Malaria vaccines for travelers

l 3 R family, members of government missions, employ- members of the armed forces returning to the ing to malaria cases resulting from international these persons visited malaria-endemic areas, then combinations for preventing malaria. Although some, such as chloroquine, are now less useful failure to prevent malaria in travelers cannot be What, then, is responsible for the ongoing Travel Medicine and Infectious Disease (2004) 2, 193–210 travel are those resulting from within-country attributed to the lack of effective drugs. Soviet Union following deployment to Afghanistan, a country where the risk is not particularly high, imported 7683 cases of P. vivax malaria during the 9-year military engagement in that country.6 Add- due to the development of resistance by the malaria parasite, several, including mefloquine, doxycyline and atovaquone/proguanil, are highly efficacious for prophylaxis in most areas. Thus, the ees of non-government organizations, missionaries, immigrants, refugees and military personnel, indi- cating that malaria is a potential risk for nearly every type of traveler. It has been a particularly serious problem for the military: for example, approximately 10 million international tourists were at risk in 2003 while traveling to the areas listed. Travelers remain at risk despite the existence for over 50 years of highly effective drugs and drug sons visiting countries of birth to see friends and million; to Central America, 4.9 million.9 If 10–15% of REVIEW Malaria vaccines for trave Thomas L. Richie* Naval Medical Research Center Malaria Program, 50 MD 20910-7500, USA Received 7 July 2004; accepted 7 July 2004 Rationale for a travelers’ malaria vaccine Malaria is the most important infectious threat facing travelers to malaria-endemic areas of the tropics, if one considers both risk and potential severity. The problem is illustrated by the many travelers diagnosed with this protozoal infection after returning home from visits to malaria-ende- mic countries: in the USA, travelers account for 1000–1500 imported cases per year, resulting in 8 deaths in 2002;1 in Canada, 300–1000 cases per year;2 in Europe, roughly 8000,3 with the number of imported cases to France reaching 7500 in 2000;4 in Japan, 100–150 cases per year.5 These infections occur among tourists and business travelers, per- ers obert Grant Avenue, Silver Spring, travel, for example residents of malaria-free capital cities traveling to rural locations to visit friends or family. Areas where a traveler can acquire malaria include the tropical portions of Africa, Asia, South America, Central America, Oceania and the Middle East (Fig. 1) and also subtropical areas, such as in parts of China7 and Afghanistan.8 The number of travelers at risk has increased during the last decade in association with the globalization of business and tourism. The World Tourism Organization estimated that the number of international tourist arrivals to Southeast Asia in 2003 was 35.7 million; to sub-Saharan Africa, 19.8 million; to Oceania, 9.4 million; to South Asia, 6.8 www.elsevierhealth.com/journals/tmid the parasite and the vector all contribute to 1477-8939/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tmaid.2004.07.004 * Tel.: C1-301-319-7584. E-mail address: [email protected]. toll of malaria in travelers? The human host, the latent stages and there is often uncertainty amongst health care providers regarding when to use primaquine and how to dose it appropriately, and also there is often poor compliance with re 1 T.L. Richie194 the problem. In order to be protected, the traveler must visit a physician’s office, pharmacy or health care facility prior to travel, obtain a drug that is effective in the area to be visited, accept the complexity and inconvenience of drug admin- istration, tolerate often troublesome side effects,10 and foot the expense (Fig. 2). These obstacles are accentuated for within-country travelers such as refugees for whom it may be Figu difficult to find or afford health care. Evidence for the disconnect between travelers and medical care is illustrated by the results of knowledge, attitude and practice surveys administered to international travelers in the departure lounges of major airports around the world. Recent surveys reveal that generally !50% of international travelers to malarious areas are carrying anti- malaria medications.11–15 On the parasite side, there is the problem of drug resistance, necessitating careful choice of drugs for prophylaxis or treatment that are appropriate for the geographical area visited. In addition, there is the problem that clinical presentations often occur weeks to months after returning home,16 when the patient is back in a non-endemic area served by a health system unfamiliar with the clinical manifes- tations, diagnosis and treatment of this infection.17 In cases of infection with P. vivax or P. ovale, both of which exhibit resting stages in the liver that can lead to a relapse of infection weeks to months after the primary infection is cured, supplemental treatment with primaquine may be required to kill Figure 2 Although no malaria