract

本发明提供了一种寨卡病毒表位肽及其应用。具体地，本发明提供了一种针对寨卡病毒的CD4+、CD8+T细胞表位肽及其组合，包括SEQ ID NO.1‑27所示的多肽。本发明的表位肽既包括CD4+T细胞表位，又包括CD8+T细胞表位，均能够在小鼠体内激起较强的T细胞免疫反应，并提供针对寨卡病毒的保护作用。本发明的寨卡病毒表位肽还可以用于寨卡病毒的检测，具有广泛的应用。The present invention provides a Zika virus epitope peptide and its application. Specifically, the present invention provides a CD4+, CD8+ T cell epitope peptide and a combination thereof against Zika virus, including the polypeptides shown in SEQ ID NO. 1-27. The epitope peptide of the present invention includes both CD4+ T cell epitopes and CD8+ T cell epitopes, and both can stimulate a strong T cell immune response in mice and provide protection against Zika virus. The Zika virus epitope peptide of the present invention can also be used for the detection of Zika virus, and has a wide range of applications.

Description

Zika virus dominant T cell epitope peptide and application thereof in vaccines and diagnosis

Technical Field

The invention relates to the fields of biomedicine and immunology, in particular to a dominant epitope peptide of a CD4+ and a CD8+ T cell of Zika virus and application thereof.

Background

Zika virus was first discovered in 1947 in Wuganzhai Kasanlin, but it has not received wide attention from international society until the epidemic outbreak in south America in 2015. Zika virus belongs to the flavivirus family of flaviviridae, and is widely prevalent with dengue virus in tropical and subtropical regions. The surface of the virus is covered with an envelope, and the inner nucleocapsid forms an icosahedral structure, and the genome of the icosahedral structure is positive-sense single-stranded RNA with the size of about 11 kb. In general, Zika virus infection causes mild symptoms in humans, including fever, macula, arthralgia, and conjunctivitis, but it has also recently been found that Zika virus is associated with neurological complications, including neonatal ocular vision disorders, microcephaly, hydrocephalus, and epilepsy due to maternal infections during pregnancy, and Guillain-Barre syndrome associated with Zika virus, which is manifested in adults. The possible vertical transmission, sexual transmission, blood transmission and other transmission ways of the Zika virus also cause that the Zika virus is harder to prevent and treat and is more likely to cause serious public health crisis.

Currently, there is no commercially licensed Zika vaccine available, and most of the Zika vaccines in the clinical research stage focus on enhancing the production of neutralizing antibodies specific to Zika viruses. However, antibody immunization with Zika virus may have a negative effect, i.e., antibodies to Zika virus can assist dengue virus in enhancing infection (ADE) of a body or cells in vivo or in vitro, and vice versa. Therefore, the vaccine based on antibody reaction is used alone in the epidemic area where two viruses are co-epidemic, and a great hidden danger is inevitably caused. On the other hand, memory CD4+ and CD8+ T cell responses of Zika virus have been shown to have protective effects against Zika virus. Therefore, the determination of the T cell epitope of Zika virus and the development of T cell vaccines will be an important complement to the existing Zika T cell immune research and vaccine development. In addition, researches show that the distribution of dominant T cell epitopes stimulated by Zika virus infection and dengue virus infection has large difference, and the method can be applied to diagnosis and differentiation of infection of the two viruses in the future.

The functions and action mechanisms of CD8+ T cells and CD4+ T cells (including Th1 and Th2 cells) are different, and previous researches show that the CD8+ T cells can assist the body to eliminate viruses in the first infection process of the Zika virus and play a role in protection. CD4+ T cells were shown to produce protection against secondary infection only by pro-inflammatory cytokines and supporting antibody responses. Therefore, in the course of studying the T cell responses of zika virus and developing T cell vaccines, two types of epitopes and responses still need to be studied separately.

In conclusion, the development of CD4+, CD8+ T cell dominant epitope peptide of Zika virus and application thereof are urgently needed in the field.

Disclosure of Invention

The invention aims to provide CD4+ and CD8+ T cell dominant epitope peptide of Zika virus and related application thereof.

In a first aspect of the present invention, there is provided a combination of T cell epitope peptides against zika virus, said combination of epitope peptides comprising two or more polypeptides selected from the group consisting of:

(a) a polypeptide as set forth in SEQ ID No. 1-27;

(b) 1-27 amino acid sequence through one, two or three amino acid residue substitution, deletion or addition, and can be combined with T cell surface receptor;

(c) and SEQ ID NO:1-27, and can be combined with T cell surface receptor;

wherein the epitope peptides in the combination of epitope peptides are not directed against a T cell surface receptor (TCR).

