Role of immunoglobulin A monoclonal antibodies against P23 in controlling murine Cryptosporidium parvum infection

Abstract

Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer's patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72. 7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum infection.

FIG. 1

Sporozoites stained by IFA with IgA MAb G9H4. Note the reactivity of MAb G9H4 with the sporozoite pellicle and Ag trails (arrows). The staining patterns of MAbs G9H4, H8H12, G9H9, H8H2, F3H6, and H8H6 were the same. Bar, 5 μm.

FIG. 2

Western immunoblot demonstrating the monomeric-polymeric states of anti-C. parvum IgA MAbs. Lanes: 1, SP2/O hybridoma supernatant; 2, F3H6; 3, G9H4; 4, G9H9; 5, H8H2; 6, H8H6; 7, H8H12; 8, MOPC-318 monomeric IgA control. Double bands typical of dimeric IgA at >200 kDa are indicated by the paired arrowheads. IgA monomers migrating at 116 to 120 kDa are indicated by the single arrowhead. The values on the left are molecular masses in kilodaltons.

FIG. 3

Western immunoblot demonstrating reactivity of anti-C. parvum IgA MAbs with sporozoite P23. Lanes: 1, F3H6; 2, H8H2; 3, H8H6; 4, H8H12; 5, G9H4; 6, G9H9; 7, C6B6; 8, MOPC-318 monomeric IgA control; 9, dimeric IgA isotype-matched control MAb 92.07t. The values on the left are molecular masses in kilodaltons.