Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes

Vaccine 21 (2003) 3143–3148

Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes Takeshi Arakawa a , Takafumi Tsuboi b,∗,1 , Ayano Kishimoto c , Jetsumon Sattabongkot d , Nantavadee Suwanabun d , Thanaporn Rungruang b , Yasunobu Matsumoto e , Naotoshi Tsuji f , Hajime Hisaeda g , Anthony Stowers h , Isao Shimabukuro i , Yoshiya Sato a,i , Motomi Torii b a

f

Division of Molecular Microbiology, Center of Molecular Biosciences, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan b Department of Molecular Parasitology, Ehime University School of Medicine, Shigenobu-cho, Ehime 791-0295, Japan c Department of Bio-production, School of Agriculture, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan d Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand e 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 g Department of Immunology and Parasitology, School of Medicine, University of Tokushima, Tokushima 770-8503, Japan h Laboratory of Parasitic Diseases, Malaria Vaccine Development Unit, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 208521, USA i Department of Parasitology, School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan Received 25 October 2002; received in revised form 20 March 2003; accepted 24 March 2003

Abstract Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control. To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT). Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response). Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes. The observed transmission-blocking effect was dependent on immune sera dilution. This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; Malaria; Mucosal

1. Introduction Malaria remains a leading cause of high morbidity and mortality in human populations, particularly in sub-Saharan Africa. Although Plasmodium falciparum is responsible for the highest mortality rate among the four species of human malaria parasites, vivax malaria, which exhibits significantly ∗

Corresponding author. Tel.: +81-89-960-5286; fax: +81-89-960-5287. E-mail address: [email protected] (T. Tsuboi). 1 Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. Tel.: +81-89-927-8277; fax: +81-89-927-9941.

lower mortality than that caused by falciparum malaria, is responsible for the most recurrent form of malaria, causing high morbidity for millions of people in tropical and subtropical countries outside of sub-Saharan Africa [1]. Significant efforts have been made to control malaria through several strategies such as the development of effective anti-malaria drugs and the use of insecticide-impregnated mosquito nets in highly endemic areas. However, the emergence of drug-resistant parasites and insecticide-resistant mosquitoes has made chemotherapy and vector control difficult [2]. In response to the complex life cycle of malaria parasites and the discrete nature of effective immune responses to each developmental stage, considerable efforts have

0264-410X/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00258-5

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been made to develop effective vaccines targeting each developing stage of the parasite. Although not providing direct protection for vaccines, vaccines targeting antigens expressed on the surface of the sexual/sporogonic (gametocyte, gamete, zygote, ookinete and oocyst) stages of malaria parasites are considered one promising strategy for malaria control [3–5]. Such vaccines, called transmission-blocking vaccines (TBVs), induce antibodies in the vertebrate host, which inhibit parasitic development within the mosquito midgut, consequently reducing or preventing parasite transmission to another host. Thus, TBVs may function to prevent the spread of escape mutants resistant to vaccines developed against the other stages of the parasite. Target antigens fall into two classes. The first involves major proteins found on the surface of both gametocytes and gametes of malaria parasites and they are referred to as pre-fertilization target antigens. Two proteins in this family are the Pfs48/45 [6] and Pfs230 [7] molecules that are expressed on the surface of male and female gametocytes and gametes of P. falciparum. Monoclonal antibodies against them can be potent in blocking the transmission of the parasites [8,9]. Pfs48/45 is responsible for male gamete fertility [10]. A TBV based on these antigens would be boosted by the following natural infection and their development as candidate antigens for malaria TBV is currently underway. The second class involves the major surface proteins of zygote and ookinete stages of the parasite. Antibodies induced by vaccines against ookinete surface proteins have been demonstrated to block ookinete development into oocysts in mosquito midguts in animal models [11–15]. The leading candidates for a TBV in P. vivax are the ookinete surface proteins, Pvs25 and Pvs28 [16]. Mice immunized with yeast-produced Pvs25 and Pvs28 adsorbed to aluminum hydroxide (alum) intraperitoneally developed strong antibody responses. The antisera completely inhibited the development of oocysts in mosquitoes when the antisera were ingested with the P. vivax-parasite [17]. However, there are at least three major problems in the development of TBVs: (1) no post-infection booster effect is expected for TBVs based on the ookinete surface proteins; (2) subunit proteins alone may have reduced immunogenicity and the response may be short-lived; and (3) population-level delivery with very high compliance levels is required. Thus a safe, non-invasive (painless), frequently and easily administrable vaccine is necessary for TBVs to be successful. Although considerable efforts have been made for the development of mucosal vaccines against mucosal pathogens [18], there are few reports describing mucosal vaccines’ potential usefulness against non-mucosal infectious agents like malaria. In this study, we have demonstrated that intranasal immunization of mice with a P. vivax ookinete surface protein, Pvs25 co-administered with cholera toxin (CT), induced strong serum antibodies, which when mixed with parasitemic blood of vivax patients, completely block parasite transmission to Anopheles dirus mosquitoes. This is the first demonstration that mucosal vaccination with a TBV can induce complete