In another preferred embodiment, the polypeptides (b) and (c) are immunogenic.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise one or more than one polypeptide selected from the group consisting of the polypeptides shown in SEQ ID NO. 1-11.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise one or more than one polypeptide selected from the group consisting of the polypeptides shown in SEQ ID No. 5, 6, 7, 8 and 10.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise the polypeptides shown in SEQ ID No. 1-11, preferably comprise the polypeptides shown in SEQ ID No. 5, 6, 7, 8 and 10, and the epitope peptide combination is a CD4+ T cell epitope peptide combination.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise one or more than one polypeptide selected from the group consisting of the polypeptides shown in SEQ ID No.1, 7, 11-27.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise one or more than one polypeptide selected from the group consisting of the polypeptides shown in SEQ ID No.11, 16, 20, 21 and 22.

In another preferred embodiment, the epitope peptide and the epitope peptide combination thereof at least comprise the polypeptide shown in SEQ ID No.1, 7, 11-27, preferably comprise the polypeptide shown in SEQ ID No.11, 16, 20, 21, 22, and the epitope peptide combination is a CD8+ T cell epitope peptide combination.

In another preferred example, the epitope peptide and the epitope peptide combination thereof at least comprise the polypeptide shown in SEQ ID No.1, 7 and 11.

In another preferred example, the epitope peptides shown in SEQ ID NO.1, 7 and 11 are CD4+ T cell epitope peptide and CD8+ T cell epitope peptide.

In another preferred embodiment, the epitope peptide combination comprises both CD4+ T cell epitope peptide and CD8+ T cell epitope peptide.

In a second aspect of the present invention, there is provided a T cell epitope peptide against zika virus selected from the group consisting of:

(a) a polypeptide as set forth in SEQ ID No. 1-27;

(b) 1-27 amino acid sequence is substituted, deleted or added by one, two or three amino acid residues to form derivative polypeptide with immunogenicity;

(c) and SEQ ID NO:1-27, and can be combined with T cell surface receptor;

In another preferred embodiment, the epitope peptide is a CD4+ T cell epitope peptide or a CD8+ T cell epitope peptide.

In a third aspect of the invention, there is provided an isolated or concatenated combination of polynucleotides, the polynucleotides of said combination of polynucleotides encoding the epitope peptide of the epitope peptide combination of the first aspect of the invention or encoding the epitope peptide of the second aspect of the invention, respectively.

In another preferred embodiment, the polynucleotide comprises deoxyribonucleotides and messenger ribonucleotides.

In another preferred embodiment, the polynucleotide is in a naked form or in a wrapped form.

In a fourth aspect of the invention, there is provided a vector comprising a combination of polynucleotides according to the third aspect of the invention.

In another preferred embodiment, the combination comprises two or more of said polynucleotides.

In a fifth aspect of the invention, there is provided a genetically engineered host cell comprising a vector according to the fourth aspect of the invention or a chromosome into which a polynucleotide of a combination of polynucleotides according to the third aspect of the invention has been integrated.

In a sixth aspect of the present invention, there is provided a method for producing an epitope peptide or a combination of epitope peptides against zika virus, the method comprising the steps of:

(a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression;

(b) epitope peptides against Zika virus were isolated from the cultures.

In another preferred embodiment, the method further comprises mixing the epitope peptides obtained in (b) to prepare an epitope peptide combination against Zika virus.

In another preferred embodiment, the epitope peptide combination can be a mixture or a combination containing a plurality of epitope peptides connected in series.

In a seventh aspect of the present invention, there is provided a fusion protein comprising the epitope peptide combination of the first aspect of the present invention or the epitope peptide of the second aspect of the present invention.

In another preferred embodiment, the fusion protein comprises one or more (e.g. 2, 3, 4, 5) epitope peptides of the combination of epitope peptides according to the first aspect of the invention.

In another preferred embodiment, the fusion protein comprises a p-MHC tetramer.

In another preferred embodiment, the fusion protein may further comprise other proteins derived from Zika virus or dengue virus (other Zika virus or dengue virus epitope peptides).

In another preferred embodiment, the fusion protein further comprises a carrier protein.

In another preferred embodiment, the carrier protein is a pathogen protein, including a viral protein, a bacterial protein, a chlamydia protein, a mycoplasma protein, or a combination thereof.

In another preferred embodiment, the carrier protein comprises BSA, KLH, MHC tetramer complex, and nanoparticles.

In another preferred embodiment, the fusion protein further comprises a linker peptide sequence.

In another preferred embodiment, the fusion protein further comprises a tag sequence.

