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Infection and Immunity, April 2006, p. 2043-2051, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2043-2051.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Infection with Plasmodium berghei Boosts Antibody Responses Primed by a DNA Vaccine Encoding Gametocyte Antigen Pbs48/45

Diana Haddad, Jorge Maciel, and Nirbhay Kumar*

Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205

Received 23 September 2005/ Returned for modification 1 November 2005/ Accepted 5 January 2006


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ABSTRACT
 
An important consideration in the development of a malaria vaccine for individuals living in areas of endemicity is whether vaccine-elicited immune responses can be boosted by natural infection. To investigate this question, we used Plasmodium berghei ANKA blood-stage parasites for the infection of mice that were previously immunized with a DNA vaccine encoding the P. berghei sexual-stage antigen Pbs48/45. Intramuscular immunization in mice with one or two doses of DNA-Pbs48/45 or of empty DNA vaccine as control did not elicit detectable anti-Pbs48/45 antibodies as determined by enzyme-linked immunosorbent assay. An infection with P. berghei ANKA 6 weeks after DNA vaccination elicited comparable anti-Pbs48/45 antibody levels in mice which had been primed with DNA-Pbs48/45 or with empty DNA vaccine. However, a repeat infection with P. berghei ANKA resulted in significantly higher anti-Pbs48/45 antibody levels in mice which had been primed with the DNA-Pbs48/45 vaccine than the levels in the mock DNA-vaccinated mice. In parallel and as an additional control to distinguish the boosting of Pbs48/45 antibodies exclusively by gametocytes during infection, a separate group of mice primed with DNA-Pbs48/45 received an infection with P. berghei ANKA clone 2.33, which was previously described as a "nongametocyte producer." To our surprise, this parasite clone too elicited antibody levels comparable to those induced by the P. berghei gametocyte producer clone. We further demonstrate that the nongametocyte producer P. berghei clone is in fact a defective gametocyte producer that expresses Pbs48/45, much like the gametocyte producer clone, and is therefore capable of boosting antibody levels to Pbs48/45. Taken together, these results indicate that vaccine-primed antibodies can be boosted during repeat infections and warrant further investigation with additional malaria antigens.


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INTRODUCTION
 
Malaria continues to be a devastating disease, affecting hundreds of millions of people living in areas where it is endemic in the developing world (41). Over 2 billion people are presently exposed to the threat of malaria, resulting in 1 to 2 million deaths annually (15). Clearly, more and/or better means of control are needed to eventually eradicate this disease. The design of a vaccine that is effective in all affected regions and affordable to even the poorest countries remains a priority. The malaria parasite, Plasmodium sp., has a multistage life cycle, and for a vaccine to be effective in controlling the disease and ultimately in conferring protection against infection, it should ideally target more than one stage of this parasite's complex life cycle (31).

In trying to interrupt the life cycle of the parasite, a promising approach is the blockage of transmission between vertebrate and invertebrate hosts. Transmission occurs via the mature sexual forms of Plasmodium species (the gametocytes), and a vaccine targeting these and the subsequent stages, particularly the gamete and/or zygote stages, could curtail transmission by interfering with sexual development or fertilization (5). Immunity against sexual stages is believed to be mostly mediated by antibodies recognizing the surface antigens in these parasite stages (7, 19).

An essential yet unresolved issue in the development of a malaria vaccine is whether the protective antibodies that are elicited by vaccination can be boosted through natural infection. This is an important logistical consideration for vaccination programs, particularly in areas that are difficult to access and to monitor. Moreover, natural boosting is of utmost importance for the maintenance of effective transmission-blocking immunity, which depends on the continuous presence of high levels of antibodies (7, 19).

Indeed, few studies exist where the boosting of antimalaria immune responses in vaccinated individuals is demonstrated to occur through infection (3, 40). It is, however, well established that humans living in areas of malaria endemicity develop clinical immunity against malaria under the conditions of premunition and that this immunity is antibody mediated, antigen specific, and long lived (10, 25).

In this study, we investigated whether repeated infections with a rodent malaria parasite, Plasmodium berghei, could boost antibody responses to the sexual stage antigen Pbs48/45 (38) in mice primed with a DNA vaccine encoding the antigen. Antigen Pbs48/45 is a well-conserved orthologue of antigen Pfs48/45 from Plasmodium falciparum (37). Moreover, both antigens, Pfs48/45 (6, 21, 22, 32, 39) and Pbs48/45 (38), are present on the surfaces of gametocytes and gametes of the corresponding species and have been shown to be targets of transmission-blocking antibodies.


