CN112574319B

CN112574319B - African swine fever virus P12 protein nanoparticle and preparation method and application thereof

Info

Publication number: CN112574319B
Application number: CN202011533399.1A
Authority: CN
Inventors: 林坚; 朱新杰; 李紫晨; 郑梦竹; 沈显贵
Original assignee: Non Zero Sum Beijing Investment Management Co ltd
Current assignee: Non Zero Sum Beijing Investment Management Co ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2023-04-18
Anticipated expiration: 2040-12-23
Also published as: CN112574319A

Abstract

The invention relates to an African swine fever virus P12 protein nanoparticle and a preparation method and application thereof, wherein the protein nanoparticle is formed by self-assembly of protein monomers, and the protein monomers are fusion proteins sequentially containing metallothionein, glutathione-S-transferase and P12 protein from amino acid to carboxyl terminal. The GSTP1-MT3 protein and the P12 antigen of African swine fever virus are subjected to fusion expression, and the protein nanoparticles can be spontaneously formed in a pichia pastoris expression system through the induction of ferrous ions. Through immunogenicity determination in mice, the P12 protein nanoparticles can cause very strong immune response, and have great potential to be developed into a novel, safe and effective African swine fever virus vaccine.

Description

African swine fever virus P12 protein nanoparticle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an African swine fever virus P12 protein nanoparticle and a preparation method and application thereof.

Background

African Swine Fever (ASF) is an acute, hemorrhagic and highly-contact infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs or wild pigs, and is characterized by short course of disease, high fever and hemorrhagic lesions, the death rate of acute infection reaches 100 percent, and the swine industry worldwide is seriously threatened.

In the 60's of the 20 th century, people first tried inactivated vaccines of ASF, but most of them were not protected. In 2014, blome et al still cannot resist strong toxic attack by using the latest adjuvant in combination with ASF inactivated vaccine for immunization. The subunit vaccines prepared with protective antigens (p 72, p54, CD2v, etc.) only provide partial protection in immunized pigs, and combined immunization does not provide protection. Attempts have been made to use DNA vaccines that express protective antigens and viral vector vaccines that utilize viral vectors that provide immunity similar to subunit vaccines, but that provide only partial protection or no protection.

The genome of ASFV is about 170-193kb, contains 150-167 Open Reading Frames (ORFs), encodes 150-200 proteins, of which about 50 are structural proteins of virus. The ASFV virion has an icosahedral structure, has a diameter of about 200nm, and is composed of nucleocapsid protein-coated viral genome DNA, a viral inner envelope, a viral capsid, and an outer envelope. The envelope protein is a main structural protein constituting virus particles, is also an important surface antigen and is closely related to host cell tropism, pathogenicity and immunogenicity. According to the current research, the functional ASFV envelope proteins are mainly CD2v, p54, p12, p30, p17, p22 and the like, however, the proteins can not obtain effective protection effect after being used as antigens to immunize pigs. For example, neilan et al use P22 and P30, P54, P72 proteins as antigens for "cocktail" mixed immunization of pigs without effective protection.

By discussing and analyzing the prior art, the protective efficacy of the current African swine fever virus subunit vaccine, nucleic acid vaccine and virus live vector vaccine is low. Therefore, the immunogenicity of the African swine fever virus protein needs to be further researched, and a safe and effective ASF vaccine is constructed by using a new technology and a new method.

Disclosure of Invention

The invention aims to provide an African swine fever virus P12 protein nanoparticle and a preparation method and application thereof. The GSTP1-MT3 protein and the P12 antigen of African swine fever virus are subjected to fusion expression, and the protein nanoparticles can be spontaneously formed in a pichia pastoris expression system through the induction of ferrous ions. Through immunogenicity measurement in mice, the P12 protein nanoparticles can cause strong immune response, and have great potential to be developed into a novel, safe and effective African swine fever virus vaccine.

To this end, in a first aspect, the present invention provides an african swine fever virus P12 protein nanoparticle, wherein the protein nanoparticle is formed by self-assembly of protein monomers, and the protein monomers are fusion proteins which sequentially comprise metallothionein, glutathione S-transferase and P12 protein from amino acid to carboxyl terminal.

