Click here to load reader

Host-Parasite Interactions of Cryptosporidium

  • View
    50

  • Download
    1

Embed Size (px)

DESCRIPTION

Host-Parasite Interactions of Cryptosporidium. Molecular Basis of Attachment and Invasion. Ultrastructural Aspects of Cryptosporidium Attachment and Invasion. Zoites attach to host cells by their anterior pole Rhoptries and micronemes discharge their contents - PowerPoint PPT Presentation

Text of Host-Parasite Interactions of Cryptosporidium

  • Host-Parasite Interactions of CryptosporidiumMolecular Basis of Attachment and Invasion

  • Ultrastructural Aspects of Cryptosporidium Attachment and Invasion Marcial and Madara, 1986; Lumb et al, 1988; Tzipori, 1988; Fayer et al, 1990, Yoshikawa and Iseki, 1992; Fayer et al, 1997; Griffiths and Tzipori, 1998, Zoites attach to host cells by their anterior pole Rhoptries and micronemes discharge their contents Electron-dense bands form in host cell cytoplasm Zoites invaginate the host cell plasma membrane which eventually engulfs the parasite within the parasitophorus vacuole Parasite remains in parasitophorus vacuole in unique intracellular but extracytoplasmic location Unique feeder organelle membrane forms at the site of attachment

  • Electron Micrograph of Cryptosporidium Sporozoite Attaching to Intestinal Microvillus MembraneTzipori, 1988

  • Electron Micrograph of Cryptosporidium Merozoite Invading Intestinal Epithelial Cell MembraneTzipori, 1988

  • Factors affecting Cryptosporidium sporozoite attachment in vitro Hamer et al, 1994; Joe et al, 1998; Chen et al 1998; Chen et al 2000 Time Number of sporozoites Temperature Divalent cations pH Host cell type Differentiation status of host cells Host plasma membrane domain

  • Role of Parasite and Host Cytoskeletal Elements in Cryptosporidium Motility, Attachment and Invasion in vitro Forney et al, 1998, Forney et al, 1999, Yu and Lee, 1996; Bonin et al, 1999, Chen et al 2000, Elliot and Clark, 2000 Sporozoite motility is powered by actin- myosin motor system Host cell actin is recruited to the host-parasite interface during invasion. Filamentous actin is assembled into a plaque-like structure Host cytoskeletal molecules may be involved in parasitophorus vacuole formation

  • Surface/Apical Proteins of Cryptosporidium >20 sporozoite surface proteins 11-~1300 kDa identified Surface/apical proteins implicated in attachment and/or invasion many identified by antibodies which inhibit infection in vitro and/or in vivo in animal models many proteins glycosylated many proteins shed in trails during gliding motility

  • Surface/Apical Proteins of Cryptosporidium >200 kDa-~1300 kDaPetersen et al 1992; Doyle et al, 1993; Barnes et al, 1998; Langer and Riggs, 1996; Riggs, 1997; Riggs et al, 1997; Langer et al 1999; McDonald et al, 1995, Robert et al, 1994) 40-47 kDa Nesterenko et al, 1999; Cevallos et al, 2000; Strong et al, 2000 20-27 kDa Ungar and Nash, 1986; Mead et al, 1988, Arrowood et al, 1989; Arrowood et al, 1991; Perryman et al, 1996; Perryman et al, 1999; Enriquez and Riggs, 1998; Lumb et al, 1989; Tilley et al, 1993; Tilley and Upton, 1994 15-17 kDa Tilley et al, 1991; Tilley et al, 1993,; Tilley and Upton, 1994; Jenkins et al 1993; Jenkins and Fayer, 1995; Khramtsov et al, 1993; Sagodira, 1999; Gut and Nelson, 1994; Strong et al, 2000; Cevallos et al, 2000; El Shewy et al, 1994; Mead et al, 1988; Moss et al 1994, 1998; Reperant et al 1992, 1994; Peeters et al, 1992; Ortega-Mora et al 1994; Priest et al, 1999; Priest et al, 2000 TRAP C1 (Spano et al, 1998) Gal/GalNAc-specific lectin/s (Joe et al. 1994; Joe et al, 1998; Chen et al, 2000)

  • Effect of MAb 4E9 IgM on C. parvum infection of Caco-2A cells Infection (A405nm)Cevallos et al, 2000 IgM g/ml 4E9 B9A4

    Chart1

    0.44350.0128281916

    0.01666666670.0061237244

    0.07333333330.028347178

    0.14616666670.0339051987

    0.23583333330.001931105

    0.44266666670.0349869023

    26-27 feb

    CytotoxicityAttachmentInfection

    123456789101112123456789101112123456789101112

    AAA

    B1.5271.4961.4761.3661.5081.3721.4511.4331.4531.038B0.1930.6690.4560.8620.9780.8690.2710.1930.1420.096B0.2080.4810.4880.5820.4290.2930.2220.2290.2270.221

    C1.5571.5261.7621.4251.4091.3971.3401.3001.3931.311C0.1680.6620.4770.7830.7570.6220.2650.1990.1820.091C0.2280.4850.4840.5580.4830.3800.3040.2790.2390.244

    D1.2441.7311.6471.4951.4471.2921.1771.1821.3741.372D0.1690.6470.4860.8550.7960.6880.3170.2260.1750.095D0.2520.4370.4480.6190.4810.3440.3270.3050.2520.261

    E1.4961.6711.6561.5721.3951.4301.2451.3111.5081.493E0.1610.6460.4430.7940.8770.8000.3180.2430.1940.086E0.2680.5370.4780.6380.5400.3460.3270.3030.2690.253

    F1.4301.6101.4701.6661.4001.3991.2741.3121.4371.490F0.1820.6620.5350.8290.8540.7800.3060.2780.2360.095F0.2650.4640.4680.6900.6640.4360.3780.3280.2780.285

    G1.5221.5471.6401.4591.3901.2761.3251.4111.5841.812G0.1980.2610.3190.3420.2830.2300.1660.1390.1330.097G0.2530.4630.4160.5710.5770.3500.3600.3300.2360.242

    HHH

    blank1.530blank0.179blank0.246

    Mabs4E9Mabs4E9

    Mabs4E9meanSEMmeanSEMmeanSEMmeanSEM

    4E91.6091001.609Positive0.4790.0051000.3010.016Positive0.2320.0141000.2180.011

    4G121.497501.3024E90.3010.016500.1170.0114E90.2180.011500.0740.022

    B4A91.425101.3254G120.6460.016100.0490.0154G120.3640.020100.0500.015

    4D11.36151.458B4A90.6740.03850.0070.015B4A90.2830.03450.0050.008

    4D10.5730.0434D10.1130.019

    attachmentinfectionSEMSEM4E90.4910.2780.6840.8000.6910.0930.015-0.037-0.0830.2350.2420.3360.1830.047-0.024-0.017-0.019-0.025

    Positive0.4790.2320.0050.014attachmentinfectionSEMSEM(2)0.4840.2990.6050.5790.4440.0870.0210.004-0.0880.2390.2380.3120.2370.1340.0580.033-0.007-0.002

    4E90.3010.2180.0160.0111000.3010.2180.0160.0110.4690.3080.6770.6180.5100.1390.048-0.004-0.0840.1910.2020.3730.2350.0980.0810.0590.0060.015

    4G120.6460.3640.0160.020500.1170.0740.0110.0220.4680.2650.6160.6990.6220.1400.0650.016-0.0930.2910.2320.3920.2940.1000.0810.0570.0230.007

    B4A90.6740.2830.0380.034100.0490.0500.0150.0150.4840.3570.6510.6760.6020.1280.1000.058-0.0840.2180.2220.4440.4180.1900.1320.0820.0320.039

    4D10.5730.1130.0430.01950.0070.0050.0150.0080.2170.1700.3250.3310.1040.1140.084-0.010-0.004

    26-27 feb

    000.01371232210.004554119

    000.01117636390.0158322456

    000.02022649530.0158259281

    000.0341906420.0378584205

    000.0193191040.0434584859

    attachment

    infection

    Sheet2

    000.01371232210.004554119

    000.01117636390.0158322456

    000.02022649530.0158259281

    000.0341906420.0378584205

    attachment

    infection

    13-mar-98

    ATTACHMENT4E9

    blankPos4e9blank

    0.2220.3320.2550.2270.1990.2941.2190.9130.5710.7470.469

    0.2310.3140.260.2390.2180.3181.2640.8750.5520.7780.477

    0.2250.2870.240.2080.2150.3521.1900.8800.5720.7020.391

    0.2360.30.2480.2260.2260.3001.2560.8800.6000.7130.408

    0.2240.3030.2470.2290.2410.2901.3750.9130.5840.7090.394

    0.2130.3350.2580.2410.2280.3071.4480.9720.5610.7220.409

    0.2250.310

    Control4E9

    Positve0.0870.982

    4E90.0260.5950.41870.3

    0.2630.11543.5

    13-mar-98

    00

    00

    00

    Control

    4E9

    Infection

    19-mar-98

    0

    0

    Attachment5x104/well

    26-mar-98

    InfectionAttachment

    123456789101112123456789101112

    AA

    B0.2710.3600.2890.3170.3200.342B0.1940.3240.2040.2180.2260.244

    C0.2860.3440.2960.2740.3300.370C0.1880.3490.1830.1940.2390.273

    D0.2820.3480.2740.3040.3270.328D0.2030.3750.2500.2540.2610.241

    E0.3560.3320.2580.3000.2950.318E0.1890.3500.2450.2710.2590.249

    F0.2980.3350.3350.2970.2950.320F0.1940.3660.2370.2650.2700.311

    G0.2570.3800.2670.2500.3020.299G0.1790.3680.2670.2710.2570.270

    HH

    blank0.292blank0.191

    4E9positive4E9positive

    1000.0560.0581000.1700.164

    500.057500.174

    100.057100.170

    50.06650.176

    26-mar-98

    00

    00

    00

    00

    4E9

    positive

    Infection

    3 Apr 98

    00

    00

    00

    00

    4E9

    positive

    Attachment

    HW-data

    Attachment 1 hAttachment 1.5 hInfection

    0.2580.2580.7370.6900.3980.3880.2560.2410.6660.6760.3670.3570.3150.2770.5860.5930.3310.377

    0.2620.2850.7510.6190.3850.3490.2500.2370.6330.6140.3500.3210.2750.2440.5450.5350.3630.390

    0.2510.2460.6840.6610.3470.3570.2530.2300.6510.6280.3250.3480.2820.2990.6940.5530.3390.368

    0.2710.2590.6290.6070.3410.3620.2580.2680.6930.6580.3640.3680.2960.3150.6290.5910.3530.334

    Blank0.261Blank0.249Blank0.288

    Positive0.411Positive0.403Positive0.303

    4E90.1054E90.1014E90.069

    AttachmentInfection

    Positive0.4110.303

    4E90.1050.069

    Inhibition74.577.2

    HW-data

    00

    00

    Positive

    4E9

    HW-data (2)

    Attachment 1 hInfection

    blankpositive10050105blankpositive10050105

    0.1740.4390.2180.2390.2290.2220.3420.4280.3490.3630.3800.320

    0.1870.4640.2610.2980.2390.2230.3370.3870.3960.3690.3770.405

    0.1770.4200.2510.3050.2710.2230.2970.4150.3610.3470.3530.330

    0.1900.4420.2880.2750.2670.2430.3210.4380.3930.3710.3660.362

    0.1850.4210.2550.2630.2300.2560.3100.5410.4180.4090.4240.407

    0.1760.5210.2410.2580.2300.2660.2920.4470.3740.3300.3520.323

    Blank0.1820.317

    AttachmentInfection

    4E9Positive0.4510.126

    1000.2520.065

    500.2730.048

    100.2440.059

    50.2390.041

    HW-data (2)

    00

    00

    00

    00

    00

    Attachment

    Infection

    O.D.