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MATERIALS AND METHODS
 
Cloning of Pbs48/45 gene from Plasmodium berghei into a DNA vaccine plasmid using Gateway technology. Genomic DNA from Plasmodium berghei ANKA strain, clone 2.34, was used for amplification of the Pbs48/45 gene (38). Primers were designed so that a fragment of the Pbs48/45 gene lacking its signal sequence (encoding amino acids 52 to 449) was amplified. A 5' start and a 3' stop codon were added to the forward (5'-AATGAGTATGTTTCTCCAGATGAA-3') and reverse (5'-CATAAAACCAGTTATTTTATCCAT-3') primer sequences, respectively. Additionally, the forward and reverse primers were preceded by the Gateway recombination sequences (5'-GCAGGCTCCACC-3' and 5'-AGAAAGCTGGGT-3', respectively). Thus, the entire forward and reverse primer sequences used here in the amplification of the Pbs48/45 gene were 5'-GCAGGCTCCACCATGAATGAGTATGTTTCTCCAGATGAA-3' and 5'-AGAAAGCTGGGTTTACATAAAACCAGTTATTTTATCCAT-3', respectively. PCR amplification and Gateway cloning was carried out using conditions described previously (14). A fragment of approximately 1.2 kb was obtained by PCR amplification for 35 cycles consisting of 94°C for 30 s, 52°C for 60 s, and 68°C for 2 min. The product of this first PCR amplification was subjected to a second PCR with Gateway forward primer attB1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACC-3') and reverse primer attB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3') to extend each recombination tag, as indicated by the manufacturer (Invitrogen Inc., Carlsbad, CA). Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) was used in the first and second PCR amplification.

Cloning of the Pbs48/45 fragment into the DNA vaccine vector DV1020 (1) was carried out by means of an intermediary Gateway entry vector (pDONR207) as described before (14). Entry and DV1020 clones were analyzed by PCR and sequencing using vector-specific primers (14). Plasmid DNA from a confirmed DV1020-Pbs48/45 clone was prepared and purified using an endotoxin-free plasmid purification Giga kit (QIAGEN Inc., Valencia, CA).

Parasite clones. P. berghei ANKA clone 2.34, a normal gametocyte producer clone (11, 28), and clone 2.33, described as a nongametocyte producer clone (11, 12, 28) from the same strain, were used for the infection of mice and for immunofluorescence studies.

Immunization and parasite infection. All animal experiments were conducted in accordance with the guidelines indicated in the National Institutes of Health Guide to Laboratory Animal Care and were approved by the Johns Hopkins University Animal Care and Use Committee. Six-to-8-week-old female BALB/c mice were used. Four groups of five mice were immunized as indicated in Fig. 1. Plasmid DNA was administered intramuscularly (i.m.) by using a total of 100 µg of DNA in 100 µl of phosphate-buffered saline (PBS). Half of this dose was injected into each gastrocnemius muscle with a 29-gauge needle. Infection with parasites was by the intraperitoneal (i.p.) route using approximately 2.5 x104 parasites per mouse. Serum was collected by tail bleeding at the time points indicated in Fig. 1.


Figure 1
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  		FIG. 1. Schematic outline showing the immunization and infection schedule for each group of mice (n = 5). Groups 1, 2, and 3 were injected i.m. with 100 µg of DNA vaccine encoding antigen Pbs48/45, as described in Materials and Methods, while group 4 received 100 µg of the empty DNA vaccine vector. Six weeks after DNA injection, group 1 received a second i.m. injection with the DNA-Pbs48/45 vaccine (100 µg), while groups 2 and 4 were infected i.p. with approximately 2.5 x 104 P. berghei clone 2.34 parasites. In parallel, group 3 was infected i.p. with a similar number of P. berghei clone 2.33 parasites. Six weeks after the first infection (groups 2, 3, 4) or second DNA immunization (group 1), all four groups of mice were infected a second time with 2.5 x 104 P. berghei clone 2.34. Time points for DNA injections or parasite infections are indicated by arrows. All mice were bled at the indicated time points. Thus, the first bleed (B1) was 5 weeks after the first DNA injection, B2 was 18 days after the first parasite infection (groups 2, 3, and 4) or the second DNA injection (group 1), B3 was just prior to the second parasite infection (i.e., 5 weeks after the first parasite infection for groups 2, 3, and 4 or the second DNA injection for mice in group 1), and B4 was 2 weeks after the second parasite infection.