vaccines are currently on the market, an estimated cost of $250 for an immuni- zation series seems reasonable. Comparing this to the actual retail costs of chemoprophylactic drugs currently available in the USA* indicates that a vaccine could result in significant cost savings, depending upon which drug is chosen for comparison. Savings increase in proportion to the length of the trip (assuming that the cost of a vaccine would be independent of the length of the trip). A full cost equation should also include the expense of visiting a travel clinic and the effectiveness of the preventive strategy (in that acquiring malaria would clearly add significantly to the cost of any modality). (*Provided by Giant Pharmacy, Clarksville, Maryland, May 2004; Brand name drugs capitalized, generic drugs lower case. Vibramycin, doxycycline; Lariam, mefloquine; Aralen, chloroquine; no generic form of Malarone is available.) administration. This problem was experienced by US Marines redeploying to the USA from Somalia in 1993; there were 97 cases of P. vivax, nearly all of which could have been prevented with a properly administered course of primaquine.18 The vector itself interferes with effective drug prophylaxis because of its nocturnal and inapparent bite. Not aware of the presence of the vector, and thinking that the risk of malaria is small, the traveler may be lured into non-compliance with prophylaxis, especially if experiencing side effects. It is fairly common for travelers returning with malaria to claim that they did not receive any mosquito bites! Recognizing these problems, medical commu- nities concerned with travelers’ health have struggled to enhance understanding and compli- ance with malaria prevention. For example, in North America both the US Center for Disease chemoprophylaxis (Fig. 2), vaccination would represent a one-time expense with the enviable property ‘fire and forget,’ meaning that once administered, no further thought or action would be required for malaria prevention during the period of protection afforded by the vaccine. Even if efficacy as measured in controlled trials lagged behind that of drug prophylaxis, the real world effectiveness would likely be superior due to automatic compliance following vaccine administration. Travelers would be saved the inconveniences of carrying drugs, tolerating side effects, and continuing administration after travel has ended. The goal of vaccinating travelers against malaria infection complements the important goal of protecting the hundreds of millions of persons living in endemic areas who are threatened by malaria on a daily basis throughout their lives. One of the four ebs ovi we tm tion tio ctio So ler th m alt Malaria vaccines for travelers 195 Control and Prevention and Health Canada maintain websites and hotlines for physicians and patients, and in addition several private organizations pro- vide excellent reference services for physicians that detail malaria risks throughout the tropics and recommend chemoprophylaxis that is appropriate for local drug resistance patterns (Table 1). Personal protective measures, including bednets, repellants, long-sleeved shirts, etc. are also avail- able to assist the traveler in preventing mosquito bites, although the use of such measures may be curtailed due to inconvenience and discomfort in hot tropical climates. An effective malaria vaccine would eliminate most of the obstacles to malaria prevention, excepting only thepre-travel visit(s) to receive thevaccine from a health care professional. As with vaccines against other infectious diseases, overall benefits for the traveler and for public health would be immense. Even if purchase costs were similar to those of Table 1 http://www.who.int/ith/: World Health Organization w publication ‘International Travel and Health’ and also pr http://www.cdc.gov/travel/: Center for Disease Control for travelers including information on malaria risk http://www.hc-sc.gc.ca/pphb-dgspsp/tmp-pmv/index.h health recommendations for travelers including informa http://www.nlm.nih.gov/medlineplus/malaria.html: Na of extensive information about malaria, including instru http://www.astmh.org/scripts/clinindex.asp: American travel clinics around the world where international trave http://www.travmed.com/: World Medical Guide link on recommendations for travelers, including information on https://www.tripprep.com: Provides country-specific he on malaria risk. Requires registration (no cost) species of human malaria, P. falciparum, kills 0.7–2.7 million people per year,19 the majority of these children living in sub-Saharan Africa, but including many adults, particularly in areas where transmission is more sporadic and there is little clinical immunity. Pregnant women, particularly primagravidas, are especially vulnerable. There are 400–900 million new clinical cases per year in African children alone.19 Developing a malaria vaccine to protect travelers on the one hand, and those living in endemic areas on the other, is a public health imperative. While it would be ideal to develop a vaccine that prevented malaria infection altogether in residents of endemic areas, it