    Effect of MAb 4E9 in attachment and infection

    HW-data (3)

    Attachment 1 hInfection

    blankpositive10050105blankpositive10050105

    0.1640.1800.8300.5290.3630.4110.2850.2740.5470.5600.5590.624

    0.1840.1930.7780.6170.3610.4220.2940.2310.5400.5450.6390.670

    0.1890.1550.7330.5250.4030.3490.3410.3620.5430.5560.6400.548

    0.1610.1840.7730.5220.3780.3880.2700.3060.5120.5190.6280.607

    0.1830.1820.8090.6240.3770.3890.2840.3200.6230.5630.7640.655

    0.1870.1860.7620.6060.3700.3600.2730.2730.5400.5580.6320.631

    Blank0.1780.291

    AttachmentInfection

    4E9Positive0.1800.003

    1000.7810.260

    500.5710.259

    100.3750.353

    50.3870.331

    HW-data (3)

    00

    00

    00

    00

    00

    Attachment

    Infection

    O.D.

    Effect of MAb 4E9 in attachment and infection

    June 3, 98

    Attachment ELISA for 4E9

    Control100

    1080.70.3

    2572.71.6

    5069.03.4

    10059.91.9

    June 3, 98

    0

    00.310.31

    01.621.62

    03.433.43

    01.91.9

    IgG concentration (g/ml)

    A405 nm (% of control)

    June12, 98

    Attachment ELISA for 4G12

    Control100

    5091.33.3

    10091.42.3

    20090.61.2

    June12, 98

    0

    03.33.3

    02.32.3

    01.21.2

    IgG concentration (g/ml)

    A405 nm (% of control)

    June18, 98

    Infection ELISA for 4E9

    Control100

    2566.61.7

    5050.82.8

    10050.91.0

    June18, 98

    0

    01.661.66

    02.812.81

    01.031.03

    IgG concentration (g/ml)

    A405 nm (% of control)

    June 24, 98

    Attachment 1 hInfection

    blankpositive10050105blankpositive10050105

    0.2360.3690.2970.2730.2700.2880.3110.5430.3870.3450.4070.431

    0.2230.4660.2780.2390.2700.3110.3510.4830.3370.3370.3910.439

    0.1900.3260.2470.2280.2360.2510.2960.5360.3730.3230.3960.420

    0.2010.3370.2220.2450.2400.2470.2630.5670.3190.3480.3660.406

    0.2170.3350.2400.2290.2210.2910.2650.4430.3220.3320.2970.341

    0.1700.2800.2080.2170.2090.2000.2380.4350.2810.3410.3210.320

    Blank0.2060.287

    %Attachment%infectionInfection

    4E9Control100.00.352Control100.00.214

    10070.60.24910023.00.049

    5067.70.2395023.50.050

    1068.40.2411035.40.076

    575.20.265549.30.106

    June 24, 98

    0

    0

    0

    0

    0

    %Attachment

    % attachment

    Inhibition of Attachment by 4E9

    June 24, 98 (2)

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4E9

    June 24, 98 (3)

    Attachment 1 hInfection

    blankpositive100501051blankpositive100501051

    0.4350.6700.4910.4960.4470.4510.5130.3620.6150.3800.3820.3780.4250.513

    0.3920.6390.4170.4250.4190.4240.5120.3760.6510.3600.4570.4060.4380.521

    0.3650.6270.5490.4000.4280.5010.4660.3080.6440.3700.4310.4750.3940.467

    0.3490.6310.4720.4430.5110.4490.470.3340.6240.4530.3880.4560.4000.494

    0.3710.6180.3860.4180.5180.5670.5480.3230.6190.3970.4430.4230.4280.467

    0.3700.5770.4790.4980.5450.5350.5750.3180.4940.2560.3390.3050.3430.287

    Blank0.3800.337

    %Attachment%infectionInfection

    4E9Control100.00.257Control100.00.271

    10032.20.08310012.00.033

    5021.80.0565025.80.070

    1032.80.0841026.00.070

    538.20.098525.00.068

    147.30.121144.80.121

    June 24, 98 (3)

    0

    0

    0

    0

    0

    0

    %Attachment

    % attachment

    Inhibition of Attachment by 4E9

    July 9, 98

    0

    0

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4E9

    1A-B

    Attachment 1 hInfection

    blankB4A9positive100501051blankB4A9positive100501051

    0.1490.6050.9700.8021.0900.8700.6660.8110.4480.7160.8871.0111.6321.5451.0450.751

    0.1580.6731.1880.9601.0211.1340.720.8140.3920.7730.9931.3151.5601.6101.1680.791

    0.1630.7151.0140.8471.1651.1610.7710.950.4530.7321.0941.2801.3061.3801.3660.828

    0.1600.6161.0890.8521.0971.0600.9170.8760.4600.6561.0701.1831.3901.4381.2950.702

    0.1660.6651.2260.9311.2341.5380.9270.8420.4020.6280.8341.0291.4371.4981.4451.106

    0.1570.6280.9870.6691.5201.4081.2390.9880.4680.9040.9021.5661.7501.9370.9921.026

    Blank0.1590.437

    %Attachment%infectionInfection

    4E9none100.00.939Control100.00.526

    B4A952.80.496B4A956.60.298

    10076.70.720100150.80.794

    50102.60.96350361.31.075

    10105.90.99410379.91.131

    568.30.6415262.50.781

    176.90.7211144.50.430

    1A-B

    0

    0

    0

    0

    0

    0

    0

    % attachment

    Inhibition of Attachment by 4E9

    INFECTION

    0

    0

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4E9

    ATTACHMENT

    Attachment 1 hInfection

    blankpositiveB4A9100501051blankpositiveB4A9100501051

    0.1400.3390.4250.2850.4480.5100.5420.6580.2160.2820.4050.3280.3440.3490.3240.284

    0.1480.3970.4440.3350.3570.4590.490.5250.2390.3300.4430.3460.3970.3740.3630.27

    0.1480.3970.5660.2910.4510.4420.4830.5220.2160.2710.3490.3140.2960.3470.4060.337

    0.1360.3750.5190.3090.3490.5440.5250.6120.2000.2860.3960.3460.3260.3680.3590.312

    0.1280.3520.4780.3170.3960.4760.5570.1640.2080.2930.3960.3580.3970.3180.3580.347

    0.1620.4280.6310.3050.2130.9460.2710.1260.1890.3000.4270.3190.3170.3670.350.373

    Blank0.1440.211

    %Attachment%infectionInfection

    4E9Control66.60.228Control43.00.082

    B4A9100.00.343B4A9100.00.191

    10047.80.16410064.70.124

    5074.80.2575070.50.135

    1099.90.3431074.50.143

    5109.60.376577.70.149

    1127.10.436157.10.109

    ATTACHMENT

    0

    0

    0

    0

    0

    0

    0

    % attachment

    Inhibition of Attachment by 4E9

    JCI

    0

    0

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4E9

    Attachment 1 hInfection

    blankpositiveB4A9100501051blankpositiveB4A9100501051

    0.1400.3390.4250.2850.4480.5100.5420.6580.2160.2820.4050.3280.3440.3490.3240.284

    0.1480.3970.4440.3350.3570.4590.490.5250.2390.3300.4430.3460.3970.3740.3630.27

    0.1480.3970.5660.2910.4510.4420.4830.5220.2160.2710.3490.3140.2960.3470.4060.337

    0.1360.3750.5190.3090.3490.5440.5250.6120.2000.2860.3960.3460.3260.3680.3590.312

    0.1280.3520.4780.3170.3960.4760.5570.1640.2080.2930.3960.3580.3970.3180.3580.347

    0.1620.4280.6310.3050.2130.9460.2710.1260.1890.3000.4270.3190.3170.3670.350.373

    Blank0.1440.211

    %Attachment%infectionInfection

    4E9Control66.60.228Control43.00.082

    B4A9100.00.343B4A9100.00.191

    10047.80.16410064.70.124

    5074.80.2575070.50.135

    1099.90.3431074.50.143

    5109.60.376577.70.149

    157.10.109

    0

    0

    0

    0

    0

    % attachment

    Inhibition of Attachment by 4E9

    0

    0

    0

    0

    0

    % attachment

    Inhibition of Attachment by 4E9

    Attachment 1 hInfection

    blankpositiveB4A9100501051blankpositiveB4A9100501051posneg

    0.4220.8130.7120.4470.6180.7150.7020.7471.0131.032

    0.4870.9371.0310.5200.6570.6260.6971.0260.961.016

    0.4900.8540.9320.4850.5090.5300.7030.8671.1061.179

    0.5020.9690.9810.5060.4890.5580.6951.0660.940.943

    0.4940.9110.9240.4880.4850.6950.7860.911.0320.883

    0.4600.9110.9360.5090.5370.6080.6870.8950.9070.809

    Blank0.0000.476

    %Attachment%infectionInfection

    4E9Control0.00.000Control95.50.423

    B4A90.00.000B4A9100.00.444

    1000.00.0001003.80.017

    500.00.0005016.50.073

    100.00.0001033.00.146

    50.00.000553.20.236

    10.00.000199.80.443

    Normal116.60.517

    Immune113.00.501

    0

    0

    0

    0

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4E9

    Attachment 1 hInfection

    blankpositive4D1100501051blankpositive4D1100501051

    0.3110.7020.6420.7770.5720.6580.6150.5660.4931.0190.9751.0220.9860.5770.850.801

    0.3160.5880.6690.6120.5260.5230.5720.5340.5330.9580.9381.1180.7980.8630.9260.903

    0.2870.5680.5550.6180.6170.5450.5420.5340.4911.0680.9960.9811.0930.9050.8620.85

    0.3020.5280.5850.6890.5440.5440.5480.5050.4720.9001.0410.6340.7510.7290.8260.887

    0.3400.6610.7650.6640.5460.5850.5670.4940.4851.0730.8901.0400.7541.1061.0530.807

    0.3240.6980.7940.6170.5510.6580.6820.6130.3770.8771.1110.9351.0220.9410.8760.815

    Blank0.3130.475

    %Attachment%infectionInfection

    4E9Control89.80.296Control98.20.507

    4D1100.00.3304D1100.00.517

    100108.70.35910092.90.480

    5075.10.2485082.40.426

    1078.10.2581073.20.378

    577.40.255582.00.424

    167.10.221171.40.369

    0

    0

    0

    0

    0

    0

    0

    % attachment

    Inhibition of Attachment by 4G12

    0

    0

    0

    0

    0

    0

    0

    % infection

    Inhibition of Infection by 4G12

    AB

    &L&"Times,Bold"&24Figure 1&R&"Times,Bold"&24Manuscript 1

    10010.060121454410.0601214544

    3.75798571971.55480777331.5548077733

    16.53513716656.62064940636.6206494063

    32.95753476146.73298491816.7329849181

    53.17549793313.39352561013.3935256101

    99.81210071410.576029446910.5760294469

    IgM (g/ml)

    B9A4

    4E9

    Infection (% of control)

    1001.40064081131.4006408113

    47.77280684692.15200415262.1520041526

    74.849251118510.462377014810.4623770148

    99.941645594222.780952224322.7809522243

    109.6284769512.556823191812.5568231918

    B9A4

    4E9

    IgM (g/ml)

