LANCE E. PERRYMAN,* KIMBERLY A. KEGERREIS, AND PATRICIA H. MASON. Department of Veterinary Microbiology and Pathology, Washington State ...
Vol. 61, No. 11
INFECrION AND IMMUNITY, Nov. 1993, p. 4906-4908 0019-9567/93/114906-03$02.00/0 Copyright © 1993, American Society for Microbiology
Effect of Orally Administered Monoclonal Antibody on Persistent Cryptosporidium parvum Infection in scid Mice LANCE E. PERRYMAN,* KIMBERLY A. KEGERREIS, AND PATRICIA H. MASON Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040 Received 22 June 1993/Returned for modification 19 August 1993/Accepted 1 September 1993
scid mice, persistently infected after exposure to 107 Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).
The protozoal agent Cryptosporidium parvum infects intestinal epithelial cells of most mammalian species (10). Immunocompetent humans infected with C. parvum experience diarrhea of limited duration followed by recovery within 7 to 21 days and resistance to reinfection. Immunocompromised humans develop persistent infections accompanied by severe, sometimes life-threatening diarrhea (7-11, 13, 18, 25). Management of these patients is complicated by the absence of chemotherapeutic agents that are predictably effective in clearing C. parvum infection. Given the importance of immune responses in controlling C parvum infection, we and others have produced monoclonal antibodies (mAbs) that neutralize the infectivity of merozoites and/or sporozoites, the two infectious stages of the organism (2, 5, 22, 26). Investigators have shown that these antibodies protect suckling mice from initial infection (2, 5, 20, 22, 26, 28) and reduce the severity of persistent cryptosporidial infections in nude mice (4, 19). Severe combined immune deficient (scid) mice infected with C. parvum have been used to study infection parameters and treatment protocols (14-16, 24). We report the effect of oral administration of mAb 17.41 on adult scid mice persistently infected with C. parvum. The results demonstrate a reduction of parasite numbers within intestinal epithelial cells accompanied by fewer C. parvum oocysts shed in feces. No reduction of parasite numbers in gastric epithelial cells was achieved by this protocol. Male scid mice (C.B-17/lcr Tac-scidfDF; Taconic, Germantown, N.Y.) were maintained in microisolator cages. They were assigned to two groups and infected at 6 weeks of age by oral administration of 107 C. parvum oocysts. The C. parvum isolate, originally obtained from H. Moon (Ames, Iowa), was passaged in calves and prepared for administration as previously described (4). Six weeks postinfection, mice in group 1 were treated daily for 14 (experiment 1) or 17 (experiment 2) days with 0.4 mg of immunoglobulin M in the form of ascites fluid induced by hybridoma cell line 17.41. This line produces an immunoglobulin M mAb that neutralizes the infectivity of C. parvum sporozoites and merozoites (5, 22). Mice in group 2 received 0.4 mg of control immunoglobulin M mAb according to the same schedule. Feces from individual mice was collected weekly and analyzed for the presence and number of C. parvum oocysts as follows. Individual fecal pellets were weighed prior to emulsification *
Corresponding author.