is felt by many experts that the high inoculation rate experienced in tropical areas, which can exceed 1000 infectious mosquito bites per person per year, will overwhelm any vaccine. Because long-term residents do eventually acquire a degree of clinical immunity to the infection (see below), many argue that goals for malaria ite provides a detailed chapter on malaria from the des country-specific information on malaria risk bsite provides country-specific health recommendations l: Health Canada website provides country-specific on malaria risk nal Institutes of Health website provides links to sources nal material ciety for Tropical Medicine and Hygiene website lists s can obtain medical care is page provides country-specific health alaria risk h recommendations for travelers including information vaccines designed for residents of endemic areas malaria. Data from sequential challenge infections with different malaria species, given for the purpose of malaria therapy in the 1950s,20 and should be amendable to vaccination, because there T.L. Richie196 from cross-species challenge experiments using the irradiated sporozoite vaccine in the 1970s (immunizing with one species of malaria and challenging with another),21 indicate that protec- tion against several species by a single vaccine may be difficult to achieve. Current efforts at develop- ing a vaccine are directed primarily toward P. falciparum, the species responsible for the majority of cases acquired by travelers and the large majority of malaria-related deaths in endemic areas, with P. vivax second on the list. It is assumed that if we can succeed at making vaccines against these two species, it should be relatively easy to transfer the same vaccine technologies to the less pathogenic P. malariae and P. ovale. Why a malaria vaccine is feasible In the world of vaccinology, it is often stated that ‘all the easy vaccines have been made,’ leaving behind the problem pathogens. Common to many of these agents is the characteristic of persisting chronically in the human host despite the presence of comp- lement, NK cells, antibodies, cytotoxic T cells, cytokines and all other arms of the immune system. Malaria exemplifies this ability to exist long term in the human host (recent estimate for the duration of P. falciparum, the ‘shortest duration’ species, are on the order of 2 years or longer),22 confronting vaccine developers with a conundrum: how can a vaccine stimulate immunity to eliminate a parasite already able to circumvent all facets of the human immune response? However, malaria, it turns out, could be more modest than preventing infection. If, for example, a vaccine could reduce the morbidity associated with the first several infections experi- enced during early childhood without necessarily preventing those infections, thereby accelerating the transition from clinically naı¨ve to clinically resistant, it would be of great benefit. This concept has led to the differentiation of two types of malaria vaccines: anti-infection, which would pri- marily target the traveler or other individuals with limited exposure and a need to completely prevent the infection, and anti-disease, designed for those experiencing prolonged, intense transmission and for whom the goal of vaccination is to hasten the transition to acquired clinical immunity. At this point it appears that different vaccines may be needed for the four species of human exist two models proving that the immune system can indeed be induced to kill the parasites. Liver stage immunity model. Malaria infections begin when the sporozoite stage enters a capillary during the nocturnal feeding of the female Ano- pheles mosquito. The sporozoite is carried by the blood stream until a pass through the liver allows invasion of hepatocytes and establishes the liver or ‘pre-erythrocytic’ stages of the infection. The single sporozoite undergoes asexual multiplication within the hepatocyte and 5 days to many months later (depending on the species and whether it is a standard liver stage parasite or the latent stage that occurs with P. vivax and P. ovale) up to 30,000 merozoite forms burst from the ruptured hepato- cyte and re-enter the blood stream (Fig. 3). During this initial, clinically silent incubation period, parasites developing within host hepato- cytes may be effectively killed by cell-mediated immune responses induced by immunization with irradiated sporozoites. In this first vaccine model, volunteers are immunized via the bites of 1000–2000 malaria- infected, irradiated mosquitoes, in several immu- nization sessions over the course of 3–4 months. The radiation-damaged sporozoites localize in liver cells, but they are unable to complete liver stage development, transform into merozoites and re-enter the blood. This aborted liver stage infection appears to present parasite antigens or epitopes to the immune system via major histo- compatibility complex (MCH) class I molecules expressed on the surface of infected hepatocytes.23 These antigens induce an immune response that is sterilizing to subsequent challenge with intact sporozoites (O90% efficacy against both homolo- gous and heterologous