transmission-blocking immunity against the human malaria parasite.

2. Materials and methods 2.1. Animals and immunization Seven-week-old female CAF1 (H-2d/a ) and A/J (H-2a ) mice were purchased from Japan SLC (Hamamatsu, Japan) and maintained in the animal facility at University of the Ryukyus. Mice were intranasal immunized with 25 ␮g of yeast-produced recombinant Pvs25 [17] mixed with 5 ␮g CT (Sigma, St. Louis, MO). Mice were immunized three times at weeks 0, 3 and 5 with 20 ␮l of vaccine mixture dissolved in PBS through a nasal orifice. As negative controls, 5 ␮g CT alone or PBS was administered by the same route and schedule. A week after the last immunization, immune sera were collected and used for transmission-blocking assays. Animal studies were conducted in compliance with guidelines and protocols of University of the Ryukyus Animal Care and Use Committee. 2.2. Enzyme-linked immunosorbent assay (ELISA) Flat-bottom 96-well microtiter plates (Dynex Technology Inc, Chantilly, VA) were coated with recombinant Pvs25 (200 ng/well in bicarbonate buffer, pH 9.6) at 4 ◦ C overnight. Wells were blocked with 1% BSA and serially diluted mouse sera (100 ␮l/well) with PBS (containing 0.5% BSA) were applied to antigen-coated wells in duplicates and incubated for 2 h at 37 ◦ C followed by incubation with anti-mouse immunoglobulin (alkaline phosphatase conjugate) for each isotype (IgM, IgG, IgE and IgA) and IgG subclass (IgG1, IgG2a, IgG2b and IgG3). Bound secondary antibodies were visualized by p-nitrophenyl phosphate (Sigma, St. Louis, MO), and absorbance was read at 415 nm with a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA). Anti-Pvs25 serum antibody titers were determined as the highest serum dilutions giving an optical density of 0.1. Statistical significance of differences in absorbance values was determined by the Scheffe’s F-test. 2.3. Transmission-blocking assays All human subjects research conducted in these studies was 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. Peripheral blood was collected from 13 volunteer vivax patients who came to the malaria clinics in Mae Sod and Mae Kasa districts in Tak province, northwestern Thailand. A positive infection with P. vivax malaria was confirmed by Giemsa stain of thick and thin blood smears. Blood was collected by heparinized syringes after written informed consent was obtained from the individual volunteers.