In an eighth aspect of the present invention, there is provided a pharmaceutical composition, said composition comprising the epitopic peptide combination of the first aspect of the present invention, the epitopic peptide of the second aspect of the present invention, the polynucleotide combination of the third aspect of the present invention, the vector of the fourth aspect of the present invention, the host cell of the fifth aspect of the present invention, or the fusion protein of the seventh aspect of the present invention, and a pharmaceutically acceptable carrier and/or adjuvant.

In another preferred embodiment, the composition is a vaccine.

In a ninth aspect of the invention, there is provided a vaccine composition comprising an epitopic peptide combination of the first aspect of the invention, an epitopic peptide of the second aspect of the invention, a polynucleotide combination of the third aspect of the invention, a vector of the fourth aspect of the invention, a host cell of the fifth aspect of the invention, or a fusion protein of the seventh aspect of the invention, and an immunologically acceptable carrier and/or adjuvant.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the vaccine composition is a nucleic acid vaccine composition comprising a polynucleotide according to the third aspect of the invention (including naked or encapsulated forms of deoxyribonucleotides and messenger ribonucleotides) or a vector according to the fourth aspect of the invention.

In another preferred embodiment, the vaccine comprises peptide vaccine, protein vaccine, mRNA vaccine and DNA vaccine.

In a tenth aspect of the invention, there is provided a use of the epitope peptide combination of the first aspect of the invention, the epitope peptide of the second aspect of the invention, the polynucleotide combination of the third aspect of the invention, or the fusion protein of the seventh aspect of the invention, (a) for the preparation of a vaccine against zika virus; and/or (b) is used for preparing a medicament for treating Zika virus.

In another preferred embodiment, the medicament comprises a therapeutic vaccine.

In the eleventh aspect of the present invention, there is provided a use of the epitope peptide combination according to the first aspect of the present invention, the epitope peptide according to the second aspect of the present invention, the polynucleotide combination according to the third aspect of the present invention, or the fusion protein according to the seventh aspect of the present invention, for preparing a reagent or a kit for detecting zika virus.

In a twelfth aspect of the present invention, there is provided a kit for detecting zika virus, comprising: a container or carrier; and a detection reagent comprising the epitope peptide combination of the first aspect of the present invention, the epitope peptide of the second aspect of the present invention, or the fusion protein of the seventh aspect of the present invention, in the container or on the carrier.

In another preferred embodiment, the reagent comprises a solid phase carrier and the epitope peptide of any one of SEQ ID NO.1-27 or a combination thereof or a conjugated protein thereof, such as p-MHC tetramer, coated on the solid phase carrier.

The p-MHC tetramer is formed by adding a Biotinylase Substrate Peptide (BSP) with the length of 15 amino acid residues to the carboxyl end of an MHC molecule such as an HLA-A2 heavy chain through a genetic engineering technology to form a fusion protein, and incubating the fusion protein with a specific antigen short peptide in vitro according to a certain proportion to fold the fusion protein into a correct conformation so as to form a pMHC complex. Biotin is labeled on lysine residues of a substrate peptide, so that one streptavidin is combined with four biotin-labeled pMHC complexes to form a tetramer, and the MHC-antigen peptide tetramer can be combined with antigen-specific TCR, namely, can be used for capturing or detecting antigen-specific CTL, and can be sorted for in vitro culture amplification and functional analysis.

In another preferred embodiment, the reagents are used to sort and enrich for Zika virus-specific T cells in tissue samples such as PBMCs or spleen cells.

In another preferred embodiment, the kit or the reagent is used for detecting whether the tissue sample to be detected is derived from a host infected with Zika virus.

In another preferred embodiment, the test sample is from a population in the pandemic region of Zika virus.

In another preferred embodiment, the sample to be tested is from a primate or other mammal.

In a thirteenth aspect of the present invention, there is provided a method for diagnostic or non-diagnostic detection of Zika virus infection in a sample, comprising the steps of:

(1) preparing epitope peptide of Zika virus shown in any one of SEQ ID NO.1-27 or epitope peptide combination thereof;

(2) incubating the epitope peptide or the epitope peptide combination thereof obtained in the step (1) with a sample to be detected and obtained by separation;

(3) detecting whether the IFN-gamma is highly expressed in the sample by ELISPOT or intracellular cytokine staining;

wherein, if the sample in the step (3) shows the phenomenon, the sample is infected with Zika virus.

In another preferred embodiment. The sample comprises PBMC, spleen and lymph node cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of IFN-. gamma.ELISpot assay screening for dominant T-cell epitopes. BALB/c mouse spleen cells 4 weeks after Zika virus infection were stimulated with different peptides, wherein conA stimulation was used as a positive control, PBS stimulation was used as a negative control, the peptide stimulation results of uninfected mouse spleen cells were used as a parallel comparison, the dotted line is the sum of the mean value of the stimulation results of uninfected spleen cells and three times the standard deviation, and the sequences in the figure are marked with the corresponding SEQ ID NO on the abscissa.

