Nasal Immunization with a Malaria Transmission-Blocking Vaccine Candidate, Pfs25, Induces Complete Protective Immunity in Mice against Field
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感染与免疫杂志 2005年第11期
Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan
Department of Molecular Parasitology, Ehime University School of Medicine, Toon, Ehime 791-0295, Japan
Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, Tsukuba, Ibaraki 305-0856, Japan
Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Ehime 790-8577, Japan
ABSTRACT
Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.
INTRODUCTION
Malaria causes high mortality and morbidity in tropical and subtropical countries, killing more than 3 million people annually (4, 29). The emergence of drug-resistant parasites and insecticide-resistant mosquitoes has raised continued public health problems worldwide. Given their complex life cycle and the discrete nature of immune responses to each developmental stage, the malaria parasites provide many potential targets for the development of prophylactic vaccines. Transmission-blocking vaccines the target sexual stages of the parasites (i.e., gametocyte, gamete, zygote, and ookinete) (6). Transmission-blocking antibodies ingested together with the gametocytes block parasite development in the mosquito midgut, preventing parasite transmission to other susceptible individuals. Thus, transmission-blocking vaccines are expected to prevent the spread of escape mutants that could be emerging during the course of antimalaria drug treatment or other prophylactic vaccines targeting asexual stages of the parasites. A leading transmission-blocking vaccine candidate antigen against Plasmodium falciparum is the ookinete surface protein Pfs25 (17, 18), and a clinical-grade recombinant Pfs25 expressed in Pichia pastoris is now available (33).
Mucosal vaccination with nonreplicating particles or recombinant proteins in combination with effective mucosal adjuvants has demonstrated their ability to induce local protective immunity against mucosal pathogens (32). Nasal vaccines in particular are by far the most effective mucosal vaccines, capable of priming a full range of local as well as systemic immune responses against protective antigenic epitopes (13, 14). In addition, this type of topically administrable, needle-free, noninvasive vaccine may be safer than injection-based parenteral vaccines by reducing the risk of infection from blood-borne pathogens, and may also be cost-effective because administration does not require highly trained medical or veterinary personnel.
Although mucosal vaccines have several attractive features over parenteral vaccines, their targets had been almost exclusively limited to mucosal infections, and their potential applicability to nonmucosal pathogens such as arthropod vector-borne parasites and viruses seemed to be unappreciated. However, previous studies with malaria parasites (1, 5, 15, 23, 24, 27, 30) and Japanese encephalitis virus (unpublished data), which are prototypical mosquito-borne infectious protozoa and virus, respectively, indicated that mucosal vaccines could be effective alternative immunization methods.
In this study we evaluated the ability of transmission-blocking mucosal vaccines against field isolates of P. falciparum. Our results indicate that recombinant Pfs25 is sufficiently immunogenic when coadministered intranasally with a mucosal adjuvant to achieve robust immune protection against parasite transmission, suggesting that noninvasive mucosal vaccines are a promising alternative approach for malaria prevention.
MATERIALS AND METHODS
Mice and immunization schedule. Six-week-old female BALB/c (H-2d) and A/J (H-2a) mice (Japan SLC, Hamamatsu, Japan) were used for intranasal immunization experiments. Groups of six to seven mice were intranasally immunized three times at weeks 0, 3, and 5 with 20 μg of Pichia pastoris-expressed recombinant Pfs25 (33) mixed with 1 μg of cholera toxin (CT; Sigma-Aldrich) as a mucosal adjuvant. The volume of the mixture was adjusted to 15 μl with phosphate-buffered saline (PBS) and administered to external nares with micropipette without anesthesia. As negative controls mice were intranasally administered with 1 μg of CT alone or PBS.
Serum and mucosal sample collection. Blood was collected from immunized mice one week after the third immunization by cardiac puncture under complete anesthesia confirmed by eyelid reflex responses. Immune sera prepared from the collected blood were used for antibody titer analysis and transmission-blocking assays. Nasal secretions were collected from exsanguinated animals immediately after sacrifice by washing the nasal cavities several times with 200 μl of PBS. The samples were centrifuged to remove insoluble debris and supernatant was immediately analyzed for specific antibodies. For collection of intestinal secretory antibodies, a fraction of the small intestine (approximately 3 cm long) was excised and cut perpendicularly to open the intestinal tubes. The excised samples were immersed in 0.5 ml of PBS and vigorously vortexed, followed by centrifugation to remove insoluble debris. Supernatant was used for antibody analysis.