strain challenges).24 The protective efficacy of the irradiated sporozoite vaccine has been demonstrated for both P. falci- parum and P. vivax malaria, as well as for murine25 and simian26 malaria species. At this point, protec- tive efficacy has only been demonstrated with regard to experimental sporozoite challenge, but anecdotal information regarding immunized volun- teers who have traveled to malaria-endemic areas without taking chemoprophylaxis indicates that vaccination also prevents natural infection. The highly efficacious irradiated sporozoite vaccine model suggests two approaches for devel- oping a vaccine. One is to mass produce irradiated sporozoites as an attenuated organism vaccine to be administered via syringe. A biotechnology company, Sanaria, Inc., in Gaithersburg, Maryland, USA, is attempting to develop the irradia- ted sporozoite vaccine for manufacturing and to utilize this information to select protective Malaria vaccines for travelers 197 and licensure, and is systematically addressing the many technical barriers that hinder the develop- ment of such a vaccine, including how to achieve sterility, purity, cryostorage and syringe adminis- tration.27 A second approach is to identify the antigens and immune responses underlying irra- diated sporozoite-induced protection, and aim to reproduce the antigenic stimulus with a multi- component (‘antigen cocktail’) sub-unit vaccine. Currently, the US Navy Malaria Program in Silver Spring, Maryland, maintains a protocol for irra- diated sporozoite immunization of human volun- teers via mosquito bite, and actively seeks to identify the underlying mechanisms of protection Figure 3 One reason for the difficulty of making a malaria vaccine is that at each stage of the life cycle, patterns of protein expression change, meaning that the immunity induced by a vaccine will be stage-specific. If a few parasites escape the immunity induced by a pre- erythrocytic stage vaccine, for example, they will grow in the blood unchecked. A second reason is that different forms of immunity are needed to destroy different parasite stages, will cell-mediated immunity particularly important for destroying developing liver stage parasites. Thus a vaccine may need to induce both antibody and cellular responses to multiple stages simultaneously. (Modified with permission from Richie TL, Saul A. Progress and challenges for malaria vaccines. Nature 2002;415:694–701.) antigens or epitopes and to combine these antigens as a complex, multi-component sub-unit vaccine that mimics irradiated sporozoites in the breadth and potency of the immune responses it induces (see Genomes-to-vaccines, below). Blood stage immunity model. The debilitating clinical manifestations of malaria infection result from the cycle of erythrocyte invasion and destruc- tion caused by merozoite stages that are released from the liver. These parasites invade and then multiply within erythrocytes, consuming hemo- globin and plasma-derived nutrients such as glucose while multiplying 5–10-fold during a maturation period within the erythrocyte that lasts 48–72 h, depending upon the parasite species. The blood stage cycle is particularly pathogenic when the infection is due to P. falciparum, which causes infected erythrocytes to adhere to endothelial cells and also to uninfected erythrocytes, leading to sequestration of the parasites and blood cells and disruption of microcirculatory flow in the brain, uterus and other organs. Because many malaria antigens are presented to the immune system, particularly the spleen, during blood stage infec- tion, residents of malaria-endemic areas gradually develop antibody responses capable of limiting both parasite multiplication and the severity of clinical disease. These immune responses may inhibit merozoite invasion of erythrocytes by attacking the merozoite surface or the apical organelles responsible for invasion, or may cause the destruc- tion of infected erythrocytes by binding to malaria proteins expressed on the surface of erythrocytes, or they may neutralize toxic malarial compounds released during erythrocyte rupture. The effectiveness of the humoral immune response to blood stage infection has been demonstrated by the reductions in parasitemia and the clinical improvement observed in children stricken with malaria who are given immunoglobu- lin transfusions derived from adult residents of malaria-endemic areas.28–30 These antibodies appear capable of transferring the clinically pro- tected state that follows repeated exposure to malaria. This clinical immunity, termed naturally acquired immunity (NAI), does not prevent malaria infection or transmission, but does restrict both parasite density and infection-related morbidity.31 NAI constitutes the second model, indicating the feasibility of a vaccine. As with the irradiated sporozoite vaccine, the list of antigens recognized by these protective immune responses remains largely unknown, but, in theory, it should be possible to design a sub-unit vaccine capable of eliciting the same immunological responses, thereby protecting against blood stage infection. and also the goal of protecting those