Antisporozoite Antibodies with Protective and Nonprotective Activities: In Vitro and In Vivo Correlations Using Plasmodium gallinaceum, an Avian Model

ABSTRACT. A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (1) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense ISI to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P. gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial ISI to SL-29 and macrophage cells and partial inhibition to the exoerythrocytic development of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce ISI or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed.     

In vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages

Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. the host cells were macrophage precursors from chicken bone marrow. At various times after introduction of Sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). the time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until Ac end of the primary exoerythrocytic schizogony.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Use of a DNA probe to measure the neutralization of Plasmodium berghei sporozoites by a monoclonal antibody

A specific DNA probe has been used to quantify the neutralizing effects of monoclonal antibodies (3D11) against the circumsporozoite protein of Plasmodium berghei sporozoites. The amount of parasite DNA was measured in the livers of Norway Brown rats at the peak of proliferation of the exoerythrocytic forms (EEF). In vitro treatment of 1.5 X 10(5) sporozoites with 0.36 microgram/0.5 ml of whole 3D11 IgG neutralized about 90% of the sporozoite infectivity. When the dose was 3.6 micrograms no signal was detected, indicating that less than ten sporozoites developed into EEF in the liver. In contrast, 3.6 micrograms of Fab obtained from 3D11 neutralized sporozoite infectivity by only 60%. Although the neutralizing effect of 3D11 was very marked, the infected rats developed parasitemias after a prolonged delay in patency, suggesting that a small proportion of sporozoites was resistant to the effects of 3D11. The sporozoites were subjected to four cycles of 3D11-mediated selection, each one involving treatment of sporozoites with the antibodies, injection of the mixture into rats, infection of hamsters with blood stage parasites obtained from the rats, feeding of Anopheles stephensi on these hamsters, and obtaining sporozoites from the salivary glands of the infected mosquitoes. After four cycles of selection, the susceptibility of the resulting sporozoites to different concentrations of 3D11 was compared with that of nonselected sporozoites. No differences were detected, indicating that the capacity of a few sporozoites to escape the neutralizing effect of 3D11 antibodies is not inherited.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells; an in vitro assay of protective antibodies

Plasmodium falciparum and P. vivax sporozoites were observed to invade cultured human hepatoma cells in vitro. Monoclonal antibodies to the circumsporozoite (CS) protein of each of these malarial species blocked invasion. Inhibition was species-specific, but was independent of the geographic origin of each strain. Because these monoclonal antibodies have been shown to diminish or abolish sporozoite infectivity to susceptible primate hosts, it is suggested that inhibition of invasion of sporozoites (ISI) into cultured cells may represent in in vitro assay for protective antibodies. This was confirmed by the finding that serum taken from volunteers immune to sporozoite challenge also totally blocked sporozoite invasion. The ISI assay also detected naturally acquired invasive-neutralizing antibodies in areas endemic for malaria. This ISI assay may therefore be useful in determining the incidence of inhibitory anti-sporozoite antibodies in general populations, and allow the monitoring of the effect of an anti-malarial vaccine using sporozoite-derived antigens.     

Monoclonal antibody produced against partially purified sporozoite antigen inhibits invasion of cells by sporozoites of avian Eimeria species

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.     

Reduced Invasion of Cultured Cells Pretreated With A Monoclonal Antibody Elicited Against Refractile Body Antigens of Avian Coccidial Sporozoites

The effect of a monoclonal antibody (1209-C2) elicited against sporozoite refractile-body antigen on invasion of cultured baby hamster kidney cells by avian Eimeria species was examined in vitro. Pretreatment of sporozoites with 1209-C2 for 45 min before inoculation into cultures or simultaneous introduction of sporozoites and 1209-C2 into cultures had no significant effect on invasion. However, pretreatment of cultures for 45 min with 1209-C2 (also with media from other cloned 1209 cell lines) significantly inhibited invasion by sporozoites of Eimeria tenella and E. adenoeides. Pretreatment of cultures with 2 unrelated monoclonal antibodies with the same isotype as 1209-C2 did not inhibit invasion by E. tenella. There was a significant correlation between time of exposure of the cultures to 1209-C2 and invasion (r = -0.80924; p = 0.0001), with inhibition of invasion occurring after 20 min exposure, but not after 10 min. There was also a significant correlation between the titer of 1209-C2 and invasion (r = 0.62291; p = 0.0305). Monoclonal antibody 1209-C2 cross-reacted with epitopes of baby hamster kidney cells by both immunofluorescence and Western blot. The fluorescent labeling of the cells differed according to the fixative that was used. In formalin-fixed cultures labeled with 1209-C2, fluorescent foci were distributed over the entire cell; after methanol fixation, 1209-C2 reacted with only discrete foci in the nucleus. On Western blots of sporozoites, 1209-C2 reacted with antigens having molecular sizes of approximately 8, 17, 23, 30, and 45-60 kDa, and with several minor bands. On baby hamster kidney cells, the antibody reacted primarily with bands of approximately 30, 45-60, and slightly with other bands. The data suggest that interactions among similar molecules in the sporozoites and host cells may play a role in cellular invasion.     