Antibody titer determination by enzyme-linked immunosorbent assay (ELISA) for serum, nasal and intestinal secretions. Serum and mucosal samples were analyzed for the presence of specific antibodies by ELISA as described previously (1). Briefly, 96-well ELISA plates (Sumilon, Sumitomo Bakelite, Japan) were coated with recombinant Pfs25 proteins (33). The plates were washed with PBS containing 0.05% Tween-20 (PBST) three times and blocked with 1% BSA in PBS. Serial dilutions of serum samples were applied to wells in duplicates. Secondary antibodies specific for each mouse antibody isotype (immunoglobulin G [IgG], IgM and IgA) and IgG subclass (IgG1, IgG2a, IgG2b, and IgG3) was used for detection. Optical density (OD) was measured by microplate reader (Bio-Rad Laboratories) at 415 nm. The OD415 value of 0.1 was used as the baseline to determine the endpoint titers for specific serum IgG. In some experiments, serum antibody levels were expressed as OD415 measurement after making appropriate dilutions as indicated. Antibodies present in nasal secretions were analyzed by diluting the nasal washings by 15-fold with PBS before applying the samples to microtiter plates. To analyze specific antibodies in intestinal secretions, intestinal washings collected as described above were diluted with PBS by 2-fold prior to ELISA. Student's t test was performed to compare antibody levels of serum and mucosal samples between different test groups.
Recognition of native parasite by immunofluorescence assay. All human materials used in this study were reviewed and approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and the Human Subjects Research Review Board of the United States Army. For purification of gametocytes, peripheral blood was collected by heparinized syringes under written informed consent from patients who came to the malaria clinics in the Mae Sod district in the Tak province of northwestern Thailand. Infection with P. falciparum was confirmed by Giemsa stain of thick and thin blood smears. Cultured P. falciparum parasite preparations rich in zygotes and small numbers of ookinetes were spotted on slides and fixed with acetone as previously described (25). The slides were blocked with PBS containing 5% nonfat milk and incubated with Pfs25/CT immune sera. The slides were washed with ice-cold PBS for 5 min and incubated with fluorescein isothiocyanate-conjugated anti-mouse antibody, followed by washing with ice-cold PBS. Slides were examined by confocal scanning laser microscope (Nikon C-1).
Transmission-blocking assays. Peripheral blood was collected from four volunteer patients as described above. Their parasitemia were ranging from 0.04 to 0.18%, and gametocytemia from 0.002% to 0.011%. Collected blood was aliquoted into tubes (300 μl/tube) and plasma was removed. Mouse immune sera were diluted (2-, 8- and 32-fold) with heat-inactivated normal human AB serum prepared from malaria nave donors. Each diluted test serum was mixed with P. falciparum-infected blood cells as described above (1:1 vol/vol ratio) and incubated for 15 min at room temperature. The mixture was placed in a membrane feeding apparatus kept at 37°C to allow starved Anopheles dirus A mosquitoes (Bangkok colony, Armed Forces Research Institute of Medical Sciences) to feed on the blood meals for 30 min. Unfed mosquitoes were removed and only fully engorged mosquitoes were maintained for a week by giving 10% sucrose water in the insectary.
For each mouse test immune serum, 20 mosquitoes (i.e., a total of 80 mosquitoes for four patients' blood samples) were dissected and analyzed by staining with 0.5% mercurochrome to count the number of oocysts developed within the mosquito midgut under the microscope. Mann-Whitney U test was used to examine the difference in oocyst counts per mosquito between control and immunized groups. Fisher's exact probability test was used to examine the difference of infection rates between control and immunized groups. P values less than 0.05 were considered statistically significant.