FIG. 2 shows the results of dominant T-cell epitope specific Intracellular Cytokine Staining (ICS) of memory T-cells. BALB/c mouse spleen cells 4 weeks after Zika virus infection were stimulated with 10 peptides, with PBS as a negative control. CD4 positive T cells and CD8 positive T cells were delineated and their IL-2 and IFN- γ expression is shown.

FIG. 3 shows the results of mice splenocytes after recovery from infection stimulated by dominant T cell epitopes. Wherein, FIG. 3A and FIG. 3B show the proportion of IFN-. gamma.expressing cells in CD4+ and CD8+ T cells, respectively, and the dotted line is the sum of the mean and three-fold standard deviation of the spleen cell stimulation results of uninfected mice.

Figure 4 shows the immunogenicity of different groups of epitope peptides in mice. The results show that the mixture of the CD4T cell epitope peptide of the G2 group, the CD8T cell epitope peptide of the G3 group and the G4 group can stimulate specific T cell reaction in mice after the mice are immunized in the form of peptide fragment vaccines. Wherein the G1 group is a PBS immune control group. The strength and type of the memory response of the related peptide fragments can be determined through the determination results of (A, B) IFN-gamma ELISpot assay and (C, D) ICS experiment of mouse splenocytes.

FIGS. 4A and 4C show the number of IFN-. gamma.enzyme linked spots produced by stimulating splenocytes from mice in the group G2/G4 with epitope monopeptide CD4T cells, and the proportion of IFN-. gamma.or IL-2 expressing CD4T cells, respectively.

FIGS. 4B and 4D show the number of IFN-. gamma.enzyme linked spots produced by stimulating splenocytes from mice in the group G3/G4 with epitope monopeptide CD8T cells, and the proportion of IFN-. gamma.or IL-2 expressing CD8T cells, respectively.

Figure 5 shows that the peptide fragment combination vaccine provides protection against zika virus in mice. Mice immunized by the peptide fragment combination are subjected to virus challenge by using Zika virus subcutaneous injection, and serum or tissues are collected three days and seven days after the virus challenge. FIG. 5A shows the results of the determination of the titer of viremia by plaque assay, FIGS. 5B, 5C, 5D, 5E, 5F, 5G, 5H show the results of the determination of viral RNA copies in tissues by quantitative PCR, and the dotted line is the lower limit of detection of the experimental system.

Fig. 6A shows that the peptide fragment combination vaccine does not produce neutralizing antibodies in mice. Mice were immunized with peptide fragment combinations and challenged with zika virus. Serum of each group of mice is collected on the third day after the challenge to carry out a plaque inhibition neutralization experiment.

Fig. 6B shows that in vitro experiments verify that the peptide fragment combination vaccine does not produce ADE effects after immunization of mice. Mice were immunized with the peptide fragment combinations and blood was collected 14(pre-infection) days after the last immunization and 3 days after infection (post-infection). Serum was diluted in a gradient and assayed for the ability of serum to cause an increase in K562 infection in vitro with zika virus. Flavivirus broad spectrum antibody 4G2 (dilutions 0.01, 0.1, 1, 10, 100, 100 μ G/ml) was used as a positive control. The results shown are representative of two replicates.

Detailed Description

The present inventors have extensively and intensively studied and, for the first time, have unexpectedly found an epitope peptide combination against Zika virus, comprising the polypeptides shown in SEQ ID Nos. 1 to 27. The Zika virus epitope peptide not only comprises a CD4+ T cell epitope, but also comprises a CD8+ T cell epitope, can stimulate stronger T cell immune response in a mouse body, and provides a protective effect against Zika virus. The Zika virus epitope peptide of the invention can also be used for detecting Zika virus. The present invention has been completed based on this finding.

Specifically, the invention screens CD4+ and CD8+ T cell dominant epitopes based on the synthesis of whole protein peptide fragments. The invention proves that the vaccine containing the T cell epitope peptide provides theoretical basis and lead products for developing T cell vaccines of Zika virus and other viruses based on CD4+ and CD8+ T cell epitopes. At present, the Zika virus has no vaccine on the market, so the Zika virus T cell peptide fragment vaccine is complementary and perfected to the current Zika virus candidate vaccine.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

Zika virus T cell epitope peptide

The invention provides an immunodominant Zika virus CD4T cell epitope peptide fragment, wherein the epitope peptide comprises any one or more sequences of SEQ ID NO.1-SEQ ID NO. 11.

The invention also provides an immunodominant Zika virus CD8T cell epitope peptide segment, wherein the epitope peptide comprises one or more sequences of SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.11-SEQ ID NO. 27.