Further, the protein nanoparticles are formed by self-assembly of the protein monomers through induction of metal ions, and the metal ions are Fe ²⁺ 。

Metallothionein is a protein with low molecular weight, high metal content, rich cysteine and ubiquitous in biology. Generally, metallothioneins can self-assemble to form fusion proteins induced by metal ions, such as Cd ²⁺ 、Gd ³⁺ 、Cr ³ ⁺ 、Ni ²⁺ 、Fe ²⁺ 、Mn ²⁺ 、Co ²⁺ And the like, and the prior art has no general guiding principle for whether the self-assembly proteins induced by different metal ions have structural and performance differences or what structural and performance differences exist. In the research process, the invention discovers that the fusion protein provided by the invention is in Fe relative to other metal ions ²⁺ The protein nanoparticles formed by self-assembly under the induction of ions have remarkably better immune performance, which is probably related to the structures formed by self-assembly.

Further, the metallothionein is MT3, and the amino acid sequence of the metallothionein is SEQ ID NO:1 is shown.

Further, the glutathione-S-transferase is GSTP1, and the amino acid sequence thereof is SEQ ID NO:2, respectively.

Further, an optional first connecting peptide is also included between the metallothionein and the glutathione-S-transferase, and an optional second connecting peptide is also included between the glutathione-S-transferase and the P12 protein.

Further, the first and second linker peptides are each independently selected from a rigid linker peptide or a flexible linker peptide, preferably a flexible linker peptide. In a specific embodiment, the linker peptide is (GGGGS) _n And n is an integer of 1 to 4.

According to the sequence information of Chinese epidemic strains of African swine fever virus (African swine fever virus isolate China/2018/AnhuiXCGQ, complete genome, genBank: MK 128995.1), the amino acid sequence of the P12 protein is SEQ ID NO:3, respectively.

Further, the amino acid sequence of the protein monomer is SEQ ID NO:4, respectively.

Further, the protein monomer further comprises a signal peptide, thereby facilitating secretory expression of the protein monomer in a host cell. In certain embodiments, a protein monomer of the invention comprises a signal peptide at its amino terminus. Since the present invention has found in the course of research that expression of the protein monomer by a eukaryotic expression system, such as yeast, is more advantageous for its expression, modification, secretion and self-assembly than a prokaryotic expression system, such as E.coli, in a preferred embodiment, the signal peptide may be an alpha-factor secretion signal peptide.

Further, the protein monomer further comprises a protein tag. Such protein tags are well known in the art, e.g., his, flag, MBP, HA, myc, etc., and one skilled in the art knows how to select an appropriate protein tag for a desired purpose (e.g., purification, detection, or tracking). In certain embodiments, a protein monomer of the invention comprises a His-tag. In certain embodiments, a protein monomer of the invention comprises a protein tag at its carboxy terminus.

In a second aspect of the invention, there is provided a nucleic acid encoding a protein monomer according to the invention.

Further, the nucleic acid is SEQ ID NO:5, position 13-987 of SEQ ID NO: bits 13-1005 of 5.

In a third aspect of the invention, there is provided a vector comprising a nucleic acid according to the invention. In certain embodiments, the vector is a plasmid, cosmid, or phage.

In a fourth aspect of the invention, there is provided a host cell comprising a nucleic acid and/or vector according to the invention and/or expressing a protein monomer according to the invention. Such host cells include prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.), and the like. In a preferred embodiment, the host cell is a yeast cell.

In a fifth aspect of the present invention, there is provided a method for preparing the protein nanoparticle of the present invention, which comprises culturing the host cell of the present invention under conditions allowing the expression of the protein monomer; adding metal ions in the culture process for induction; and carrying out protein purification on the cultured host cell culture to prepare the protein nanoparticles formed by self-assembly of the protein monomers.

Further, the metal ion is Fe ²⁺ The concentration of the metal ion is 0.01 to 0.5mM, preferably 0.5mM.