RESULTS
Systemic and mucosal antibody responses induced in mice by intranasal immunizations. Immunization with Pfs25/CT resulted in a significant increment of specific anti-Pfs25 serum IgG responses (P < 0.01) (Fig. 1A). Higher IgG responses were induced in A/J than BALB/c mice, but the difference did not reach statistically significant level (P = 0.23). We also evaluated specific reactivity of immune sera against denatured forms of the recombinant Pfs25 proteins. We found that mucosally induced antibodies exhibited strict conformation-dependent reactivity, in which both A/J and BALB/c mouse immune sera recognized only native proteins, while no specific reaction was observed against denatured forms of the proteins (P < 0.001) (Fig. 1B). Mucosal immunization induced comparable levels of IgG1, IgG2a and IgG2b, but not IgG3 (Fig. 1C). Immunoglobulin isotypes other than IgG (i.e., IgM, IgE, and IgA) were also detected, although at relatively low levels in Pfs25/CT immune sera, but not in immune sera derived from intranasal administration of CT alone or PBS (Fig. 1D).
Next we analyzed mucosal antibody responses (Fig. 2). Intranasal immunization with Pfs25/CT induced significant levels of anti-Pfs25 IgA and IgG (P < 0.01) in nasal washings, whereas control mice given CT alone or PBS did not develop Pfs25-specific mucosal antibodies (Fig. 2A). IgG subclass analysis for nasal washings revealed a similar pattern seen for serum IgG subclasses (Fig. 2B). Mucosal adjuvant CT-specific IgA and IgG were also detected in nasal washings of mice immunized with Pfs25/CT or CT alone, but not in mice given PBS (Fig. 2C). Intranasal immunization also resulted in low but detectable levels of Pfs25-specific IgA and IgG in intestinal secretions of Pfs25/CT immunized group, but not in CT or PBS group, and the induced IgG subclass patterns were similar to that for nasal IgGs (data not shown).
Recognition of native Pfs25 proteins expressed at ookinete stage of P. falciparum. To evaluate antibody specificity of mucosally induced immune sera to native Pfs25 proteins, Pfs25/CT immune sera were reacted with cultured ookinete preparations. As indicated in Fig. 3, the immune sera specifically recognized ookinetes but not gametocytes. The fluorescent signal was localized on the surface of ookinetes, confirming that induced antibodies were capable of recognizing native Pfs25 proteins on the surface of ookinetes. The result was consistent with the expression patterns of the Pfs25 proteins in the parasites.
Evaluation of transmission-blocking activity. Transmission-blocking assays were performed using pooled immune sera of mice intranasally immunized with Pfs25/CT, CT alone or PBS. Pfs25/CT immune sera, but not control CT or PBS serum, had significant transmission-blocking activities, indicated by profound reduction of the numbers of oocysts developed in mosquito midgut (Table 1). Dilution of the immune sera resulted in reduction of transmission-blocking activities. In addition, Pfs25/CT immune sera significantly reduced mosquito infection rate defined as percent of infected mosquitoes in a total number of mosquitoes examined (Table 1). Serum dilution resulted in an increase in the infection rate.
We also analyzed the complete transmission-blocking rate, defined as the percentage of volunteers whose infected blood did not establish any parasite infection in mosquitoes (Table 1). All four human blood samples failed to transmit any parasite to mosquitoes when mixed with Pfs25/CT immune sera, but dilution of the immune sera gradually reduced the complete transmission-blocking rate.
We found strong correlations of Pfs25-specific serum antibody levels (Fig. 1) with the oocyst counts (correlation coefficient r = –0.717), with the mosquito infection rate (r = –0.832), and with the complete transmission-blocking rate (r = 0.878). In contrast, no correlation was observed between CT-specific antibody levels and the transmission-blocking activities.
DISCUSSION
With the prospect of developing noninvasive malaria transmission-blocking vaccines, we have previously demonstrated that intranasal immunization of recombinant Pvs25, an orthologous gene product of Pfs25 and a vaccine candidate against P. vivax malaria, induced a robust systemic immune response, conferring a significant protection against parasite transmission to mosquitoes (1). In this study we demonstrated a potent mucosal immunogenicity and protective efficacy of recombinant Pfs25 proteins (33). In addition, this is the first report indicating that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites in a malaria-endemic area.
