# Patent

## Title
Use of coccidian

## Abstract

The present invention provides a novel use of coccidian, specifically relates to the use of coccidian as a vaccine live vector. The present invention further provides a live vaccine with coccidian as a vector, which is transgenic coccidian capable of expressing exogenous protein or stably transfected coccidian that contain expression vector and can express exogenous coccidian. The present coccidian vector live vaccine can induce organisms to simultaneously generate protective humoral and cellular immune responses (including the mucosal immune response), as well as generate memory responses, which can be readily carried out and has stable effect and high biological safety without generating immune tolerance.

## Background
TECHNICAL FIELD
The present invention relates to a novel use of coccidia, specifically relates to the use of the coccidia as a live vector of vaccine.
BACKGROUND ART
The recombinant vector vaccine developed by the use of transgenic technology with pathogenic micro-organisms as the vector, can prevent the attenuated vaccine strain getting strong again, but also generate cellular immune responses and has incomparable advantages over traditional vaccines. Currently, research at home and abroad is focusing on genetic recombinant vaccine with a live vector and its mechanism of action. An effective live vector vaccine I is required to have two conditions: first, being safe to host, second, being able to effectively express and deliver the protective antigen having immunocompetence.
At present, research-focused vaccine vectors comprise two categories: one category is attenuated viral vectors, such as the pox virus and adenovirus; the other is enteric pathogenic attenuated bacteria and symbiotic bacteria, such as Salmonella and lactic acid bacteria. Live virus vector can cause effective specific immune response through displaying exogenous antigen on the host cell surface or releasing it into the extracellular environment to make it be recognized by the host immune system. It is possible for the enteric pathogenic attenuated bacteria and symbiotic bacteria after the genetic modification to sustainably and efficiently express autoantigens and exogenous antigens in the host body, thus effectively stimulating protective mucosal immunity, humoral and cellular immune responses against pathogens and foreign antigens. There are inherent weaknesses for the attenuated viruses, enteric pathogenic bacteria and symbiotic bacteria as a live vaccine vector. As a vaccine vector, exogenous genetic fragment that viruses and bacteria can accommodate is small and difficult to carry out large-scale genetic modification. Viral vectors readily trigger host immune tolerance and are difficult to achieve the desired effect by oral administration. Enteric pathogens vectors such as Salmonella easily spread to other parts of the body in the host body, damage other organs and tissues, and cause immune tolerance due to persistent infection; some strains are zoonotic pathogens, which are released into the environment and pose a potential threat to the health of human and animals. In addition, bacterial vectors can not perform glycosylation for the expressed exogenous protein and other modifications, thus causing lack of immune activity of the expressed exogenous protein.
The use of eukaryotes as a carrier will undoubtedly have a greater advantage over viruses and prokaryotes. A larger eukaryotic vector genome can be carried out large-scale genetic modification. And in eukaryotic cells, proteins can be correctly folded and modified to express the target protein with normal activity. Although the challenge use of eukaryotic vectors faces is far greater than viruses and bacteria carriers, because it involves a more complex biological interactions, but since genomic and proteomic research has been relatively mature today, such research can be turned into reality from imagine.
It has been several decades since live oocyst vaccines prepared by blending a variety of attenuated chicken coccidian became available. At present its annual sales have been more than 300 million U.S. dollars, and in the prevention and treatment of coccidiosis in chickens it is playing an increasingly important role. Now study on the interaction between the host and the live oocysts of chicken coccidia vaccine, as well as the immune response mechanism against coccidiosis in the host has been in-depth. However, so far there is no report on application of chicken coccidia as live vector vaccine and application of other Eimeriidae generic coccidia and Cryptosporidium coccidia as live vector vaccine.
SUMMARY OF INVENTION
The object of the present invention is to provide a use of coccidiosis as live vaccine vector;
The other object of the present invention is to provide a live coccidia vector vaccine. Use of live coccidia vector vaccine according to the present invention to express the target protein can induce organism to simultaneously generate protective humoral and cellular immune responses (including the mucosal immune response) and generate memory responses.
