VIOLIN: Vaccine Investigation and Online Information Network

Cryptosporidium parvum

Table of Contents

General Information
Vaccine Related Pathogen Genes
1. Cp12 (Protective antigen)
2. CP15 (Protective antigen)
3. CP15/60 (Protective antigen)
4. CP15/60 (Protective antigen)
5. Cp21 (Protective antigen)
6. CP23 (Protective antigen)
7. CpP2 (Protective antigen)
8. p23 (Protective antigen)
Vaccine Related Host Genes
1. Ighg1
2. Ighv1-9
Vaccine Information
References

I. General Information

1. NCBI Taxonomy ID:

5807

2. Disease:

Cryptosporidiosis

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).

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).

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).

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

1. Cp12

Gene Name : Cp12
Sequence Strain (Species/Organism) : Cryptosporidium parvum Iowa II
NCBI Gene ID : 3372891
NCBI Protein GI : 66357286
Locus Tag : cgd4_2330
Genbank Accession : AAEE01000008
Protein Accession : XP_625821
Taxonomy ID : 353152
Chromosome No : 4
Gene Starting Position : 554385
Gene Ending Position : 554699
Gene Strand (Orientation) : -
Protein pI : 4.35
Protein Weight : 11648.13
Protein Length : 104

DNA Sequence : Show Sequence

>NC_006983.1:554385-554699 Cryptosporidium parvum Iowa II chromosome 4, whole genome shotgun sequence
ACTATATTTGTTCATTCATCTGTTTCTTTTCTATGTTCCTTTCAATTGTTCTTGCCATTTCTGAAGGCGA
CTGTAGCCCTCCCATCACACTATTAAAATCGGTTGGAACTTGAATATAGTTATATTGATTACCATTGGTA
AAATTTTGCGCATCGTTTCTTTCAGGTTGCCACTGGAGCCTATCCTGTTCGAATACGTTGTGCCAAAGTA
CTCCTTCATACCAGTATGTCAATGAAATGCCGGTAGGGAGTAGAGCTACAAATATCGCAGCATAGAAACC
TATTGAAAACTTCCAATCTATTGATGCATCTGACA

Protein Sequence : Show Sequence

>XP_625821.1 hypothetical protein [Cryptosporidium parvum Iowa II]
MSDASIDWKFSIGFYAAIFVALLPTGISLTYWYEGVLWHNVFEQDRLQWQPERNDAQNFTNGNQYNYIQV
PTDFNSVMGGLQSPSEMARTIERNIEKKQMNEQI

Molecule Role : Protective antigen
Related Vaccine(s): pVAX1-C-Cp12-Cp21

2. CP15

Gene Name : CP15
NCBI Protein GI : ABO61338.1
Other Database IDs : CDD:185442
Taxonomy ID : 5807
Gene Strand (Orientation) : ?
Protein Name : 15 kDa sporozoite surface antigen
Protein pI : 10.74
Protein Weight : 12412.8
Protein Length : 202
Protein Note : 40S ribosomal protein S15; Provisional

Protein Sequence : Show Sequence

>ABO61338.1 15 kDa sporozoite surface antigen, partial [Cryptosporidium parvum]
MKLDEVVELLPARKRRKIARGCLNRRTAAFIAKLRKSKAECPMGEKPVAVRTHLRNMVILPEMVGAVAGV
YNGKTYATVEIKPEMIGMYLGEFSITYKPVRHGKPGVGSTSSSRFIPLK

Molecule Role : Protective antigen
Related Vaccine(s): Cp15-23 , pVAX-15-23

3. CP15/60

Gene Name : CP15/60
Sequence Strain (Species/Organism) : Cryptosporidium parvum
VO ID : VO_0011309
NCBI Gene ID : 3371947
NCBI Nucleotide GI : 170676501
NCBI Protein GI : 170676502
Protein Accession : ACB30404.1
Taxonomy ID : 5807
Gene Strand (Orientation) : ?
Protein Name : sporozoite surface antigen CP15/60
Protein pI : 4.64
Protein Weight : 15777.88
Protein Length : 224

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

Protein Sequence : Show Sequence

>ACB30404.1 sporozoite surface antigen CP15/60 [Cryptosporidium parvum]
MGNLKSCCSFADEHSLTPTQLVVGNGSGASETASNHPQEEVNDINTFNVKLTMQDRSKLDCEVVFDSTSI
SLSGDGKCRNIALDEIHQLLYSKEELSRVESSAGISDSDNCVAIHLKESGNCIPLFFNNSQDKERFVATA
NKFKPNFN