residing in endemic areas. meet the needs of travelers, the vaccine has T.L. Richie198 Protecting against a multistage, heterogeneous parasite The most frequent approach to vaccine design has been to focus on single malarial proteins or individual epitopes derived from single malarial proteins. These include well-characterized antigens from the pre-erythrocytic stages, such as the circumsporozoite protein (CSP), which is the dominant surface molecule of sporozoites, and also from the erythrocytic stages, such as merozoite surface protein 1 (MSP1) or the apical membrane antigen 1 (AMA1).33 The most effective of these single antigen vaccines, called RTS,S/AS02A, is based on CSP. RTS,S/AS02A can protect between 42% and 86% of immunized adult volunteers against experimental, homologous-strain, sporozoite chal- lenge for several weeks34,112 and in a key field efficacy trial conducted in The Gambia, it was shown to protect 34% (95% CI:8% to 53%; PZ0.014) of semi-immune African adults against natural, heterologous-strain, sporozoite challenge (as measured by new-onset parasitemia), over one malaria transmission season.35 While it is unlikely that this level of efficacy will be sufficient to While the irradiated sporozoite vaccine relies primarily upon cell-mediated immune responses directed against liver stage parasites, NAI relies primarily upon antibody-mediated immune responses directed against blood stage parasites, although antibody dependent cellular mechanisms may also play a role in blood stage immunity.32 These two models, immunization with irradiated sporozoites and NAI, indicate the feasibility of anti- infection (liver stage) and anti-disease (blood stage) vaccines, respectively. It is important to note, however, that a liver stage vaccine could, in theory, reduce disease severity and thereby help residents of endemic areas, by reducing liver stage burden and the size of the parasite inoculum into the blood, a prediction based on the reduction in morbidity and mortality associated with the use of insecticide impregnated bednets (which also reduce parasite inoculum), and, likewise, a highly effective blood stage vaccine could prevent all manifestations of infection and thus protect tra- velers. Because of the broad overlap in benefits between the two types of vaccines, it may turn out that a liver stage, a blood stage or a combined stage vaccine could meet the goal of protecting travelers the potential to reduce the frequency and severity of clinical malaria episodes in children by reducing the initial sporozoite and liver stage burden and thus the infectious inoculum of liver stage mer- ozoites released into the blood, and is currently being developed for that purpose. Other vaccine developers believe that a multi- component vaccine will ultimately be required, based on the supposition that the effectiveness of both the irradiated sporozoite vaccine and NAI results from summed immune responses against multiple antigenic determinants. This rationale for the polyantigen approach appears particularly cogent when one considers that the early stages of the life cycle take place within hepatocytes and therefore are not accessible to antibodies, and would be most effectively contained by cell- mediated immunity. Because of the human leuko- cyte antigen (HLA) restriction of cell-mediated immunity, protective immune responses in volun- teers immunized with irradiated sporozoites likely recognize a different set of genetically restricted epitopes derived from several different antigens in each protected individual, depending upon HLA haplotype. According to this reasoning, it may be possible to induce effective liver stage immunity in the general population only by including a variety of antigens within a vaccine, in order to provide ‘something for everyone,’ thereby achieving broad population coverage.36 A multivalent vaccine may also be needed in order to protect against the antigenic heterogen- eity of the malaria parasite. First, there may be multiple, antigenically variant alleles for a given gene, as exemplified by the gene encoding MSP1.37, 38 As a second reason for parasite heterogeneity, some antigens are polymorphic due to the existence in the genome of multiple, variant copies of the same gene. For example, 50 odd genes encode the immunodominant erythrocyte membrane protein 1 of P. falciparum (EMP1), and members of this gene family are transcribed and translated in sequence during the course of an infection, leading to antigenic variation and continuous side-stepping of acquired humoral immunity even if the individual is infected with a single, genetically homogeneous clone.39 It is thought that NAI may depend upon a repertoire of responses to all the variants of EMP1, which develop gradually after multiple infections, perhaps complemented by the gradual acquisition of variant-transcendent responses that cross-pro- tect among the EMP1 variants.40,41 The most ambitious vaccine developers anticipate a multivalent vaccine derived from sporozoite, liver and blood stage antigens in combination. The liver stage component of the vaccine would be designed to induce primarily cell-mediated immune