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Blood was aliquoted into tubes (300 ␮l/tube) and plasma was removed. Mouse immune sera were diluted to 1:2, 1:8, and 1:32 with normal human AB serum (prepared from malaria na¨ıve donors). Each diluted serum was mixed with P. vivax-infected blood cells (1:1 (v/v) ratio) obtained from the donor and incubated for 15 min at room temperature. The mixture was placed in a membrane-feeding apparatus kept at 37 ◦ C to allow starved An. dirus A (Bangkok colony, AFRIMS) mosquitoes to feed on the blood meals for 30 min. Unfed mosquitoes were removed and only fully engorged mosquitoes were maintained in the insectary at 25 ◦ C for a week and fed water containing 10% sucrose. For each experimental group, 20 mosquitoes were dissected and midguts were stained with 0.5% mercurochrome and examined under the light microscope to count the number of oocysts developed. Statistical significance of differences in oocyst numbers was determined by the Scheffe’s F-test. 3. Results 3.1. Serum anti-Pvs25 antibody levels Intranasal immunization of mice with 25 ␮g yeastproduced Pvs25 co-administered with 5 ␮g CT induced significant levels of serum IgG in both CAF1 and A/J mouse strains as compared with control mice given PBS (P < 0.0001) or 5 ␮g CT (P < 0.0001) (Fig. 1a). The immunization also induced low but significant IgM antibodies in comparison with control groups (PBS and CT) in both mouse strains (data not shown). Slightly higher IgG titers were observed for A/J mouse sera than CAF1 sera but the difference was not significant (68666.7 ± 7575.9 S.E.M. versus 57916.7 ± 11551.6 S.E.M., P = 0.8695). The vaccine induced immune sera showed a high IgG1/IgG2a ratio (CAF1: 20.7; A/J: 6.3), but no significant difference in antibody titer was observed between IgG2a, IgG2b and IgG3 (Fig. 1b). When CAF1 mice were immunized intraperitoneally with yeast-produced Pvs25 adsorbed to alum, the immune sera also showed a high IgG1/IgG2a ratio (IgG1: 715461/IgG2a: 2543, unpublished data). Intranasal immunization with Pvs25/CT also induced low but detectable levels of Pvs25-specific serum IgA (CAF1: 21.5; A/J: 22.5), however, no increase in Pvs25-specific serum IgE was detected in Pvs25/CT immunized mice in comparison with sera from pre-immune mice (Fig. 1c). Significant elevation of the serum antibody was detected only when the vaccine antigen was co-administered with CT through the nasal route (data not shown). 3.2. Transmission-blocking effects To evaluate the ability of anti-Pvs25 mouse immune sera to block vivax malaria parasite transmission to vector mosquitoes, two-fold diluted pooled sera were mixed with infected packed blood cells and An. dirus mosquitoes were

Fig. 1. Anti-Pvs25-specific serum antibody responses following mucosal vaccination of CAF1 or A/J mice. (a) Total serum IgG antibody titers. These were calculated as reciprocals of the highest serum dilutions giving an optical density value of 0.1 at 415 nm. Error bars indicate the S.E.M. values obtained from six to seven mice for each immunization group. (b) Serum IgG titers by subclass. Each subclass of serum IgG was determined by mouse IgG subclass-specific antibodies and the titers were calculated as in Fig. 1a. (c) Serum IgA and IgE titers. Serum IgA and IgE antibodies were determined by antibodies specifically recognizing ␣Fc and εFc regions of mouse immunoglobulin. For IgA and IgE the antibodies titers were calculated as reciprocals of the highest dilutions of pooled immune sera, which gave an optical density value of 0.1 at 415 nm. Results are expressed as average values of two separate measurements.

allowed to feed on the mixture from a membrane-feeding apparatus. Approximately 20 fully engorged mosquitoes per experimental group were dissected about a week after blood meals and the number of oocysts in their midguts was counted. We found a significant reduction in the number of oocysts per midgut in mosquitoes given immune sera from mice immunized with Pvs25/CT (CAF1: 0.034±0.015 S.E.M.; A/J: 0) as compared with PBS (CAF1: 6.828 ± 1.164 S.E.M., P < 0.0001; A/J: 6.326 ± 0.904 S.E.M., P < 0.0001) or CT (CAF1: 10.051 ± 1.341 S.E.M., P < 0.0001; A/J: 4.971 ± 0.645 S.E.M., P = 0.004) immunized groups (Fig. 2a). We also observed a significant difference in the number of oocysts between CAF1 and A/J mice for the CT immunized group (10.051 ± 1.341 S.E.M. versus 4.971 ± 0.645 S.E.M., P = 0.0032). The observed transmission-blocking effects of the anti-Pvs25 immune

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Fig. 2. Effect of mucosal vaccination with Pvs25/CT on oocyst numbers. The data are expressed as mean number of oocyst ± standard error in a midgut of dissected mosquito. (a) Effect on oocyst numbers of two-fold diluted immune sera obtained from PBS, CT or Pvs25/CT immunized mice. (b) Effect on oocyst numbers of 1:2, 1:8 and 1:32 dilutions of sera from CAF1 or A/J mice immunized with Pvs25/CT.