Molecule Role : Protective antigen
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 10^3 C. parvum oocysts per mouse (Jenkins et al., 1999).
Related Vaccine(s): C. parvum DNA vaccine encoding CP15/60

4. CP15/60

Gene Name : CP15/60
Sequence Strain (Species/Organism) : Cryptosporidium parvum
NCBI Protein GI : AAA28294
Taxonomy ID : 5807
Protein Name : CP15/60
Protein pI : 5
Protein Weight : 16900.14
Protein Length : 216

Protein Sequence : Show Sequence

>AAA28294.1 CP15/60, partial [Cryptosporidium parvum]
ARVLIKEKQNMGNLKSCCSFADEHSLTSTQLVVGNGSGASETASNHPQEEVNDINTFNVKLIMQDRSKLD
CEVVFDSTSISLSGDGKCRNIALDEIHQLLYSKEELSRVESSAGISDSDNCVAIHLKESGNCIPLFFNNS
QDKERFVATANKFKPNFN

Molecule Role : Protective antigen
Molecule Role Annotation : (Roche et al., 2013)

5. Cp21

Gene Name : Cp21
Sequence Strain (Species/Organism) : Cryptosporidium parvum Iowa II
NCBI Gene ID : 3373643
NCBI Protein GI : 66358562
Locus Tag : cgd2_2570
Genbank Accession : AAEE01000005
Protein Accession : XP_626459
Taxonomy ID : 353152
Chromosome No : 2
Gene Starting Position : 519155
Gene Ending Position : 519697
Gene Strand (Orientation) : -
Protein pI : 7.81
Protein Weight : 19446.76
Protein Length : 180

DNA Sequence : Show Sequence

>NC_006981.1:519155-519697 Cryptosporidium parvum Iowa II chromosome 2, whole genome shotgun sequence
GCTATTCATCTGTTTGAACGTCAGAATCAGAATTATATATTTCTATTACTCTCTCGTATACAAAATAAGG
GGATGCGCCTGGAATATTACTTGGATTCCTCCCTTTTATTTGTTTTCTAGGTCTTACTGGTCTTGGGGAA
CTGATAGCATTTCGAATTTTCCTTAAATGAGTGGCATTAGATAAAAGTTCTGGTATATGAACTAGCTGAT
AGATAGGTACTTTAGTTAGGTTTTGATAAATTATATTTTCATAGGTTTCTTTAAGTGTGCTTTCACTCAC
ATCAAAATAATCATGTTCGCTGGATTTAGCAACCAATTGAAAGTCTTCTGGAATTATTTTCCGAACAAGA
TATATTATTTGTTCCACTGAAAGAAGGGTCTTGAGTCTAGATGCTAATTCTTTCAAACTTATTTCGCTGT
TTTCAAAAGATAACGCTTCAGCTCTACTCACAGCCAACTTTGGGGAAACCTCATGTTGTGAAGTTGCAAA
AATGTCAAACAATACGAAGAAAATAAAAATAAATAGTGATGCTCTTTTAGACA

Protein Sequence : Show Sequence

>XP_626459.1 hypothetical protein [Cryptosporidium parvum Iowa II]
MSKRASLFIFIFFVLFDIFATSQHEVSPKLAVSRAEALSFENSEISLKELASRLKTLLSVEQIIYLVRKI
IPEDFQLVAKSSEHDYFDVSESTLKETYENIIYQNLTKVPIYQLVHIPELLSNATHLRKIRNAISSPRPV
RPRKQIKGRNPSNIPGASPYFVYERVIEIYNSDSDVQTDE

Molecule Role : Protective antigen
Related Vaccine(s): pVAX1-C-Cp12-Cp21

6. CP23

Gene Name : CP23
Sequence Strain (Species/Organism) : Cryptosporidium parvum
VO ID : VO_0011308
NCBI Gene ID : 3372811
NCBI Nucleotide GI : U34390
NCBI Protein GI : AAB17724.1
Taxonomy ID : 5807
Gene Strand (Orientation) : ?
Protein Name : Cp23 antigen
Protein pI : 4.69
Protein Weight : 11901.22
Protein Length : 177
Protein Note : genotype: cervine

Protein Sequence : Show Sequence

>AAB17724.1 protein with neutralization-sensitive epitopes [Cryptosporidium parvum]
MGCSSSKPETKVAENKSAADANKQRELAEKKAQLAKAVKNPAPISNQAQQKPEEPKKSEPAPNNPPAADA
PAAQAPAAPAEPAPQDKPADAPAAEAPAAEPAAQQDKPADA