responses capable of destroying most developing parasites during the asymptomatic hepatic stages, and the sporozoite and blood stage components would induce primarily antibody-mediated immune responses capable respectively of neutralizing sporozoites prior to liver cell invasions and of restricting blood stage multiplication in case parasites break past the defenses in the liver. This tiered approach is illustrated in Fig. 4. A small number of combined pre-erythrocytic/erythrocytic vaccines of this sort have been tested,42,43 as well as several combined sporozoite/liver stage vaccines. The development of multi-antigen vaccines lags that of single antigen vaccines, but for all the reasons given, may be critical to achieving the high levels of efficacy needed to protect travelers against malaria. transmission-blocking vaccine would not directly Figure 4 In this figure, the solid line (A) represents the development of the parasite in an unvaccinated host: Malaria vaccines for travelers 199 hepatocytes are infected according to the size of the sporozoite inoculum, and rupture after 5 days, releasing merozoites into the blood. These multiply logarithmi- cally, increasing in numbers roughly 10-fold every 2 days (P. falciparum), until there are enough parasites to cause clinical symptoms. A pre-erythrocytic stage vaccine (dotted lines, B) will reduce liver stage burden. If all parasites are killed, there will be no infection. If a few parasites get through this first tier of defense, the overall clinical severity of the infection may be reduced because the immune system, even if not primed by a vaccine, has more time to respond to the blood stage infection, causing it to peak at a lower density. A blood stage vaccine will decrease the rate of growth in the blood (dashed lines, C), preventing disease even if not the infection. Actual parasite population growth curves (here expressed as the number of infected host cells) are not actually smooth as indicated by the figure. (Modified with permission from Perlman P, Troye-Blomberg M (eds): Malaria Immunology. Chem Immunol. Basel, Karger, 2002:80:262–86.) assist the vaccinated individual, it might signifi- cantly reduce malaria transmission in a given area if a large proportion of the population was immu- nized, and in the case of liver or blood stage vaccines, parasite variants escaping the vaccine- induced immune responses to liver or blood stage antigens might be prevented from spreading if a transmission-blocking component was included in the vaccine. Blocking transmission is probably less important for travelers’ vaccines, because the majority of malaria infections acquired by inter- national travelers become manifest after the traveler returns to his or her country of origin and thus are generally not transmitted. On occasion, however, there are mini-epidemics of introduced malaria follow the arrival of a malaria-infected traveler into a non-endemic area,47,48 and these could be prevented in the case of vaccine-resistant parasites by the addition of transmission-blocking components to a travelers’ vaccine. Genomes-to-vaccines There are two key components to a vaccine: the right antigens, meaning antigens that are both safe for administration to humans and capable of inducing protective immune responses, and the right vaccine delivery system, meaning a way to formulate the antigens and administer the vaccine so that it induces the desired protective responses. Until recently, vaccine programs have focused more on how to formulate the antigen rather than questioning the intrinsic protective potential of the antigen itself. The development pathway leading to RTS,S/AS02A is an example of this: CSP was selected as the antigen, and then many years of effort expended to identify a formulation that was Transmission-blocking vaccines The last stage of the parasite in the human host is the gametocyte, which also develops inside an erythrocyte but in this case does not multiply or cause rupture of the host cell. Rather, the gametocyte-infected erythrocyte circulates in the blood stream attendant upon ingestion by a mosquito. Some vaccine developers, such as the US National Institutes of Health Malaria Vaccine Development Branch, are constructing vaccines based on the antigens expressed by gametocytes or later stages of the parasite that are found in the mosquito.44,45 Antibodies to these antigens, if ingested during the mosquito blood meal, can block development within the insect vector and thus interrupt transmission.46 Although a sufficiently immunogenic. This focus on vaccine formulation and delivery stems from the fact there has been no alternative; the technology simply has not existed to allow a systematic search for new antigens. However, with the publication of the P. falciparum genome in 2002, researchers are now able to explore various approaches for the systema- tic identification of the antigens and epitopes that are the target of protective cellular and humoral immune responses to malaria, utilizing the entire 23 megabase P. falciparum genome as the starting point, rather than the current list of available antigens, which represents less than 1% of