Further, in the course of the cultivation, the pH of the medium used is 6.5 to 8.0, preferably 8.0.

In a sixth aspect of the invention, there is provided an immunogenic composition or vaccine comprising a protein nanoparticle according to the invention, and/or a nucleic acid according to the invention, and/or a vector according to the invention, and/or a host cell according to the invention.

Further, the active ingredient of the immunogenic composition or vaccine is the protein nanoparticle of the present invention, and/or the nucleic acid of the present invention, and/or the vector of the present invention, and/or the host cell of the present invention.

Further, the immunogenic composition or vaccine further comprises one or more of an adjuvant, a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

In a seventh aspect of the invention, there is provided a protein nanoparticle according to the invention, a nucleic acid according to the invention, a vector according to the invention, a host cell according to the invention or an immunogenic composition or vaccine according to the invention for use in the manufacture of a product for inducing a specific immune response against african swine fever virus in a subject and/or for preventing and/or treating african swine fever virus in a subject.

Further, the product is a vaccine.

Further, the specific immune response comprises an antibody response.

Further, the subject is a mammal, such as a pig, monkey, rat, human.

Since the GSTP1-MT3 protein is disclosed for the first time, the inventor of the patent finds that the protein has the functions of targeting mitochondria, regulating macrophage function phenotype and resisting viruses, particularly single-stranded RNA viruses through continuous research. In the latest research published in the present patent, the inventors coupled GSTP1-MT3 protein with protein of ASFV, constructed and expressed nanoparticles of ASFV structural proteins PP220, CD2v, P54, P30, P17, P12, P49, P22, j18L, non-structural protein EP153R and unknown functional protein CP312R, respectively, and analyzed immunogenicity of these nanoparticle antigens through animal experiments. The research result shows that most of the nano-particle proteins do not obtain good effect, and the nano-particle vaccine obtained by fusing GSTP1-MT3 and P12 can generate P12 specific antibody with higher titer after being used for immunizing mice.

Compared with the prior art, the invention has the following remarkable progress:

the GSTP1-MT3 protein and the P12 protein of African swine fever virus are fused, and the protein nanoparticles can be spontaneously formed in a pichia pastoris expression system through the induction of ferrous ions. The protein nanoparticles can cause strong immune response by performing immunogenicity measurement in mice. The protein nanoparticle provided by the invention overcomes the defect of low protective efficacy of African swine fever virus subunit vaccines, nucleic acid vaccines and virus live vector vaccines in the prior art, and has great potential to be developed into new safe and effective African swine fever virus vaccines.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 is a diagram of the spectrum of plasmid pPICZaA-GSTP1-MT 3-P12;

FIG. 2 is a graph showing the results of characterization of GSTP1-MT3-P12 protein nanoparticles; the left side is the result of SDS-PAGE identification, and the right side is the result of Western Blot analysis;

FIG. 3 is the result of particle size analysis of GSTP1-MT3-P12 protein nanoparticles;

FIG. 4 shows the result of detecting the titer of antibodies specific to GSTP1-MT3-P12 protein nanoparticles.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

According to the sequence information of Chinese epidemic strains of African swine fever virus (African swine fever virus isolate China/2018/AnhuiXCGQ, complete genome, genBank: MK 128995.1), the invention designs and synthesizes the amino acid sequence and the DNA sequence of the GSTP1-MT3-P12 fusion protein, MW 36kD, and MT3, linker1, GSTP1, linker2 and P12 proteins are arranged from the N end to the C end in sequence. In a specific embodiment, for the convenience of purification, a His tag is fused to the C-terminal of GSTP1-MT3-P12, and a yeast expression plasmid is constructed, wherein the expression vector is pichia pastoris expression vector pPICZ α a (Invitrogen), and the map of the constructed plasmid is shown in fig. 1.