Molecule Role : Protective antigen
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 , Cp15-23 , pVAX-15-23

7. CpP2

Gene Name : CpP2
Sequence Strain (Species/Organism) : Cryptosporidium parvum
NCBI Gene ID : 3372370
NCBI Protein GI : AAF21857.1
Other Database IDs : CDD:100111
Taxonomy ID : 5807
Gene Strand (Orientation) : ?
Protein Name : acidic ribosomal protein P2
Protein pI : 4.15
Protein Weight : 11050.47
Protein Length : 179
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

Protein Sequence : Show Sequence

>AAF21857.1 acidic ribosomal protein P2 [Cryptosporidium parvum]
MGMKYVAAYLLCVSSGKEQPTASDIKKVLESVGIEYDQSIIDVLISNMSGKLSHEVIASGLSKLQSVPTG
GVAVSGGAAAASGAAQDSAPAEKKKEEEEEEEGDLGFSLFD

Molecule Role : Protective antigen
Related Vaccine(s): C. parvum DNA vaccine encoding CpP2

8. p23

Gene Name : p23
Sequence Strain (Species/Organism) : Cryptosporidium parvum
NCBI Protein GI : AEJ22854
Taxonomy ID : 5807
Protein Name : p23
Protein pI : 4.72
Protein Weight : 11352.56
Protein Length : 164
Protein Note : subtype: IIm

Protein Sequence : Show Sequence

>AEJ22854.1 p23, partial [Cryptosporidium parvum]
MGCSSSKPETKVAENKSAADANKQRELAEKKAQLAKAVKNPAPISNQAQQKPEEPKKSEPAPNNPPAADA
PAAQAPAAPAEPAPQDKPADAPAAEAPAAEPAAQQDKPADS

Molecule Role : Protective antigen
Molecule Role Annotation : (Askari et al., 2016)

III. Vaccine Related Host Genes

1. Ighg1

Gene Name : Ighg1
Sequence Strain (Species/Organism) : Mus musculus
NCBI Gene ID : 16017
Genbank Accession : AC160982
Molecule Role : Vaximmutor
Related Vaccine(s): C. parvum DNA vaccine encoding CP23

2. Ighv1-9

Gene Name : Ighv1-9
Sequence Strain (Species/Organism) : Mus musculus
NCBI Gene ID : 668478
Genbank Accession : AC073561
Taxonomy ID : 10090
Chromosome No : 12
Gene Starting Position : 114583568
Gene Ending Position : 114583861
Protein Name : immunoglobulin heavy variable V1-9
Protein Note : Also known as Igg2a; Gm16697

DNA Sequence : Show Sequence

>gi|372099098:114583568-114583861 Mus musculus strain C57BL/6J chromosome 12, GRCm38 C57BL/6J
GTCTTGCACAGTAATAGATGGCAGAGTCCTCAGTTGTCAGGCTGCTGAGTTGCATGTAGGCTGTGTTGGA
GGATGTATCTGCAGTGAATGTGGCCTTGCCCTTGAACTTCTCATTGTAGTTAGTACTACCACTTCCAGGT
AAAATCTCTCCAATCCACTCAAGGCCATGTCCAGGCCTCTGCTTTACCCACTCTATCCAGTAGCCAGTGA
ATGTGTAGCCAGTAGCCTTGCAGGAAAGCTTCACTGAGGCCCCAGGCTTCATCAGCTCAGCTCCAGACTG
CTGCAGCTGAACCT

Molecule Role : Vaximmutor
Related Vaccine(s): C. parvum DNA vaccine encoding CP23

IV. Vaccine Information

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.

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 10^3 C. parvum oocysts per mouse (Jenkins et al., 1999).

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.

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.

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.