Wherein SEQ ID NO.1, SEQ ID NO.7 and SEQ ID NO.11 have the characteristics of both CD4T cell epitope peptide and CD8T cell epitope peptide.

The invention provides a conjugate which contains one or more epitope peptides, or both the CD4T cell epitope peptide and the CD8T cell epitope peptide. The conjugated moiety may be BSA, KLH or other forms of carrier proteins or nanoparticles, or may be an MHC tetrameric complex, or other forms of detectable labels.

The invention also provides a fusion protein, which comprises

(a) One or more of the epitope peptides described above, with or without other proteins. The other proteins may be derived from Zika virus or dengue virus, or may be various conjugate proteins described above.

(b) And the connecting sequence peptide connecting each epitope peptide and each protein comprises single copies or multiple copies of GGGS and GPGPG.

(c) Various tags to aid in purification.

Another objective of the invention is to provide a vaccine design scheme using Zika virus epitope peptide as an immunogen composition.

Such vaccines include the aforementioned protein or particle immunogens, as well as DNA and RNA vaccines having as a core the polynucleotide encoding the aforementioned polypeptide. The vaccine can be used alone or in combination with other vaccines.

In an embodiment of the present invention, the technical solution for achieving the above object is as follows: a peptide fragment vaccine of Zika virus comprising as an active ingredient an immunodominant peptide selected from the group consisting of the peptides mentioned above.

Prior to the present invention, although partially zika virus protective immunodominant epitopes have been identified, immunodeficient B6, AG6 or AG129 mice were the subject of primary study, and most experimentally verified based on software predicted sequences, focusing on one of CD4+, CD8+ T cell epitopes. Therefore, the immunodominance of the obtained epitope can not completely reflect the state in a wild mouse, the number of the epitope is limited, and the epitope can not be applied to BALB/c mice with a more extensive immune system in the field of vaccine research. The invention is based on the synthesis of the whole protein peptide segment, and directly screens the dominant epitope peptide segment through experimental detection. The peptide segment is designed to be 18 amino acids in length and overlapped by 10 amino acids, so that the vast majority of CD8+ T cell epitopes and the main CD4T cell epitope can be screened and identified simultaneously. The invention proves that the vaccine containing the T cell epitope peptide can stimulate stronger T cell immune response in a mouse body and provides a protective effect against Zika virus. The invention provides theoretical basis and lead products for developing the T cell vaccines of the Zika virus and other viruses based on CD4+ and CD8+ T cell epitopes. At present, the Zika virus has no vaccine on the market, so the Zika virus T cell peptide fragment vaccine is complementary and perfected to the current Zika virus candidate vaccine.

It is to be understood that the epitope peptide of the present invention also includes a derivative polypeptide formed by substituting, deleting or adding one, two or three amino acid residues of the amino acid sequence of SEQ ID Nos. 1 to 27, which has a binding activity to a T cell surface receptor. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also provides an epitopic peptide combination comprising at least two epitopic peptides of the invention or polypeptides derived therefrom, as described in the first aspect of the invention.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If a polypeptide in a natural state in a living cell is not isolated and purified, the same polypeptide is isolated and purified if it is separated from other substances coexisting in the natural state.

As used herein, "isolated peptide" means that the polypeptide of the present invention is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify the polypeptides of the invention using standard protein purification techniques. The substantially purified polypeptide (fusion protein) is capable of generating a single major band on a non-reducing polyacrylamide gel. In the present invention, the polypeptide of the present invention includes a short peptide conforming to the structural formula of formula I or a polypeptide containing a core sequence X.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.

Once the relevant peptide sequences have been identified, they can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating the relevant peptide (fusion protein) from the propagated host cells by conventional methods.

In addition, fragments of the relevant peptide sequences can also be synthesized directly by chemical methods.

ADE Effect

ADE effect, i.e., antibody-dependent enhancement (ADE). Viral infection begins with adhesion to the cell surface, which is accomplished by the interaction of viral surface proteins with specific receptor and ligand molecules on the target cell. Antibodies specific for viral surface proteins can often suppress this step, and "neutralize" the virus, rendering it incapable of infecting cells. However, in some cases, antibodies play opposite roles during viral infection: they assist virus entry into target cells and increase infection rates, a phenomenon known as antibody-dependent enhancement.

In the case of Zika virus, antibody immunization against Zika virus may have a negative effect, i.e., antibodies against Zika virus can assist dengue virus in enhancing infection of the body or cells in vivo or in vitro (ADE effect).