The target gene is cloned into the expression vector having the drug selection marker or fluorescent selection marker, the recombinant vector is introduced into the cells of coccidia by electroporation or other transfection methods, then the integrated transgenic coccidia expressing target protein or stably transfected coccidia that contain expression vector and can express exogenous protein are obtained through drug screening, or flow cytometry screening. After inoculation of chicken or other poultry and mammals with transgenic coccidia or stably transfected coccidia as vaccine, when the coccidia express the target protein in the developing process, the protein expression in time and space are subjected to the regulation of the upstream and downstream sequences of the coccidia phased expression gene.
The coccidia vector in the present invention can be used to express spike glycoprotein such as Eimeria maxima TFP250 protein, avian influenza virus HA, and the like, and other external and internal antigen protein (HA, NA, NP, M1, M2, NS1, NS2, PA, PB1, and PB2 proteins), important antigen protein against pathogens such as Newcastle disease virus F protein and so on, thus causing the host's immune responses.
Expression vectors for transforming coccidia can be any expression vectors that can express exogenous protein in coccidian. The recombinant vectors in the present invention include pH4sp-HA1-EYFP-ACTIN, pH4SP-M2e-EYFP-ACTIN, pHgra8-E-HA-A3′, pHgra8-E-NA-A3′, pHgra8-E-NP-A3′, pHDEA-TFP and so on. These vectors are used to transform coccidia, which are screened in order to obtain transgenic coccidia or stably transfected coccidian that can stably express the target protein. The obtained coccidia are orally administrated to immunized animals. As a result, it was found that the obtained coccidian have a significant immune effect on the coccidian and/or virus infection.
Live vaccine vector in the present invention is precocious attenuated or wild-type coccidia, preferably Eimeria Wenyonella, Cryptosporidium and so on. Chicken coccidia represented by Eimeria tenella are the preferred species, which can be used to carry out transgenic research and act as live vaccine vector.
Because coccidian haploid has about 14 chromosomes, its genome size reaches 60 Mb, therefore there are a considerable number of sites that can be used for integration, and the genome can allow insertion of large fragments of exogenous gene. Genetic flanking sequence of coccidia can be used to regulate the transcription of exogenous protective antigen gene, which will periodically express and secrete in the period of growth and development of the coccidia from the coccidia (using signal peptide of coccidia), thus obtaining transgenic coccidia that can stably express the target protein and stimulate a strong host humoral and cellular immune responses (including the mucosal immune response). Compared with bacterial and viral vaccine vectors, transgenic coccidia as a live vaccine vector has a unique advantage.
First, nuclear genome size of coccidia is about 60 Mb, which can accommodate large fragments of exogenous gene. Second, the coccidia are eukaryotes, which can express antigen protein modified by glycosylation. Third, the coccidia enter intestinal epithelial cells to form parasitophorous vacuole, causing the separation from host cell nucleus, so its genetic fragments holds little probability to insert or integrate into the host cell genome. Fourth, due to the protection of oocyst wall, after oral administration live oocyst vaccine of transgenic coccidia will effectively reach specific sites of intestinal or respiratory vaccines to play a role as vaccine and will not reduce the activity of the vaccine because of acid environment of the crop and the stomach and so on. Fifth, oral vaccination is simple and applicable to groups to carry out drinking water immunization or feed preparation immunization; since oocysts can be repeatedly infected under natural breeding conditions, one shot immunity is equivalent to several times of vaccination. Sixth, the coccidia have a short breeding cycle, and the infections are self-limiting, the coccidia are eliminated from the body after generation of oocysts, which does not cause immunotolerance problems. Seventh, various hosts of the coccidia have a strong specificity without cross-species transmission, and they are widely distributed in fields, release of transgenic coccidia in the field will not cause the spread of the kind of pathogen, hence have a high bio-security.
The establishment of live vaccine vector of the present invention marks the birth of a new type of vaccine. The invention, which expresses another one or more (type) antigens used in vaccine with live vaccine (live coccidia oocysts) as vector and simultaneously stimulate cellular immunity and humoral immunity (including mucosal immunity) against a variety of pathogens, is an a useful complement to the eukaryotic expression system; to express the target protein of organisms in coccidia adds a new research direction for Parasitology; the coccidia gene engineering vaccine based on the present invention can also be used in production, and can in a planned way control the incidence of coccidiosis in China, and make survival rate, weight gain and feed conversion of the immune animal be higher than or equal to production performance of animals that use anticoccidial drugs, thus allowing the broad masses of farmers to benefit, and the said vaccine is pollution-free without residue.



