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Previous Article | Next Article 
Infection and Immunity, August 2003, p. 4375-4381, Vol. 71, No. 8
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.8.4375-4381.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Biomedical Primate Research Centre, Department of Parasitology, 2280 GH Rijswijk,1 U-Cytech bv, Utrecht University, 3584 CJ Utrecht The Netherlands,3 Institute of Primate Research, National Museums of Kenya, Karen, Nairobi, Kenya2
Received 14 August 2002/ Returned for modification 14 November 2002/ Accepted 19 May 2003
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ABSTRACT |
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) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasite Plasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites. P. knowlesi parasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN- under the control of the heterologous P. berghei apical membrane antigen 1 promoter, produced bioactive IFN- in a developmentally regulated manner. IFN- expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN- was shown through inhibition of virus cytopathic effect and confirmed by using M. mulatta peripheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) have been shown to modulate immune responses, leading to enhanced protection (15-17, 19, 35, 43, 47). Vaccinia virus and simian immunodeficiency viruses expressing a range of host cytokines were attenuated in vivo, leading to enhanced immune responses (15-17), and Leishmania major expressing host IFN- was significantly attenuated in nude mice (47). These data indicate that in vivo expression of host cytokines by pathogens can manipulate the host-pathogen interaction and generate protective host responses. Thus far, expression of host cytokines by malaria parasites has not been examined. The development of transfection technology for malaria parasites (18, 49, 51, 54) now enables expression of recombinant host proteins, such as cytokines in Plasmodium.
IFN- is one of the central effector cytokines in host response to malaria infection, especially during the liver stage (14, 21, 33, 38-40) and hence is attractive for expression in malaria parasites. In vitro and in vivo studies in rodent models of malaria have demonstrated that IFN- plays a central role in protection against malaria liver-stage infection, possibly by inducing the infected hepatocyte to produce nitric oxide that kills parasites (31, 33). In clinical vaccination studies with an attenuated sporozoite vaccine (reviewed in reference 32), vaccinated humans were protected from subsequent infection through IFN--dependent responses. In separate studies in mice and monkeys, sterile protection was achieved through IFN--dependent responses after exogenous treatment with interleukin-12 (IL-12) (21, 39). Studies using rodent and human malaria models have demonstrated that IFN- also plays a role in protection against malaria blood stages when either endogenously produced (11, 20, 28, 44, 45, 55) or exogenously administered (2, 8, 9, 41).
Although cytokines have been shown to mediate protection against malaria infection after exogenous delivery (8, 21, 39, 41), systemically delivered cytokines are short-lived and require repetitive administration (often in large doses that could be toxic to the host) (6, 9, 22), and only a small portion reaches the site of infection (6, 26). Alternatively, cytokine expression by the pathogen itself will ensure that the cytokine is released where its activity is required, as long as the infection persists and in proportion to the level of infection. In this report, Plasmodium knowlesi, a natural malaria parasite of macaque monkeys (5) and an experimental system for human malaria, was transfected in vitro to express Macaca mulatta IFN-. In vitro expression and bioactivity of P. knowlesi-expressed rhesus monkey IFN- (rhIFN-) was characterized, showing for the first time that malaria parasites can produce a bioactive recombinant host cytokine.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transfection constructs and procedures.