the total repertoire.49 The annotated genomic sequence of P. falci- parum encodes more than 5300 putative proteins, many with multiple open reading frames (ORFZ uninterrupted sequence of DNA that can be trans- lated into a protein).50 A centralized database called PlasmoDB (www.plasmodb.org) has been established by David Roos and colleagues at the University of Pennsylvania to facilitate access to the P. falciparum genomics sequence as well as to relevant data, such as information on stage-specific protein expression (‘proteomics’),51,52 gene expression profiles,53 bioinformatic software and predictive algorithms.54 PlasmoDB forms the start- ing point for an initial process of computer-based antigen selection and prioritization, which involves characterizing the ORFs contained in the genome according to specific criteria in order to select the ORFs associated with a parasite stage (for example, ORFs encoding proteins contained in the sporozoite proteome),51 and/or those possessing other desired characteristics, such as sequences suggesting sur- face anchoring or host cell binding. This initial, computer-based step generates a prioritized list of antigens that may contain several hundred candi- dates, a more workable number for screening than 5300 (Fig. 5). da s (d chn de r m re id D as his hi pat por age T.L. Richie200 Figure 5 Methodologies for utilizing genomic sequence vaccines require the complex interplay of bioinformatic (denoted by solid arrows), many of which involve novel te (see text for descriptions). Open reading frames (ORF) are by solid boxes. There are multiple alternative pathways fo example, either transcriptionally active polymerase chain encode as expressed in cell-free systems, or circular plasm transfect or transduce antigen presenting cells (APCs) such to screen either sera or cells from protected volunteers. T sequence information to develop malaria vaccines,’61 w Research Center Malaria Program. MHC, major histocom peripheral blood mononuclear cell; ISI, inhibition of s development assay; GIA, growth inhibition assay (blood st ta to identify new candidate antigens for use in malaria enoted by dotted arrows) and molecular manipulations ologies that are amenable to high-throughput processing noted by open boxes, and their expressed protein product any of the steps, only some of which are illustrated. For action (TAP) fragments (linear DNA) or the proteins they NA generated by recombinatorial cloning, can be used to dendritic cells (DC); similarly, protein arrays can be used figure is derived from the review ‘Utilization of genomic ch outlines approaches explored by the Naval Medical ibility complex; HLA, human leukocyte antigen; PBMC, ozoite invasion assay; ILSDA, inhibition of liver stage ); IFAT, immunofluorescent antibody test. The next step is to utilize an appropriate plat- of the antigen (see below). If adapted to robotic Malaria vaccines for travelers 201 form technology for expressing the theoretical protein(s) encoded by the selected ORFs. This may involve traditional cloning methods, in which the ORF is amplified via polymerase chain reaction (PCR) utilizing specific primers and then ligated into the expression cassette of a suitable DNA plasmid. The recombinant plasmid, a double-stranded ring of DNA, serves as a vector for carrying the malaria gene and allowing its expression in cells that are transfected by the plasmid. Efficient cloning sys- tems have been developed (e.g. Gatewaye system, InVitrogen Inc.)55 that can utilize a 96 well format and allow high-throughput processing. Recombi- nant plasmids can be used to immunize mice, providing antibodies with specificities recognizing the novel antigen. Antibodies so generated may permit an assessment of antigen function (e.g. inhibition of sporozoite invasion (ISI assay), inhibition of liver stage development (ILSDA assay), or blood stage growth inhibition (GIA)) and may also allow confirmation of stage-specificity and indicate subcellular localization (e.g. via immunofluores- cence antibody testing).56 In addition, recombinant plasmids can be used to transfect standard cell lines or antigen presenting cells (such as dendritic cells) or lymphocytes derived from malaria-immune hosts for immunoscreening to see the protective value of the encoded protein (see section on Immunoscre- ening below). However, although the recombina- torial cloning approach exemplified by the Gatewaye system is efficient compared with traditional cloning methods, it is nevertheless labor-intensive and in addition it is difficult to be certain that the immune responses generated by immunization with the recombinant DNA plasmids in mice will generate immune responses with the required specificities and sufficient intensity to elucidate the potential protective efficacy of the antigen in ISI, ILSDA, GIA or other assays or will truly anticipate how protective the antigen will be in humans. Alternative approaches have been developed that bypass the cloning step. One example is transcriptionally active PCR (TAP), which generates directly from the ORF, via a nested PCR reaction, amplified products with functional eukaryotic or prokaryotic promoter