Parasitemia monitoring. Parasitemias in three to five mice from each infected group were monitored daily by conventional Giemsa staining starting on day 4 after infection. In the first days of the infection, all five mice from each group were bled, but as the parasitemias increased and the mice became anemic, three mice from each group were randomly selected each time for bleeding and monitoring. Thin blood films were prepared by tail bleeding, air dried, and methanol fixed before staining. When the parasitemias reached approximately 5% (day 6 after infection), all mice were treated by using a subcurative dose consisting of 15 mg of sulfadiazine per liter of drinking water for 17 days. To clear remaining asexual parasites in mice which were not completely cured with the subcurative treatment, a curative dose consisting of an intraperitoneal injection of chloroquine (300 µg/mouse), combined with the sulfadiazine treatment given in water, was administered daily for 4 days, starting on day 8 after sulfadiazine treatment.

ELISA and Western blotting. Recombinant Pbs48/45 protein (38) used in enzyme-linked immunosorbent assay (ELISA) and Western blot assays was a kind gift from Melissa R. van Dijk. Serum antibody reactivity to recombinant Pbs48/45 was conducted by conventional ELISA essentially as described earlier (23). Briefly, 96-well Immulon-2 plates were coated with 2 µg/ml of recombinant Pb48/45 in bicarbonate buffer (4 mM Na2CO3, 8 mM NaHCO3, pH 9.6) and incubated overnight at 4°C. Wells were blocked with 200 µl of 5% nonfat milk in diluent (0.01% Tween 20 in PBS, for 1 h at 37°C). After a brief rinse with washing buffer (0.05% Tween 20 in PBS), 100 µl of serum dilutions in 1% milk in diluent were added to duplicate wells and incubated for 2 h at room temperature. Plates were washed 5 times and incubated for 1 h at room temperature with 100 µl of a goat anti-mouse immunoglobulin G-horseradish peroxidase conjugate (Kierkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:2,500 in 1% milk. Plates were developed with the 2,2'-azinobis-(3-ethylbenzthiazolinesulfonic acid) (ABTS) single-reagent substrate (Kierkegaard & Perry Laboratories), and absorbance was read at 405 nm. Normal mouse serum tested at a dilution of 1:50 was used as negative control, and the mean optical density (OD) value multiplied by 2 was used as cutoff.

Mouse sera were also probed against recombinant Pbs48/45 by Western blot analysis. Electrophoresis was performed by running 5 µg of recombinant Pbs48/45 on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigel under reducing condition. Blotted nitrocellulose membranes were cut into strips and probed with pooled mouse sera obtained after the fourth bleed (Fig. 1) or with rabbit anti-Pbs48/45. Strips were developed using chemiluminescent reagents (Amersham, Inc., Piscataway, NJ) following the manufacturer's instructions.

RT-PCR. Mice were infected with P. berghei ANKA clone 2.34 or clone 2.33, and gametocyte-enriched blood was used for the extraction of RNA by using TRIzol (Invitrogen, Inc.). Total RNA was treated with RNase-free DNase (Ambion, Carlsbad, CA) for 1 h and subjected to a reverse transcription (RT) reaction by using Omniscript reverse transcriptase (QIAGEN, Inc.). RNA from each clone without reverse transcription was included in each PCR amplification reaction as a negative control. Following reverse transcription, PCR amplification of the Pbs48/45 transcript from each clone was performed using Taq polymerase and the forward and reverse primer sequences designed for amplification of the Pbs48/45 gene but lacking the Gateway recombination tag (5'-ATGAATGAGTATGTTTCTCCAGATGAA-3' and 5'-TTACATAAAACCAGTTATTTTATCCAT-3', respectively). Genomic DNA from P. berghei ANKA clone 2.34 was used as a positive control.

Immunofluorescence. A polyclonal rabbit antiserum generated by injection with recombinant Pbs48/45 protein (38) (kind gift from Melissa van Dijk) was used for the detection of Pbs48/45 in ANKA clones 2.34 and 2.33. Briefly, parasites were obtained from infected Swiss mice following the method described by Beetsma et al. (4). Immunofluorescence assay (IFA) slides were prepared when gametocytemias and asexual parasitemias were approximately 3 and 8%, respectively. Parasites were fixed by air drying and stored desiccated at –70°C until needed. Slides were thawed at room temperature for 1 to 2 h in a desiccator and fixed with 100% high-pressure liquid chromatography-grade cold methanol (Fisher Scientific, Fairlawn, NJ) for 20 min at –20°C. After rehydrating with PBS, the slides were blocked with 5% milk in PBS for 30 min at 37°C. Incubations with serum and secondary antibodies were performed for 1 h at room temperature. DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) (Molecular Probes, Eugene, Oregon) stain was included at 1 µg/ml in the last two washing steps to stain nuclei. Slides were quickly dried and mounted with the Gel Mount aqueous mounting medium (Sigma, St. Louis, MO) before being viewed under UV light.

