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Cryptosporidium parvum |
| Table of Contents |
- General Information
- NCBI Taxonomy ID
- Disease
- Introduction
- Microbial Pathogenesis
- Host Ranges and Animal Models
- Host Protective Immunity
- Vaccine Related Pathogen Genes
- CP15/60
(Protective antigen)
- CP15/60
(Protective antigen)
- CP23
(Protective antigen)
- CpP2
(Protective antigen)
- p23
(Protective antigen)
- Vaccine Related Host Genes
- Ighg1
- Ighv1-9
- Vaccine Information
- C. parvum DNA vaccine encoding CP15/60
- C. parvum DNA vaccine encoding CP23
- C. parvum DNA vaccine encoding CpP2
- References
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| I. General Information |
| 1. NCBI Taxonomy ID: |
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5807 |
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2. Disease: |
| Cryptosporidiosis |
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3. Introduction |
Cryptosporidium parvum is one of several species that cause cryptosporidiosis, a parasitic disease of the mammalian intestinal tract. Primary symptoms of C. parvum infection are acute, watery, and non-bloody diarrhoea. C. parvum infection is of particular concern in immunocompromised patients, where diarrhea can reach 10–15L per day. Other symptoms may include anorexia, nausea/vomiting and abdominal pain.
Infection is caused by ingestion of sporulated oocysts transmitted by the fecal-oral route. In healthy human hosts, the median infective dose is 132 oocysts. The general C. parvum life cycle is shared by other members of the genus. Invasion of the apical tip of ileal enterocytes by sporozoites and merozoites causes pathology seen in the disease. Infection is generally self-limiting in immunocompetent people. In immunocompromised patients, such as those with AIDS or those undergoing immunosuppressive therapy, infection may not be self-limiting, leading to dehydration and, in severe cases, death.
The diagnosis of C. parvum consists of serological tests and microscopic evaluation of oocysts in stools using Kinyoun acid-fast staining. C. parvum is considered to be the most important waterborne pathogen in developed countries. It is resistant to all practical levels of chlorination, surviving for 24hrs at 1000 mg/L free chlorine (Wiki: Cryptosporidium parvum). |
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4. Microbial Pathogenesis |
| Invasion of the apical tip of ileal enterocytes by sporozoites and merozoites causes pathology seen in the disease. Infection is generally self-limiting in immunocompetent people. In immunocompromised patients, such as those with AIDS or those undergoing immunosuppressive therapy, infection may not be self-limiting, leading to dehydration and, in severe cases, death (Wiki: Cryptosporidium parvum). |
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5. Host Ranges and Animal Models |
| Cryptosporidiosis is an enteric diarrheal disease caused mainly by Cryptosporidium parvum, an obligate intracellular protozoan parasite of the intestinal epithelium that infects a variety of mammalian species, including rodents, livestock, and humans (Ehigiator et al., 2007). |
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6. Host Protective Immunity |
| Cryptosporidium sp. infection induces both cellular and humoral immune responses in infected hosts, but protection is mediated primarily through cellular immunity (Ehigiator et al., 2007). |
| II. Vaccine Related Pathogen Genes |
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1. CP15/60 |
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Gene Name :
CP15/60
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Sequence Strain (Species/Organism) :
Cryptosporidium parvum
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VO ID :
VO_0011309
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NCBI Nucleotide GI :
170676501
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NCBI Protein GI :
170676502
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Protein Accession :
ACB30404.1
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Taxonomy ID :
5807
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Gene Strand (Orientation) :
?