All transfection constructs contained a heterologous selection cassette based on a mutagenized Toxoplasma gondii dihydrofolate reductase/thymidine synthase gene (dhfr/ts) conferring pyrimethamine resistance, flanked by P. berghei dhfr/ts flanking sequences (25). To construct the rhIFN- expression vector, the open reading frame (ORF) of M. mulatta IFN- was isolated by XbaI and SpeI restriction digestion of the M. mulatta IFN- cloning vector (a gift from F. Villinger) (52), Klenow polymerase treatment, and purification with the Qiagen gel extraction kit (Qiagen, Chartsworth, Calif.). The IFN- gene was cloned into the blunted BamHI site of plasmid pDB.DTM.DB/AB.-.DB (25) to generate pDB.DTM.DB/AB.Mm.DB (Fig. 1A). The construct was used for episomal transfection of P. knowlesi (25). The 140-kDa merozoite surface antigen was shown to be nonessential during the blood-stage development of P. knowlesi (23). Therefore, we designed the rhIFN- expression construct for integration into the P. knowlesi 140-kDa locus. The M. mulatta IFN- ORF was isolated as described above, and through a series of cloning steps, plasmid p140K/DB.DTM.DB/AB.Mm.DB/140K was generated (Fig. 1B). Given that integration into the P. knowlesi genome by a double-crossover mechanism requires linear constructs (24), the construct for integration into the 140-kDa locus was linearized by restriction digestion with PvuI and NotI prior to transfection. As controls, parasites were transfected separately with pDB.DTM.DB/AB.-.DB and p140K.DB.DTM.DB/AB.-.DB/140K constructs. Parasite cultivation and in vitro transfection and selection procedures were performed as described elsewhere (24).
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(Fig. 1B). Primers A and B amplify the ORF of rhIFN- from nucleotide positions + 24 to + 478. In order to discount the presence of episomes following integration-dependent transfection, primers C (5'-GTCATAGCTGTTTCCTG-3') and D (5'-GTGTCTATATTACCAACTC-3') were used to amplify the plasmid backbone of the integration construct. Primers E (5'-GAATTCCATTTATGAATATCC-3') and D (Fig. 1B) were subsequently used to confirm integration into the 140-kDa locus, by amplifying the downstream region of the disrupted locus.
In vitro analysis of rhIFN- expression. Transfected and control parasite cultures were expanded in vitro (24), and culture supernatants were harvested and frozen at -80°C. The culture supernatants were analyzed for the presence of rhIFN- by enzyme-linked immunosorbent assay (ELISA, using a macaque IFN- ELISA kit (U-Cytech, Utrecht, The Netherlands) according to the manufacturer's instructions.
To monitor release of rhIFN-, control and rhIFN--expressing P. knowlesi cultures were synchronized by alanine treatment (4). Schizont-stage parasites (5 x 108) were inoculated into 20 ml of culture medium with 0.1 µM pyrimethamine and 5% hematocrit and cultured in vitro (24). Culture supernatants for determining release of IFN- over 6-h time spans during the life cycle of the parasite were harvested by centrifugation of parasite cultures. Subsequently, fresh culture medium was added to the parasites. All harvested supernatants were stored at -80°C until assayed.
In order to determine the in vitro stability of rhIFN-, 500 µl of parasite culture supernatant containing parasite-produced rhesus IFN- was incubated for 30 h with wild-type P. knowlesi-infected erythrocytes or in culture medium only. Parasite-containing cultures had a hematocrit of 2.5% and a starting parasitemia of 3% (90% rings, 4% schizonts, and 6% trophozoites). Aliquots of the culture supernatant were harvested at 6-h intervals and stored at -80°C.
To assay for rhIFN- present in the culture medium, supernatants were thawed on ice, and the rhIFN- concentration was determined by ELISA (U-Cytech).
Antiviral cytopathic effect assay.
The bioactivity of rhIFN- was quantified by its ability to inhibit the cytopathic effect of vesicular stomatitis virus (VSV) in human HEp2 cell lines (48). Human HEp2 cells (48) were plated in duplicate wells at a concentration of 2 x 104 per well and incubated at 37°C for 24 h in 1% fetal calf serum (FCS)-RPMI medium with culture supernatants of rhIFN--expressing parasites. The cells were challenged with an appropriate dilution of VSV (48) and cultured for a further 24 h in 10% FCS-RPMI medium. As a control for IFN- specific activity, parasite culture supernatants containing rhIFN- were incubated with 15 µg of neutralizing antibody per ml (U-Cytech) for 30 min at room temperature prior to incubation with the HEp2 cells. Supernatants from parasite-free culture medium and wild-type parasite cultures were also used as controls. Recombinant human IFN- (U-Cytech) was used as a standard for calibration of antiviral cytopathic effect. One antiviral unit of rhIFN- was defined as the inverse of the dilution that conferred 50% protection to the monolayer.
