A liver-stage specific antigen of P. berghei identified by a monoclonal antibody* L. Winger, A. Suhrbier, C.A. O'Dowd, K.J. Hodivala, & R.E. Sinden Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each stage are being tested for their potential as vaccine candidates. Recently it has become clear that the liver stage can also be the target of protective immune responses; however, only the circumsporozoite protein has been identified as a protective liver antigen. It is critical for vaccine evaluation and development to identify other liver antigens and assess their potential role in immunity. In this paper we describe a monoclonal antibody, which recognizes a liver-specific antigen of Plasmodium berghei (referred to as Pbll). Passive immunization studies using this antibody suggest that it may influence the course of sporozoite-induced infections.

Introduction Current research into malaria vaccines has highlighted two candidates which have attracted intense analysis: the precursor of the major merozoite antigens (PMMSA) (1, 2) and the circumsporozoite protein (CSP) (3, 4). Whilst in previous years the liver stage has been ignored as a potential target it is now clear that both the PMMSA (5, 6) and CSP (7,8) are also expressed in the liver stage. The CSP, presented on the infected hepatocyte in association with the major histocompatibility complex, appears to be the target recognized by protective cytotoxic T cells (9-12). The low number of polymorphic T-cell epitopes on the CSP (13) and the realization that the liver stage may be subject to a whole range of other immune responses (cytokines (14); phagocytes (15, 16); antibody (17)) have highlighted the need to identify novel liver-stage antigens, which may be relevant to the development of an anti-malarial vaccine. As part of the search for such antigens and the elucidation of their role in immunity, it is a prerequisite to have monospecific reagents which recognize liver-specific antigens. One successful approach has been to use naturally occurring antisera to select genomic libraries (18). However, because of recent advances in culture techniques (8, 19) it is now possible to grow sufficient liver-stage parasites for direct stage-specific immunization and for subsequent screening. In this report we describe a liver-specific antigen (Plasmodium berghei liver 1, Pbl 1) recognized by a monoclonal antibody. Passive immunization studies using this antibody suggested that it may * From the Molecular and Cellular Parasitology Research Group, Department of Pure and Applied Biology, Imperial College, London SW7 2BB, England. Correspondence to Dr L. Winger at this address.


influence the course of sporozoite-induced infections in vivo although it had no detectable effect on parasites grown in vitro.

Materials and methods Monoclonal antibody production BALB/c mice were first immunized with HepG2 cells alone and were then treated with cyclophosphamide to suppress the subsequent response against the HepG2 cells (20). After the three-week recovery period the animals were immunized three times with freezethawed 48-hour exo-erythrocytic (EE) schizonts grown in HepG2 cells (approximately 2.5 x 10i cells with a 10-20% infection rate) given at 2-week intervals. Assay of the serum by indirect immunofluorescence on acetone-fixed cultured EE schizonts in HepG2 cells showed that although some HepG2 background reactivity occurred, individual mice exhibited significant antibody reactivity to the parasite. After 5 months the mice were inoculated intravenously with 104 sporozoites on each of two succeeding days. On day 6 and/or 7 after sporozoite inoculation, when a Giemsa-stained blood smear showed positive blood parasitaemia, the mouse was sacrificed and the spleen removed for fusion. The fusion protocol was performed as described by Winger et al. (21). The supernatants of the resulting hybridomas were screened by indirect immunofluorescence on live and acetone-fixed cultured liver schizonts, washed parasitized blood smears, and smeared air-dried sporozoites. Ascites fluid of selected monoclonal antibodies, which had immunofluorescent titres of between 1/100000 and 1/500000 on liver parasites, were used at 1/50 dilutions on thick films of highly parasitized blood to verify the liver specificity of the monoclonal antibodies. Bulletin of the World Health Organization, 66 (Suppl.): 172-177 (1990)