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Protein Name :
sporozoite surface antigen CP15/60
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Protein pI :
4.64
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Protein Weight :
15777.88
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Protein Length :
224
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DNA Sequence : Show Sequence
>gi|170676501|gb|EU532137.1| Cryptosporidium parvum sporozoite surface antigen CP15/60 mRNA, complete cds
ATGGGTAACTTGAAATCCTGTTGTTCTTTTGCCGATGAACACTCCCTAACCCCTACTCAACTAGTAGTTG
GAAATGGTTCAGGAGCTTCAGAAACTGCTTCCAACCACCCCCAAGAAGAAGTTAATGATATTAATACTTT
TAATGTAAAGTTAACAATGCAAGATAGAAGTAAGCTTGACTGTGAGGTAGTATTTGATAGCACAAGTATT
TCGCTTTCTGGAGATGGAAAATGCAGAAATATTGCTTTGGATGAAATCCACCAATTATTATATTCAAAGG
AAGAGCTCTCTAGAGTTGAAAGTAGTGCTGGAATCAGTGATTCCGACAATTGTGTTGCAATTCATCTCAA
AGAATCAGGAAACTGTATTCCCCTTTTCTTTAATAATTCGCAAGACAAAGAAAGATTTGTTGCAACAGCA
AACAAATTCAAACCAAACTTTAACTAAAACAGGAATTTCATTAATTCTGGATTTGTTTTTTCCAAACCCT
ATAAATAAAATCCAAATAGCTTTTCTTCAATAACAGGGAG
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Protein Sequence : Show Sequence
>ACB30404.1 sporozoite surface antigen CP15/60 [Cryptosporidium parvum]
MGNLKSCCSFADEHSLTPTQLVVGNGSGASETASNHPQEEVNDINTFNVKLTMQDRSKLDCEVVFDSTSI
SLSGDGKCRNIALDEIHQLLYSKEELSRVESSAGISDSDNCVAIHLKESGNCIPLFFNNSQDKERFVATA
NKFKPNFN
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Molecule Role :
Protective antigen
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Molecule Role Annotation :
Preparturient cows were immunized three times over a six-week period with recombinant plasmid DNA encoding the Cryptosporidium parvum CP15/60 antigen by injecting the DNA in the mammary gland. Colostrum from these cows contained Ab specific for C. parvum sporozoites and oocysts as indicated by immunofluorescence Ab (IFA) staining. Mice receiving immune colostrum showed partial protection (about 50% reduction) against intestinal C. parvum development compared to mice receiving control colostrum. This protection was evident at a challenge dose of 103 C. parvum oocysts per mouse (Jenkins et al., 1999).
- Related Vaccine(s):
C. parvum DNA vaccine encoding CP15/60
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2. CP15/60 |
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Gene Name :
CP15/60
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Sequence Strain (Species/Organism) :
Cryptosporidium parvum
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NCBI Protein GI :
AAA28294
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Taxonomy ID :
5807
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Protein Name :
CP15/60
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Protein pI :
5
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Protein Weight :
16900.14
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Protein Length :
216
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Protein Sequence : Show Sequence
>AAA28294.1 CP15/60, partial [Cryptosporidium parvum]
ARVLIKEKQNMGNLKSCCSFADEHSLTSTQLVVGNGSGASETASNHPQEEVNDINTFNVKLIMQDRSKLD
CEVVFDSTSISLSGDGKCRNIALDEIHQLLYSKEELSRVESSAGISDSDNCVAIHLKESGNCIPLFFNNS
QDKERFVATANKFKPNFN
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Molecule Role :
Protective antigen
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Molecule Role Annotation :
(Roche et al., 2013)
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3. CP23 |
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Gene Name :
CP23
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Sequence Strain (Species/Organism) :
Cryptosporidium parvum
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VO ID :
VO_0011308
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NCBI Protein GI :
88659549
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Taxonomy ID :
5807
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Gene Strand (Orientation) :
?
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Protein Name :
Cp23 antigen
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Protein pI :
4.69
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Protein Weight :
11901.22
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Protein Length :
177
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Protein Note :
genotype: cervine
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Protein Sequence : Show Sequence
>ABD47677.1 Cp23 antigen [Cryptosporidium parvum]
MGCSSSKPETKVADNKSAADANKQRELAEKKAQLAKAVKNPAPISNQPQAKPEEPKKAEPAPVPAPAPVP
APAPAPVPTPAPVPEAPAAEAPSNQAPAPVPAEAPAAEAPVDQNGVPQEKPADA
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Molecule Role :
Protective antigen
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Molecule Role Annotation :
In this study, Cp23 antigen was investigated as a vaccine candidate using the DNA vaccine model in adult interleukin-12 (IL-12) knockout (KO) mice, which are susceptible to C. parvum infection. Cp23-DNA vaccination induced a 50-60% reduction in oocysts shedding, indicating a partial protection against C. parvum infection in IL-12 KO mice (Ehigiator et al., 2007).