DESCRIPTION OF DRAWINGS
FIG. 1 is a constitutive schematic diagram of vector pHDEA-TFP, in which H4: Eimeria tenella histone 4 upstream regulatory sequence; DHFR: Toxoplasma gondii DHFR-TS gene coding region (for pyrimethamine resistance); EYFP2: Enhanced yellow-fluorescent protein gene coding region; Actin: Eimeria Actin gene downstream regulatory sequences. wherein, TFP is Eimeria maxima TFP250 genes, the insertion position of other antigen protein gene can be the same or different.
FIG. 2 is a constitutive schematic diagram of vector pH4sp-HA1-EYFP-ACTIN, in which, H4: Eimeria tenella histone 4 upstream regulatory sequence; SP: a signal peptide sequence of antigen 2 on the surface of Eimeria tenella, said sequence is 69 base pairs in length; HA1: H5N1 avian influenza virus HA antigen HA1 subunit; EYFP: enhanced yellow fluorescent protein gene coding region; Actin poly A: Eimeria tenella Actin gene downstream regulatory sequences.
FIG. 3 is a constitutive schematic diagram of vector pH4SP-M2e-EYFP-ACTIN, in which, H4: Eimeria tenella histone 4 upstream regulatory sequence; SP: a signal peptide sequence of antigen 2 on the surface of Eimeria tenella, said sequence is 69 base pairs in length; M2e: extracellular domain of H5N1 avian influenza virus M2 antigen; EYFP: enhanced yellow fluorescent protein gene coding region; Actin poly A: Eimeria tenella Actin gene downstream regulatory sequences.
FIG. 4 is a constitutive schematic diagram of vector pHGRA8-E-HA-A3′, in which, H4: Eimeria tenella histone 4 upstream regulatory sequence; GRA8: Toxoplasma gondii dense granule protein GRA8 signal peptide coding sequence; EYFP: yellow fluorescent protein gene coding region; HA: the gene encoding area of H5N1 avian influenza virus HA protein; A 3′: Eimeria tenella Actin gene downstream regulatory sequences.
FIG. 5 is a constitutive schematic diagram of vector pHGRA8-E-NA-A3′, in which, H4: Eimeria tenella histone 4 upstream regulatory sequence; GRA8: Toxoplasma gondii dense granule protein GRA8 signal peptide coding sequence; EYFP: yellow fluorescent protein gene coding region; NA: the gene encoding area of H5N1 avian influenza virus NA protein; A 3′: Eimeria tenella Actin gene downstream regulatory sequences.
FIG. 6 is a constitutive schematic diagram of vector pHGRA8-E-NP-A3′, in which, H4: Eimeria tenella histone 4 upstream regulatory sequence; GRA8: Toxoplasma gondii dense granule protein GRA8 signal peptide coding sequence; EYFP: yellow fluorescent protein gene coding region; NP: gene coding area of H5N1 avian influenza virus NP protein; A 3′: Eimeria tenella Actin gene downstream regulatory sequences.