The development of Zika virus vaccines at present needs to avoid the occurrence of ADE phenomenon, which was first discovered during dengue virus infection in recent 50 years ago and is considered as one of the important factors causing the aggravation of secondary infection. Several studies have demonstrated a complex serological interaction between DENV and ZIKV. Although the sequence of Zika virus differs from that of dengue virus by about 41-46% (E protein), the similarity is sufficient to allow antibodies against dengue virus to cross-react with Zika virus and drive the ADE phenomenon. This factor must therefore be taken into account in the vaccine approach against both viruses. On the other hand, memory CD4+ and CD8+ T cell responses of Zika virus have been shown to have protective effects against Zika virus. Therefore, the determination of the T cell epitope of Zika virus and the development of T cell vaccines will be an important complement to the existing Zika T cell immune research and vaccine development. The ADE phenomenon is avoided when Zika vaccine relies primarily on T cell responses to provide protection.

Pharmaceutical compositions and modes of administration

The invention also provides a pharmaceutical composition. The pharmaceutical compositions of the invention may be therapeutic or prophylactic (e.g. vaccines). The pharmaceutical composition of the invention comprises an effective amount of the epitope peptide or the combination of epitope peptides of the invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.

In the present invention, these (vaccine) compositions comprise an immunizing antigen (including the short peptides, collections of peptides, or derivatives thereof of the present invention), and are typically combined with a "pharmaceutically acceptable carrier", which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Examples of suitable carriers include, but are not limited to, proteins, lipid aggregates (e.g., oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants").

Furthermore, the (vaccine) composition of the invention may also contain additional adjuvants. Representative vaccine adjuvants include (but are not limited to) the following classes: inorganic adjuvants such as aluminum hydroxide, alum, etc.; synthetic adjuvants such as artificially synthesized double-stranded polynucleotides (double-stranded polyadenylic acid, uridylic acid), levamisole, isoprinosine, and the like; oil agents, such as Freund's adjuvant, peanut oil emulsion adjuvant, mineral oil, vegetable oil, etc.

Typically, the vaccine composition or immunogenic composition can be prepared as an injectable formulation, such as a liquid solution or suspension; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

The composition can be made into unit or multi-component dosage form. Each dosage form contains a predetermined amount of active material calculated to produce the desired therapeutic effect, together with suitable pharmaceutical excipients.

The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous, intratumoral, intramuscular, intraperitoneal, subcutaneous, intradermal, paracancerous, or topical administration.

In the case of a (vaccine) composition, a safe and effective amount of an epitope peptide or collection of peptides of the invention is administered to a human, wherein the safe and effective amount is generally at least about 1 microgram of peptide per kilogram of body weight, and in most cases does not exceed about 8 milligrams of peptide per kilogram of body weight, preferably the dose is from about 1 microgram to 1 milligram of peptide per kilogram of body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

(a) the invention has completed the in vitro identification of T cell epitope and has carried on the in vivo protection experiment of BALB/c mouse, the result shows, the peptide fragment screened out of the invention has stronger immunogenicity after immunizing the mouse, and can provide the protective action against Zika virus in the mouse.

(b) Unlike conventional B cell vaccines, the epitope peptides selected by the present invention are distributed in the non-structural protein region, and the protection is not generated by neutralizing antibodies, which may circumvent ADE reactions.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Reagents and starting materials used in the examples were commercially available unless otherwise specified.

Example 1 screening of the dominant T cell epitope of Zika in BALB/c mice

The precursor protein sequence of Zika virus was obtained from the NCBI database (GenBank ID: KU 866423.2). 427 peptides covering the full-length precursor protein were synthesized based on this sequence, each 18 amino acids long and the adjacent peptides overlapped by 10 amino acids. All peptide fragments were synthesized by Gill Biochemical company, and the synthetic peptides were dissolved in DMSO (Sigma) at a concentration of 40mg/ml and stored as a stock solution in an ultra-low temperature freezer at-80 ℃.

Since mice with a healthy immune system are difficult to infect with Zika virus, we injected 6-8 weeks old mice with antibodies to type I interferon receptors in advance and subcutaneously 10 days after⁵A short-term infection with the Zika virus was established by the amino acid sequence of the peptide fragment of the Zika virus (SZ-01) of PFU. 28 days after infection, spleen cells of the mice were isolated, and IFN-. gamma.secretion from the spleen cells of the mice was detected using an IFN-. gamma.ELISpot kit (Mabtech). Freshly isolated BALB/c mouse splenocytes (3 x 10) were taken according to kit instructions⁵Cells/well) were added to elispot plates (Mil lopore) pre-coated with IFN- γ capture antibody and stimulated with zika virus peptides (10 μ g/ml) while ConA (5 μ g/ml) was stimulated as a positive control and PBS was treated as a negative control. The wells were incubated at 37 ℃ for 48 hours, the cells washed away, IFN-. gamma.detected with biotin-labeled diabodies, and developed with Strep-HRP and TMB substrates. The number of spots formed at the bottom of the well represents the number of cells activated and secreting IFN-. gamma.s. Spots were counted using a CTL immuno-spot apparatus (Cellular Technology Ltd.). The screening criteria for peptide fragments of positive epitopes specific to Zika virus were the sum of the mean value and three times the standard deviation of the PBS group.