In vitro whole-blood-cell activation assay.
The bioactivity of rhIFN- was also determined by measuring release of tumor necrosis factor alpha (TNF-
) from whole-blood-cell cultures incubated with parasite culture supernatants. Blood was obtained by venipuncture from a naïve rhesus monkey and used immediately. The blood was washed three times in RPMI 1640, diluted 1:1 in 10% FCS-RPMI medium, and plated at 1 ml per well in 24-well culture plates containing parasite culture supernatants and controls. Whole-blood-cell culture supernatants were harvested 6 h later and stored at -80°C. Frozen culture supernatants were thawed on ice and assayed for TNF- with a monkey TNF- ELISA kit (U-Cytech) according to the manufacturer's instructions. Controls included parasite culture supernatants that were preincubated with 15 µg of IFN- neutralizing antibody per ml (U-Cytech) for 30 min at room temperature prior to the activation assay, culture supernatants from wild-type parasites, and bacterial lipopolysaccharide (LPS).
FACS analysis.
IFN- stimulates monocytes to upregulate CD64 and major histocompatibility complex (MHC) class II DR expression in humans and rodents (3, 27, 46, 53). To determine whether parasite culture supernatants from rhIFN--producing parasites could produce a similar effect on rhesus monkey whole blood cells, rhesus mokey venous blood was obtained, processed, and cultured as described above, except that the cultures were incubated for 48 h. The blood cells were harvested, lysed with fluorescence-activated cell sorter (FACS) lysis solution (BD, Heidelberg, Germany), and stained with antibodies against human CD14, CD64, and MHC class II DR (BD). These antibodies cross-react with rhesus homologues. The number of CD64+ MHC class II DR+ cells within the CD14+ cell population was analyzed by FACS as previously described (34).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production.
P. knowlesi blood-stage parasites were transfected with rhIFN- expression constructs pDB.DTM.DB/AB.Mm.DB and linearized p140K/DB.DTM.DB/AB.Mm.DB/140K to determine the capacity of P. knowlesi to express host IFN- from episomal and integrated genes, respectively. Pyrimethamine-resistant parasites were observed in transfected cultures at 8 days postelectroporation. Plasmid rescue experiments and PCR analysis using primers A and B amplifying the IFN- ORF showed that plasmid pDB.DTM.DB/AB.Mm.DB was intact and therefore stable in P. knowlesi and that the gene was also present in parasites transfected with the integration construct. Integration into the 140-kDa locus was confirmed by PCR using primers D and E (Fig. 1C), and analysis for circular plasmids following integration-dependent transfection using primers C and D was negative (Fig. 1D). This showed that gene targeting into the 140-kDa locus in P. knowlesi by double-crossover mechanisms was feasible.
To determine rhIFN- production in transfected P. knowlesi, parasite cultures were expanded, and their supernatants were harvested and analyzed for the presence of rhIFN- by ELISA with a macaque IFN- ELISA kit. rhIFN- was detected in culture supernatants from parasites transfected with plasmid pDB.DTM.DB/AB.Mm.DB, and the integration construct but not from control cultures (Table 1). The results further suggested that both episomal and chromosomal expression of IFN- produced similar amounts (Table 1) and that empty vector-transfected parasites are equivalent to wild-type parasites. In all subsequent experiments, episomally transfected parasites were used.
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production was evaluated in order to define the kinetics of rhIFN- release into culture medium. Cultures of rhIFN--producing parasites were initiated at the schizont stage; supernatants were harvested at 6-h intervals and assayed for rhIFN- by ELISA. Culture medium was completely replaced at each harvest time point. rhIFN- was detected in the culture supernatants only after the rupture of schizonts (Fig. 2A).