- Related Vaccine(s):
C. parvum DNA vaccine encoding CP23
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4. CpP2 |
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Gene Name :
CpP2
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Sequence Strain (Species/Organism) :
Cryptosporidium parvum
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NCBI Protein GI :
AAF21857.1
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Other Database IDs :
CDD:100111
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Taxonomy ID :
5807
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Gene Strand (Orientation) :
?
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Protein Name :
acidic ribosomal protein P2
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Protein pI :
4.15
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Protein Weight :
11050.47
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Protein Length :
179
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Protein Note :
Ribosomal protein P2. This subfamily represents the eukaryotic large ribosomal protein P2. Eukaryotic P1 and P2 are functionally equivalent to the bacterial protein L7/L12, but are not homologous to L7/L12. P2 is located in the L12 stalk, with proteins...; cd05833
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Protein Sequence : Show Sequence
>AAF21857.1 acidic ribosomal protein P2 [Cryptosporidium parvum]
MGMKYVAAYLLCVSSGKEQPTASDIKKVLESVGIEYDQSIIDVLISNMSGKLSHEVIASGLSKLQSVPTG
GVAVSGGAAAASGAAQDSAPAEKKKEEEEEEEGDLGFSLFD
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Molecule Role :
Protective antigen
- Related Vaccine(s):
C. parvum DNA vaccine encoding CpP2
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5. p23 |
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Gene Name :
p23
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Sequence Strain (Species/Organism) :
Cryptosporidium parvum
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NCBI Protein GI :
AEJ22854
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Taxonomy ID :
5807
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Protein Name :
p23
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Protein pI :
4.72
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Protein Weight :
11352.56
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Protein Length :
164
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Protein Note :
subtype: IIm
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Protein Sequence : Show Sequence
>AEJ22854.1 p23, partial [Cryptosporidium parvum]
MGCSSSKPETKVAENKSAADANKQRELAEKKAQLAKAVKNPAPISNQAQQKPEEPKKSEPAPNNPPAADA
PAAQAPAAPAEPAPQDKPADAPAAEAPAAEPAAQQDKPADS
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Molecule Role :
Protective antigen
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Molecule Role Annotation :
(Askari et al., 2016)
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| III. Vaccine Related Host Genes |
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1. Ighg1 |
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Gene Name :
Ighg1
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Sequence Strain (Species/Organism) :
Mus musculus
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NCBI Gene ID :
16017
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Genbank Accession :
AC160982
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Molecule Role :
Vaximmutor
- Related Vaccine(s):
C. parvum DNA vaccine encoding CP23
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2. Ighv1-9 |
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Gene Name :
Ighv1-9
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Sequence Strain (Species/Organism) :
Mus musculus
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NCBI Gene ID :
668478
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Genbank Accession :
AC073561
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Taxonomy ID :
10090
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Chromosome No :
12
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Gene Starting Position :
114583568
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Gene Ending Position :
114583861
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Protein Name :
immunoglobulin heavy variable V1-9
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Protein Note :
Also known as Igg2a; Gm16697
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DNA Sequence : Show Sequence
>gi|372099098:114583568-114583861 Mus musculus strain C57BL/6J chromosome 12, GRCm38 C57BL/6J
GTCTTGCACAGTAATAGATGGCAGAGTCCTCAGTTGTCAGGCTGCTGAGTTGCATGTAGGCTGTGTTGGA
GGATGTATCTGCAGTGAATGTGGCCTTGCCCTTGAACTTCTCATTGTAGTTAGTACTACCACTTCCAGGT
AAAATCTCTCCAATCCACTCAAGGCCATGTCCAGGCCTCTGCTTTACCCACTCTATCCAGTAGCCAGTGA
ATGTGTAGCCAGTAGCCTTGCAGGAAAGCTTCACTGAGGCCCCAGGCTTCATCAGCTCAGCTCCAGACTG
CTGCAGCTGAACCT
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Molecule Role :
Vaximmutor
- Related Vaccine(s):
C. parvum DNA vaccine encoding CP23
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| IV. Vaccine Information |
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1. C. parvum DNA vaccine encoding CP15/60 |
| a. Vaccine Ontology ID: |
| VO_0011502 |
| b. Type: |
| DNA vaccine |
| c. Status: |
| Research |
| d. Antigen |
| C. parvum sporozoite surface antigen CP15/60 |
| e. Gene Engineering of
CP15/60 |
- Type:
DNA vaccine construction
- Description:
Recombinant pCP15/60 plasmid DNA was prepared using standard procedures and resuspended to 1 mg/ml in 0.15 M NaCl. Sera was collected from multiparous Holstein–Fresian cows at 8–12 weeks prior to parturition and tested by IFA for presence of Ab to C. parvum oocysts (Jenkins et al., 1999).