Our recent studies with rodent malaria P. yoelii also demonstrated that intranasal immunization of mice with recombinant Pys25 proteins (25) provided complete transmission-blocking immunity in both active and passive immunization regimens (unpublished data). Unlike the results of our previous studies with P. vivax (1) and P. yoelii (unpublished data), we found that the serum IgG subclasses induced by Pfs25/CT immunization were not strongly biased towards IgG1. It rather induced comparable levels of IgG1, IgG2a, and IgG2b (Fig. 1C), implying that mixed Th1 and Th2 responses were induced. Malkin et al. observed that the presence of heat-labile components in the membrane feeder enhanced the transmission-blocking activity of Pvs25 antisera (21). Because all heat-labile components were inactivated by heat treatment during the transmission-blocking assays performed in our present and previous studies, we might have underestimated the levels of actual transmission-blocking activity of mucosally induced immune sera. However, regardless of the types of immunity induced, specific antibody titers were the best correlate for protection, and no such correlation was found between CT-specific antibody titers and protective efficacy: these observations were consistent with other transmission-blocking vaccine studies (2, 7, 10, 11, 16, 18-20). Taken together, transmission-blocking activity was clearly correlated with levels of vaccine antigen-specific serum IgG, regardless of the repertoire of IgG subclasses induced, strain of mouse used, difference in Plasmodium species targeted, or type of immunization method employed.
In contrast to the general perception that mucosal vaccines are much less effective for the induction of systemic antibody responses than parenteral vaccines, we found that intranasal vaccines, when coadministered with a strong mucosal adjuvant like CT, are not necessarily considered inferior to parenteral vaccines at least in a murine model (1, 2, 16, 18; unpublished data). Systemic antibodies raised against Pichia pastoris-expressed recombinant Pfs25 proteins (33) by intranasal immunization specifically recognized native proteins expressed on ookinete surface (Fig. 3), but barely recognized heat-denatured or reduced form of proteins (Fig. 1B), indicating that at least some conformational epitopes were retained in the course of intranasal immunization and correctly presented to the immune system in an intact form for the induction of biologically active antibodies that functioned as transmission-blocking agents within the mosquito midgut.
The highly efficacious transmission-blocking activity observed with mucosally induced immune sera supports the potential of applying mucosal vaccines to malaria prophylactics. Further, our recent mucosal immunization studies with various antigens such as formalin-inactivated Japanese encephalitis virus vaccine and the paramyosin antigen of Schistosoma japonicum demonstrated that mucosal vaccines induced strong and long-lasting humoral as well as cellular immunity comparable to that with parenteral vaccines when strong adjuvants like CT were coadministered (unpublished data). These results indicate that CT is a strong immune potentiator that may be able to induce immunological memory against heterologous antigens in a rodent model. However, CT needs to be precluded from clinical use due to its enterotoxicity and potential hazardous effects on olfactory nerves (12). Therefore, the particular vaccination regimen presented in this study using CT as an adjuvant needs to be considered as a model system to prove the effectiveness of mucosal vaccines against malaria transmission.
Since the mucosal immunogenicity of Pfs25 may not depend on a particular mucosal adjuvant or delivery system, specific targeting or immunomodulation of professional antigen-presenting cells such as dendritic cells and B cells with other, potentially safer agents (3, 8, 9, 22, 26, 28, 31) than CT may offer new approaches for the development of malaria vaccines and warrant further evaluation of mucosal and other less invasive vaccination regimens as alternative strategies for malaria control in the future.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid for Scientific Research 16390125, 16406009, and 14770111 and Grant-in-Aid for Scientific Research on Priority Areas 16017273 from the Ministry of Education, Culture, Sports, Science and Technology, and a Grant for International Health Cooperation Research (15C-5) from the Ministry of Health, Labor and Welfare, Japan. This work also received financial support from the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.
We thank Jeeraphat Sirichaisinthop and the staffs of the Office of Vector-Borne Disease Control 1, Saraburi, Thailand, for constant help in setting up the field sites, and the staffs of the Department of Entomology, AFRIMS, Bangkok, Thailand. We also thank the Malaria Vaccine Initiative at the Program for Appropriate Technology in Health for their constant help with transmission-blocking vaccine development.