    Attachment (% of control)

    Infection

    blankpositiveB4A9100501051B4A9100501051

    0.4220.8130.7120.4470.6180.7150.7020.7470.1420.226

    0.4870.9371.0310.5200.6570.6260.6971.0260.1500.2210.550

    0.4900.8540.9320.4850.5090.5300.7030.8670.4560.0090.0330.0540.2270.391

    0.5020.9690.9810.5060.4890.5580.6951.0660.5050.0300.0130.219

    0.4940.9110.9240.4880.4850.6950.7860.910.4480.0120.2190.434

    0.4600.9110.9360.5090.5370.6080.6870.8950.4600.0330.0610.1320.419

    0.476100%0.444

    %infectionInfection

    0.423

    100100.00.444101000.4440.013B4A9100.00050.00010.0005.0001.000

    1003.80.01721000.0170.006

    5016.50.0737500.0730.0280.4560.0090.1420.1500.2260.550

    1033.00.1467100.1460.0340.5050.0300.0330.0540.2210.391

    553.20.236350.2360.0020.4480.0120.0130.2190.2270.434

    199.80.4431110.4430.0350.4600.0330.0610.1320.2190.419

    No MAb95.50.0005

    0.000

    010.060121454410.0601214544

    01.55480777331.5548077733

    06.62064940636.6206494063

    06.73298491816.7329849181

    03.39352561013.3935256101

    010.576029446910.5760294469

    IgM (g/ml)

    B4A9

    4E9

    Infection (% of control)

    00.0128281916

    00.0061237244

    00.028347178

    00.0339051987

    00.001931105

    00.0349869023

    IgM (g/ml)

    B4A9

    4E9

    Infection (A 405 nm)

    Attachment 1 h0.343

    blankpositiveB4A9100501051control100501051B4A9100501051

    0.1400.3390.4250.2850.4480.5100.5420.65840.84883.2130.7148.8158.1192.00.3040.3660.398

    0.1480.3970.4440.3350.3570.4590.490.52543.18297.7104.2133.9143.0153.20.3000.2130.3150.3460.381

    0.1480.3970.5660.2910.4510.4420.4830.52243.18284.9131.6129.0140.9152.30.4220.1470.3390.378

    0.1360.3750.5190.3090.3490.5440.5250.61239.68190.2101.8158.7153.2178.60.3750.1650.2050.4000.3810.468

    0.1280.3520.4780.3170.3960.4760.5570.16437.34792.5115.5138.9162.547.90.3340.1730.2520.3320.020

    0.1620.4280.6310.3050.2130.9460.2710.12647.26789.062.1276.079.136.80.161

    Blank0.144

    %Attachment

    4E9Control66.60.228B4A9100501051

    100100.00.3431.4011000.3430.026

    10047.80.1642.1521000.1640.0040.3000.1470.3040.3660.3980.381

    5074.80.25710.462500.2570.0230.4220.1650.2130.3150.3460.378

    1099.90.34322.781100.3430.0190.3750.1730.2050.4000.3390.468

    5109.60.37612.55750.3760.0190.3340.1610.2520.3320.3810.020

    1.00093.80.32227.456

    01.40064081131.4006408113

    02.15200415262.1520041526

    010.462377014810.4623770148

    022.780952224322.7809522243

    012.556823191812.5568231918

    B4A9

    4E9

    IgM (g/ml)

    Attachment (% of control)

    00.0263387389

    00.0044409709

    00.0226292583

    00.0188475242

    00.0188475242

    1000.44350.0128281916B4A9100501051

    1000.0170.006B4A9100501051

    500.0730.0280.4560.0090.1420.1500.2260.5501000.3430.026

    100.1460.0340.5050.0300.0330.0540.2210.3911000.1640.0040.3000.1470.3040.3660.3980.381

    50.2360.0020.4480.0120.0130.2190.2270.434500.2570.0230.4220.1650.2130.3150.3460.378

    10.4430.0350.4600.0330.0610.1320.2190.419100.3430.0190.3750.1730.2050.4000.3390.468

    50.3760.0190.3340.1610.2520.3320.3810.020

    0.44350.0128281916

    0.01666666670.0061237244

    0.07333333330.028347178

    0.14616666670.0339051987

    0.23583333330.001931105

    0.44266666670.0349869023

    0.34273333330.0263387389

    0.16373333330.0044409709

    0.25653333330.0226292583

    0.34253333330.0188475242

    0.37573333330.0188475242

    B4A9

    4E9

    IgM (g/ml)

    Attachment (A405 nm)

  • Effect of MAb 4E9 IgM on C. parvum infection of neonatal Balb/c miceNo. of oocysts/5lHamer, Ward and Tzipori,

    Chart1

    20.833.63.6

    0.3330.3330.333

    Sheet1

    Control20.833.6

    MAb 4E90.3330.333

    Sheet1

    3.63.6

    0.3330.333

    Sheet2

    Sheet3

  • Reactivity of MAb 4E9 with C. parvum developmental stages by immunofluorescenceCevallos et al, 2000

  • Reactivity of MAb 4E9 with C. parvum developmental stages by immunoelectron microscopyCevallos et al, 2000

  • 12kDa20097.466.2453121.5116.3Immunoblot analysis of C. parvum antigens recognized by MAb 4E91, Oocysts2, Sporozoites

    Cevallos et al, 2000

  • GP900 >900kDa glycoprotein present in sporozoites and merozoites; shed from surface of sporozoites during gliding motility localized to micronemes of invasive stages by IEMencoded by 7.5kb gene locus, 5.5kb ORF, corresponding to predicted 1832 amino acid protein deduced amino acid sequence shows a mucin-like protein containing cysteine-rich and polythreonine domains native GP900 binds to intestinal epithelial cells and competitively inhibits infection in vitro cysteine-rich domain of recombinant GP900 as well as antibodies to it inhibit infection in vitro Petersen et al 1992, Barnes et al, 1998, Ward and Cevallos, 1998

  • CSL (circumsporozoite precipitate-like) glycoprotein identified by surface and apical-reactive MAbs C4A1, 3E2 localized to surface and apical region (micronemes and dense bodies) of sporozoites and merozoites MAb 3E2 elicits CSP-like reaction (formation, posterior movement and release of membraneous Ag-MAb precipitates MAb 3E2 neutralizes sporozoite infectivity and prevents infection in neonatal Balb/c mice soluble glycoprotein exoantigen comprised of multiple ~1300 kDa molecular species with differing pIs isolated native CSL binds to intestinal epithelial cells and inhibits sporozoite attachment to and invasion of these cells Langer and Riggs, 1996, Riggs, 1997, Riggs et al, 1997, Langer et al 1999,

  • 1, total proteins2, shed proteinsReactivity of MAb 4E9 with C. parvum shed proteinsCevallos et al, 2000

  • GP900 is not related to gp40MAb4E9anti-gp40Silver stain of HPA-affinity purified glycoproteinsImmunoblot of GalNAc eluatelysateeffluentGalNAceluateCevallos et al, 2000

  • gp40-specific antisera inhibit C. parvum infection of intestinal epithelial Caco 2A cellsInfection (A405nm)Cevallos et al, 2000

    Chart1

    0.21966666670.02083569680.0208356968

    0.09350.02590527620.0259052762

    0.21508333330.02156776670.0215677667

    0.04133333330.01393491360.0139349136

    final

    final

    10000

    36.92287693278.85825481698.8582548169

    18.07536987454.88614334414.8861433441

    % of infection

    Infection 1

    10000

    59.17691579944.14.1

    51.56102175971.81.8

    % attachment

    Infection 2

    10 minControlPre-immAPre-immBPre-nonePre-leftPost-immAPost-immBPost-nonePost-left

    0.2630.3560.4090.390.4110.4320.3450.2620.2690.261

    0.2780.3880.3850.4340.4550.460.3870.2970.260.258

    0.2950.3560.3980.4590.4580.440.4060.3340.2940.274

    0.2560.3460.3780.4080.430.4070.3760.3030.3030.296

    0.2590.3390.3790.3680.4370.4110.3860.2940.3060.318

    0.2590.3220.3470.3610.4110.4050.3410.3220.2970.278

    blank0.268

    0.1430.1640.001-0.007

    0.1870.192-0.008-0.010

    0.1900.1720.0260.006

    0.1620.1390.0350.028

    0.1690.1430.0380.050

    0.1430.1370.0290.010

    blank0.268blank0.000

    Control0.083100.0Control0.0000.0

    Pre-immA0.114138.0Pre-immA0.0000.0

    Pre-immB0.135163.0Pre-immB0.0000.0

    Pre-none0.165199.6Pre-none0.0000.0

    Pre-left0.158190.1Pre-right0.0000.0ownaverage

    Post-immA0.105127.092.084.4Post-immA0.0000.00.00.0

    Post-immB0.03440.624.927.0Post-immB0.0000.00.00.0

    Post-none0.02023.912.012.3Post-none0.0000.00.00.0

    Post-left0.01315.17.97.7Post-right0.0000.00.00.0

    900-A92.0900-A0.0

    900-B24.9900-B0.0

    40-N12.040-N0.0

    40-L7.940-R0.0

    Infection 2

    0

    0

    0

    0

    Infection (A450 nM)

    Inhibition of infection by immune sera

    infection 1 and 2 gp40 only

    10 minControlPre-immAPre-immBPre-nonePre-leftPost-immAPost-immBPost-nonePost-left15 min

    0.3260.4880.5480.5310.5920.540.4740.4960.4260.3350.3600.5620.7330.7320.7600.7040.6150.6100.5450.426

    0.3840.490.5790.7040.660.670.5560.5440.5350.4240.3710.5770.6970.8320.9450.8380.7140.6570.6730.546

    0.3570.5380.6060.7430.5890.6680.650.5610.4520.4460.3920.6120.7090.8660.7570.8400.7850.6960.7090.562

    0.3740.5310.6310.7710.7140.6560.6010.5480.5540.4510.3630.5630.8190.9200.8970.8380.7430.6750.6900.570

    0.30.4980.5460.6420.6260.6330.5290.5480.6030.4640.3470.5910.6530.7770.7620.8360.6920.7090.7470.579

    0.3290.4810.50.5920.5330.5390.5310.5020.5030.3710.4080.5890.6520.7900.6860.6960.6690.6790.6370.499

    blank0.3450.2470.1950.081-0.010

    0.3150.3250.1900.079

    0.2440.3230.1070.101

    0.3690.3110.2090.106

    0.2810.2880.2580.119

    0.1880.1940.1580.026

    0.2740.27266666670.16716666670.0701666667

    blank0.345blank0.374

    mean pre-immune

    Control0.159100.0Control0.209100.00.4

    Pre-immA0.223140.2Pre-immA0.337161.4

    Pre-immB0.319200.1Pre-immB0.446213.6

    Pre-none0.274172.0Pre-none0.428204.8

    Pre-left0.273171.1Pre-right0.419200.4ownaverage

    Post-immA0.212132.994.978.1Post-immA0.330157.897.880.9

    Post-immB0.188118.159.069.4Post-immB0.298142.566.773.0

    Post-none0.167104.961.061.2Post-none0.293140.568.672.0

    Post-left0.07044.025.725.7Post-right0.15775.137.538.5

    900-A94.9900-A80.9

    900-B59.0900-B73.0

    40-N61.040-N72.0

    40-L25.740-R38.5

    infection 1 and 2 gp40 only

    0

    0

    0

    0

    Infection (A450 nM)