A liver-sage specific antigen of P. berghel

Cuiture of EE stage of P. b.rghel and immunofluoroscent staining The culture of the liver stage of P. berghei in HepG2 cells and the immunofluorescent antibody staining technique was performed as described previously (6,8). Segmenters, fully mature EE merozoites held together in a stroma consisting in part of host cell cytoplasm, were prepared as described previously (22). Passive Immunization trials Monoclonal antibodies 17.9.15 (specific to an ookinete surface antigen (21)), 17.6.1 (binding to an internal antigen of EE merozoites and all blood-stage parasites (23)), 23.8.1 (an anti-circumsporozoite protein antibody, data not shown), and anti-Pb 1l were purified from ascites fluid according to the method of Reik et al. (24). All the antibodies were of the IgG 1 subclass. Protein levels were determined using a Biorad Protein standard and by spectrophotometric analysis at 280 nm. Using an ELISA for murine gamma-interferon no gamma-interferon was detected in these preparations to levels of 97 pcg or 1U per mg IgG. In one experiment the antibodies were further purified on a Protein G affinity column (Biorad). In a series of six experiments, 1 mg of purified monoclonal antibody or PBS was injected intravenously into groups of 3 or 4 mice 24 hours after infection with equal numbers of sporozoites (approx. 5 x 103) and blood parasitaemias were monitored on days 5, 6, 7 and 8 post infection. The experiments were performed in a double-blind manner and Giemsa-stained smears were randomized and coded prior to reading (parasites per 104 RBC/slide). To investigate the sensitivity of using subsequent blood-stage parasitaemia as a measure of intervention at the liver, 4000, 20000 and 100000 sporozoites were injected into groups of animals and their parasitaemia monitored as described above.

In vitro assays for Inhibitory effects of antibody 13 mm coverslips with a confluent monolayer of HepG2 cells were infected with approximately 2 x 104 sporozoites in the presence of 1 mg/ml of antiPb 11 or the same concentration of control antibody 17.6.1. The parasites were then cultured for 48 h with the antibodies before they were fixed and parasite numbers, size and maturity determined by light microscopy (using methods described previously (25)).

Results The majority of clones derived from the fusion had reactivities against both the blood- and liver-stage parasites. Of these, several initially appeared to be WHO Bulletin OMS: Supplement Vol. 68 1990

liver specific when the culture supernatants were being screened; however, using low dilutions of ascites fluids on highly parasitized blood, cross-reactivity was often demonstrated. This may reflect the greater ease in detecting low abundance antigens on the relatively much larger liver-stage parasites using indirect immunofluorescent staining. A monoclonal antibody, designated anti-Pbll, was found to be completely specific to the EE stage not reacting with the sporozoite or the blood stage. The epitope bound by anti-Pbll was first detected in/on the intra-hepatic differentiating 'sporozoite' 1-2 hours post invasion. This staining became brighter over the next 10 hours (Fig. 1 shows several intracellular sporozoites at 3 hours) and developed into a peripheral pattern around the newly formed trophozoite. This pattern was often associated with one or more irregular protrusions from the parasite apparently into the cytoplasm of the infected hepatocyte (Fig. 2). This peripheral pattern persisted to the end of the EE development. At 45-50 hours, however, the irregular protrusions became less apparent. At 55 hours (when the parasite plasmalemma and parasitophorous vacuole are invaginated, first to form cytomeres [recognizable by phase optics, Fig. 3 on right] and then merozoites), the peripheral pattern was maintained (Fig. 3). In the free segmenters an irregular speckled pattern was observed (Fig. 4). No staining was associated with the EE merozoites or the surface of the infected hepatocyte. The addition of anti-Pbl 1 to cultured liver-stage parasites during or after sporozoite invasion had no effect on the EE parasite number or size (data not shown). Pooled results from each antibody group from all the passive immunization experiments are presented in Fig. 5. The data suggest a possible reduction in parasitaemia on day 7 when anti-Pbl 1 or 23.8.1 are compared to the control monoclonal antibodies (values from Student t-test for difference range from 0.77 to 0.91) or to phosphate-buffered saline (value from Student t-test for difference is 0.97).

Discussion The persistence of a peripheral staining pattern during the segmentation (8) of the parasite suggested that Pbl 1 is located on the parasitophorous vacuole membrane. The pattern seen in the free segmenters (Fig. 4) is consistent with the parasitophorous vacuole membrane being disrupted by the emerging merozoites. Comparing sera from sporozoite-induced and transfusion malaria, Druilhe et al. suggested that liverspecific antigens seen in the course of a natural infection are located at the periphery of EE schizonts (26). Although it remains to be clearly established 173

L. Wlnger et al.

Fig. 1-4. ImmunoHluorescent staining of dirnt-ag liver parasite with anti-Pbil are shown on the eft on the rght of each figure Is a phase picture of th same field. (Scale 10 pm) (1) 3 hours post infection. (2) 24 h parasite. (3) 55 h parasite. (4) Free segmenters and liver-stage merozoites.