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Department of Molecular Parasitology, Ehime University School of Medicine, Toon, Ehime 791-0295, Japan
Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Laboratory of Global Animal Resource Science, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, Tsukuba, Ibaraki 305-0856, Japan
Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852
Cell-Free Science and Technology Research Center, Ehime University, Matsuyama, Ehime 790-8577, Japan
ABSTRACT
Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.
INTRODUCTION
Malaria causes high mortality and morbidity in tropical and subtropical countries, killing more than 3 million people annually (4, 29). The emergence of drug-resistant parasites and insecticide-resistant mosquitoes has raised continued public health problems worldwide. Given their complex life cycle and the discrete nature of immune responses to each developmental stage, the malaria parasites provide many potential targets for the development of prophylactic vaccines. Transmission-blocking vaccines the target sexual stages of the parasites (i.e., gametocyte, gamete, zygote, and ookinete) (6). Transmission-blocking antibodies ingested together with the gametocytes block parasite development in the mosquito midgut, preventing parasite transmission to other susceptible individuals. Thus, transmission-blocking vaccines are expected to prevent the spread of escape mutants that could be emerging during the course of antimalaria drug treatment or other prophylactic vaccines targeting asexual stages of the parasites. A leading transmission-blocking vaccine candidate antigen against Plasmodium falciparum is the ookinete surface protein Pfs25 (17, 18), and a clinical-grade recombinant Pfs25 expressed in Pichia pastoris is now available (33).
Mucosal vaccination with nonreplicating particles or recombinant proteins in combination with effective mucosal adjuvants has demonstrated their ability to induce local protective immunity against mucosal pathogens (32). Nasal vaccines in particular are by far the most effective mucosal vaccines, capable of priming a full range of local as well as systemic immune responses against protective antigenic epitopes (13, 14). In addition, this type of topically administrable, needle-free, noninvasive vaccine may be safer than injection-based parenteral vaccines by reducing the risk of infection from blood-borne pathogens, and may also be cost-effective because administration does not require highly trained medical or veterinary personnel.
Although mucosal vaccines have several attractive features over parenteral vaccines, their targets had been almost exclusively limited to mucosal infections, and their potential applicability to nonmucosal pathogens such as arthropod vector-borne parasites and viruses seemed to be unappreciated. However, previous studies with malaria parasites (1, 5, 15, 23, 24, 27, 30) and Japanese encephalitis virus (unpublished data), which are prototypical mosquito-borne infectious protozoa and virus, respectively, indicated that mucosal vaccines could be effective alternative immunization methods.
In this study we evaluated the ability of transmission-blocking mucosal vaccines against field isolates of P. falciparum. Our results indicate that recombinant Pfs25 is sufficiently immunogenic when coadministered intranasally with a mucosal adjuvant to achieve robust immune protection against parasite transmission, suggesting that noninvasive mucosal vaccines are a promising alternative approach for malaria prevention.
MATERIALS AND METHODS
Mice and immunization schedule. Six-week-old female BALB/c (H-2d) and A/J (H-2a) mice (Japan SLC, Hamamatsu, Japan) were used for intranasal immunization experiments. Groups of six to seven mice were intranasally immunized three times at weeks 0, 3, and 5 with 20 μg of Pichia pastoris-expressed recombinant Pfs25 (33) mixed with 1 μg of cholera toxin (CT; Sigma-Aldrich) as a mucosal adjuvant. The volume of the mixture was adjusted to 15 μl with phosphate-buffered saline (PBS) and administered to external nares with micropipette without anesthesia. As negative controls mice were intranasally administered with 1 μg of CT alone or PBS.
Serum and mucosal sample collection. Blood was collected from immunized mice one week after the third immunization by cardiac puncture under complete anesthesia confirmed by eyelid reflex responses. Immune sera prepared from the collected blood were used for antibody titer analysis and transmission-blocking assays. Nasal secretions were collected from exsanguinated animals immediately after sacrifice by washing the nasal cavities several times with 200 μl of PBS. The samples were centrifuged to remove insoluble debris and supernatant was immediately analyzed for specific antibodies. For collection of intestinal secretory antibodies, a fraction of the small intestine (approximately 3 cm long) was excised and cut perpendicularly to open the intestinal tubes. The excised samples were immersed in 0.5 ml of PBS and vigorously vortexed, followed by centrifugation to remove insoluble debris. Supernatant was used for antibody analysis.