Example 1 plasmid construction

The present example provides the amino acid sequence of african swine fever virus P12 protein nanoparticle (GSTP 1-MT 3-P12) as shown in SEQ ID NO:4, respectively. Entrusting the Onychoma biology company to synthesize a DNA fragment of GSTP1-MT3-P12 fused with His tag at the C end, wherein the nucleic acid sequence is shown as SEQ ID NO:5 (wherein, the 13 th to 987 th sites code the sequence shown in SEQ ID NO:4, the 988 th to 1005 th sites code His tag), and is constructed into a plasmid vector pPICZaA, the map of the constructed plasmid is shown in figure 1, and the plasmid is named as pPICZaA-GSTP1-MT3-P12. The plasmid vector pPICZaA contains an alpha-factor secretion signal peptide sequence before the insertion fragment. Culturing the monoclonal strain with correct sequencing, and extracting plasmid pPICZaA-GSTP1-MT3-P12 for later use.

The amino acid sequence of GSTP1-MT3-P12 (SEQ ID NO: 4):

DPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCSCCPAECEKCAKDCVCKGGEAAEAEAEKCSCCQGGGGSMPPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQEGSLKASCLYGQLPKFQDGDLTLYQSNTILRHLGRTLGLYGKDQQEAALVDMVNDGVEDLRCKYISLIYTNYEAGKDDYVKALPGQLKPFETLLSQNQGGKTFIVGDQISFADYNLLDLLLIHEVLAPGCLDAFPLLSAYVGRLSARPKLKAFLASPEYVNLPINGNGKQGGGGSMALDGSSGGGSNMPRQQKKCSKAEECTCNNGSCSLKTS

example 2 induced expression of protein nanoparticles

After linearization, the plasmid prepared in example 1 is transferred to X-33 yeast competent cells for induced expression, and the specific steps are as follows:

(1) Plasmid linearization

The plasmid pPICZaA-GSTP1-MT3-P12 prepared in example 1 was digested with the linearized enzyme Sac I as follows:

after mixing, the mixture was centrifuged at 12000rpm for 1min at room temperature to throw the liquid to the bottom of the tube and then in a 37 ℃ water bath for 4 hours.

And (3) after the enzyme digestion, taking 2 mu L of the plasmid, carrying out 1% agarose gel electrophoresis on the 2 mu L of the plasmid and the original plasmid to identify a linearization result, identifying that the plasmid is completely digested, and recovering the digested plasmid by using a plasmid kit for subsequent test steps.

(2) Electric transfer (sterile operation)

Adding 10 mu L of the linearized plasmid obtained in the step (1) into 90 mu L of X-33 yeast competent cells, uniformly mixing, and adding into an electric rotating cup; 2000v,5ms electric shock, then immediately adding 800 microliter of sorbitol, sucking all the sorbitol out, transferring the sorbitol into a 15mL centrifuge tube, and carrying out shake culture at 25 ℃ and 200rpm for 2 hours; then 3mL of non-resistance YPD medium was added at 25 ℃ and shaking cultured at 200rpm for 2 hours. The resulting culture was plated (YPD bleomycin resistant plate) and cultured in an inverted manner in an incubator at 28 ℃ for 2 days.

After the culture is finished, selecting monoclone to be marked on a new bleomycin resistant plate, numbering the new bleomycin resistant plate, carrying out inverted culture in an incubator at 28 ℃ for 24 hours, taking out and storing at 4 ℃ for later use.

(3) Inducible expression

Selecting the single colony prepared in the step (2), inoculating the single colony in 2.5mL YPD, culturing at 28 ℃,250rpm until OD600 is 6, and culturing for about 16-18h; then 150 mul of the bacterial liquid is taken and transferred to 3mL BMGY culture medium with pH 8.0, and the BMGY culture medium is cultured at 28 ℃ and 250rpm until OD600 is 1 for about 8-12h; the cells were suspended in BMGY medium (3 mL BMMY medium) at 1100rpm at room temperature for 5min, and then subjected to induction expression by adding 0.5% final methanol and 0.1mM ferrous sulfate ions, to the cells, at pH 8.0. Wherein, methanol is added once every 24h, yeast extract and peptone with the final concentration of 1 percent are supplemented, and metal ions are not required to be added repeatedly. After 72 hours of culture, the culture product was collected, identified by SDS-PAGE in example 3, and then protein was purified according to example 4.