SPECIFIC MODE OF CARRYING OUT THE INVENTION
The present invention will be further illustrated in combination with specific examples. It should be understood that these examples are only intended to explain the present invention without limiting the scope of protection of the invention.
Example 1
The example is intended to use transgenic Eimeria tenella as a live vaccine vector to express the target gene, thus achieving simultaneous immunization of both the coccidia and certain pathogens.
Eimeria maxima TFP250 gene is taken as an example:
1. Constructing Expression Vector and Cloning the Target Gene into the Expression Vector
10-day-old chickens are inoculated with Eimeria tenella strain BJ125 (Suo Xun, Qu Hong-fei, LIU Zong-quan, Lin Kun-hua, Lu Yan-li. Late-maturing strains breeding and immunogenicity study of Eimeria tenella. 1996, 4 Journal of Agricultural Biotechnology (2), 78-79), after 6-9 days manure or intestinal contents of sick chicken are collected to obtain coccidia oocyst, after the sporogenesis the sporangia are extruded on slide glass, a sporangium is sucked up using capillary pipette under the microscope, namely, a single sporangium of Eimeria coccidia isolated therefrom, the proliferated oocysts are collected after oral infection of the chicken with the isolated single sporangia of coccidia, that is, purified genetic relatively consistent coccidia strains system is obtained. After purification of the obtained genetic relatively consistent Eimeria tenella, coccidia genome DNA is extracted with the general methods, coccidia gene regulatory sequence (histone 4 upstream regulatory sequence H4) is cloned using PCR method, Eimeria maxima TFP250 gene sequence is obtained using RT-PCR. The regulatory sequence H4, drug screening marker gene DHFR-TS, TFP250 and other components are constructed into the yellow fluorescence vector (purchased from BD Company), expression vector pHDEA-TFP required for Eimeria tenella transgene is constructed, the detailed contents are as follows:

Cloning Toxoplasma gondii DHFR-TS gene coding region: extracting total RNA of Toxoplasma gondii, amplifying and obtaining DHFR-TS gene using RT-PCR method, introducing restriction sites SacII and BamHI respectively at both ends, designing upstream and downstream primers (DHFR-UP: 5′-CATCCGCGGATGCAGAAACCGGTGTG-3′ (SEQ ID NO.7); DHFR-L: 5′-CTGGATCCAAGACAGCCATCTCCATC-3′ (SEQ ID NO.8)), obtaining DNA fragments with the size of 1.8 Kb, recovering target fragment and ligating it with the vector PGEM-T, transforming after overnight, extracting plasmids, identifying by enzyme digestion, introducing mutant site against pyrimethamine by PCR after DNA sequencing of the obtained candidate positive plasmids.