Antibody titer determination by enzyme-linked immunosorbent assay (ELISA) for serum, nasal and intestinal secretions. Serum and mucosal samples were analyzed for the presence of specific antibodies by ELISA as described previously (1). Briefly, 96-well ELISA plates (Sumilon, Sumitomo Bakelite, Japan) were coated with recombinant Pfs25 proteins (33). The plates were washed with PBS containing 0.05% Tween-20 (PBST) three times and blocked with 1% BSA in PBS. Serial dilutions of serum samples were applied to wells in duplicates. Secondary antibodies specific for each mouse antibody isotype (immunoglobulin G [IgG], IgM and IgA) and IgG subclass (IgG1, IgG2a, IgG2b, and IgG3) was used for detection. Optical density (OD) was measured by microplate reader (Bio-Rad Laboratories) at 415 nm. The OD415 value of 0.1 was used as the baseline to determine the endpoint titers for specific serum IgG. In some experiments, serum antibody levels were expressed as OD415 measurement after making appropriate dilutions as indicated. Antibodies present in nasal secretions were analyzed by diluting the nasal washings by 15-fold with PBS before applying the samples to microtiter plates. To analyze specific antibodies in intestinal secretions, intestinal washings collected as described above were diluted with PBS by 2-fold prior to ELISA. Student's t test was performed to compare antibody levels of serum and mucosal samples between different test groups.
Recognition of native parasite by immunofluorescence assay. All human materials used in this study were reviewed and approved by the Institutional Ethics Committee of the Thai Ministry of Public Health and the Human Subjects Research Review Board of the United States Army. For purification of gametocytes, peripheral blood was collected by heparinized syringes under written informed consent from patients who came to the malaria clinics in the Mae Sod district in the Tak province of northwestern Thailand. Infection with P. falciparum was confirmed by Giemsa stain of thick and thin blood smears. Cultured P. falciparum parasite preparations rich in zygotes and small numbers of ookinetes were spotted on slides and fixed with acetone as previously described (25). The slides were blocked with PBS containing 5% nonfat milk and incubated with Pfs25/CT immune sera. The slides were washed with ice-cold PBS for 5 min and incubated with fluorescein isothiocyanate-conjugated anti-mouse antibody, followed by washing with ice-cold PBS. Slides were examined by confocal scanning laser microscope (Nikon C-1).
Transmission-blocking assays. Peripheral blood was collected from four volunteer patients as described above. Their parasitemia were ranging from 0.04 to 0.18%, and gametocytemia from 0.002% to 0.011%. Collected blood was aliquoted into tubes (300 μl/tube) and plasma was removed. Mouse immune sera were diluted (2-, 8- and 32-fold) with heat-inactivated normal human AB serum prepared from malaria nave donors. Each diluted test serum was mixed with P. falciparum-infected blood cells as described above (1:1 vol/vol ratio) and incubated for 15 min at room temperature. The mixture was placed in a membrane feeding apparatus kept at 37°C to allow starved Anopheles dirus A mosquitoes (Bangkok colony, Armed Forces Research Institute of Medical Sciences) to feed on the blood meals for 30 min. Unfed mosquitoes were removed and only fully engorged mosquitoes were maintained for a week by giving 10% sucrose water in the insectary.
For each mouse test immune serum, 20 mosquitoes (i.e., a total of 80 mosquitoes for four patients' blood samples) were dissected and analyzed by staining with 0.5% mercurochrome to count the number of oocysts developed within the mosquito midgut under the microscope. Mann-Whitney U test was used to examine the difference in oocyst counts per mosquito between control and immunized groups. Fisher's exact probability test was used to examine the difference of infection rates between control and immunized groups. P values less than 0.05 were considered statistically significant.