sera were dependent on serum dilution as mean numbers of oocysts increased when the dilution factor increased [CAF1 (1/2: 0.034 ± 0.015 S.E.M., 1/8: 1.196 ± 0.195 S.E.M., 1/32: 2.182 ± 0.365 S.E.M.); A/J (1/2: 0; 1/8: 0.637 ± 0.103 S.E.M., 1/32: 0.981 ± 0.152 S.E.M.)] (Fig. 2b). The anti-Pvs25 immune sera were also found to be effective in reducing the oocyst positive rate (Fig. 3a). The oocyst positive rate was expressed as the percentage of oocyst positive mosquitoes out of the total mosquitoes dissected. A significant reduction in the oocyst positive rate was observed for Pvs25/CT immune sera (CAF1: 2.55%; A/J: 0%) in comparison with sera obtained from PBS (CAF1: 51.52%; A/J: 40.77%) or CT (CAF1: 55.09%; A/J: 48.54%) immunized groups. As in the case for the mean number of oocysts developed per midgut (Fig. 2b), the oocyst positive rate increased for both CAF1 and A/J immunized mice as the serum dilution factor increased [CAF1 (1/2: 2.55%, 1/8: 26.94%, 1/32: 35.41%); A/J (1/2: 0%, 1/8: 23.72%, 1/32: 28.02%)] (Fig. 3b). The effect of immune sera on the inhibition of oocyst development was also evaluated based on the complete transmission-blocking rate, defined as the percentage of volunteer patients whose blood, when mixed with the immune sera, resulted in no oocyst development in all mosquitoes dissected. A significantly higher complete transmission-blocking rate was observed for immune sera derived form Pvs25/CT immunized mice (CAF1: 76.92%; A/J: 100%) than sera derived from PBS (CAF1: 25%; A/J: 23.08%) or CT (CAF1: 33.33%; A/J: 30.77%) immunized group (Fig. 4a). Similar to both the number of oocysts developed per infected midgut, and the oocyst positive rate

Fig. 3. Effect of Pvs25/CT mucosal vaccine on oocyst positive rate. Oocyst positive rate was defined as the percentage of mosquitoes in which one or more oocysts developed from among the total mosquitoes dissected. (a) Effect on oocyst positive rate of 1:2 dilution of immune sera from CAF1 or A/J mice immunized with Pvs25/CT, CT or PBS. (b) Effect on oocyst positive rate of 1:2, 1:8 and 1:32 dilutions of sera from CAF1 or A/J mice immunized with Pvs25/CT. For both (a) and (b) numbers provided for each data point are numbers of infected mosquitoes in total numbers of mosquitoes dissected.

Fig. 4. Effects of mucosal vaccination with Pvs25/CT as measured proportion of mosquitoes without infection. Complete transmission-blocking rate is the percentage of vivax patients, whose blood when mixed with anti-Pvs25/CT immune sera yielded no oocysts among all dissected mosquitoes. (a) Complete blocking rate mediated by sera at a 1:2 dilution from CAF1 or A/J mice immunized with Pvs25/CT, CT or PBS. (b) Complete blocking rate mediated by sera at a 1:2, 1:8 and 1:32 dilution from CAF1 or A/J mice immunized with Pvs25/CT. For both (a) and (b) numbers provided for each data point are numbers of vivax volunteer patients whose blood fed to mosquitoes did not result in any oocyst development in the total number of volunteers.

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(Figs. 2b and 3b), dilution of the immune sera resulted in a reduction in the complete transmission-blocking ability, although 1/32 A/J serum showed a slightly higher blocking rate than 1/8 A/J serum [(CAF1: 1/2: 76.92%, 1/8: 38.46%, 1/32: 27.27%); (A/J: 1/2: 100%, 1/8: 38.46%, 1/32: 45.45%)].