    Infection in presence of immune sera

    December 9 (10 min)

    Pre-nonePre-leftPost-nonePost-leftPre-nonePre-leftPost-nonePost-left

    0.1430.1640.001-0.0070.1430.1640.001-0.007

    0.1870.192-0.008-0.0100.1870.192-0.008-0.010

    0.1900.1720.0260.0060.1900.1720.0260.006

    0.1620.1390.0350.0280.1620.1390.0350.028

    0.1690.1430.0380.0500.1690.1430.0380.050

    0.1430.1370.0290.0100.1430.1370.0290.010

    0.2470.1950.081-0.0100.2470.1950.081-0.010

    0.3150.3250.1900.0790.3150.3250.1900.079

    0.2440.3230.1070.1010.2440.3230.1070.101

    0.3690.3110.2090.1060.3690.3110.2090.106

    0.2810.2880.2580.1190.2810.2880.2580.119

    0.1880.1940.1580.0260.1880.1940.1580.026

    mean a0.1650.1580.0200.013mean a0.1650.1580.0200.013

    0.2740.27266666670.16716666670.07016666670.2740.27266666670.16716666670.0701666667

    mean total0.2200.2150.0940.041

    no negative numbers0.0210.0220.0260.014no negative numbers

    0.4-4.70.40.00.4-4.70.40.0

    -5.0-6.60.00.0-5.0-6.60.00.0

    15.53.615.53.615.53.615.53.6

    21.017.621.017.621.017.621.017.6

    22.831.522.831.522.831.522.831.5

    17.36.117.36.117.36.117.36.1

    29.6-3.729.60.029.6-3.729.60.0

    69.329.069.329.069.329.069.329.0

    39.137.039.137.039.137.039.137.0

    76.338.976.338.976.338.976.338.9

    94.243.694.243.694.243.694.243.6

    57.79.557.79.557.79.557.79.5

    mean a12.89.8mean a12.89.8

    meanb61.026.3meanb61.026.3

    4.15.14.15.1

    9.87.29.87.2

    mean t36.918.1mean t36.918.1

    sem8.94.9sem8.94.9

    preimmune1000preimmune-10.2200.021

    anti-gp40 136.98.9anti-gp40 10.0940.026

    anti-gp40 218.14.9preimmune-20.2150.022

    anti-gp40 20.0410.014

    December 9 (10 min)

    000

    08.85825481698.8582548169

    04.88614334414.8861433441

    % of infection

    attach

    0.02083569680.0208356968

    0.02590527620.0259052762

    0.02156776670.0215677667

    0.01393491360.0139349136

    Sheet3

    InfectionAttachment

    BlankPositivePre-immune4E9GP900 1:100GP40 NoneGP40 LeftGP40 RightBlankPositivePre-immune4E9GP900 1:100GP40 NoneGP40 LeftGP40 RightPositive

    0.4861.7721.8621.0201.6001.6101.6181.3840.3560.3440.2580.5990.6320.3370.4310.4760.4840.5010.4230.192

    0.4521.7741.8981.1591.3151.7791.7691.5230.3580.3430.2630.5580.6160.3150.4550.5380.4580.3900.4150.204

    0.5632.2411.9021.0881.7811.6741.6931.5730.3460.3560.2750.6490.6550.3500.4080.4620.4540.4610.4420.184

    0.4751.8571.7631.3911.4961.5731.6521.6560.3750.3520.2740.6430.6010.3480.4510.4760.4510.4340.4430.195

    0.4872.2062.3201.3851.9311.8831.8301.6240.3540.3440.2860.5990.6290.3720.4110.4360.4370.4800.4210.200

    0.5042.2712.2871.2591.8441.7911.8541.7100.3260.3430.2840.5860.6210.3560.4080.5030.4460.4410.4190.195

    blank0.495blank0.273

    Positive1.526101.0Positive0.33294.3

    Pre-immune1.511100.0Pre-immune0.352100.0

    4E90.72347.84E90.07320.7

    GP900 1:1001.16777.2GP900 1:1000.15443.7

    GP40 None1.22481.0GP40 None0.20959.2

    GP40 Left1.24282.2GP40 Left0.18251.6

    GP40 Right1.08471.7GP40 Right0.17850.5

    Positive0.15443.7

    Positive101.0Positive94.3

    Pre-immune100.0Pre-immune100.0

    4E947.84E920.7

    GP900 1:10077.2GP900 1:10043.7

    GP40 None81.0GP40 None59.2

    GP40 Left82.2GP40 Left51.6

    GP40 Right71.7GP40 Right50.5

    00.0Positive43.7

    Sheet3

    100.9817981247

    100

    47.8212906784

    77.2200772201

    81.0038610039

    82.1731936018

    71.7374517375

    0

    % infection

    Infection

    0

    0

    0

    0

    0

    0

    0

    0

    % attachment

    Attachment

    AttachmentAttachment

    BlankPositivePre-immune4E9GP900 1:100GP40 NoneGP40 LeftGP40 RightPositiveBlankPositivePre-immune4E9GP900 1:100GP40 NoneGP40 LeftGP40 RightPositive

    0.2580.5990.6320.3370.4310.4760.4840.5010.4230.1920.2580.5990.6320.3370.4310.4760.4840.5010.4230.192

    0.2630.5580.6160.3150.4550.5380.4580.3900.4150.2040.2630.5580.6160.3150.4550.5380.4580.3900.4150.204

    0.2750.6490.6550.3500.4080.4620.4540.4610.4420.1840.2750.6490.6550.3500.4080.4620.4540.4610.4420.184

    0.2740.6430.6010.3480.4510.4760.4510.4340.4430.1950.2740.6430.6010.3480.4510.4760.4510.4340.4430.195

    0.2860.5990.6290.3720.4110.4360.4370.4800.4210.2000.2860.5990.6290.3720.4110.4360.4370.4800.4210.200

    0.2840.5860.6210.3560.4080.5030.4460.4410.4190.1950.2840.5860.6210.3560.4080.5030.4460.4410.4190.195

    blank0.273blank0.273

    Positive0.606Positive0.606

    Pre-immune0.626Pre-immune0.626

    4E90.3464E90.346

    GP900 1:1000.427GP900 1:1000.427

    GP40 None0.482GP40 None0.482

    GP40 Left0.455GP40 Left0.455

    GP40 Right0.451GP40 Right0.451

    Positive0.427Positive0.427

    Pre-immuneGP40 NoneGP40 LeftPre-immuneGP40 NoneGP40 Left

    0.3590.2030.2110.3590.2030.211

    0.3430.2650.1850.3430.2650.185

    0.3820.1890.1810.3820.1890.181

    0.3280.2030.1780.3280.2030.178

    0.3560.1630.1640.3560.1630.164

    0.3480.2300.1730.3480.2300.173

    0.3520.2090.1820.3520.2090.182

    %101.857.559.8%101.857.559.8

    97.375.152.497.375.152.4

    108.353.551.3108.353.551.3

    93.057.550.493.057.550.4

    100.946.246.5100.946.246.5

    98.765.249.098.765.249.0

    2.14.11.80.010.010.0

    Pre-immune100.00Pre-immune0.3520.01

    gp40 159.24.1gp40 10.2090.01

    gp40 251.61.8gp40 20.1820.01

    000

    04.14.1

    01.81.8

    % attachment

    0.0073831640.007383164

    0.01433507740.0143350774

    0.00651152820.0065115282

    Attachment (A405 nm)

    PercentageAbsorbance

    10000

    36.92287693278.85825481698.8582548169

    18.07536987454.88614334414.8861433441

    0.21966666670.02083569680.0208356968

    0.09350.02590527620.0259052762

    0.21508333330.02156776670.0215677667

    0.04133333330.01393491360.0139349136

    Infection (A405 nm)

  • b-galactosidaseHPA-glycoproteinsShed proteinsBinding (A405nm)gp40 binds to intestinal epithelial Caco 2A cellsCevallos et al, 2000

    Chart9

    -0.02925-0.007-0.014250.00141421360.00141421360.00424264070.00424264070.0017677670.001767767

    0.05425-0.01350.001750.02863782460.02863782460.0017677670.0017677670.00883883480.0088388348

    0.597250.19350.048750.06682159080.06682159080.03500178570.03500178570.03500178570.0350017857

    1.050250.78750.104750.0081317280.0081317280.04065863990.04065863990.01096015510.0109601551

    1.252751.1740.139250.01131370850.01131370850.03394112550.03394112550.00707106780.0070710678

    1.317251.44450.130250.00530330090.00530330090.06045762980.06045762980.00919238820.0091923882

    1.294251.54350.123250.03358757210.03358757210.04136574670.04136574670.0063639610.006363961

    1.149251.5090.103250.01803122290.01803122290.1113693180.1113693180.00919238820.0091923882

    HPA- glycoproteins

    Shed proteins

    b-galactosidase

    gp40 (g/ml)

    12 AUG-99 10 MIN

    USUAL1 WEEK

    0.130.1360.1580.1730.1980.2330.2160.2380.2370.2730.2810.310.3080.3620.4930.5510.4840.5740.6190.583

    0.1420.1720.1780.2060.2420.2570.2260.2520.2760.3040.2920.3030.3440.380.440.5030.4560.4640.5170.507

    0.1550.1380.1510.1750.1860.1860.1770.2120.210.2080.2760.2620.2720.2990.3250.3580.3210.3760.390.389

    0.1060.110.110.110.1210.1190.130.1210.1310.1260.2220.220.2050.2390.2310.2420.240.2450.2590.234

    0.1190.1310.120.1320.1260.1370.1320.1360.1390.1380.2340.2140.2170.2450.250.2370.2410.2660.2650.261

    0.1170.1210.1270.1290.1470.1480.130.1560.1530.150.2380.2270.2380.2650.2960.2720.2790.2810.2970.329

    blank 400.255blank 400.601

    blank 150.290blank 150.512

    blank 4E90.209blank 4E90.390

    blank gal0.139blank gal0.263

    blank oval0.152blank oval0.313

    blank no 1st0.118blank no 1st0.234

    -0.125-0.119-0.097-0.082-0.057-0.022-0.039-0.017-0.320-0.291-0.293-0.239-0.108-0.050-0.117-0.027

    -0.148-0.118-0.112-0.084-0.048-0.033-0.064-0.038-0.220-0.209-0.168-0.132-0.072-0.009-0.056-0.048

    -0.054-0.071-0.058-0.034-0.023-0.023-0.0320.003-0.114-0.128-0.118-0.091-0.065-0.032-0.069-0.014

    -0.020-0.008-0.019-0.007-0.013-0.002-0.007-0.003-0.029-0.049-0.046-0.018-0.013-0.026-0.0220.003

    -0.035-0.031-0.025-0.023-0.005-0.004-0.0220.005-0.075-0.086-0.075-0.048-0.017-0.041-0.034-0.032

    12 AUG-99 30MIN

    USUAL1 WEEK

    0.1680.1760.2070.2690.2950.3640.3490.3530.3720.4410.3930.4260.4630.6270.9070.8550.8070.8270.8910.91

    0.1790.2280.2620.3150.4080.430.3830.4170.4180.4970.3980.4510.4920.6180.8070.8970.7370.7770.8720.815

    0.2170.1670.1960.2490.2670.2550.2460.2650.2780.2720.390.3640.3880.4960.530.5730.5240.6110.6020.589

    0.1150.120.1180.1180.1310.1270.1420.1320.1470.1410.2720.2850.270.3280.3110.3220.3180.3130.3180.291