WHO Bulletin OMS: Supplement Vol. 66 1990

A liver-stage spcif antigen of P. brgh.I Fig. 5. Average course of parasitmeml when 24 hours aftr receiving sporozofte Inocula.

mice are

passively Immunized with antl-Pbll and control antibodies

I lonn






17.6.1 (anti-blood) anti-Pbll 23.8.1 (anti-CS) 17.9 (anti-ookinete)













Days after sporozoite inoculation

Fig. 6. The effect of different sporozoite Inocula on the course of the subsequent blood-stage infections.


m c0



4000 sporozoites

20,000 sporozoites =i// g- 100,000 sporozoites

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Days after sporozoite inoculation WHO Bulletin OMS: Supplement Vol. 68 1990


L. Winger et al.

whether animals make a response against Pbl 1 during natural infections, it is likely that such a response occurs because the final immunization, used prior to the fusion reported here, consisted of viable sporozoites. The exact location and role of this antigen is unknown; it may, for example, be involved in the transfer of nutrients from the host to the parasite. It is not inconceivable that it may represent host-cell protein made in response to or altered by the parasite. Despite the fact that liver-specific antibody responses are made by the host (18, 26) no demonstration of their protective activity has been reported. In six separate experiments in vivo, passive immunization with monoclonal antibodies administered 24 hours after sporozoite inoculation showed no consistent protective effect. If all the data are taken together, however, there is an indication that the parasitaemia is slightly delayed in animals treated with anti-Pbll or 23.8.1 compared to the control monoclonal antibodies or PBS. Both Pbll and the circumsporozoite antigens are stromal antigens and would only be seen by antibodies at the end of EE development, when the segmenters are released into the blood stream. The antibodies may cause opsinization of EE merozoites clumped together in the stroma. The phagocytic activity of resident Kupffer cells and infiltrating neutrophils and macrophages at the time of parasite rupture is well known (27, 28). These data are not consistent with earlier reports that polyclonal anti-sporozoite immune serum had no effect on EE merozoites (29). However, the length of the pre-patent period is a very insensitive measure of the efficiency of intervention at the liver. Monkeys given 10' or 107 sporozoites did not show significant differences in their prepatent period (30). Comparisons of the parasite growth curve may be a slightly more sensitive assay (see Fig. 6). These curves also illustrate that an 80% reduction in sporozoite inoculuin only results in a half-day delay in parasitaemia growth kinetics. Pbl 1 may also be a target for CD8 + T cells. The amount of CSP, a known target for such cells, decreases during liver-stage development (28). In contrast, PB11 is synthesized in high amounts throughout liver-stage development, although it is as yet unclear whether it is accessible to the host-cell cytoplasmic processing enzymes. Many more liver-stage antigens are under active investigation, and their inclusion in a multistage antimalaria vaccine may eventually be considered.

Acknowledgements We would like to thank S. Slade and Dr J. Langhorne (Max Planck Institut Fur Immunbiologie, Freiberg, Zaehringen, Federal Republic of Germany) for the interferon assays 176

and Dr P. Clay (Department of Chemical Engineering, Imperial College) for his assistance with the irradiation of HepG2 cells. This work was funded by the MRC, Leverhulme Trust, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

References 1. Patarroyo, M.E. et al. A synthetic vaccine protects humans against challenge with asexual blood stages of P. falciparum. Nature, 332: 158-161 (1988). 2. Holder, A.A. The precursor to major merozoite surface antigens; structure and role in immunity. Prog. allergy, 41: 72-97 (1988). 3. Herrlngton, D.A. et al. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature, 328: 257-259 (1987). 4. Nussenzwelg, V. & R.S. Num.enzweIg. Experimental basis for the development of a synthetic vaccine against Plasmodium falciparum malaria sporozoites. In: Porter, R. & Whelan, J., ed. Synthetic peptides as antigens (CIBA Foundation Symposium 119). Chichester, John Wiley, 1985, pp. 150-159. 5. Szarfman, A. et al. Allelic forms of gp195, a major blood stage antigen of Plasmodium falciparum, are expressed in liver stages. J. exp. med., 167: 231-236