Amaxa transfection of mammalian cells. To assess whether the DV1020-Pbs48/45 DNA plasmid expressed the Pbs48/45 protein and whether the mouse antiserum that was generated after DNA immunization or after parasite infection recognized the DNA vaccine-encoded Pbs48/45 protein, mammalian HEK293 cells were transfected using the Amaxa V kit (Amaxa, Inc., Gaithersburg, MD) following the manufacturer's specifications. Briefly, cells were grown to 80 to 90% confluence before transfection and harvested by trypsinization. Approximately 1 x 106 cells were transfected either with DV1020-Pbs48/45 or with empty DV1020 plasmid. Approximately, 20,000 to 30,000 transfected cells were added to each of eight wells in a microchamber culture slide (Labtek, Campbell, CA) and cultured at 37°C with 5% CO2 for 24 and 48 h. Slides were gently washed with PBS, air dried briefly, and fixed with cold (100%) methanol as described above. Slides were processed for IFA immediately after fixation to assess the expression of Pbs48/45. Protein expression was first assessed at different time points using the rabbit anti-Pbs48/45 polyclonal antiserum at 1:200. Reactivity of mouse antisera to Pbs48/45 transfectants was assessed at different dilutions by IFA as described above.

Statistical analysis. Statistical differences between geometric means of the ELISA absorbance values of each immunization group were analyzed using an unpaired, two-tailed Student's t test.


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RESULTS
 
Boosting of anti-Pbs48/45 antibody levels by parasite infection in mice primed with a DNA vaccine encoding Pbs48/45. To investigate whether an infection with malaria blood-stage parasites is capable of specifically boosting antibodies to a parasite protein, we primed mice by immunizing them with a DNA vaccine encoding antigen Pbs48/45 and subsequently infected them with live Plasmodium berghei ANKA parasites as outlined in Fig. 1. Mice in groups 1, 2, and 3 received an intramuscular injection with DNA vaccine plasmid encoding Pbs48/45, while control group 4 received an injection with the empty DNA vaccine DV1020 plasmid. Six weeks after DNA injection, mice in groups 2 and 4 were infected intraperitoneally with P. berghei ANKA clone 2.34 parasites, followed by a second similar infection after a 6-week interval. In parallel, mice in group 3 received the same dose of live P. berghei ANKA clone 2.33 parasites, followed by subsequent infection with P. berghei clone 2.34 parasites. Group 1 received a second DNA-Pbs48/45 injection, followed by an infection with P. berghei clone 2.34 6 weeks after the second DNA injection (Fig. 1). Serum was obtained at the time points indicated in Fig. 1.

Figure 2 shows the antibody reactivity to recombinant Pbs48/45 protein displayed by the different groups of mice at each time point. After one dose of either DNA-Pbs48/45 vaccine (groups 1, 2, and 3) or of empty DNA vector (group 4), the anti-Pbs48/45 reactivity was undetectable by ELISA in serum from all groups of mice at even a 1:50 dilution (Fig. 2). A second similar dose of DNA-Pbs48/45 given to mice in group 1 did not appear to increase the anti-Pbs48/45 antibody levels. Thus, Pbs48/45 DNA vaccination (two doses) on its own does not seem to induce detectable anti-Pbs48/45 antibodies.


Figure 2
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  		FIG. 2. Antibody reactivity to recombinant Pbs48/45 protein of antiserum from each immunization group taken at the time points indicated in Fig. 1 and measured by ELISA. Bars 1 though 4 correspond to bleedings 1 through 4 in Fig. 1. Values indicated for each time point correspond to the geometric mean of the absorbance values (405 nm) from five individual mouse antisera within each group (tested at a 1:50 dilution). The cutoff OD value of 0.22 was obtained by multiplying the average of five different OD values that were obtained from a pool of normal serum (1:50) by two and is indicated by the segmented horizontal line. Error bars indicate standard deviations.

To evaluate the boosting of DNA-primed immune responses during infection, mice in groups 2 and 4 were infected with the P. berghei clone 2.34 6 weeks after priming with a single dose of DNA-Pbs48/45 or with empty DNA vector, respectively. Eighteen days after this first infection, the anti-Pbs48/45 antibody levels in mice from groups 2 and 4 were detectable at dilutions of up to 1:100 (not shown) and their geometric mean OD values did not differ statistically (P > 0.1).

Six weeks after the first parasite infection, the anti-Pbs48/45 antibody levels in groups 2 and 4 had decreased to almost background values; results for group 2 presented geometric mean OD values that were slightly higher than the cutoff value (0.22), while results for control group 4 had OD values just below this value. A second infection with P. berghei ANKA clone 2.34 parasites given 6 weeks after the first infection evoked a significant increase in anti-Pb48/45 antibody levels in both groups of mice. However, the antibody levels in group 2 (primed with DNA vaccine) were statistically higher than those in group 4 (immunized with control plasmid) at all serum dilutions tested (P < 0.05) (Fig. 2 and 3A). The results thus far presented in Fig. 2 suggest that a single parasite infection can induce some anti-Pbs48/45 antibody response independently of any immune priming. It is only after a second parasite infection that the boosting of antibody levels is significantly higher in the DNA-Pbs48/45-primed mice (Fig. 2).