    0.1130.130.1140.130.1260.1350.1350.1390.1420.1380.2760.2760.2770.3160.3130.3030.3090.3340.3250.322

    0.1340.130.1390.1460.1620.1670.1480.1670.1710.1740.3020.30.3180.3450.3820.3720.3670.3830.390.418

    blank 400.407blank 400.901

    blank 150.458blank 150.844

    blank 4E90.275blank 4E90.596

    blank gal0.140blank gal0.324

    blank oval0.173blank oval0.404

    blank no 1st0.129blank no 1st0.303

    -0.239-0.231-0.200-0.138-0.112-0.043-0.058-0.054-0.508-0.475-0.438-0.2740.006-0.046-0.094-0.074

    -0.279-0.230-0.196-0.143-0.050-0.028-0.075-0.041-0.446-0.393-0.352-0.226-0.0360.054-0.107-0.066

    -0.058-0.108-0.079-0.026-0.008-0.020-0.029-0.010-0.206-0.232-0.208-0.099-0.065-0.023-0.0710.016

    -0.027-0.010-0.026-0.010-0.014-0.005-0.005-0.001-0.048-0.048-0.047-0.008-0.011-0.021-0.0150.011

    -0.039-0.043-0.034-0.027-0.011-0.005-0.025-0.005-0.102-0.104-0.086-0.059-0.022-0.032-0.037-0.021