RESULTS
Systemic and mucosal antibody responses induced in mice by intranasal immunizations. Immunization with Pfs25/CT resulted in a significant increment of specific anti-Pfs25 serum IgG responses (P < 0.01) (Fig. 1A). Higher IgG responses were induced in A/J than BALB/c mice, but the difference did not reach statistically significant level (P = 0.23). We also evaluated specific reactivity of immune sera against denatured forms of the recombinant Pfs25 proteins. We found that mucosally induced antibodies exhibited strict conformation-dependent reactivity, in which both A/J and BALB/c mouse immune sera recognized only native proteins, while no specific reaction was observed against denatured forms of the proteins (P < 0.001) (Fig. 1B). Mucosal immunization induced comparable levels of IgG1, IgG2a and IgG2b, but not IgG3 (Fig. 1C). Immunoglobulin isotypes other than IgG (i.e., IgM, IgE, and IgA) were also detected, although at relatively low levels in Pfs25/CT immune sera, but not in immune sera derived from intranasal administration of CT alone or PBS (Fig. 1D).
Next we analyzed mucosal antibody responses (Fig. 2). Intranasal immunization with Pfs25/CT induced significant levels of anti-Pfs25 IgA and IgG (P < 0.01) in nasal washings, whereas control mice given CT alone or PBS did not develop Pfs25-specific mucosal antibodies (Fig. 2A). IgG subclass analysis for nasal washings revealed a similar pattern seen for serum IgG subclasses (Fig. 2B). Mucosal adjuvant CT-specific IgA and IgG were also detected in nasal washings of mice immunized with Pfs25/CT or CT alone, but not in mice given PBS (Fig. 2C). Intranasal immunization also resulted in low but detectable levels of Pfs25-specific IgA and IgG in intestinal secretions of Pfs25/CT immunized group, but not in CT or PBS group, and the induced IgG subclass patterns were similar to that for nasal IgGs (data not shown).
Recognition of native Pfs25 proteins expressed at ookinete stage of P. falciparum. To evaluate antibody specificity of mucosally induced immune sera to native Pfs25 proteins, Pfs25/CT immune sera were reacted with cultured ookinete preparations. As indicated in Fig. 3, the immune sera specifically recognized ookinetes but not gametocytes. The fluorescent signal was localized on the surface of ookinetes, confirming that induced antibodies were capable of recognizing native Pfs25 proteins on the surface of ookinetes. The result was consistent with the expression patterns of the Pfs25 proteins in the parasites.
Evaluation of transmission-blocking activity. Transmission-blocking assays were performed using pooled immune sera of mice intranasally immunized with Pfs25/CT, CT alone or PBS. Pfs25/CT immune sera, but not control CT or PBS serum, had significant transmission-blocking activities, indicated by profound reduction of the numbers of oocysts developed in mosquito midgut (Table 1). Dilution of the immune sera resulted in reduction of transmission-blocking activities. In addition, Pfs25/CT immune sera significantly reduced mosquito infection rate defined as percent of infected mosquitoes in a total number of mosquitoes examined (Table 1). Serum dilution resulted in an increase in the infection rate.
We also analyzed the complete transmission-blocking rate, defined as the percentage of volunteers whose infected blood did not establish any parasite infection in mosquitoes (Table 1). All four human blood samples failed to transmit any parasite to mosquitoes when mixed with Pfs25/CT immune sera, but dilution of the immune sera gradually reduced the complete transmission-blocking rate.
We found strong correlations of Pfs25-specific serum antibody levels (Fig. 1) with the oocyst counts (correlation coefficient r = –0.717), with the mosquito infection rate (r = –0.832), and with the complete transmission-blocking rate (r = 0.878). In contrast, no correlation was observed between CT-specific antibody levels and the transmission-blocking activities.
DISCUSSION
With the prospect of developing noninvasive malaria transmission-blocking vaccines, we have previously demonstrated that intranasal immunization of recombinant Pvs25, an orthologous gene product of Pfs25 and a vaccine candidate against P. vivax malaria, induced a robust systemic immune response, conferring a significant protection against parasite transmission to mosquitoes (1). In this study we demonstrated a potent mucosal immunogenicity and protective efficacy of recombinant Pfs25 proteins (33). In addition, this is the first report indicating that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites in a malaria-endemic area.