Figure 3
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  		FIG. 3. Antibody reactivity to recombinant Pbs48/45 in pooled sera from each immunization group obtained 2 weeks after the second parasite infection (bleeding 4). (A) Antibody titration by ELISA. Shown are the mean absorbance values (405 nm) corresponding to reciprocal of serum dilution. (B) Western blot reactivity to reduced recombinant Pbs48/45. Lanes are as indicated. Pooled antisera from each immunization group (I to IV) and normal mouse serum (N) were tested at 1:50, while rabbit anti-Pbs48/45 (R) was tested at 1:2,500. Recombinant Pb48/45 protein was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, where it runs at about 45 kDa, as indicated by the arrow. Molecular masses in kilodaltons are indicated at the right.

The repeat infection appeared to boost effectively in spite of the fact that the overall asexual and sexual parasitemia values in all reinfected mice, regardless of whether they were primed with DNA-Pbs48/45 vaccine, were lower than those for the parasitemias displayed after only one infection. Thus, during the first infection, which lasted 14 days, asexual parasitemia values ranged from 0.03 to 27%, while gametocytemias were between 0.01 and 1.5%. After a second infection, the asexual parasitemias and gametocytemias did not go beyond 9 and 0.2%, respectively.

Recognition of Pbs48/45 expressed in mammalian cells by antisera from immunized and boosted mice. Since we did not observe detectable antibody responses against recombinant Pbs48/45 by ELISA after one or two DNA injections in any of the immunization groups, we wondered whether Pbs48/45 is encoded by the DNA vaccine plasmid. Protein expression was thus investigated by in vitro transfection of mammalian cells followed by IFA analysis using the control rabbit anti-Pbs48/45 antibody. The intense immunofluorescence signal, observed with this control antiserum in approximately 90% of all cells in a monolayer, indicated strong expression of Pbs48/45 by the DNA plasmid in mammalian cells (Fig. 4A). We next sought to determine recognition of Pbs48/45 expressed in eukaryotic cells by antisera from the immunized mice. We tested antisera from mice in groups 1 and 2 at different dilutions, and results from the best-reacting dilution, 1:25, are shown. As expected, antisera from mice in group 2 obtained after one and two parasite infections were both reactive, with a proportion of transfected cells (80 to 90%) similar to that of the control antibody (used at 1:200), albeit with a weaker signal (Fig. 4B and C, respectively). However, antisera from the same group obtained after one DNA injection did not show appreciable reactivity with the transfected cells (not shown). Similarly, antisera from mice in group 1 were reactive with the transfectants only after a parasite infection (not shown) but not after one or two DNA injections (Fig. 4D). Normal mouse serum did not react with the DNA-Pbs48/45-transfected cells (Fig. 4E), nor did any of the immune antisera tested against cells transfected with empty plasmid DV1020 (not shown). These results indicate that DNA plasmids encoding Pbs48/45 express Pbs48/45 in eukaryotic cells and suggest that its expression in mice after one or two DNA injections by the immunization method used here may be suboptimal to trigger a detectable antibody response. Nonetheless, as suggested by the results for group 2 (Fig. 2), the amount of Pbs48/45 produced after DNA vaccination was enough to prime an antibody response that could be further boosted by repeated parasite infections.


Figure 4
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  		FIG. 4. Expression of Pbs48/45 protein in HEK293 mammalian cells by the DNA-Pbs48/45 vaccine plasmid and recognition of the expressed protein by various antisera. Cells were transfected with DNA-Pbs48/45 using the Amaxa method as indicated in Materials and Methods. Transfected cells were probed by IFA with the following antisera: polyclonal rabbit anti-Pbs48/45 antiserum (A) and pooled antisera from immunization group 2 obtained after the first (bleeding 2) (B) and second (bleeding 4) (C) parasite infection; pooled antisera obtained after two DNA-Pb48/45 injections (group 1, bleeding 2) (D); and normal mouse serum (E). Rabbit and mice sera were tested at 1:200 and 1:25 dilutions, respectively.