- Detailed Gene Information: Click here.
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| f. Vector: |
| pCP15/60 (Jenkins et al., 1999) |
| g. Immunization Route |
| Parenchymal injection |
| h.
Mouse Response |
- Host Strain:
C57BL/6NCr
- Vaccination Protocol:
Cows (n=2) with negligible titers (<1:50) to C. parvum oocysts were immunized at 6, 4 and 2 weeks prior to parturition with 1 mg of plasmid DNA by needle-syringe injection into the parenchymal tissue of the mammary gland. Colostrum from the cows was also tested for conferring passive immunity against C. parvum infection by oral administration to immunosuppressed adult inbred mice. Immune colostrum and control colostrum were administered to separate groups of dexamethasone (DEX)-treated adult C57BL/6NCr mice beginning 12 h before and at 12 h intervals for 3 days after oral C. parvum oocyst infection (Jenkins et al., 1999).
- Challenge Protocol:
In three separate experiments, DEX-treated mice (n=4 per group, six groups per treatment) received 250 μl of HBC or NBC by gastric intubation using a 26 ga. gavage needle 12 h prior to C. parvum infection. At 0 h, the mice were infected with either 103 or 104 C. parvum oocysts and given 250 μl of either HBC or NBC. Control mice were given H2O and either infected or not infected with C. parvum oocysts (Jenkins et al., 1999).
- Efficacy:
Mice receiving immune colostrum showed partial protection (about 50% reduction) against intestinal C. parvum development compared to mice receiving control colostrum. This protection was evident at a challenge dose of 103 C. parvum oocysts per mouse (Jenkins et al., 1999).
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2. C. parvum DNA vaccine encoding CP23 |
| a. Vaccine Ontology ID: |
| VO_0011362 |
| b. Type: |
| DNA vaccine |
| c. Status: |
| Research |
| d. Antigen |
| Cp23 antigen |
| e. Gene Engineering of
CP23 |
- Type:
DNA vaccine construction
- Description:
The C. parvum 27-kDa antigen coding sequence (GenBank accession number U34390) was amplified from a Cp23- pGEX 4T-2 clone. The fragment was cloned into the EcoRI and NotI restriction enzyme sites of the pUMVC4b expression vector (Adevron, Fargo, ND, USA). This vector has a cytomegalovirus promoter and immunoadjuvant site that enhances immune responses. Plasmids containing inserts (pUMVC4b-Cp23) and plasmids without inserts (pUMVC4b) were transformed into UltraMAX™ DH5a-FT™ Competent Cells (Invitrogen, Carlsbad, CA, USA), purified, and dissolved in sterile Tris–borate–ethylenediamine tetraacetic acid (TBE). After cloning, the sequence of the resulting clone was confirmed by automated DNA sequencing (Ehigiator et al., 2007).
- Detailed Gene Information: Click here.
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| f. Vector: |
| pUMVC4b expression vector |
| g. Immunization Route |
| Subcutaneous injection |
| h.
Mouse Response |
- Host Strain:
C57BL/6 KO
- Vaccination Protocol:
Mice (ten per group) were injected subcutaneously in the ear with 100-μg plasmid pUMVC4b-Cp23 in TBE on days 0, 14, and 29. Mice in the control group were injected with either the vector pUMVC4b without the insert or with PBS (Ehigiator et al., 2007).
- Challenge Protocol:
The different treatment groups of IL-12p40KO mice were challenged with a dose of 1,000 C. parvum oocysts by oral gavage 2 weeks after the last immunization (Ehigiator et al., 2007).
- Efficacy:
Cp23-DNA vaccination induced a 50-60% reduction in oocysts shedding, indicating a partial protection against C. parvum infection in IL-12 KO mice (Ehigiator et al., 2007).