After three rounds of identification and screening, the results showed that the T cell epitope peptides specific to zika virus were widely distributed throughout the precursor protein (as shown in table 1), and ELISpot results for the most significant 27 positive T cell epitope peptide fragments (SEQ ID No.1-SEQ ID No.27) are presented in fig. 1.

Example 2 validation and typing of immunodominant T cell epitope peptides

Immunodominant T cell epitopes were verified by intracellular cytokine staining and the CD4 and CD8 attributes of the epitopes were identified.

BALB/c mouse splenocytes taken 28 days after infection were stimulated with positive peptides (50. mu.g/ml), PMA/ionomycin (ebioscience) and PBS, respectively, selected in example 1 for 2 hours before adding Golgi plug (BD Biosciences). Cells were harvested 4 hours later for intracellular cytokine staining. The collected cells were first treated with anti-mouse CD3 (Alexa)

488, Biolegend), anti-mouse CD4(BV786, BD Bioscience), anti-mouse CD8(APC, Biolegend), Aqua fluorescent (Life technologies) for cell membrane surface and live cell staining. After fixation of the permeant membrane, staining with intracellular cytokines was performed with anti-mouse IL-2(PE/Cy7, Biolegend), anti-mouse IFN-. gamma. (PE, Biolegend). The antibody-washed cells were resuspended in PBS and analyzed by a Fortessa flow cytometer (BD Biosciences).

The results of intracellular cytokine staining experiments show that 27 peptide fragments can stimulate memory T cells to secrete IL-2 or IFN-gamma, and the peptide fragments are verified to be T cell epitope peptides. Further cell typing results show that 8 peptide fragments are CD4+ T cell epitope peptide, 16 peptide fragments are CD8+ T cell epitope peptide, and the other 3 peptide fragments simultaneously comprise CD8+ and CD4+ T cell epitope. FACS results for 10 representative peptides are presented in FIG. 2, and IFN- γ responses in CD4+ or CD8+ T cells stimulated by all 27 peptides are summarized in FIG. 3.

Thus, 27T-cell epitope peptides specific to zika virus have been identified by the present invention through examples 1 and 2, and the sequence distribution is shown in tables 1 and 2.

TABLE 1 immunodominant CD4+ T cell epitope peptide specific for Zika virus to which the present invention relates

Example 3 immunogenicity testing of immunodominant epitope peptides

Selecting 10 immunodominant T cell epitope peptides, and detecting the feasibility and immunogenicity of the T cell vaccine in a peptide fragment combination mode. The peptide fragment combinations were classified into three groups according to table 3, G2, according to the CD4 or CD8 properties of T cell epitope peptides: CD4+ T cell epitope peptide combination, G3: CD8+ T cell epitope peptide combination, G4: CD4+ and CD8+ T cell epitope peptide combination-supplemented with adjuvant CpG, mice were immunized by subcutaneous injection and the G1: PBS immunization group was set as a control.

The immunization process of the peptide fragment combination (peptide fragment vaccine) is to immunize once every two weeks for 4 times.

TABLE 3 epitope peptide combination immunization

Two weeks after the last immunization, splenocytes from mice were isolated and stained with IFN-. gamma.ELISpot and intracellular cytokines and FACS detected for memory T cell responses to determine the immunogenicity of the epitope peptides. The procedure was as in examples 1 and 2.

As shown in FIG. 4, two weeks after immunization, the mice were able to detect a strong memory T cell response against most epitope peptides, but the NS4B (92-109) peptide fragment showed only weak immunogenicity or no immunogenicity in either group G3 or group G4. The immunogenicity of each peptide fragment in different combinations is different, which may be related to the interplay and relative immunodominance between the peptide fragments. Secondly, the immunogenicity of the CD4T epitope peptide is generally more potent than that of the CD8T epitope peptide, which is also associated with the intracellular or extracellular presentation pathway of MHC-I/II molecules. Although more elaborate regulation is still required to obtain a more comprehensive T cell response, in general, we have demonstrated that T cell epitope peptides in the form of peptide fragment vaccines are able to provoke specific T cell responses in vivo.