Our recent studies with rodent malaria P. yoelii also demonstrated that intranasal immunization of mice with recombinant Pys25 proteins (25) provided complete transmission-blocking immunity in both active and passive immunization regimens (unpublished data). Unlike the results of our previous studies with P. vivax (1) and P. yoelii (unpublished data), we found that the serum IgG subclasses induced by Pfs25/CT immunization were not strongly biased towards IgG1. It rather induced comparable levels of IgG1, IgG2a, and IgG2b (Fig. 1C), implying that mixed Th1 and Th2 responses were induced. Malkin et al. observed that the presence of heat-labile components in the membrane feeder enhanced the transmission-blocking activity of Pvs25 antisera (21). Because all heat-labile components were inactivated by heat treatment during the transmission-blocking assays performed in our present and previous studies, we might have underestimated the levels of actual transmission-blocking activity of mucosally induced immune sera. However, regardless of the types of immunity induced, specific antibody titers were the best correlate for protection, and no such correlation was found between CT-specific antibody titers and protective efficacy: these observations were consistent with other transmission-blocking vaccine studies (2, 7, 10, 11, 16, 18-20). Taken together, transmission-blocking activity was clearly correlated with levels of vaccine antigen-specific serum IgG, regardless of the repertoire of IgG subclasses induced, strain of mouse used, difference in Plasmodium species targeted, or type of immunization method employed.
In contrast to the general perception that mucosal vaccines are much less effective for the induction of systemic antibody responses than parenteral vaccines, we found that intranasal vaccines, when coadministered with a strong mucosal adjuvant like CT, are not necessarily considered inferior to parenteral vaccines at least in a murine model (1, 2, 16, 18; unpublished data). Systemic antibodies raised against Pichia pastoris-expressed recombinant Pfs25 proteins (33) by intranasal immunization specifically recognized native proteins expressed on ookinete surface (Fig. 3), but barely recognized heat-denatured or reduced form of proteins (Fig. 1B), indicating that at least some conformational epitopes were retained in the course of intranasal immunization and correctly presented to the immune system in an intact form for the induction of biologically active antibodies that functioned as transmission-blocking agents within the mosquito midgut.
The highly efficacious transmission-blocking activity observed with mucosally induced immune sera supports the potential of applying mucosal vaccines to malaria prophylactics. Further, our recent mucosal immunization studies with various antigens such as formalin-inactivated Japanese encephalitis virus vaccine and the paramyosin antigen of Schistosoma japonicum demonstrated that mucosal vaccines induced strong and long-lasting humoral as well as cellular immunity comparable to that with parenteral vaccines when strong adjuvants like CT were coadministered (unpublished data). These results indicate that CT is a strong immune potentiator that may be able to induce immunological memory against heterologous antigens in a rodent model. However, CT needs to be precluded from clinical use due to its enterotoxicity and potential hazardous effects on olfactory nerves (12). Therefore, the particular vaccination regimen presented in this study using CT as an adjuvant needs to be considered as a model system to prove the effectiveness of mucosal vaccines against malaria transmission.
Since the mucosal immunogenicity of Pfs25 may not depend on a particular mucosal adjuvant or delivery system, specific targeting or immunomodulation of professional antigen-presenting cells such as dendritic cells and B cells with other, potentially safer agents (3, 8, 9, 22, 26, 28, 31) than CT may offer new approaches for the development of malaria vaccines and warrant further evaluation of mucosal and other less invasive vaccination regimens as alternative strategies for malaria control in the future.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid for Scientific Research 16390125, 16406009, and 14770111 and Grant-in-Aid for Scientific Research on Priority Areas 16017273 from the Ministry of Education, Culture, Sports, Science and Technology, and a Grant for International Health Cooperation Research (15C-5) from the Ministry of Health, Labor and Welfare, Japan. This work also received financial support from the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.
We thank Jeeraphat Sirichaisinthop and the staffs of the Office of Vector-Borne Disease Control 1, Saraburi, Thailand, for constant help in setting up the field sites, and the staffs of the Department of Entomology, AFRIMS, Bangkok, Thailand. We also thank the Malaria Vaccine Initiative at the Program for Appropriate Technology in Health for their constant help with transmission-blocking vaccine development.
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