4. Discussion In this study we demonstrated for the first time that recombinant P. vivax ookinete surface protein, Pvs25 effectively induced transmission-blocking immunity in at least two strains of mice (CAF1 and A/J) when given intranasally with CT. The mucosal immunization of the vaccine antigen induced levels of transmission-blocking serum antibodies, which were comparable with those, induced by intraperitoneal immunization with alum-absorbed Pvs25 vaccine antigen [17]. The immune sera obtained from the nasal route showed a high IgG1/IgG2a ratio similar to that from intraperitoneal route with alum as an adjuvant. These results confirmed that mucosal immunization successfully induced Th-2 subtype humoral immune responses against Pvs25, which is required for effective malaria TBV. Since the molecules are expressed by parasites mainly in the mosquito vector, one would not expect infection-induced booster effect by the vaccines. In addition, unlike live attenuated or killed vaccines, long-term immunity is generally difficult to attain by recombinant polypeptide vaccines. Moreover, to be successful, TBVs require population-level delivery with very high compliance levels. One possible strategy to overcome these problems is to develop a type of vaccine that is safe, non-invasive (painless), readily available and repeatably administrable to the public in malaria endemic areas. Development of effective mucosal TBVs may be able to overcome these problems of TBVs. In this study we used CT as a mucosal adjuvant to augment immune responses against recombinant Pvs25. However, the use of CT is not allowed in humans due to its potential enterotoxicity. Recently, a non-toxic mutant of CT and a related heat-labile toxin from enterotoxigenic Escherichia coli were developed and their efficacy as mucosal adjuvant, though lower than parental toxins, was demonstrated [19,20]. The mucosal carrier property of the non-toxic B subunit of these toxins (CTB or LTB) has also been investigated in the form of chemically or genetically conjugated vaccine antigens. These mucosal carrier molecules have recently been successfully produced in Gram-positive bacterial [21], transgenic plant [22–24] and yeast (Arakawa, unpublished data) gene expression systems. We expect that these non-toxic mucosal adjuvant and mucosal carrier systems will contribute to the development of novel mucosal TBVs. If the significant malaria transmission-blocking effects demonstrated in this study can be reproduced in human vaccine trials using mutant forms of enterotoxins or fusion proteins with a mucosal carrier like CTB or other bacterial and viral proteins

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with mucosal carrier properties, mucosal malaria TBVs could play an important part of malaria control program.

Acknowledgements This work was financially supported by a basic science research grant provided by Bio-oriented Technology Research Advancement Institution (BRAIN) and Grants-in-Aid for Scientific Research (14770111, 13576007, 14570215) and Scientific Research on Priority Areas (13226087, 14021082) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant for Research on Emerging and Re-emerging Infectious Diseases (H12-Shinkou-17) from the Ministry of Health, Labor and Welfare, Japan. This work also received financial support from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). We thank Jeeraphat Sirichaisinthop and staff members of the Office of Vector Borne Disease Control 1, Saraburi, Thailand for their generous support to set up the field sites, and staff members of Department of Entomology, AFRIMS, Bangkok, Thailand for their technical assistance. We also thank the Malaria Vaccine Initiative at PATH for their help with the TBV development project. References [1] Mendis K, Sina BJ, Marchesini P, Carter R. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 2001;64:97–106. [2] Breman JG. The ears of the hippopotamus: manifestations, determinants, and estimates of the malaria burden. Am J Trop Med Hyg 2001;64:1–11. [3] Tsuboi T, Cao YM, Hitsumoto Y, Yanagi T, Kanbara H, Torii M. Two antigens on zygotes and ookinetes of Plasmodium yoelii and Plasmodium berghei that are distinct targets of transmission-blocking immunity. Infect Immun 1997;65:2260–4. [4] Carter R. Transmission blocking malaria vaccines. Vaccine 2001;19:2309–14. [5] Tsuboi T, Tachibana M, Kaneko O, Torii M. Transmission-blocking vaccine of vivax malaria. Parasitol Int 2003;52:1–11. [6] Kocken CH, Jansen J, Kaan AM, et al. Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum. Mol Biochem Parasitol 1993;61:59–68. [7] Williamson KC, Criscio MD, Kaslow DC. Cloning and expression of the gene for Plasmodium falciparum transmission-blocking target antigen, Pfs230. Mol Biochem Parasitol 1993;58:355–8. [8] Carter R, Graves PM, Keister DB, Quakyi IA. Properties of epitopes of Pfs 48/45, a target of transmission blocking monoclonal antibodies, on gametes of different isolates of Plasmodium falciparum. Parasite Immunol 1990;12:587–603. [9] Read D, Lensen AH, Begarnie S, Haley S, Raza A, Carter R. Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement-fixing. Parasite Immunol 1994;16:511–9. [10] van Dijk MR, Janse CJ, Thompson J, et al. A central role for P48/45 in malaria parasite male gamete fertility. Cell 2001;104:153–64. [11] Kaslow DC, Bathurst IC, Lensen T, Ponnudurai T, Barr PJ, Keister DB. Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum. Infect Immun 1994;62:5576–80.

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