Example 3SDS-PAGE identification

1mL of the culture product prepared in example 2 was aspirated, centrifuged at maximum speed for 2min at room temperature in a centrifuge, and the supernatant was collected and mixed with a Loading buffer to prepare a sample, which was subjected to SDS-PAGE identification and analyzed for expression level. The result of SDS-PAGE is shown in FIG. 2 (left), and the target protein is successfully expressed.

Example 4 protein purification

After SDS-PAGE identification, the culture product prepared in example 2 was subjected to protein purification by the following specific steps:

collecting the culture product, centrifuging at 4000rpm for 20min, taking the supernatant, adding protease inhibitor according to 1M Tris 500, and adjusting the pH to 8.0 with 1M Tris; then, the mixture was centrifuged at 12000rpm, the supernatant was collected and subjected to suction filtration with filter paper, and the supernatant after the suction filtration was purified with a Ni column. Collecting the purified GSTP1-MT3-P12 protein nanoparticles, and performing Western Blot analysis, wherein the result is shown in FIG. 2 (right); dynamic light scattering particle size measurement of GSTP1-MT3-P12 protein nanoparticles by a Zeta potential analyzer showed that the particle size was 15nm and the particles had good uniformity, as shown in FIG. 3.

Example 5 mouse immunization

Mouse immunization experiments were performed using the GSTP1-MT3-P12 protein nanoparticles prepared in example 4.

10 mu g of GSTP1-MT3-P12 protein nanoparticles are taken, 20 mu g of aluminum hydroxide alum adjuvant (Thermo; 77161) is added, and PBS is mixed until the final volume is 100 mu L for mouse immunization. Taking 5-week-old BALB/C female mice (purchased from Spbefof corporation), and carrying out intramuscular injection immunization on each mouse; the immune is carried out once every two weeks and 3 times, the serum antibody is detected on 21 days and 35 days respectively, and the specific steps of ELISA for detecting the serum antibody are as follows:

(1) Experimental materials:

PBS：Solarbio；P1020-500

1 × Washing buffer: from ELISPOT kit; 1

ELISA coating solution: solarbio; c1050-100ml

5% bovine serum albumin BSA: jingmei bioengineering, inc.; RF101-100

HRP-labeled goat anti-mouse IgG: china fir gold bridge; ZB-2305

Soluble one-component TMB substrate solution: TIANGEN; PA107-01

ELISA stop solution: solarbio; c1058-100ml

ELISA plate sealing membrane: AXYGEN; platemax CyclerSeal Sealing Film

1×8Flat Bottom，Certified High Binding：Costar；42592

(2) The experimental steps are as follows:

1. antigen pre-coating: the P12 antigen was diluted to 1. Mu.g/mL with coating solution, coated with 100. Mu.L/well and coated overnight at 4 ℃.

2. Washing the plate: discarding the coating solution, patting dry on filter paper, adding 200 μ L of 1 × Washing buffer, standing for 1min, discarding the Washing solution, patting dry, and repeating for 3 times.

3. And (3) sealing: 5% bovine serum albumin BSA (0.5 g/10mL,4 ℃ temporary storage), 100. Mu.L/well, 37 ℃ blocking for 1h.

4. Washing the plate: discard the blocking solution, pat dry on filter paper, add 200. Mu.L of 1 × Washing buffer, stand for 1min, discard the Washing solution, pat dry, repeat 3 times.

5. Adding a primary antibody: the serum to be detected is diluted in a multiple ratio, 100. Mu.L/well and incubated at 37 ℃ for 1h.

Serum dilution: 3 μ L of serum +297 μ L of PBS as 1, and 150 μ L +150 μ L of PBS as 1, in order to be diluted in two-fold ratio to 1.

6. Washing the plate: discard primary antibody, pat dry on filter paper, add 200. Mu.L of 1 × Washing buffer, stand for 1min, discard Washing solution, pat dry, repeat 3 times.