Cloning Eimeria tenella histone 4 upstream regulatory sequences and Eimeria tenella Actin gene downstream regulatory sequences: allowing the coccidia genomic DNA as a template, at both ends of the upstream regulatory sequence introducing restriction site NdeI and SacII (H4-UP: 5′-CAGCATATGAACCAGCAAAGGTAGCAAC-3′ (SEQ ID.9); H4-L: 5′-CTACCGCGGGATACCCTGGATGTTGTC-3′ (SEQ ID NO.10)) for amplification of H4 upstream regulatory sequence; at both ends of the yellow fluorescent protein gene and the downstream regulatory sequences introducing BamHI and MfeI sites, respectively designing primers (E2-UP: 5′-CATCCAGGGTATCGGATCCTGTC G-3′ (SEQ ID NO.11); Actin-L: 5′-CGCAATTGCTTCACATGGAACCCCTGG-3′ (SEQ ID NO.12)), which are amplified by PCR to obtain a yellow fluorescent protein gene and the downstream regulatory sequences. Respectively obtaining upstream regulatory sequence with the size of 2.0 kb and 2.9 kb and the DNA fragments of the downstream regulatory sequences fused with the yellow fluorescent protein, recovering target fragment and ligating it with the vector PGEM-T, transforming after overnight, extracting plasmids, identifying by enzyme digestion, carrying out DNA sequencing of the obtained candidate positive plasmids. cloning Eimeria maxima TFP250 gene: extracting the total RNA from Eimeria maxima and carrying out reverse transcription to obtain cDNA, during PCR amplication respectively introducing Not I and fused SacII and BamHI sites (T-UP: 5′ATCCCGCGGGCCCGGGATCCTGTCGCCACCATGGAATTGCACCCCATTCC AG 3′ (SEQ ID NO.13); T-L: 5′ ATTTGCGGCCGCCTACTGAATGTCGCCGCTGTCG 3′ (SEQ ID NO.14)) at both ends of TFP250 and performing amplication, obtaining DNA fragments with the size of 660 bp, recovering target fragment and ligating it with the vector PGEM-T (purchased from Promega company), transforming after overnight, extracting plasmids, identifying by enzyme digestion, carrying out DNA sequencing for the obtained candidate positive plasmids.
Ligating each gene after digestion with yellow fluorescent vector after corresponding digestion, eventually obtaining the expression vector containing each component with its nucleotide sequence as shown in sequence list SEQ ID NO.1.
2. Transformation and Screening of Coccidia
The proliferated oocysts are collected after oral infection of the chicken with single oocysts of the preservative Eimeria tenella, 15 days old chickens are inoculated with the above-mentioned oocysts, 200-500 oocysts for each chicken. The discharged coccidia oocysts are collected and purified after 6 to 9 days, in this way continuous passages are done, the obtained oocysts are the oocysts required for constructing a vector.
By electroporation the constructed pHDEA-TFP is transfected into the sporozoites of Eimeria tenella, which is inoculated in ileocecal opening of the chicken through cloaca, while specific pyrimethamine resistance screening drugs are added. Specifically, 1×107 precocious strain sporozoites are taken, 50 μg of pHDEA-TFP plasmids are added, in which shock condition is 2 kv, capacitance is 25, shock time is 0.3 ms, after standing for 20 min, the transfected sporozoite are inoculated again in ileocecal opening of the chicken through cloaca. After 24 hours of incubation the selected pressure is exerted, namely, the specific pyrimethamine resistance screening drugs are added, the test is observed under fluorescence microscope.
Transgenic coccidia line or stably transfected coccidia line capable of stable expression can be obtained by repeating this process. Under fluorescence microscope it will be observed that transgenic coccidia capable of stable expression or the polypide of stably transfected coccidia line can emit yellow fluorescence, luminescence rate is higher than 99%.
3. Inoculation of Chickens
First, the oocysts are sporulated in vitro, the 1-5 day-old healthy chickens are choosed to be orally inoculated with the breeding early-maturing strains or wild-type transgenic coccidia or sporulated oocysts of stably transfected coccidia Coccidia sporulated coccidia, 200-500/chicken, after 5-7 days the chickens are detected for clinical manifestations, the number of oocysts per gram (OPG), pathological grading and other indices.
4. The Effect
After inoculation it is founded that the transgenic coccidia or stably transfected coccidia have good immunogenicity against Eimeria tenella and Eimeria maxima, or other pathogenic infections,
Seven-day-old AA broiler chickens are inoculated with the oocysts of transgenic coccidia, 1.0×103 oocyst/chicken, at the fourteenth day after inoculation, after attacking-coccidia, the number of oocysts discharged from per chicken was significantly lower than those in non-immunized groups without attacking-coccidia (P<0.05), and there is no significant difference between the attacking-coccidia group and the group immunized with parent strain oocysts with the same dose (P>0.05). It indicates that inoculation with the transgenic coccidian, 1.0×103 oocyst/chicken, can provide seven-day-old chicks with immunoprotection. Another immune procedure is to use low-dose immunization of transgenic coccidia oocysts, 200 oocysts/chicken, to immunize 4-day-old AA broiler chickens, at the fourteenth day after immunizing two times, the AA broiler chickens are attacked with transgenic coccidia and parent strain oocysts, 1.0×103 oocyst/chicken, only to find the minimal amount of fecal oocysts are discharged along with Faeces, the oocyst production per chicken is significantly lower than those in non-immunized groups without attacking-coccidia (P<0.01), similar with the groups immunized with parent strains, the chickens in the group immunized with the transgenic coccidian two times are completely protected. The data from two kinds of immune schemes indicate that the expression of exogenous genes does not affect the immunogenicity of the transgenic coccidia. After inoculation, the pathological grading, oocyst production of coccidia in the infected chickens and cecal lesion values all decrease, the decline of weight gain of chickens is improved, OPG value is 6×104-6×106, a good immune protection is generated to prevent lethal dose of coccidia attack or the challenged infections induced by other pathogens.
Example 2
pH4sp-HA1-EYFP-ACTIN, pH4SP-M2e-EYFP-ACTIN, pHgra8-E-HA-A3′, pHgra8-E-NA-A3′ and pHgra8-E-NP-A3′ expression vector are constructed respectively according to the method similar to the above-mentioned methods, which are then transformed into the coccidia, followed by obtaining the coccidia that can stably express the target gene through screening. Cohabitation infection test indicates that the obtained coccidia have high protective rate against coccidian infection and virus infection (>90%).
INDUSTRIAL APPLICABILITY
The present invention provides a novel use of coccidia, specifically relates to the use of coccidia as a live vaccine vector. The present invention further provides a live vaccine with coccidia as a vector, which is transgenic coccidia capable of expressing exogenous protein or stably transfected coccidia that contain expression vector and can express exogenous coccidia. The present coccidia vector live vaccine can induce organisms to simultaneously generate protective humoral and cellular immune responses (including the mucosal immune response) and generate memory responses, which can be readily carried out and has stable effect and high biological safety without generating immune tolerance. The coccidia gene engineering vaccine based on the present invention can also be used in production, and can in a planned way control the incidence of coccidiosis in China, and make survival rate, weight gain and feed conversion of the immune animal be higher than or equal to production performance of animals that use anticoccidial drugs, thus allowing the broad masses of farmers to benefit, and the said vaccine is pollution-free without residue.
The description of the sequences:
SEQ ID NO.1 ̃6 are in sequence: full sequences of expression vector pHDEA-TFP, pH4sp-HA1-EYFP-ACTIN, pH4SP-M2e-EYFP-ACTIN, pHgra8-EYFP-HA-A3′, pHgra8-EYFP-NA-A3′ and pHgra8-EYFP-NP-A3′.