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.     

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.     

Effect of Conditioned Media from Several Cell Types on Invasion by Eimeria adenoeides Sporozoites1

ABSTRACT. The effect of conditioned media (media aspirated from a variety of cell cultures after 4 d of growth) on cellular invasion by sporozoites of the turkey coccidium, Eimeria adenoeides, was examined. Conditioned medium from turkey kidney cells and baby hamster kidney cells failed to alter invasion. However, conditioned medium from turkey cecal cell cultures produced a significant (P ≤ 0.05), two-fold increase in invasion over control medium in a variety of cell types. Retentates of conditioned medium from the turkey cecal cells that were passed through microconcentrators having molecular mass cutoffs of 50, 100, and 300 kDa similarly enhanced invasion over retentates from control medium. However, retentates from microconcentrators with a cutoff of 1,000 kDa failed to enhance invasion. Pretreatment in conditioned medium, followed by washing of sporozoites prior to inoculation into cultures, did not result in enhanced invasion. Moreover, when the interval between inoculation of sporozoites into cells and fixation of cultures was reduced to less than 3 h, no enhancement of invasion occurred. Conditioned medium from turkey cecal cells that were grown in the presence of ³⁵S-translabel had at least two labeled bands at 150 kDa and > 200 kDa that were absent in conditioned media from turkey kidney and baby hamster kidney cells.     

Immunoradiometric assay to measure the in vitro penetration of sporozoites of malaria parasites into hepatoma cells

We describe here an immunoradiometric assay to quantitate the in vitro invasion of hepatoma cells by sporozoites. The assay measures levels of circumsporozoite (CS) antigen that remain associated with the hepatoma cells after their incubation with the parasites. Several observations show that these measurements reflect internalized rather than extracellular antigen. For example, when incubations were performed with nonviable parasites (sonicated or heated), or in the presence of metabolic inhibitors, such as sodium azide and deoxyglucose, the amounts of CS antigen found in hepatoma cell extracts were greatly diminished. Moreover, Western blotting experiments revealed a striking difference in the pattern of CS proteins of infected cell extracts as compared with those of free parasites. The assay was used to measure the amounts of intracellular CS antigen for several days after infection of the hepatoma cells. The results confirmed previous microscopic observations, made by using immunofluorescence techniques, showing that the CS antigen in the host's liver cells diminishes progressively while the parasite develops into the exoerythrocytic stage. The immunoradiometric assay should facilitate the evaluation of the effects of drugs on sporozoites and also on studies aimed at the identification of a sporozoite receptor on the hepatocyte.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

Cell surface antigens during submerged development of Myxococcus xanthus examined with monoclonal antibodies

Eighteen monoclonal antibodies directed against cell surface antigens of Myxococcus xanthus were followed by enzyme-linked immunosorbent assay. Three of the monoclonal antibodies were specifically directed against antigens present only on cells undergoing fruiting body development. These cell surface antigens became detectable by the early preaggregation stage (2 to 4 h) of development and increased until early aggregation (9 to 10 h), after which the concentrations of two of the cell surface antigens remained constant and the concentration of the third decreased. The remaining 15 monoclonal antibodies recognized cell surface antigens that were shared by vegetative and developing cells. Based on their relative concentrations during development, these shared antigens can be grouped into three classes. In the first class antigen concentration remained constant, in the second it decreased, and in the third it increased. Western blots of cell surface antigens were probed with monoclonal antibodies. Five monoclonal antibodies reacted with material in distinct bands, five monoclonal antibodies reacted with multiple, diffuse bands, and eight monoclonal antibodies were not reactive in Western blots.