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protects the protein from rapid degradation by proteases (36). However, malaria parasites have severely reduced capacity for N glycosylation of proteins (12), prompting us to determine the in vitro stability of P. knowlesi-produced IFN-. It would be expected that highly unstable rhIFN- would be detected only immediately following schizont rupture. Evaluation of the stability of rhIFN- showed that, in the presence or absence of infected erythrocytes, the concentration of rhIFN- in the supernatant did not change significantly over a 30-h period (Fig. 2B).
P. knowlesi-produced rhIFN- is bioactive.
The standard procedure for determining bioactivity of IFN- is to test the antiviral cytopathic effect (16, 29, 37, 48). To determine bioactivity of rhIFN- in P. knowlesi culture supernatants using an antiviral cytopathic effect assay, HEp2 cells (48) were incubated overnight with parasite culture supernatants. The cells were subsequently challenged with VSV and observed 24 h later for cytopathic effect. The HEp2 cells were protected from VSV cytopathic effect by culture supernatants from parasites transfected with the rhIFN- expression plasmid, but not by control supernatants (Fig. 3A). Protection from cytopathic effect was abrogated by preincubation with a neutralizing anti-IFN- antibody (48), demonstrating that the protection was mediated by rhIFN-. The concentration of rhIFN- in parasite culture supernatants was calculated at 125 antiviral units per 5 x 108 schizonts.
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activates blood cells to release TNF- (3). To determine whether culture supernatants from rhIFN--producing P. knowlesi activate rhesus whole blood cells to release TNF-, blood was obtained from a rhesus monkey and incubated with culture supernatants from rhIFN--producing parasites. Whole-blood-cell culture supernatants were harvested and assayed for TNF- release. Significant TNF- was released by whole blood cells incubated with culture supernatants from rhIFN--producing parasites (Table 2). The release was blocked when culture supernatants from rhIFN--producing parasites were mixed with neutralizing antibody against human IFN- prior to incubation with whole blood cells (Table 2), confirming that activation of rhesus whole blood cells to release TNF- was mediated via rhIFN-. TNF- release from whole blood cells incubated with culture supernatants from control cultures was below detection levels (Table 2).
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activates monocytes and macrophages to upregulate CD64 and MHC class II DR antigens (3, 27, 46, 53). To determine the effect of culture supernatants from IFN--producing P. knowlesi on the expression of CD64 and MHC class DR by monocytes from naïve rhesus monkeys, the whole blood was incubated with parasite culture supernatants, harvested, stained, and analyzed by FACS for expression of CD14, CD64, and MHC class II DR antigens. Incubation of rhesus monkey blood cells with culture supernatant from rhIFN--expressing P. knowlesi resulted in an increased number of CD14+ cells expressing CD64 and MHC class II DR (Fig. 3B). Neutralization of IFN- inhibited this effect. Supernatants from control parasites and cell culture medium had no effect on the number of CD14+ cells expressing CD64 and MHC class II DR.
P. knowlesi-produced rhIFN- had no marked effect on in vitro parasite multiplication.
The in vitro growth rate of rhIFN--producing parasites was comparable to that in wild-type and control cultures (data not shown), indicating that the production of rhIFN- had no marked effect on parasite production.
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DISCUSSION |
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gene under control of the heterologous P. berghei apical membrane antigen 1 promoter, express bioactive IFN-. This is the first example of a host molecule being produced by a malaria parasite. It demonstrates that the malaria parasite transcription and translation machinery can effectively express a bioactive host cytokine.