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 × 10^3 C. parvum oocysts by oral gavage 2 weeks after the last immunization. (Benitez et al., 2011)

4. Cp15-23

a. Type:

Subunit vaccine

b. Status:

Research

c. Host Species for Licensed Use:

Human

d. Antigen

Cp15: 15 kDa sporozoite surface antigen (Liu et al., 2010); Cp23: a glycoprotein, geographically conserved among C. parvum isolates, present in both the sporozoite and merozoite stages (Liu et al., 2010)

e. Gene Engineering of CP15

Type: Recombinant protein preparation
Description:
Detailed Gene Information: Click here.

f. Gene Engineering of CP23

Type: Recombinant protein preparation
Description:
Detailed Gene Information: Click here.

g. Immunization Route

Intramuscular injection (i.m.)

h. Description

C. parvum recombinant protein vaccine that uses Cp15-23 fused protein as antigen and uses Freund’s adjuvant. (Liu et al., 2010)

i. Mouse Response

Host Strain: BALB/c mice (Liu et al., 2010)
Vaccination Protocol: The mice were randomly divided into different groups and were immunized subcutaneously with 10 μg of rCp15–23, rCp23, or crude extract of C. parvum in complete Freund’s adjuvant. Subsequent immunizations on days 14 and 28 were performed with 10 μg proteins in incomplete Freund’s adjuvant. The control group were given adjuvant alone. (Liu et al., 2010)
Immune Response: Humoral: All immunized groups have specific antibody responses. The concentrations of IgG remained at low levels until days 14 after the first vaccination, whereas the second dose of vaccine rapidly and significantly boosted the responses. A peak concentration was observed after third boost. Both rCp15–23and rCp23 induced stronger antibody response than crude extract (P<0.05). (Liu et al., 2010)
Cellular: The number of CD4+ and CD8+ T cells was increased in all immunized groups compared with the control (P<0.01), whereas the number of CD4+ T cells was much more than that of CD8+ T cells. The stimulation of cells from rCp15–23 immunized mice generated higher CD4+, CD8+ T cells and the ratio of CD4+/CD8+ than the other two immunized groups (P<0.01) (Liu et al., 2010)
Cytokine: Significantly higher concentrations of IFN-γ or IL-12 were found in the spleen cells of all immunized groups. The IFN-γ and IL-12 levels were significantly higher in rCp15–23 immunized mice compared with the crude extract immunized mice (P<0·05). No significant difference was observed in crude extract group compared with the control group. Very low level of IL-4 was found in all the groups and no difference was found between different groups. (Liu et al., 2010)
Challenge Protocol: 10 mice were randomly selected from each group and were challenged with 1×10^6 oocysts orally 2 weeks after the last immunization. (Liu et al., 2010)

5. pVAX-15-23

a. Type:

DNA vaccine

b. Status:

Research

c. Host Species for Licensed Use:

Human

d. Antigen

Cp15: 15 kDa sporozoite surface antigen (Wang et al., 2010); Cp23(Wang et al., 2010): a glycoprotein, geographically conserved among C. parvum isolates, present in both the sporozoite and merozoite stages (Liu et al., 2010)

e. Gene Engineering of CP15

Type: DNA vaccine construction
Description: Amplified by PCR using a reverse primer containing a synthetic linker sequence. The amplified Cp15 was linked to Cp23 and was inserted into the pVAX1 expression vector. (Wang et al., 2010)
Detailed Gene Information: Click here.

f. Gene Engineering of CP23

Type: DNA vaccine construction
Description: Amplified by PCR. The amplified Cp23 was linked to Cp15 and was inserted into the pVAX1 expression vector. (Wang et al., 2010)
Detailed Gene Information: Click here.

g. Vector:

pVAX1: plasmid vector encoding antigen DNA (Wang et al., 2010); pMEM12R: plasmid vector encoding adjuvant sequence (Wang et al., 2010).

h. Immunization Route

Intramuscular injection (i.m.)

i. Description

C. parvum DNA vaccine that express Cp15 and Cp23 in pVAX1 as antigen and express IL-12 cytokine in pMEM12R as adjuvant. (Wang et al., 2010)

j. Mouse Response

Host Strain: C57BL/6 interleukin-12p40 (IL-12p40) knockout (KO) mice (Wang et al., 2010)
Vaccination Protocol: Each mouse was vaccinated with with 100 μg of multivalent (pVAX-15–23) or single gene (pVAX-15 or pVAX-23) DNA vaccine alone or plus pMEM12R or in TBE on days 0, 14, and 28. Non-recombinant pVAX-1 plasmid was used as negative control. (Wang et al., 2010)
Immune Response: Cellular: splenocytes from mice vaccinated with pVAX15–23 expressed significant INF-γ levels in response of both antigens. IFN-γ expression was significantly boosted by co-administration of pMEM12R, inducting Th1 type immune response. (Wang et al., 2010)
Humoral: Splenocytes from vaccinated mice exhibited a strong lymphoproliferation responses 2 and 10 weeks after the final immunization, revealing sustained response of to stimulating proteins on the term of time. Stimulation of splenocytes derived from mice immunized 10 weeks before cell harvest showed significant (p < 0.01) pVAX15–23-specific proliferative response in vitro culture. pVAX15–23-DNA vaccine could induce a more sustained cellular immune response than pVAX15 and pVAX23. (Wang et al., 2010)
Challenge Protocol: Mice were orally challenged with a single dose of 1 × 10^5 C. parvum oocysts in 200 μL of 0.15 M phosphate-buffered saline (PBS, pH 7.2) 2 weeks after the last immunization. (Wang et al., 2010)
Efficacy: The pattern of oocysts shedding was similar in all experimental groups: shedding peak on day 10-12 post challenge and steady decline with the time course. DNA vaccination mitigated the intensity of the infection with significant decrease for mice in pVAX-15-23 group. Mice injected pVAX-15-23 and pMEM12R resolved the infection earlier than other groups. (Wang et al., 2010)