An effective antibody response against Pbs48/45 is expected to suppress the infectivity of gametocytes in the mosquitoes (6, 32). To test this, mosquitoes were fed on mice from all groups 5 days after the first infection, when P. berghei gametocytes are most infective (33). We did not detect any differences in infectivity (number of oocysts) between the groups. The mosquito infection was performed before knowing the postinfection antibody levels, and we believe the antibody titers generated after one infection were possibly insufficient for infectivity reduction, as high-titer antibodies appear to be a requirement for effective transmission blocking (8, 23). Likewise, for the same reason, we did not pursue a transmission test after the second parasite infection.

Induction of anti-Pbs48/45 antibody responses by P. berghei ANKA clone 2.33. Our experimental immunization design included a group of mice (group 3) that were primed with DNA-Pbs48/45 vaccine, followed by infection with P. berghei ANKA clone 2.33, which is described in the literature as a nongametocyte producer clone (11, 12, 28). Our original intention was to include this parasite clone in the infection experiment as a negative control to define the boosting of Pbs48/45 antibodies as specifically induced by gametocytes expressing Pbs48/45 (P. berghei clone 2.34). To our surprise, the anti-Pbs48/45 antibody levels in the DNA-Pbs48/45-primed mice were, after one infection with P. berghei 2.33 (group 3), comparable to those induced by an infection with P. berghei 2.34, regardless of whether a DNA-Pbs48/45 prime had been given or not (groups 2 and 4, respectively) (Fig. 2). Indeed, both the individual antibody titers exhibited by mice in each of these groups (groups 2, 3, and 4) as well as their geometric mean OD values were similar after one infection with either P. berghei clone 2.34 or 2.33 (P > 0.2). A second infection with P. berghei 2.34 induced a further increase in anti-Pbs48/45 levels in group 3 that was comparable to the increase exhibited by group 4 after a second infection with the same parasite clone but lower than what was induced in group 2 by a second similar infection (P = 0.1) (Fig. 2 and 3A). This difference in antibody levels between groups 2 and 3 was, however, not statistically significant. Only antisera from groups 2 and 3 that were generated after this second infection recognized the reduced recombinant Pbs48/45 protein in Western blot analysis (Fig. 3B), suggesting that the DNA-Pbs48/45-primed antibody responses can be further boosted by repeated infection. These results prompted us to investigate whether P. berghei clone 2.33 actually does produce gametocytes and, if so, whether these gametocytes express the Pbs48/45 protein to account for the antibody boosting.

P. berghei ANKA clone 2.33 produces defective gametocytes and expresses Pbs48/45. A careful analysis of Giemsa-stained blood smears prepared from mice that were infected with P. berghei clone 2.33 revealed parasite forms that distantly resembled P. berghei gametocytes (Fig. 5A). These forms were not perfectly rounded, presented multiple internal vacuoles, and lacked the dotted appearance which is characteristic of a P. berghei gametocyte in Giemsa-stained smears (Fig. 5B). In order to investigate whether these abnormal forms express Pbs48/45, we next examined the presence of Pbs48/45 mRNA transcripts by RT-PCR (Fig. 5C). As a positive control, we used genomic DNA or cDNA obtained from P. berghei clone 2.34. Both clone 2.34 (Fig. 5C) and clone 2.33 revealed a doublet band of approximately 1.2 kb in size corresponding to Pbs48/45. No band was present after PCR amplification of clones 2.34 or 2.33, where reverse transcriptase had been omitted during the reverse transcription step.


Figure 5
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  		FIG. 5. Identification of abnormal gametocytes expressing Pbs48/45 in P. berghei ANKA clone 2.33. Several tests were performed to investigate whether this parasite strain, previously believed to be a nongametocyte producer, is in fact an abnormal or defective gametocyte producer strain. (A) Giemsa-stained blood smears from mice infected with P. berghei ANKA clone 2.34 or clone 2.33. Gametocytes are indicated by arrows. Note the morphological differences. Images are photographed at x1,000 magnification. (B) RT-PCR amplification of Pbs48/45 using RNA from P. berghei clone 2.34 or clone 2.33 gametocytes. Presence of the Pbs48/45 RNA transcript was detected in lanes marked +RT from each P. berghei clone, but not in the corresponding negative control lanes marked –RT, as indicated. Genomic DNA from P. berghei clone 2.34 was included as a positive control for the PCR amplification. (C) Expression of Pbs48/45 by P. berghei clone 2.33 gametocytes. Detection of Pbs48/45 expression in blood smears from mice infected with P. berghei clone 2.34 as control (first row), or P. berghei clone 2.33 (second row) was by IFA using polyclonal rabbit anti-Pbs48/45 antiserum at a 1:50 dilution, followed by a goat anti-rabbit immunoglobulin G-Alexa Fluor 488 (Molecular Probes). Normal rabbit serum used at a 1:50 dilution did not recognize P. berghei clone 2.33 gametoyctes (third row). For visual clarity, a red filter was used to change the green fluorescent signal given by Alexa Fluor 488 to red. Images are photographed at x400 magnification.