- Host Gene Response of
Ighg1
- Gene Response:
Six weeks post immunization, mice were bled and antibody responses were measured by ELISA. IgG1 responses were significantly higher in Cp23 vaccinated mice than vector (control) immunized mice (Ehigiator et al., 2007).
- Detailed Gene Information: Click here.
- Host Gene Response of
Ighv1-9
- Gene Response:
Six weeks post immunization, mice were bled and antibody responses were measured by ELISA. IgG2a responses were significantly higher in Cp23 vaccinated mice than vector (control) immunized mice (Ehigiator et al., 2007).
- Detailed Gene Information: Click here.
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3. C. parvum DNA vaccine encoding CpP2 |
| a. Type: |
| DNA vaccine |
| b. Status: |
| Research |
| c. Host Species for Licensed Use: |
| Human |
| d. Antigen |
| CpP2: acidic ribosomal proteins P2 of C. parvum (Benitez et al., 2011) |
| e. Gene Engineering of
CpP2 |
- Type:
Recombinant vector construction
- Description:
CpP2 antigen coding sequence was amplified by PCR and was ligated into the EcoRI and NotI restriction enzyme sites of the pUMVC4b expression vector. The ligation mix was then transformed into UltraMAX™ DH5a-FT™ Competent Cells and were selected on LB agar containing kanamycin (50 μg/ml). (Benitez et al., 2011)
- Detailed Gene Information: Click here.
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| f. Vector: |
| pUMVC4b (Benitez et al., 2011) |
| g. Immunization Route |
| Intramuscular injection (i.m.) |
| h.
Mouse Response |
- Host Strain:
C57BL/6 interleukin-12p40 (IL-12p40) knockout (KO) mice (Benitez et al., 2011)
- Vaccination Protocol:
Mice (10 per group) were injected with 100 μg plasmid pUMVC4b-CpP2 in TBE on days 0, 14, and 29. After primary immunization, mice in prime-boost regime were immunized with 3 μg of rP2 in Hunter’s TiterMax:PBS on days 43 and 57. The control group mice were injected with the empty pUMVC4b vector. (Benitez et al., 2011)
- Immune Response:
Humoral: Anti-P2 antibody response was detected four weeks after primary dose injection and two weeks after the first booster dose injection. The response was further enhanced after the injection of the third dose. The anti-CpP2 antibody response was not observed in the control. Poor responses to the recombinant plasmid in wildtype mice were observed. Only IgG1 response was induced. IgG2a and IgA responses were not observed. (Benitez et al., 2011)
Cellular: Stimulation of splenocyte cultures from immunized mice with different concentrations of rCpP2 protein resulted in a dose-dependent proliferative response. Increased levels of IFN-γ were observed in splenocyte cultures from immunized mice compared with both groups of controls (P<0.05). (Benitez et al., 2011)
- Challenge Protocol:
The different treatment groups were challenged with a dose of 1 × 103 C. parvum oocysts by oral gavage 2 weeks after the last immunization. (Benitez et al., 2011)
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| V. References |
1. Askari et al., 2016: Askari N, Shayan P, Mokhber-Dezfouli MR, Ebrahimzadeh E, Lotfollahzadeh S, Rostami A, Amininia N, Ragh MJ. Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum. Parasite immunology. 2016; 38(5); 282-289. [PubMed: 27012710].
2. Ehigiator et al., 2007: Ehigiator HN, Romagnoli P, Priest JW, Secor WE, Mead JR. Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum. Parasitology research. 2007; 101(4); 943-950. [PubMed: 17487508].
3. Jenkins et al., 1999: Jenkins MC, O'Brien C, Trout J, Guidry A, Fayer R. Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice. Vaccine. 1999; 17(19); 2453-2460. [PubMed: 10392628].
4. Roche et al., 2013: Roche JK, Rojo AL, Costa LB, Smeltz R, Manque P, Woehlbier U, Bartelt L, Galen J, Buck G, Guerrant RL. Intranasal vaccination in mice with an attenuated Salmonella enterica Serovar 908htr A expressing Cp15 of Cryptosporidium: impact of malnutrition with preservation of cytokine secretion. Vaccine. 2013; 31(6); 912-918. [PubMed: 23246541].
5. Wiki: Cryptosporidium parvum: Cryptosporidium parvum [http://en.wikipedia.org/wiki/Cryptosporidium_parvum]
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