7. Adding a secondary antibody: HRP-labeled goat anti-mouse IgG as secondary antibody 1.

8. Washing the plate: discard secondary antibody, pat dry on filter paper, add 200. Mu.L of 1 × Washing buffer, stand for 1min, discard Washing solution, pat dry, repeat 3 times.

9. Color development: adding soluble single-component TMB developing solution at a concentration of 100 μ L/well, and developing in dark for 15min.

10. And (4) terminating: preheating the microplate reader in advance, adding stop solution to stop color development, measuring the value at the wavelength of 450nm of the microplate reader immediately after 100 mu L/hole.

The experimental result shows that after the GSTP1-MT3-P12 protein nanoparticles are used for immunizing a mouse for 35 days, specific antibodies can be stimulated to be generated in the mouse, the average titer is as high as 1 (50000) (figure 4), and the GSTP1-MT3-P12 protein nanoparticles are proved to have good immunogenicity.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

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Claims

1. An African swine fever virus P12 protein nanoparticle, which is characterized in that the protein nanoparticle is formed by self-assembly of protein monomers, wherein the protein monomers are fusion proteins sequentially comprising metallothionein, glutathione S-transferase and P12 protein from an amino terminal to a carboxyl terminal; the metallothionein is MT3, and the glutathione-S-transferase is GSTP1; the amino acid sequence of the metallothionein is SEQIDNO:1 is shown in the specification; the amino acid sequence of the glutathione-S-transferase is SEQ ID NO:2 is shown in the specification; the amino acid sequence of the P12 protein is SEQIDNO:3 is shown in the specification;

the protein nanoparticles are formed by self-assembly of the protein monomers through induction of metal ions, and the metal ions are Fe ²⁺ 。

2. The protein nanoparticle of claim 1, further comprising a first linker peptide between the metallothionein and the glutathione-S-transferase.

3. The protein nanoparticle of claim 2, further comprising a second linker peptide between the glutathione-s-transferase and the P12 protein.

4. The protein nanoparticle according to claim 3, wherein the first linker peptide and the second linker peptide are each independently selected from a rigid linker peptide or a flexible linker peptide.

5. The protein nanoparticle of claim 1, wherein the protein monomer further comprises a signal peptide and/or a protein tag.

6. A biomaterial, wherein the biomaterial is:

(i) Nucleic acid encoding a protein monomer according to any one of claims 1 to 5;

(ii) A vector comprising the nucleic acid of (i); or

(iii) A host cell comprising (i) said nucleic acid and/or (ii) said vector.

7. The biomaterial of claim 6, wherein the nucleic acid is seq id no: position 13-987 of 5, or seq id no: bits 13-1005 of 5.

8. The method of producing a protein nanoparticle according to any one of claims 1 to 5, wherein the production method comprises: culturing the host cell of claim 6 under conditions that allow expression of the protein monomer; adding metal ions in the culture process for induction; and carrying out protein purification on the cultured host cell culture to prepare the protein nanoparticles formed by self-assembly of the protein monomers.

9. The method of claim 8, wherein the metal ion is Fe ²⁺ The concentration of the metal ions is 0.01-0.5mM.

10. The method according to claim 8, wherein the pH of the medium used during the culturing is 6.5 to 8.0.

11. An immunogenic composition or vaccine, comprising the protein nanoparticle of any one of claims 1-5.

12. The immunogenic composition or vaccine of claim 11, wherein the active ingredient of the immunogenic composition or vaccine is the protein nanoparticle of any one of claims 1 to 5.

13. The immunogenic composition or vaccine of claim 11, further comprising one or more of an adjuvant, a pharmaceutically acceptable carrier, and a pharmaceutically acceptable excipient.

14. Use of a protein nanoparticle according to any one of claims 1-5, a biomaterial according to any one of claims 6-7, or an immunogenic composition or vaccine according to any one of claims 11-13, for the manufacture of a product for inducing a specific immune response against African swine fever virus in a subject and/or for the prevention and/or treatment of African swine fever virus in a subject.

15. The use of claim 14, wherein the product is a vaccine.

16. The use of claim 14, wherein the specific immune response comprises an antibody response.

17. The use of claim 14, wherein the subject is a mammal.