    summary

    Blanks

    Usual1 week

    10 min

    blank 400.2550.601

    blank 150.2900.512

    blank 4E90.2090.390

    blank gal0.1390.263

    blank oval0.1520.313

    blank no 1st0.1180.234

    30 min

    blank 400.4070.901

    blank 150.4580.844

    blank 4E90.2750.596

    blank gal0.1400.324

    blank oval0.1730.404

    blank no 1st0.1290.303

    delta

    blank 400.1520.300

    blank 150.1680.332

    blank 4E90.0660.206

    blank gal0.0020.061

    blank oval0.0210.091

    blank no 1st0.0110.069

    18 AUG-99 10 min

    USUAL

    0.1260.1370.150.1430.1360.1410.1280.1310.1270.125

    1.2551.1951.1741.0430.9090.8620.8340.7830.7180.755

    1.2411.0881.0220.8860.670.3680.2650.2170.2010.193

    0.1470.1580.1550.1490.1680.1720.1580.1750.1680.16

    0.4490.4740.4360.3620.3350.340.2750.2660.280.272

    0.1260.1280.1310.1430.1310.140.1390.1350.1310.128

    blank 400.737

    blank 4E90.197

    blank gal0.164

    blank oval0.276

    blank no 1st0.134

    Binding protein

    gp400.5190.4590.4380.3070.1730.1260.0980.047

    4E91.0440.8910.8250.6890.4730.1710.0680.020

    galactosidase-0.017-0.006-0.009-0.0150.0040.008-0.0060.011

    ovalbumin0.1730.1980.1600.0860.0590.064-0.001-0.010

    gp400.3460.2610.2780.2210.1140.0620.0990.057

    4E90.8710.6930.6650.6030.4140.1070.0690.030

    0.0780.1560.3130.6251.2502.5005.00010.000

    gp400.0470.0980.1260.1730.3070.4380.4590.519

    4E90.0200.0680.1710.4730.6890.8250.8911.044

    galactosidase0.011-0.0060.0080.004-0.015-0.009-0.006-0.017

    ovalbumin-0.010-0.0010.0640.0590.0860.1600.1980.173

    18 AUG-99 30 min

    USUAL

    0.1460.1560.1710.1590.150.1540.1440.1550.1520.15

    2.3322.2852.2952.1431.9481.8161.7451.681.5861.647

    2.3842.1892.1131.8491.480.7220.4630.3630.3290.307

    0.220.2280.2260.220.2470.2480.2370.2560.2490.238

    0.9350.8610.8210.670.5790.6030.5030.5290.5330.512

    0.1430.1410.1450.1610.1480.1560.1560.1570.1470.149

    blank 401.617

    blank 4E90.318

    blank gal0.244

    blank oval0.523

    blank no 1st0.152

    gp400.7160.6690.6790.5270.3320.2000.1290.063

    4E92.0661.8711.7951.5311.1620.4040.1450.045

    galactosidase-0.024-0.016-0.018-0.0240.0040.005-0.0070.013

    ovalbumin0.4130.3390.2990.1480.0570.081-0.0200.007

    gp400.3030.3300.3800.3790.2750.1190.1480.057

    4E91.6541.5331.4971.3841.1060.3240.1650.038

    20 AUG-99 10 min

    USUAL

    0.1870.2150.2250.3460.5040.6050.650.6540.6150.614

    0.2040.2220.2280.3370.4130.4270.4220.4220.390.382

    0.2080.2340.310.5090.6590.5950.5970.5790.5390.53

    0.1280.1360.1490.1450.1580.1580.1790.1840.1710.164

    0.1630.1640.1680.1850.2610.3080.2990.3210.3290.319

    0.1290.1370.1350.1450.140.1490.1560.1570.160.145

    blank 150.6145

    blank 40 (1:1000)0.386

    blank 40 (1:500)0.535

    blank gal0.168

    blank oval0.324

    blank no 1st0.145

    gp15-0.428-0.400-0.390-0.269-0.111-0.0100.0360.040

    gp40-0.182-0.164-0.158-0.0490.0270.0410.0360.036

    4E9-0.327-0.301-0.225-0.0260.1250.0610.0630.045

    galactosidase-0.040-0.032-0.019-0.023-0.010-0.0100.0120.017

    ovalbumin-0.161-0.160-0.156-0.139-0.063-0.016-0.025-0.003

    gp40-0.021-0.004-0.0020.0900.0900.0570.0610.039

    4E9-0.166-0.141-0.0690.1140.1880.0770.0880.048

    0.0780.1560.3130.6251.2502.5005.00010.000

    gp400.0360.0360.0410.027-0.049-0.158-0.164-0.182

    4E90.0450.0630.0610.125-0.026-0.225-0.301-0.327

    galactosidase0.0170.012-0.010-0.010-0.023-0.019-0.032-0.040

    ovalbumin-0.003-0.025-0.016-0.063-0.139-0.156-0.160-0.161

    27 Aug 99

    USUAL

    0.8120.7640.8060.730.6930.5250.5570.510.4870.422

    0.7660.6680.6720.6320.5620.5710.5760.5150.5060.497

    0.7430.6210.5780.5130.5290.5060.4310.4490.3980.431

    0.5590.450.4530.4840.5550.4070.4580.4780.4760.432

    0.1870.1980.1980.2090.2260.2120.2030.20.20.201

    0.1200.1330.1360.1440.1490.1610.140.1490.1570.158

    blank 40N1 (1:500)0.455

    blank 40N1 (1:500)0.502

    blank 40L (1:1000)0.415

    blank 40L (1:1000)0.454

    blank gal0.201

    blank no 1st0.145

    gp40 N1 +HPA0.3110.3100.3520.2760.2390.0710.1030.056

    gp40 N10.2650.1670.1710.1310.0610.0690.0740.014

    gp40 L + HPA0.3290.2070.1640.0990.1150.0920.0170.035

    gp40 L0.105-0.004-0.0010.0300.101-0.0470.0040.024

    galactosidase-0.014-0.003-0.0030.0080.0260.0120.003-0.001

    0.0190.0780.1560.3130.6251.2502.5005.000

    gp40 N1 +HPA0.0560.1030.0710.2390.2760.3520.3100.311

    gp40 N10.0140.0740.0690.0610.1310.1710.1670.265

    gp40 L + HPA0.0350.0170.0920.1150.0990.1640.2070.329

    gp40 L0.0240.004-0.0470.1010.030-0.001-0.0040.105

    galactosidase-0.0010.0030.0120.0260.008-0.003-0.003-0.014

    27 Aug 99

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    gp40 N1 +HPA

    gp40 N1

    gp40 L + HPA

    gp40 L

    galactosidase

    HPA binding protein concentration

    A 405 nm

    30 Aug 99 (5min)

    USUAL

    0.9770.9860.8710.7490.6730.7160.6780.50.4850.487

    0.8540.7480.7040.6120.6750.6780.620.5360.5210.477

    0.8990.8870.6830.720.5360.3770.4610.5760.4740.439

    0.5710.6440.5450.5340.4290.5560.5030.4160.4660.488

    0.1580.1560.1610.1560.1550.1670.1480.1360.1610.147

    0.1220.1160.1240.120.120.1390.1290.1310.1390.128

    blank 40N1 (1:500)0.486

    blank 40N1 (1:500)0.499

    blank 40L (1:1000)0.457

    blank 40L (1:1000)0.477

    blank gal0.154

    blank no 1st0.127

    gp40 N1 +HPA0.4780.5000.3850.2630.1870.2300.1920.014

    gp40 N10.3550.2490.2050.1130.1760.1790.1210.037

    gp40 L + HPA0.4430.4310.2270.2640.080-0.0800.0050.120

    gp40 L0.0940.1670.0680.057-0.0480.0790.026-0.061

    galactosidase0.0040.0020.0070.0020.0010.013-0.006-0.018

    0.0190.0780.1560.3130.6251.2502.5005.000

    gp40 N1 +HPA0.0140.1920.2300.1870.2630.3850.5000.478

    gp40 N10.0370.1210.1790.1760.1130.2050.2490.355

    gp40 L + HPA0.1200.005-0.0800.0800.2640.2270.4310.443

    gp40 L-0.0610.0260.079-0.0480.0570.0680.1670.094

    galactosidase-0.018-0.0060.0130.0010.0020.0070.0020.004

    30 Aug 99 (5min)

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    gp40 N1 +HPA

    gp40 N1

    gp40 L + HPA

    gp40 L

    galactosidase

    HPA binding protein concentration

    A 405 nm

    30 Aug 99 (10min)

    USUAL

    1.3921.3711.2621.161.0060.9420.9370.7350.7110.802

    1.091.1271.0520.9751.0260.9560.9540.8340.8590.725

    1.2841.2931.0441.0650.830.5640.6980.8370.7210.687

    0.8080.8590.7950.7930.640.770.7860.6890.7590.692

    0.1950.1910.1940.1930.1940.2060.1830.1740.1960.177

    0.1290.130.1330.1340.1340.1520.1410.1460.1520.142

    blank 40N1 (1:500)0.757

    blank 40N1 (1:500)0.792

    blank 40L (1:1000)0.704

    blank 40L (1:1000)0.726

    blank gal0.187

    blank no 1st0.139

    gp40 N1 +HPA0.6000.6150.5060.4040.2500.1860.181-0.022

    gp40 N10.2980.3350.2600.1830.2340.1640.1620.042

    gp40 L + HPA0.5800.5890.3400.3610.126-0.140-0.0060.133

    gp40 L0.0830.1340.0700.068-0.0860.0450.061-0.037

    galactosidase0.0090.0050.0080.0070.0080.020-0.004-0.013

    0.0190.0780.1560.3130.6251.2502.5005.000

    gp40 N1 +HPA-0.0220.1810.1860.2500.4040.5060.6150.600

    gp40 N10.0420.1620.1640.2340.1830.2600.3350.298

    gp40 L + HPA0.133-0.006-0.1400.1260.3610.3400.5890.580

    gp40 L-0.0370.0610.045-0.0860.0680.0700.1340.083

    galactosidase-0.013-0.0040.0200.0080.0070.0080.0050.009

    30 Aug 99 (10min)

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    gp40 N1 +HPA

    gp40 N1

    gp40 L + HPA

    gp40 L

    galactosidase

    HPA binding protein concentration

    A 405 nm

    7 Sep 99 (10min)

    LIVE

    0.380.3890.4050.4530.4160.460.4510.4370.420.403

    0.3890.4280.4510.4280.4460.4530.4540.4390.4480.427

    0.4370.4230.4310.4230.4160.4330.4550.450.4480.466

    0.4770.4590.4620.4360.4260.4690.4530.4680.4740.457

    0.1760.1880.1990.2040.2040.2120.2270.2290.2340.225

    0.1640.1720.180.1890.1990.1940.1970.2080.2170.222

    blank 40N1 (1:500)0.412

    blank 40N1 (1:500)0.438

    blank 40L (1:1000)0.457

    blank 40L (1:1000)0.466

    blank gal0.230

    blank no 1st0.194

    gp40 N1 +HPA-0.058-0.023-0.0060.0420.0050.0490.0400.026

    gp40 N1-0.049-0.0100.014-0.0100.0090.0160.0170.002

    gp40 L + HPA-0.020-0.034-0.026-0.034-0.041-0.024-0.002-0.007

    gp40 L0.012-0.007-0.004-0.030-0.0400.003-0.0130.003

    galactosidase-0.054-0.042-0.031-0.026-0.026-0.018-0.003-0.001

    0.0190.0780.1560.3130.6251.2502.5005.000

    gp40 N1 +HPA0.0260.0400.0490.0050.042-0.006-0.023-0.058

    gp40 N10.0020.0170.0160.009-0.0100.014-0.010-0.049

    gp40 L + HPA-0.007-0.002-0.024-0.041-0.034-0.026-0.034-0.020

    gp40 L0.003-0.0130.003-0.040-0.030-0.004-0.0070.012

    galactosidase-0.001-0.003-0.018-0.026-0.026-0.031-0.042-0.054

    7 Sep 99 (10min)

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    gp40 N1 +HPA

    gp40 N1

    gp40 L + HPA

    gp40 L

    galactosidase

    HPA binding protein concentration

    A 405 nm

    7 Sep 99 (10min) (2)

    FIXED

    0.2640.2750.2710.3150.2880.3430.3060.3680.3360.374

    0.2540.2750.2760.2950.2910.30.3530.3750.3330.35

    0.2760.2590.2670.2920.3190.3020.3220.3130.330.369

    0.2960.2960.2770.2930.330.3110.330.3130.3620.309

    0.1470.1550.1370.1410.1510.1530.1470.1470.1550.141

    0.1330.1550.1470.1540.1530.1420.1520.1470.1470.169

    blank 40N1 (1:500)0.355

    blank 40N1 (1:500)0.342

    blank 40L (1:1000)0.350

    blank 40L (1:1000)0.336

    blank gal0.148

    blank no 1st0.150

    gp40 N1 +HPA-0.078-0.080-0.084-0.040-0.067-0.012-0.0490.013

    gp40 N1-0.088-0.067-0.066-0.047-0.051-0.0420.0120.034

    gp40 L + HPA-0.074-0.091-0.083-0.058-0.031-0.048-0.028-0.037

    gp40 L-0.040-0.040-0.059-0.043-0.006-0.025-0.006-0.023

    galactosidase-0.0010.007-0.011-0.0070.0030.005-0.001-0.001

    0.0190.0780.1560.3130.6251.2502.5005.000

    gp40 N1 +HPA0.013-0.049-0.012-0.067-0.040-0.084-0.080-0.078

    gp40 N10.0340.012-0.042-0.051-0.047-0.066-0.067-0.088

    gp40 L + HPA-0.037-0.028-0.048-0.031-0.058-0.083-0.091-0.074

    gp40 L-0.023-0.006-0.025-0.006-0.043-0.059-0.040-0.040

    galactosidase-0.001-0.0010.0050.003-0.007-0.0110.007-0.001

    7 Sep 99 (10min) (2)

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    00000

    gp40 N1 +HPA

    gp40 N1

    gp40 L + HPA

    gp40 L

    galactosidase

    HPA binding protein concentration

    A 405 nm

    23 Sep 99 (10min)

    Shed proteins - Fixed cells

    0.4250.4830.5560.5040.3490.1770.1460.1650.170.164

    0.4820.5370.5990.490.3890.1820.1690.1620.1670.169

    2.0672.3781.9441.3490.490.1750.1620.1560.1690.163

    0.9692.2222.0991.2090.8470.2430.1790.160.1770.167

    0.2090.2010.1740.1670.1630.1520.1480.1470.1380.138

    0.1840.1860.1740.1910.1570.1550.1430.1420.1470.141

    blank N10.168

    blank 4E90.169

    blank gal0.141

    gp40 N10.2860.3430.4100.3300.2020.012-0.010-0.004

    gp40 4E91.8982.1311.8531.1100.5000.0400.001-0.011

    galactosidase0.0560.0530.0330.0380.0190.0130.0040.003

    0.160.310.631.252.505.0010.0020.00

    gp40 N1-0.004-0.0100.0120.2020.3300.4100.3430.286

    gp40 4E9-0.0110.0010.0400.5001.1101.8532.1311.898

    galactosidase0.0030.0040.0130.0190.0380.0330.0530.056

    23 Sep 99 (10min)

    000

    000

    000

    000

    000

    000

    000

    000

    gp40 N1

    gp40 4E9

    galactosidase

    Shed proteins concentration

    A 405 nm

    23 Sep 99 (10min) (2)

    N1 sera - Live cells (Shed vs HPA)

    0.4390.4970.5250.4950.4740.330.1920.1910.1870.173

    0.4520.5040.4940.4750.4760.3650.2060.1950.1880.176

    0.5670.5220.4760.4320.3670.2340.1960.1710.1860.173

    0.6520.5820.5430.4040.3620.2320.1790.1880.1870.17

    0.1730.1760.1790.1890.1830.1740.170.1540.1740.162

    0.1820.1760.1910.1910.1960.1710.1680.1620.1570.169

    blank N10.181

    blank 4E90.179

    blank gal0.166

    HPA0.2650.3200.3290.3040.2940.1670.0180.012

    Shed0.3880.3730.3310.2390.1860.0540.0080.000

    galactosidase0.0120.0110.0200.0250.0240.0070.004-0.008

    0.160.310.631.252.505.0010.0020.00

    HPA0.0120.0180.1670.2940.3040.3290.3200.265

    Shed0.0000.0080.0540.1860.2390.3310.3730.388

    galactosidase-0.0080.0040.0070.0240.0250.0200.0110.012

    23 Sep 99 (10min) (2)

    0.0120.0005-0.0075

    0.0180.00850.0035

    0.16650.0540.007

    0.2940.18550.024

    0.3040.2390.0245

    0.32850.33050.0195

    0.31950.3730.0105

    0.26450.3880.012

    HPA

    Shed

    galactosidase

    gp40 concentration

    A 405 nm

    23 Sep 99 (1 h) (a)

    Shed proteins, fixed cells (N1 vs 4E9)

    1.631.8061.8111.7021.2730.410.2920.3640.430.411

    1.8271.8531.881.6211.2740.4120.3320.3420.410.447

    2.9632.9952.9512.8891.7440.4330.3560.3490.4030.435

    2.8772.9522.9522.8892.6450.6940.420.3340.430.383

    0.5460.5030.4750.40.3220.2830.2710.2760.2570.258

    0.4630.4530.460.4580.310.2840.2460.2540.2610.25

    blank N10.425

    blank 4E90.413

    blank gal0.257

    N11.3041.4051.4211.2370.849-0.014-0.113-0.072

    4E92.5502.5612.5392.4761.7820.151-0.025-0.071

    galactosidase0.2480.2220.2110.1730.0600.0270.0020.009

    0.160.310.631.252.505.0010.0020.00

    N1-0.072-0.113-0.0140.8491.2371.4211.4051.304

    4E9-0.071-0.0250.1511.7822.4762.5392.5612.550

    galactosidase0.0090.0020.0270.0600.1730.2110.2220.248

    23 Sep 99 (1 h) (a)

    -0.0715-0.071250.0085

    -0.1125-0.024750.002

    -0.01350.150750.027

    0.8491.781750.0595

    1.2372.476250.1725

    1.4212.538750.211

    1.4052.560750.2215

    1.3042.550250.248

    N1

    4E9

    galactosidase

    gp40 concentration

    A 405 nm

    23 Sep 99 (1 h) (b)

    N1 sera - Live cells (Shed vs HPA)

    1.5741.6971.761.7191.5120.9530.4640.4190.450.447

    1.6251.7921.7751.6871.4891.1420.5450.4230.4570.447

    1.9511.9271.8011.5681.2870.6850.4310.4290.4370.432

    2.2662.0441.9721.6641.1720.5860.4260.4410.4620.437

    0.4430.4670.470.4820.4730.4510.3670.3360.3470.347

    0.4690.4850.4960.5020.4420.3520.3420.3410.3520.365

    blank N10.450

    blank 4E90.442

    blank gal0.353

    HPA1.1491.2941.3171.2531.0500.5970.054-0.029

    Shed1.5091.5441.4451.1740.7880.194-0.014-0.007

    0.1030.1230.1300.1390.1050.0490.002-0.014

    0.020.030.060.130.250.501.002.00

    HPA- glycoproteins-0.0290.0540.5971.0501.2531.3171.2941.149

    Shed proteins-0.007-0.0140.1940.7881.1741.4451.5441.509

    -0.0140.0020.0490.1050.1390.1300.1230.103

    SEM HPA0.0010.0290.0670.0080.0110.0050.0340.018

    SEM shed0.0040.0020.0350.0410.0340.0600.0410.111

    0.0020.0090.0350.0110.0070.0090.0060.009

    23 Sep 99 (1 h) (b)

    000

    000

    000

    000

    000

    000

    000

    000

    HPA- glycoproteins

    Shed proteins

    b-galactosidase

    protein (g/ml)

    Binding (A405 nm)

    Sheet3

    Sheet3

    -0.02925-0.007-0.014250.00141421360.00141421360.00424264070.00424264070.0017677670.001767767

    0.05425-0.01350.001750.02863782460.02863782460.0017677670.0017677670.00883883480.0088388348

    0.597250.19350.048750.06682159080.06682159080.03500178570.03500178570.03500178570.0350017857

    1.050250.78750.104750.0081317280.0081317280.04065863990.04065863990.01096015510.0109601551

    1.252751.1740.139250.01131370850.01131370850.03394112550.03394112550.00707106780.0070710678

    1.317251.44450.130250.00530330090.00530330090.06045762980.06045762980.00919238820.0091923882

    1.294251.54350.123250.03358757210.03358757210.04136574670.04136574670.0063639610.006363961

    1.149251.5090.103250.01803122290.01803122290.1113693180.1113693180.00919238820.0091923882

    HPA- glycoproteins

    Shed proteins

    b-galactosidase

    gp40 (g/ml)

  • Analysis of Cpgp40/15 deduced amino acid sequenceSignal peptide326 aa /33.6 kDa protein O-glycosylation siteGPI anchor sitePolyserine region981 bpCevallos et al, 2000