Expression of the Pbs48/45 protein by P. berghei clone 2.33 parasites was next confirmed by IFA using the rabbit anti-Pbs48/45 antibody described in Materials and Methods. As shown in Fig. 5D, this antibody effectively stained the P. berghei clone 2.33 parasites in a manner similar to that of the P. berghei clone 2.34 gametocytes but did not react with any of the asexual stages (not shown). Five days after the first infection with P. berghei ANKA clone 2.33, we also performed a direct mosquito feeding test with mice from group 3 and confirmed that these defective parasite forms are indeed transmission incompetent as previously described in the literature (11). Taken together, these results indicate that P. berghei ANKA clone 2.33 produces parasites resembling gametocytes in morphology, albeit defective in morphology and in infectivity, and such parasites in defective stages express the gametocyte antigen Pbs48/45.


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DISCUSSION
 
The ideal malaria vaccine would be one that induces sterile immunity as well as complete transmission-blocking immunity, but the existing evidence indicates that this type of immunity is unlikely to be achieved by just natural infection in the host (35). The success of a vaccine against malaria in areas of endemicity will largely depend on whether the vaccine is capable of priming an immune response that can be boosted by natural infections (16, 35). It is well documented that naturally acquired immunity to the parasite is largely stage-specific and builds gradually through continuous exposure to the parasite (at times requiring up to 15 years) and is therefore only acquired by individuals living in areas of endemicity. In these individuals, immunity reduces parasite load and protects against disease and death but not against reinfection (9, 26).

Besides being directed to sporozoite and asexual blood stages, naturally acquired immunity to malaria has been shown also to target the sexual stages, where it potentially reduces transmission to mosquitoes (27, 29). This type of immunity is largely mediated by antibodies directed against the surface antigens of extracellular gametes or zygotes (7, 19, 20, 21, 29, 30, 39) and appears to be boosted in individuals who have suffered several frequent malaria attacks (27, 29). The induction of antibodies to gamete and zygote surface antigens during malarial infection can be accounted for by the presence of these antigens in gametocytes (22, 39). However, this naturally acquired transmission-blocking immunity appears to be ineffective in completely suppressing infectivity and is also short lived (27, 29, 35). One of the tasks of a transmission-blocking vaccine then should be to assist naturally acquired immunity, particularly by increasing the levels and longevity of the antibody responses. A vaccine that primes immune memory cells against protective antigens and which can be rapidly expanded upon reinfection should be effective in reducing transmission.

It is well known that serum from people who are naturally exposed to malaria or from animals infected with malaria parasites can recognize several of the leading vaccine antigens (2, 31). However, few studies exist in humans (40) or in animal models (3) showing that malaria vaccine-primed individuals or animals can be boosted through natural infection. Those studies have demonstrated an elevation of antisporozoite immunity after a sporozoite infection in humans primed with a DNA vaccine encoding a sporozoite protein (40) or an increase in antibodies to an asexual-stage antigen upon an infection with sporozoites in mice vaccinated with the asexual stage antigen (3). The present study further investigates whether the natural boosting of a transmission-blocking vaccine candidate antigen can also be achieved.

We chose antigen Pbs48/45 from the rodent malaria parasite P. berghei for our studies as it is a well-conserved orthologue of the P. falciparum transmission-blocking vaccine candidate antigen Pfs48/45 (22, 30, 37, 39). Both antigens Pfs48/45 and Pbs48/45 are expressed only by the sexual stage forms (gametocytes and gametes) of the corresponding species (6, 21, 22, 32, 39), and antibodies against Pfs48/45 are associated with the reduction of infectivity in mosquitoes (13, 22, 32, 38, 39). This is particularly evident in the vaccinated mice whose antibody levels had significantly decreased 6 weeks after one parasite infection but were rapidly boosted upon a second infection. The antibody levels in these mice were significantly higher than in control mice that had received a mock vaccination followed by two similar infections. The fact that antibody boosting occurred also in the mock-vaccinated group after the second infection indicates Pbs48/45-specific priming by the first parasite infection alone. However, the vaccinated group conceivably had an advantage in achieving higher antibody levels after infection since the premise for a vaccine is to speed up the process of immunity acquisition by priming memory B and T cells to specific vaccine candidate antigens.

It is important to note that there were few gametocytes during any given infection with the parasite strain used here. Most animals had gametocytemias below 0.1% within a period of 14 days, so the stimulatory antigenic dose during infection may have been suboptimal. Nonetheless, the boosting of anti-Pbs48/45 antibodies occurred in the vaccinated and nonvaccinated mice upon reinfection, suggesting an exquisitely specific immune recognition of this antigen. Humans living in regions of endemicity continue to experience reinfections many more times as natural immunity is slow to develop. This would imply that a vaccine-primed response will have ample opportunities to be boosted during such multiple repeat infections.