## Claims

1. A coccidian vector live vaccine, formed of a stably transfected coccidia that contains an expression vector and can express a target protein, said target protein being an antigen protein of pathogens, and said expression vector being pHDEA-TFP as shown in SEQ ID NO. 1.

2. A coccidian vector live vaccine formed of a stably transfected coccidia that contains an expression vector and can express a target protein, said target protein being an antigen protein of pathogens, and said expression vector being selected from pH4sp-HA1-EYFP-ACTIN as shown in SEQ ID NO. 2, pH4SP-M2e-EYFP-ACTIN as shown in SEQ ID NO. 3, Phgra8-E-HA-A3′, gra8pH-E-NA-A3′ as shown in SEQ ID NO. 4, or pHgra8-E-NP-A3′ as shown in SEQ ID NO. 5.

3. The coccidian vector live vaccine as claimed in claim 1, wherein the said coccidia are Eimeriidae coccidian or Cryptosporidium coccidian.

4. The coccidian vector live vaccine of claim 2, wherein the said coccidia are Eimeriidae coccidian or Cryptosporidium coccidian.

5. A method of inducing an immune response in an organism, the method comprising:
administering a vaccine live vector to an organism, wherein said vaccine live vector is the coccidian vector live vaccine as claimed in claim 1.

6. The method as claimed in claim 5, wherein the said coccidia are Eimeriidae coccidian or Cryptosporidium coccidian.

7. The method as claimed in claim 6, wherein said coccidia are Eimeriidae coccidian or Cryptosporidium coccidian of poultry or mammals.

8. The method as claimed in claim 1, wherein said stably transfected coccidia can timely and spatially control the expression of the target protein.

9. A method of inducing an immune response in an organism, the method comprising:
administering a vaccine live vector to an organism, wherein said vaccine live vector is the coccidian vector live vaccine as claimed in claim 2.