  • Analysis of Cpgp40/15 deduced amino acid sequencegp40 N-terminusgp15 N-terminusCpgp40/15MRLSLIIVLLSVIVSAVFSAPAVPLRGTLKDVPVEGSSSSSSSSSSSSSSSSSSSTSTVAPANKARTGEDAEGSQDSSGTEASGSQGSEEEGSEDDGQTSAASQPTTPAQSEGATTETIEATPKEECGTSFVMWFGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSVTFEKSDNTVKIKVNGQDFSTLSANSSSPTENGGSAGQASSRSRRSLSEETSEAAATVDLFAFTLDGGKRIEVAVPNVEDASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDLVDKDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVLgp40 N-terminusgp15 N-terminus

  • gp15/17 kDa immunodominant antigen 15 kDa protein localized to surface of sporozoites and merozoites and shed in trails during gliding motilitycontains aGalNAc residuesGut and Nelson, 1994; Strong et al 2000 15 kDa protein localized to surface of sporozoites and merozoites recognized by IgA MAbs CrA1 and CrA2 which are partially protective against C. parvum infection in scid mouse backpack tumor model Zhou et al, Cevallos et al 2000 17 kDa immunodominant antigen recognized by serum antibodies from infected humans present in TX-114 extracts of sonicated oocystsPriest et al, 1999, Priest et al, 2000

  • 123kDa3550751001502515225123kDa3550751001502515225CrA1anti-gp15

    anti-gp40gp40 and gp15 are antigenically distinct proteins1, oocysts2, sporozoites3, shed proteinsCevallos et al, 2000

  • 15253550100150751525355010015075CrA1anti-gp15kDakDa

    anti-gp40Antibodies to native gp40 and gp15 recognize the corresponding recombinant fusion proteins1, control2, r40/153, r154, r40Cevallos et al, 2000

  • SporozoitesMerozoitesReactivity of anti-gp40 antisera with C. parvum sporozoites and merozoites by immunofluorescenceCevallos et al, Infect. Immun 68: 4108-4116, 2000

  • SporozoitesMerozoitesReactivity of MAb CrA1 (anti-gp15) with C. parvum sporozoites and merozoites by immunofluorescenceCevallos et al, 2000

  • 7515253550100150kDa2257515253550100150kDa22512121212anti-gp40anti-gp15anti-r40anti-r15gp40 and gp15 are products of proteolytic cleavage of a 49kDA precursor protein Cevallos et al, 2000

  • Polymorphisms at gp15/45/60 locus sequence analysis of PCR amplified gp15/45/60 ORF from 29 diverse C. parvum isolates magnitude of sequence polymorphism identified at this locus is far greater than that detected at any other C. parvum locus identified to date 77-88% nucleotide sequence identity 67 to 80% amino acid sequence identity numerous SNPs and SAAPs in these sequences defined at least 5 distinct allelic groupings Ia, Ib, Ic, Id, (human/genotype I) II (calf/genotype II) conserved regions putative signal peptide putative GPI anchor site putative proteolytic processing siteStrong et al, 2000

  • Comparison of Type II (calf) and Type I (human) Cpgp40/15 deduced AA sequences69% identity at amino acid level, 84% identity at nucleotide level

    human gp 40/15 1 DVPVEGSSSSSSSSSSSSSSSSSSSSSSSSSTSTVAPAPK 40

    calf gp40/15 1 DVPVEGSSSSSSSSSSSSSSSS------SSSTSTVAPAN- 33

    ********************** **********

    human gp 40/15 41 KERTVEGGTEGKNEESSPGSEEQDGGKEDGGKENGEGDTV 80

    calf gp40/15 34 KARTGEDAE-GSQDSSGTEASGSQGSEEEGSEDDG----Q 68

    * ** * * .. * . * *.* . *

    human gp 40/15 81 DGEQTGSGSQVTPSGSAGTATESTATTTPKEECGTSFVMW 120

    calf gp40/15 69 ----TSAASQPTTPAQSEGATTETIEATPKEECGTSFVMW 104

    * . ** * . ** * .*************

    human gp 40/15 121 FEKGTPVATLKCGDYTIVYAPIKDQTDPAPRYISGEVTSV 160

    calf gp40/15 105 FGEGTPAATLKCGAYTIVYAPIKDQTDPAPRYISGEVTSV 144

    * *** ****** **************************

    human gp 40/15 161 SFEKSESTVTIKVNGKEFSTLSANSSSPTKDNGESSDSQV 200

    calf gp40/15 145 TFEKSDNTVKIKVNGQDFSTLSANSSSPTENGG--SAGQA 182

    .****. ** *****..************ .* * *

    human gp 40/15 201 QSRSRRSLAEENGETVATVDLFAFTLDGGRRIEVAVPKDE 240

    calf gp40/15 183 SSRSRRSLSEETSEAAATVDLFAFTLDGGKRIEVAVPNVE 222

    *******.**. *. *************.******* *

    human gp 40/15 241 NADKRSEYSLVADDKPFYTGANSGITNGVYKLDENGNLVD 280

    calf gp40/15 223 DASKRDKYSLVADDKPFYTGANSGTTNGVYRLNENGDLVD 262

    * ** ***************** *****.* *** ***

    human gp 40/15 281 KDNKVLLKDAGSSAFGFRYIVPSVFAIFAALFVL 314

    calf gp40/15 263 KDNTVLLKDAGSSAFGLRYIVPSVFAIFAALFVL 296

    *** ************ *****************

  • Southern blot analysis of Cpgp40/15Type II GCH1Type I UG502

  • Reactivity of anti-gp40 antibodies with Type I (GCH1) and Type II (UG502) isolates1525355010015075225kDaI II I II I II

  • Genotyping of C. parvum isolates from HIV-Infected Children with Persistent Diarrhea in South Africa

    C. parvum DNA PCR amplified from 21/24 stool samples Genotype of isolates determined by PCR-RFLP at TRAP C1 and COWP loci 16/21 (76%) of isolates were of the human genotype at both loci PCR amplification of Cpgp40/15 locus

  • gp40 gp40 is a mucin-like glycoprotein containing terminal aGalNAc residues gp40-specific antibodies neutralize infection in vitro and gp40 binds to intestinal epithelial cells. The gene encoding gp40 also encodes gp15, an antigenically distinct protein. gp40 is localized to the surface and apical region of invasive stages. gp40 and gp15, are products of proteolytic cleavage of a 49 kDa precursor protein expressed in intracellular stages. The Cpgp40/15 locus is highly polymorphic

  • New England Medical CenterTufts University, BostonAna Maria CevallosNajma BhatSmitha Jaison Brett Leav Roberta OConnor

    Renaud VerdonDavid Hamer

    Xiaoping ZhangMatt Waldor

    Gerald KeuschMiercio Pereira

    Childrens Hospital,Harvard University, BostonMarian NeutraXiaoyin ZhouUniversity of CaliforniaSan FranciscoCarolyn Petersen

    Bill StrongRichard NelsonUniversity of Texas, HoustonSara DannCynthia ChappellUniversity of Natal, Durban, Africa Centre for Health and Population Research, Mtubatuba Michael Bennish Nigel Rollins CDC, AtlantaJeff PriestTufts University School of Veterinary Medicine, GraftonBarry SteinGiovanni WidmerDonna AkiyoshiInderpal SinghCindy TheodosSaul TziporiACKNOWLEDGEMENTS

    I've been asked to speak about host-parasite interactions of Cryptosporidium, and these host-parasite interactions, as you can imagine, take place at a number of stages during the infection process, starting from when the infectious oocyst, is ingested by a susceptible host, going all the way through the process of excystation, attachment, infection, intracellular development, and finally, excretion of oocysts back into the environment.

    However, for the purpose of this talk, I am going to confine myself to the processes of attachment and invasion, which are primary events in establishing infection with this parasite. What I would like to do is just very briefly describe what is known about ultra structural observations about attachment and invasion, some of the factors that have been shown to affect these processes in vitro, using in vitro models, and then to focus on surface and apical complex proteins of Cryptosporidium that have been implicated as playing a role in these two processes.There is a great body of literature on the ultra structural aspects of Cryptosporidium attachment and invasion, and I'm not going to go into great detail. I just want to point out some of the similarities and differences between Cryptosporidium and other apicomplexar parasites. Invasive stages, the sporozoites and merozoites of Cryptosporidium attach to host cells by their anterior pole, and this is similar to other parasites like Toxoplasma and malaria.Like other apicomplexar parasites, Cryptosporidium have a specialized set of organelles located at the apical region, which are called apical complex organelles, and they consist of rhoptries, micronemes, and dense granules which discharge their contents during attachment and invasion. Proteins that are discharged from these organelles have been shown to play an important role in attachment and invasion, and there is evidence that in Cryptosporidium also, rhoptries and micronemes discharge their contents, although what exactly these proteins are has not been well-characterized.One of the unique aspects of Cryptosporidium attachment and invasion is that upon contact with the host cell, electron-dense bands are formed in the host cell cytoplasm, and these dense bands are believed to consist of host cytoskeletal elements. Once attachment occurs, the zoites invaginate the host cell plasma membrane, which eventually rises up and engulfs the parasite completely within the parasitiferous vacuole. Once in the parasitiferous vacuole, the parasite undergoes further intracellular development in a unique intracellular but extracytoplasmic location. So, unlike other apicomplexans like Toxoplasma and malaria, Cryptosporidium does not have direct contact with the host cell cytoplasm. And another unique aspect is this organelle called the feeder organelle membrane which forms at the site of attachment and which is believed to function to allow nutrients from the host cell to get to the parasite, although there is no evidence to support this at present.

    Here is a transmission electron micrograph taken by Doctor Saul Tzipori a while ago, showing a sporozoite that is approaching and attaching to the intestinal microvillus brush border membrane, and you can see in the apical region, the micronemes and the rhoptries and, further up there, the dense bodies. This is a merozoite, in an electron micrograph taken by doctor Tzipori, showing the merozoite actually invading the host cell, and you can see formation of the parasitiferous vacuole and formation of the parasitiferous vacuolar membrane. At the apical end of the merozoite, you can see a clear area, which is where the rhoptries and micronemes would have discharged their contents.A number of investigators, including us, have studied various factors that affect Cryptosporidium attachment and infection, using in vitro models, and we're going to hear more about some of these models later, but I'd just like to go over a few of the factors that have been described to be important in attachment in vitro. I should say that these models are somewhat artificial, because for attachment, we have to fix the cells in order to prevent invasion, so as to separate the processes of attachment and invasion. So these models are somewhat artificial, but at the present, they're all that we have to study attachment.Attachment is dependent on time. Sporozoites attach within five minutes of being added to host cells, and attachment increases rapidly until it peaks at about one to two hours, and then gradually levels off. Attachment is also dependent on the number of sporozoites added, and this varies depending on the model that is used, but with increasing number of sporozoites, you get increasing attachment until, eventually, there is a plateau.Attachment is optimal at a temperature of 37 degrees when compared to other temperatures like four degrees or room temperature. Divalent cations have also been shown to be involved in the process of attachment. One study showed that calcium and manganese were important for attachment, whereas another study showed that a high concentration of manganese could actually inhibit infection.Attachment has also been shown to be pH dependent, and a pH of 7.2 to 7.6 is optimal. Although Cryptosporidium infects a number of different host cells in vitro, in general, sporozoites attach to and invade epithelial cells. For example, a study has shown that sporozoites will bind to and invade biliary epithelial cells but not hepatocytes. The host cell differentiation status also appears to be important. In a study using intestinal epithelial CACO 2 cells, we showed a few years ago that attachment was better to cells that were less differentiated compared to those that were fully differentiated.Studies have also shown that the host plasma membrane domain is important. For example, studies showed that sporozoites would attach to the apical membrane of biliary epithelial cells but not to the basolateral membranes. I'd just like to say a few words about the role of parasite and host cytoskeletal elements in Cryptosporidium motility attachment and invasion in vitro. Sporozoite motility is powered by an actinomycin motile system, similar to other apicomplexan parasites. However, once attachment has been accomplished, the parasite cytoskeletal elements do not seem to be important for invasion.A number of studies have shown that host cell actin is recruited to the host parasite interface during the process of invasion, and a study by Elliot and Clark showed that filamentous actin is assembled into a block-like structure at the interface between the host and the parasite. At least two studies have identified host cytoskeletal molecules in the parasitiferous vacuole, suggesting that these molecules may be involved in the parasitophorus vacuole formation.