Secreted malaria parasite proteins have to be transported across three bilayer membranes before getting out of the infected erythrocyte (42). In order to determine the release characteristics of rhIFN-, time course experiments were done in vitro. The data shows that rhIFN- is released into culture medium after schizont rupture. Since the heterologous pbama-1 promoter restricts expression to late-stage schizonts in P. knowlesi (Ozwara et al., unpublished data), it is not possible to determine from these experiments whether rhIFN- is actively secreted into the culture medium or whether it is released from parasitized cells during schizont rupture. However, rhIFN- minimally has to be secreted across one set of membranes to reach the parasitophorous vacuole, suggesting that the parasite effectively recognizes higher eukaryotic secretion signals. Developmentally regulated promoters that restrict expression to the ring and trophozoite stages would be required to analyze the secretion process in more detail (10). It is noteworthy that the rhIFN- produced by the parasites has no marked effect on parasite growth in vitro. This indicates that the cytokine itself, either present in the culture supernatant or accumulated in the infected cell, has no direct effect on parasite viability.
Arakawa et al. (1) showed that bioactivity of IFN- depends on protease processing of the carboxyl end of the protein. Furthermore, stable and bioactive IFN- exists as a noncovalent homodimer (13). The fact that bioactive rhIFN- is produced in P. knowlesi suggests that the molecule is properly processed and present in the homodimeric form. Interestingly, at in vitro culture concentrations, rhIFN- was stable for at least 30 h. This duration is likely to be sufficient for in vivo activity. For example, despite clearance from rhesus monkey circulation within 6 h of exogenous administration (6), IFN- was protective in Plasmodium cynomolgi-infected rhesus monkeys (28), suggesting that stability of IFN- for 6 h could be sufficient for mediating in vivo bioactivity in the monkeys.
We expressed IFN- because it is a key effector cytokine in protection against malaria, especially during the liver stages (21, 32, 38-40). Studies in humans and animal models looking at endogenously produced and exogenously administered IFN- have shown that the cytokine is also required for protection against blood-stage infection (8, 20, 28, 41, 45, 55). In order to determine the immunomodulatory effect of rhIFN--expressing parasites during the malaria liver-stage infection, the host must be inoculated with sporozoites. Integration into the genome of the cytokine expression construct is required to express rhIFN- in the liver stages, since episomally transfected parasites undergo random segregation of plasmids (50), and in the absence of drug pressure during development in the mosquito, the plasmids could be lost. Therefore, we have generated parasites expressing rhIFN- after integration-dependent transfection. The levels of rhIFN- produced were similar to those of episomally transfected parasites.
It is now feasible to generate malaria parasites expressing bioactive host cytokines, to characterize the capacity of the parasites to immunomodulate the infection, and to characterize the role of the expressed cytokine in host responses to malaria. It is important that the safety of cytokine-expressing parasites in vivo be determined prior to such experiments. A preliminary safety study with a small number of rhesus monkeys showed that the levels of rhIFN- released by transfected malaria parasites were well tolerated by rhesus monkeys (data not shown). During this safety study, aspects of the immune responses of animals exposed to the rhIFN- parasites were compared with those of animals that had received parasites transfected with a control vector. All four animals exposed to rhIFN- parasites had a marked expansion (2.5- to 5-fold) of the 
T-cell compartment within 2 weeks of the start of infection compared with five controls (1- to 2-fold expansion). A more comprehensive analysis of host responses to rhIFN--expressing P. knowlesi in M. mulatta is ongoing. However, this study already clearly demonstrates that the rhIFN- produced by the parasites is bioactive in vivo. In conclusion, the expression of host cytokines by malaria parasites, as demonstrated in our study, offers a new approach to explore the development of attenuated and immunopotentiated malaria vaccines.
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ACKNOWLEDGMENTS |
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cloning vector and John Barnwell for sharing unpublished P. knowlesi 140-kDa sequence data. This work was supported by a grant from the European Commission (contract CT-99-10004). H.O. was funded by The Netherlands Foundation for the advancement of Tropical Research (WOTRO).
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FOOTNOTES |
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), trophozoite (
), and schizont (
) stages at the time of harvest. (B) In vitro stability of P. knowlesi-produced rhIFN-
) but not from wild-type parasites (
) and parasite culture medium (
). The antiviral activity was abrogated when culture supernatants from rhIFN-