Given that the Pbs48/45 protein was highly expressed in eukaryotic cells by the DNA vaccine and since the antibody levels after a second infection were higher in the vaccinated than in the nonvaccinated mice, we think the DNA-Pbs48/45 vaccine effectively primed immune memory cells in these mice. It is possible that after one infection, an already larger number of these immune cells existed in the vaccinated mice than in the mock-vaccinated mice. However, the readout of our assay, ELISA antibody levels, may not be on par with these cell-based differences and it is only after a second infection, when an even larger number of cells are expanded in the vaccinated mice, that this difference is reflected in the antibody levels. We did not pursue a third infection in these mice as the antiparasite immunity acquired during a primary and secondary P. berghei ANKA infection is rather effective in suppressing asexual parasite growth during repeat infections (24).

Intramuscular DNA vaccination by itself is not always efficient in inducing high antibody levels against certain antigens (34). In our study, two DNA-Pbs48/45 injections elicited antibody levels below the level of detection, suggesting that in this particular antigen model, DNA vaccination alone, although effective in priming an immune response, is not enough for boosting and that parasite reinfection may be more relevant. A vaccine that primes antigen-specific immune responses is thereby selecting a very specific repertoire of B and T cells rather than the enormous range of specificities induced by the complex antigenic mixture that is the parasite. Subsequent infections in the vaccinee would rapidly expand these selected specificities (36). Our results not only support this premise, i.e., the boosting of antibodies by natural malaria infection, but also indicate that the Pbs48/45 antigen is immunogenic and therefore a good candidate for natural boosting. We think that the results presented here using this sexual blood-stage antigen as a model are likely applicable to most other immunogenic asexual and sexual blood-stage antigens in that they too could be boosted by natural infection.

Our study also showed that the P. berghei ANKA clone 2.33 parasite, which was previously reported to be a nongametocyte producer parasite line (11, 12, 28), appeared and behaved in a manner similar to that of the gametocyte producer P. berghei ANKA clone 2.34 used in this study. Indeed, in blood smears from infected mice, P. berghei clone 2.33 revealed morphologically abnormal parasites that were reminiscent of P. berghei gametocytes. These parasite forms expressed the sexual stage antigen Pbs48/45 and also boosted anti-Pbs48/45 antibodies. Nonetheless, as previously shown, unlike P. berghei clone 2.34, clone 2.33 was incapable of infecting mosquitoes (11; data not shown). Several P. berghei laboratory strains have been shown to have irreversibly lost the ability to produce gametocytes and therefore are unable to infect mosquitoes (17, 18). Although not conclusive, it was suggested that this defect may be associated with changes in the parasite's karyotype (17, 18). However, the P. berghei ANKA clone 2.33 appears to produce gametocyte-like forms which could represent a form of incomplete or aborted sexual development since these gametocytes not only look abnormal but also are incapable of infecting mosquitoes. In this sense, it is possible that sexual commitment does occur during asexual multiplication but the resulting gametocytes may never achieve complete sexual maturity, as suggested by gene disruption studies in P. falciparum (23). We suggest that the P. berghei ANKA clone 2.33 should not be regarded as a nongametocyte producer. Instead, it should be noted that this clone produces abnormal gametocytes which are transmission incompetent.

Ideally, a malaria vaccine should rapidly induce both clinical and transmission-blocking immunity, thus reducing morbidity and mortality as it decreases the prevalence of infection until transmission is significantly reduced or completely interrupted. Furthermore, a vaccine of this sort would be both practical and economically feasible if the immunity could be enhanced by natural boosting and thus not always require repeated immunizations for its efficacy. The present study constitutes a first step forward in showing that this is feasible. But further similar studies with multiple blood-stage asexual and sexual stage antigens, optimized for higher immunogenicity, need to be undertaken in order to design the most effective subunit vaccine.


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ACKNOWLEDGMENTS
 
We thank Gregory Noland for assisting with the mosquito transmission experiments and Melanie van Dijk for the recombinant Pbs48/45 and the rabbit anti-Pbs48/45 antisera used in this study.

This study was supported by grant AI47089 from the National Institutes of Health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Johns Hopkins Malaria Research Institute, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-7177. Fax: (410) 955-0105. E-mail: nkumar{at}jhsph.edu. Back

Editor: W. A. Petri, Jr.


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Infection and Immunity, April 2006, p. 2043-2051, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2043-2051.2006
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