    Now, I'd like to talk about surface and apical proteins of Cryptosporidium that may be important in attachment and invasion. Over 20 sporozoite proteins have been identified so far, ranging from a very low molecular weight to a very high molecular weight, and many of these surface or apical complex proteins have been implicated in attachment or invasion. Some of the common aspects of these proteins are that many of them have been identified using antibodies, either monoclonal or polyclonal, which inhibit infection in vitro or in vivo in animal models. Many of these proteins are glycosylated, unlike those of other apicomplexan parasites and many of these proteins are shed from the parasite in trails during motility.I should say at this point that, although there is good evidence to suggest that some of these proteins are involved in attachment and invasion, none of them have been conclusively shown to be involved. One of the many difficulties of working with this parasite is that it's not possible to propagate the parasite in vitro, so it's difficult to do studies which can conclusively prove that a protein is involved in attachment and infection. There is no stable, or even transient, transfection system for Cryptosporidium, as there is for other apicomplexans, so we can't knock out genes that we think are important in attachment and invasion and prove their function in that way. Nevertheless, I would like to now describe a few of the surface and apical complex proteins of Cryptosporidium that are implicated in these two processes of attachment and invasion.

    There's a long list of literature here, showing various investigators who have described surface proteins of Cryptosporidium that may be involved in attachment and invasion, and I just grouped them by molecular weight. There's one group of very high molecular weight proteins, greater than 900 or 1300 kilodaltons. We don't know whether these are the same protein or whether they are different at this point.The second group is around 40 to 47 kilodaltons. We know that two of them are the same, as I'll tell you later. A group of proteins between 20 to 27 kilodaltons has also been described, and again, some of them are likely to be the same protein, but others, we're still not sure about.And then finally, there is a group of low molecular weight proteins, ranging from 15 to 17 kilodaltons in molecular weight, and there are at least three groups of 15 or 17-kilodalton proteins which are different from each other by sequence analysis, and I'll be talking about one of these groups a little later.In addition, there's a protein called TRAP C1, which has been identified by homology to similar proteins in other apicomplexan parasites, such as the malaria parasites and Toxoplasma and eimeria, and TRAP C1 homologues in these parasites have been shown to be important in attachment and invasion, and so the TRAP C1 in Crypto is also believed to mediate attachment and invasion, although there is no direct evidence of this.At least two groups, including us, have described lectins or carbohydrate-binding proteins on the parasite surface which bind to Gal and GalNAc residues on the host cell, and these might also mediate protein-carbohydrate host-parasite interactions.Now, obviously I can't describe all these proteins, but I would like to just tell you about the approach that we have taken to study proteins that are involved in attachment and invasion. Just as an example of how these proteins have been identified--and this approach has been used by many investigators. Our approach to identifying proteins that are involved in attachment and invasion was to make monoclonal antibodies, to screen them first by immunofluorescence for proteins that were localized to the surface or apical complex, and then use the monoclonal antibodies to screen for inhibition of attachment and infection, using in vitro models. The model that we used is the intestinal epithelial CACO 2 cell line. I will not go on to describe these studies in great detail, because they've been published, I'll just go rapidly through them.One of the antibodies that we identified is called 4E9, and this antibody, as you can see, compared to an isotype-matched irrelevant antibody, B9A4, inhibited sporozoite infection of host cells and attachment to host cells in a dose-dependent manner, suggesting that the proteins recognized by this antibody may be involved in this process.

    In collaboration with Doctor Tzipori, we were also able to show that this antibody prevented, or was capable of neutralizing infection of neonatal BALB-C Mice with Cryptosporidium, again suggesting that the proteins recognized by the antibody may be important.By immunofluorescence, this antibody reacted with material that was shed from the surface of oocysts during the process of excystation and also reacted with the surface of sporozoites, as you can see in panel B.When we looked at sporozoites that had been purified on a Percoll gradient or by filtration, we found that the antibody reacted with the apical end of sporozoites and also with trails. I don't know if you can see it in panel C, but there is a trail left by a sporozoite during gliding motility on a slide, and the antibody reacted with material shed in these trails. The antibody also reacted, as you can see in panel D, with intracellular stages, including merozoites, and there was also some indication that the parasitiferous vacuole was all stained.

    These finding were confirmed by electron microscopy. You can see that the sporozoites show surface as well as apical localization, and in panel D, which shows a meront from a gamma interferon knockout mouse infected with Cryptosporidium, there is also surface labeling along the merozoite.When we look by immunoblot analysis to see which proteins were recognized by this antibody, we found that it recognized mainly two proteins. The first was a very high molecular weight protein, over 200 kilodaltons, and the second was a 40-kilodalton protein. It turns out that the high molecular weight protein is the same as a protein previously describes as GP900, and this was first described by Carolyn Peterson and her group. GP900 was shown to be a glycoprotein, which is present in sporozoites and merozoites, and which is shed from the surface of sporozoites during gliding motility.It was localized by electron microscopy to micronemes of invasive stages, so it's a micronemal protein. Carolyn and her colleagues showed that this protein was encoded by a 7.5-KB gene locus, which corresponded to a predicted 1832 amino acid protein. The deduced amino acid sequence showed that GP900 is a mucin-like glycoprotein, which contained cysteine-rich and polythreonine domains, and we showed that these polythreonine domains contained sites for O-glycosylation.In collaboration with Carolyn Peterson, we also showed that native GP900 binds to intestinal epithelial cells, and Carolyn showed that the native protein could competitively inhibit infection in vitro. The cysteine rich domain of the recombinant protein, as well as antibodies to this domain, also inhibited infection in vitro, and these finding taken together suggested that GP900 is important in attachment and invasion.

    I'd just like to describe a very high molecular weight glycoprotein that was described by Mike Riggs and his colleagues, and this protein is called CSL, to stand for Circumsporozoite-like Glycoprotein. This glycoprotein was identified by antibodies that reacted with the surface and apical complex. This antibody elicited a circumsporozoite-like reaction, which is characterized by the formation and posterior movement and release of antigen membrane precipitates. The antibody neutralized sporozoite infectivity and prevented infection in neonatal Balb C mice.Mike Riggs and his colleagues showed that this is a soluble exoantigen, which is comprised of multiple 1300-kilodalton molecular species with differing isoelectric points. And he also showed that isolated native protein binds to intestinal epithelial cells and inhibits subsequent sporozoite attachment and invasion, again suggesting that this protein is important in attachment and invasion. We really don't know whether GP900 and CSL are the same or different proteins at this point.

    Now, we were interested in the other protein recognized by 4E9, which is gp40, and you can see it on the second panel there. Our approach to studying this protein was to purify it, to cut the band out of a gel and to make antibodies that recognized gp40. And as you can see on the second panel, these antibodies were specific for gp40 and did not bind to GP900, so we used these antibodies in our in vitro infection and attachment models and showed that these antibodies blocked infection in vitro, suggesting that gp40 might also be important in attachment and infection.

    We also showed that gp40, isolated from two different preparations, would bind to intestinal epithelial cells in a manner similar to GP900 and CSL, again suggesting that this protein might be important in attachment.

    [We showed that gp-40-specific antisera inhibited C. parvum infection of epithelial cells when compared to pre-immune sera.][Partially purified gp40 binds to internal epithelial cells, as shown in this slide using an ELISA-bases assay with gp-40 specific antisera.]We went ahead and cloned the gene encoding this protein, and the structure is shown up here, and I'm not going to go into it in great detail. It turns out that in addition to encoding gp40, this gene also encodes another protein called gp15. Three different groups of investigators had actually cloned gp15.[This slide shows the deduced amino acid sequence of the Cpgp40/15 gene and the sites of the gp40 and gp15 N-termini are shown with arrows.]I've listed here the three different groups that were working on the gp15 protein. The first one, Rick Nelson's group at UCSF, showed that gp15 is 15-kilodalton protein, which is localized to the surface of sporozoites and merozoites, and is shed in trails during gliding motility and also contains alpha GalNAc residues.Marian Neutra, Saul Tzipori and our group were also studying a 15-kilodalton protein, which is also localized to the surface of sporozoites and merozoites and is recognized by IgA monoclonals, which are partially protective against Cryptosporidium infection in a back-pack tumor model.And the third group was Jeff Priest and his colleagues at the CDC, who were studying a 17-kilodalton immunodominant antigen recognized by serum antibodies from infected humans. Jeff showed that this protein was membrane-associated, since it was present in triton X114 extracts of oocysts. This just shows that gp40 and gp15 are antigenically different, because antibodies that are specific for one protein don't cross-react with the other.

    We were able to show the same thing with the recombinant protein. When we looked by immunofluorescence, antibodies that were specific for gp40 recognized the apical region of isolated sporozoites. However, when we looked at intracellular stages, as you can see on the right hand panel, the antibody reacted with the surface, as well as the apical region.

    However, the antibodies to gp15 reacted exclusively with the surface of sporozoites and merozoites. [This shows processing of the precursor proteins into gp40 and gp15.]I just want to close by talking about polymorphism at the gp40-15 locus. We heard a lot about genotyping from Dr. Lal, and it turns out that the gp15--40-15 gene--is also highly polymorphic, and this was shown by Rick Nelson.This is all published, so I won't talk about it in detail, but numerous single nucleotide polymorphism and single amino acid polymorphism have been identified and define at least five different allelic groupings.

    [This slide shows SAPs from Type I and Type II isolates that we studied.]We studied an isolate that we obtained from Saul Tzipori and Donna Akiyoshi, from a child with cryptosporidiosis in Uganda. It was of the human genotype. We compared it to GCH1, which is the calf genotype, and we found by Southern blot analysis that the gene encoding gp40-15 is a single copy gene in both isolates, but the pattern of the restriction fragments is quite different, as you can see. And when we looked with antibodies, also, we found a difference in molecular weight of the gp40 protein from both isolates.

    [This slide shows variation in the size of the Cpgp40/15 PCR product amplified from isolates from South Africa.][This summarizes our findings about gp40.]So I'd just like to conclude by acknowledging all my colleagues and collaborators who have worked with us on this project. Thank you.AUDIENCE PARTICIPANT: [Off mic.] Could you comment on if any other organisms with an attachment mechanism that is most closely related, and if so, what strategies have worked in such an organism for.Dr. WARD: The question is which other Apicomplexan parasites have similar mechanisms of attachment and what strategies have been used. I would say probably the malaria and Toxoplasma systems are similar to this. Apical complex and surface proteins of these organisms have been used as targets for vaccines and for antibody therapy, so I think that's the strategy that would apply to Cryptosporidium, as well.AUDIENCE PARTICIPANT: You said that one of the factors that increased attachment was less differentiation. How do these organisms attach to transformed cells?Dr. WARD: Well, the question is how do parasites attach to transformed cells. The cell line that we used was actually a transformed cell line, so it will attach to transformed cell lines, but attachment varies from cell line to cell line, and I think you'll hear more about that in, in vitro models later on.