in 700 ,ul of phosphate-buffered saline (PBS). The resulting suspension was overlaid on 1 ml of a 1:4 dilution of Sheather's solution which was previously overlaid on 1 ml of a 1:2 dilution of Sheather's solution. Following centrifugation at 1,500 x g for 25 min, oocysts were carefully collected from the interface of the two dilutions of Sheather's solution. The oocysts were washed once and resuspended in 30 pl of PBS. They were counted in a hemocytometer at magnification of x200 with a differential interference contrast microscope. Data were converted to and expressed as oocysts per milligram of feces. Mice were terminated 8 or 8.4 weeks postinfection after 14 (experiment 1) or 17 (experiment 2) days of mAb treatment, respectively. The stomach, ileum, cecum, and colon were examined histologically for the presence and number of C. parvum organisms. Tissue infectivity scores ranging from 0 to 3 were assigned to each anatomical site, as previously described (0, no organisms observed; 1, 66% of the epithelium parasitized) (23). Results were analyzed with the Wilcoxon signed rank test to determine whether differences were significant (1). Excretion of C. parvum oocysts was readily detectable 4.4 weeks postinfection (Fig. 1). Treatment with mAb began 6 weeks postinfection. An apparent reduction in oocyst excretion was noted at week 7, but the difference was not significant until 8 weeks postinfection (P = 0.014). By completion of the experiment at 8.4 weeks postinfection, treatment with mAb 17.41 substantially reduced oocyst excretion compared with excretion with the control mAb (P = 0.001). Examination of tissue sections from treated and control mice substantiated the effect of mAb 17.41 on infection with C. parvum (Table 1). Tissue infectivity scores were significantly lower for mice treated with mAb 17.41 (P = 0.0005, experiment 1; P = 0.007, experiment 2) compared with scores with the control mAb. However, results were influenced by the anatomical site of infection. C. parvum is not typically located in the gastric epithelium of immunocompetent mice (27), but is routinely found there in scid mice (15). Oral treatment with mAb 17.41 did not reduce numbers of organisms in the stomach compared with those obtained by treatment with the control mAb. In contrast, mAb 17.41 effectively reduced the number of C. parvum organisms in the ileum, cecum, and colon (experiment 1) and in the ileum (experiment 2). The inability of mAb 17.41 to reduce organism numbers in the stomach may be explained by low gastric 4906
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with neutralizing antibodies may require targeted delivery to the biliary system and modulation of gastric pH to achieve
Group 1 *Group 2
50
desired results.
a) 40 0
0)
c)
30
c
20
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0
10
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2 3 4 4.4 5 5.4 6* 6.4 7 7.4 8 8.4 Weeks Postinfection
FIG. 1. Fecal oocyst excretion in scid mice infected with C. parvum and treated with mAbs (experiment 2). Daily oral treatment with mAb 17.41 (group 1) and the control mAb (group 2) began at 6 weeks postinfection (*). pH, which reduces binding of the mAb to a neutralizationsensitive epitope on sporozoites and merozoites. The results of this study are relevant for designing immunotherapy protocols for cryptosporidiosis. Persistent infection is predictably established in scid mice, and the dynamics of infection can be assessed by quantitating excretion of C. parvum oocysts. Oral treatment with a neutralizing mAb significantly reduced tissue infection scores and fecal oocyst excretion. Comparison of fecal oocyst counts with tissue infection scores in experiment 2 revealed a correlation coefficient of 0.48. Both assays detected the beneficial effect of mAb 17.41 treatment, and each has unique advantages. Fecal oocyst quantitation is less costly and time-consuming and allows repeated evaluation of individual animals. Tissue examination allows identification of unexpected anatomical sites of infection as well as differential effectiveness of treatment at each anatomical site. This last point is important, given the inability of oral mAb administration to reduce C. parvum infection in the biliary system (4, 19) and stomach (present study). Both locations are sites of infection in patients with AIDS (3, 6, 12, 17, 21). Therefore, treatment of persistent cryptosporidiosis in immunodeficient patients TABLE 1. C. parvum gastrointestinal infection scoresa in scid mice treated with mAbs Expt and Infection score with: p anatomical mAb 17.41 Control mAb siteb Expt 1 Stomach Ileum Cecum Colon All sites
2.83 ± 0.00 ± 1.00+ 0.75 ± 4.58 ±
0.58 0.00 0.85 0.75 1.31
2.75 ± 0.58 ± 2.33 ± 2.08+ 7.75 ±
0.62 0.79 0.49 0.67 1.77
0.74 0.03 0.002 0.002 0.0005
Expt 2 0.27 1.25 ± 0.45 1.67 ± 0.39 Stomach 0.002 Ileum 0.08 ± 0.29 1.67 ± 1.30 0.52 Cecum 0.67 ± 0.49 0.75 ± 0.87 0.08 0.50 ± 0.67 Colon 0.08 ± 0.29 0.007 2.08 ± 1.71 4.58+ 3.03 All sites Means ± standard deviations. The scores were assigned as described in the text. b n = 12 for both groups in both experiments.
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