6. pVAX1-C-Cp12-Cp21

a. Type:

DNA vaccine

b. Status:

Research

c. Host Species for Licensed Use:

Human

d. Antigen

Cp12 (Yu et al., 2010): a 12kDA surface adherence protein of C.parvum (Yao et al., 2007);Cp21: a 21kDA surface adherence protein of C.parvum (Yu et al., 2010):

e. Gene Engineering of Cp12

Type: DNA vaccine construction
Description: Amplified by PCR using a forward primer containing the booster sequence encoding for CpG-ODN. The amplified C-Cp12 was linked to Cp21 and formed C-Cp12-Cp21, and was inserted into the pVAX1 expression vector. (Yu et al., 2010)
Detailed Gene Information: Click here.

f. Gene Engineering of Cp21

Type: DNA vaccine construction
Description: Amplified by PCR using a forward primer containing a synthetic linker sequence. The amplified Cp21 with the linker sequence was inked to C-Cp12 and formed C-Cp12-Cp21, and was inserted into the pVAX1 expression vector. (Yu et al., 2010)
Detailed Gene Information: Click here.

g. Vector:

pVAX1 expression vector (Yu et al., 2010)

h. Immunization Route

Intramuscular injection (i.m.) or Nasal spray

i. Description

C. parvum DNA vaccine that express Cp12 and Cp21 in pVAX1 as antigen and use CpG-ODN as adjuvant. (Yu et al., 2010)

j. Mouse Response

Host Strain: BALB/c mice (Yu et al., 2010)
Vaccination Protocol: Two hundred mice were randomly divided into 10 groups. Mice in different groups were immunized at weeks 0, 3, and 5 with different schemes: 1) 100 μg of pVAX1-CP12 musculature, 2) 100 μg of pVAX1-CP21 musculature, 3) 100 μg of pVAX1-CP12 nasal, 4) 100 μg of pVAX1-CP21 nasal, 5) 100 μg of pVAX1-CP12-CP21 musculature, 6) 100 μg of pVAX1-C-CP12-CP21 musculature, 7) 100 μg of pVAX1-CP12-CP21 nasal, 8) 100 μg of pVAX1-C-CP12-CP21 nasal, 9) 100 μg of pVAX1, 10) 0.1mL of PBS (Yu et al., 2010)
Immune Response: Humoral: pVAX1-C-Cp12-Cp21 nasal spray group had significantly higher level of IgG compared to pVAX1 vector group and PBS group (P < 0.01). The IgG level in the pVAX1-C-Cp12-Cp21 nasal spray group was also higher than that of pVAX1-Cp12, pVAX1-Cp21, and pVAX1-Cp12-Cp21 group but was not significant.
Cellular: The percentages of CD4+ T cells in pVAX1-C-Cp12-Cp21 nasal group were significantly higher than that in the groups treated with pVAX1 vector, PBS, pVAX1-Cp12, and pVAX1-Cp21 (P < 0.01). The percentages of CD8+ T cells in pVAX1-C-Cp12-Cp21 nasal spray group was significantly higher than that in the PBS group (P < 0.01). (Yu et al., 2010)
Challenge Protocol: Each mouse was orally inoculated with 1 × 10^6 C. parvum oocysts in 0.5 ml of water 2 weeks after the last immunization. (Yu et al., 2010)
Efficacy: Mice in pVAX1-Cp12-Cp21 and pVAX1-C-Cp12-Cp21 groups excreted less numbers of oocysts than that in other groups (P < 0.05). Mice in the pVAX1-C-Cp12-Cp21 nasal spray group have a 77.5% reduction in the level of oocysts shedding. (Yu et al., 2010)

V. References

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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]