Example 4 protective experiments with T cell peptide fragment vaccine of Zika Virus

After four times of peptide fragment immunization, mice are injected with I-type interferon receptor antibody in the abdominal cavity on the 13 th day after the fourth time of immunization to block partial immune system function, and are injected with 1 x 10 subcutaneous injection on the fourteen day⁴PFU ZIKV (SZ-01). Serum is collected on the third day after infection to detect the titer of virus blood by a plaque experiment, blood, eyes, spleen, liver, brain and uterus are collected on the seventh day, tissue RNA is extracted, and ZIKV RNA copy in the tissue is determined by qPCR. Sera at day three post-infection were also used in plaque inhibition neutralization experiments to determine neutralizing antibody titers therein.

The results showed that the viremia titer of the control mice was about 10 on the third day after infection^3.5PFU, compared to which all three other experimental groups had a significant decrease (fig. 5). And on the seventh day after infection, the copy quantity of the ZIKV RNA in the blood, eyes, spleen and liver of the mice in the experimental group is obviously reduced compared with that in the control group. In the other two tissues, brain and uterus, only the ZIKV RNA copy amount of the G3 and G4 groups is obviously reduced compared with the control group, and the G2 group has no obvious difference compared with the control group.

Although the virus replication level of the experimental group was significantly lower than that of the control group, the results of the neutralization experiment showed that the neutralizing ability of the serum was not stronger than that of the control group. That is, the protective power of the mice after peptide immunization is not provided by the neutralizing antibody reaction, which suggests that the main pathway for the vaccine to function is the T cell reaction. Meanwhile, ADE experiments on serum before and after challenge prove that the T cell peptide fragment vaccine does not generate ADE-inducible antibodies in mice (figure 6).

In conclusion, the T cell vaccine with protective capacity is obtained by reasonably combining the epitopes obtained by screening, and meanwhile, the risk of ADE can be successfully avoided by the vaccine.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Claims

1. A combination of T cell epitope peptides for Zika virus, characterized in that, the combination of epitope peptides comprises two or more polypeptides selected from the following group:

(a) a polypeptide as shown in SEQ ID NO.: 1-27;

(b) a derivative polypeptide capable of binding to a T cell surface receptor formed by substitution, deletion or addition of one, two or three amino acid residues in the amino acid sequences of SEQ ID NOs: 1-27;

(c) Polypeptides that have ≥90% homology with the amino acid sequences shown in SEQ ID NOs: 1-27, and are capable of binding to T cell surface receptors,

Wherein, the epitope peptides in the epitope peptide combination target different T cell surface receptors (TCRs).

2. The epitope peptide combination according to claim 1, wherein the epitope peptide combination contains at least two or more polypeptides selected from the group consisting of SEQ ID NO.: 1-11, and The epitope peptide combination is a CD4+T cell epitope peptide combination.

3. The epitope peptide combination according to claim 1, wherein the epitope peptide combination contains at least two or more selected from the group consisting of SEQ ID NO.: 1, 7, shown in 11-27 and the epitope peptide combination is a CD8+ T cell epitope peptide combination.

4. A T cell epitope peptide for Zika virus, wherein the epitope peptide is selected from the group consisting of:

(a) a polypeptide as shown in SEQ ID NO.: 1-27;

(b) an immunogenic derivative polypeptide formed by substituting, deleting or adding the amino acid sequence of SEQ ID NO: 1-27 by one, two or three amino acid residues;

(c) A polypeptide with ≥90% homology to the amino acid sequences shown in SEQ ID NOs: 1-27 and capable of binding to T cell surface receptors;

5. An isolated or tandem polynucleotide combination, wherein the polynucleotides in the polynucleotide combination encode the epitope peptides in the epitope peptide combination according to claim 1 or claim 1 respectively. 4. The epitope peptide.

6 . A fusion protein, characterized in that, the fusion protein comprises the combination of epitope peptides of claim 1 or the epitope peptide of claim 4 . 7 .

7. A pharmaceutical composition, wherein the composition contains the epitope peptide combination of claim 1, the polynucleotide combination of claim 5, or the fusion protein of claim 6, and pharmaceutically acceptable carriers and/or excipients.

8. A vaccine composition, wherein the composition contains the epitope peptide combination of claim 1, the epitope peptide of claim 4, or the polynucleotide combination of claim 5. , or the fusion protein of claim 6, and an immunologically acceptable carrier and/or adjuvant.

9. A use of the epitope peptide combination according to claim 1, or the epitope peptide according to claim 4, or the polynucleotide combination according to claim 5, or the fusion protein according to claim 6, wherein It is characterized in that (a) for preparing a vaccine against Zika virus; and/or (b) for preparing a medicament for treating Zika virus.

10. Use of the epitope peptide combination according to claim 1, or the epitope peptide according to claim 4, or the polynucleotide combination according to claim 5, or the fusion protein according to claim 6, wherein It is characterized in that it is used for preparing a reagent or kit for detecting Zika virus.