6. Suhrbler, A. et al. Expression of the precursor of the major merozoite surface antigens during the hepatic stage of malaria. Am. j. trop. med. hyg., 40: 19-23 (1989). 7. Holllngdabl, M.R. et al. Serological reactivity of in vitro cultured exoerythrocytic stages of P. berghei in indirect immunofluorescent or immunoperoxidase antibody tests. Am. j trop. med. hyg., 32: 24-30 (1983). 8. Suhrbler, A. et al. The fate of the circumsporozoite proteins during the exoerythrocytic development of P. berghei. Europ. j. cell biol., 46: 25-30 (1988). 9. Kumar, S. et al. Cytotoxic T cells specific for the circumsporozoite protein of P. falciparum. Nature, 334: 258-269 (1988). 10. Schofield, L. et al. Gamma interferon, CD8+ T cells and antibodies required for immunity to mnalaria sporozoites. Nature, 330: 664-866 (1987). 11. Weiss, W.R. *t al. CD8+ T cells (cytotoxic/suppressors) are required for protection in mice immunised with malaria sporozoites. Proc. Natl Acad. Sci., USA, 85: 573-576 (1988). 12. Sadoff, J.C. et al. Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria. Science, 240: 336-338 (1988). 13. Good, M.F. et al. The T-cell response to the malaria circumsporozoite protein: an immunological approach to vaccine development. Ann. rev. immunol., 6: 663-688 (1988). 14. Mazier, D. et al. Pre-erythrocytic stages of plasmodia. Role of specific and nonspecific factors. Biology of the cell, 64: 165-172 (1988). 15. Garnham, P.C.C. & Bray, R.S. The influence of immunity upon the stages (including late exoerythrocytic WHO Bulletin OMS: Supplement Vol. 68 1990

A liver-stage specific antigen of P. bergh.l

16. 17. 18. 19.





schizonts) of mammalian malaria parasites. Rev. Bras. Malariol. D. Trop., 8: 152-160 (1956). Mels, J.F.G.M. et al. Cellular responses against exoerythrocytic forms of P. berghei in rats. Am. j. trop. med. hyg., 37: 506-510 (1987). Mazler, D. et al. Effects of antibodies to recombinant and synthetic peptides on development of P. falciparum sporozoites in vitro. Science, 231: 156-159 (1986). Guerln-Marchand, C. et al. A liver stage specific antigen of P. faiciparum characterized by gene cloning. Nature, 329: 164-167 (1987). Holllngdale, M.R. Malaria and the liver. Hepatology, 5: 327-335 (1985). Thomas, W.A. Production of monclonal antibodies selective for aggregation-competent chick neural retina cells; an immunosuppressive approach. J. immunol. meth., 97: 237-243. WInger, L.A. et al. Ookinete antigens of P. berghei. Appearance on the zygote of an Mr21KDa surface determinant identified by transmission blocking antibodies. Parasite immunol., 10: 193-207 (1987). Suhrbler, A. et al. The complete development in vitro of the vertebrate phase of the mammalian malarial parasite P. berghei. Trans. Roy. Soc. Trop. Med. Hyg., 81: 907-909 (1987). Suhrbler, A. et al. Contrasts in antigen expression in the erythrocytic and exoerythrocytic stages of malaria. Parasitology, 99: 165-170 (1989).

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24. Rolk, L.M. et al. A simple, non-chromatographic purification procedure for monoclonals; isolation of monoclonal antibodies against cytochrome P450 isozymes. J. immunol. meth., 100: 123-130 (1987). 25. Slnden, R.E. et al. The development and routine application of high density erythrocytic-stage cultures of Plasmodium berghei. Bull. Wld Hlth Org., 68 (Suppl.): 115-125 (1990). 26. DrulIho, P. et al. Species- and stage-specific antigens in exoerythrocytic stages of P. falciparum. Am. j. trop. med. hyg., 33: 336-341 (1984). 27. Terzakis, J.A. et al. Exoerythrocytic merozoites of Plasmodium berghei in rat hepatic Kupffer cells. J. protozool., 26: 385-389 (1979). 28. Shortt, H.E. & P.C.C. Garnham. The pre-erythrocytic development of Plasmodium cynomolgi and Plasmodium vivax. Trans Roy. Soc. Trop. Med. Hyg., 41: 785-795 (1948). 29. Vanderb.rg, J.P. Inactivity of rodent malaria antisporozoite antibodies against exoerythrocytic forms. Am. j. trop. med. hyg., 22: 573-577 (1973). 30. Schmit, L.H. et al. The characteristics of untreated sporozoite-induced and trophozoite-induced infections. Am. J. trop. med. hyg., 31 (suppl.): 612 (1982). 31. Zavala, F. et al. Immunoradiometric assay to measure the in vitro penetration of sporozoites of malaria parasites into hepatoma cells. J. immunol., 134: 1202-1205 (1985).


A liver-stage specific antigen of P. berghei identified by a monoclonal antibody.

Stage-specific immunity (to the sporozoite, the asexual blood-stages and the sexual stages of malaria) has been well documented and antigens from each...
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