and terminator sequences attached, that render the TAP product directly transcriptionally active in vivo or in vitro. Like recombinatorial cloning, TAP can be adapted for high-throughput genomic screening.57 TAP products can be used in cell-free transcription/translation systems to generate the candidate protein and can also be used to transfect dendritic cells, allowing immunoscreening to assess the protective potential work-stations, the protein products of TAP frag- ments can be used to generate protein chips, or placed into 96 well plates for testing against the sera or cells from malaria-protected individuals. There follows the most difficult aspect of the genomes-to-vaccines approach: how to functionally assess candidate antigens, once they have been expressed by plasmids or procedures such as TAP. Ideally, we require an assay that measures the ability of a candidate antigen to induce protective immune responses in humans, but there is no known high-throughput technique for doing this. Also, at the present time, in vitro surrogate markers of protective immunity against malaria have not been defined. As mentioned above, host–parasite inter- actions mediated by surface-accessible antigens may be assessed by inducing antibodies in mice which are then tested for inhibitory activity in P. falciparum ISI (sporozoite stage), ILSDA (liver stage) and GIA (blood stage) assays, but these are not high-throughput assays and do not assess the cellular responses that are likely key to the pre-erythrocytic stage vaccine needed to protect travelers. One approach to assessing functional protection is to undertake a parallel genomes-to-vaccines program utilizing an experimental model in which protection can be measured. This approach has been pioneered for malaria by the US Navy Malaria Research Program using the P. yoelii sporozoite challenge in BALB/c mice.58 The P. yoelii genome is available on PlasmoDB and a prioritized list of candidate antigens can be generated in the same way as for P. falciparum.59 Large numbers of candidate P. yoelii ORFs can be cloned using Gatewaye technology, the resulting plasmids pooled for immunization protocols, and the pro- tective effect of pooled immunizations assessed by P. yoelii sporozoite challenge.60 Plasmid pools inducing protection against challenge can be de-convoluted by evaluating their constituent plasmids one by one. Promising ORF’s can be compared (‘blasted’) against the P. falciparum genome to search for P. falciparum ORFs with a similar sequence encoding proteins of presumably similar function (orthologues), and then these P. falciparum antigens can be selected for further development. While this is a useful approach, it could be difficult to assess protection using pools of plasmids, since there may be inhibitory effects among plasmids and also pooling reduces the effective dose of each component, allowing potentially protective responses to be missed. In addition, it is not clear that orthologues will be readily identified in the P. falciparum genome, and if orthologues are present, there is no guaran- also be obtained from life-long residents of ende- example, while it was relatively easy to protect mice with synthetic peptides derived from the CSP,62,63 numerous formulations of this antigen T.L. Richie202 tee that what is protective in mice will also be protective in humans. On the other hand, pooling may reveal protective immune responses due to the combined effects of multiple antigens, protective responses which might not otherwise be identified if antigens were examined individually, and which could mirror the protective effect of the irradiated sporozoite vaccine, which we believe relies on the summed protective effects of many antigens. Immunoscreening. Probably the most effective way to assess the protective potential of candidate P. falciparum antigens is through immunoscreen- ing. As previously described, it is possible to collect peripheral blood mononuclear cells (PBMCs) and serum from protected individuals, such as those immunized with the irradiated sporozoite vaccine. It is presumed that the entire repertoire of protective sporozoite- and hepatic stage-induced T cell and B cell specificities is represented in these individuals, excluding only those malarial antigens not yet expressed at the point of developmental arrest. Lymphocytes from these volunteers should recognize the subset of protective P. falciparum proteins expressed by irradiated sporozoites on the surface of infected hepatocytes (enabling cellular immunoscreening), and similarly their antibodies should recognize the subset of P. falciparum antigens expressed on the surface of sporozoites or infected hepatocytes, or antigens released into the blood and lymph (enabling humoral immuno- screening). Because the level of protection against sporozoite challenge in a typical cohort of volun- teers immunized with the irradiated sporozoite is less than 100%, it is possible to assess PBMCs or sera from protected and non-protected individuals in parallel. Thus, the protective potential of a candidate antigen or epitope can be demonstrated by differential stimulation of recall responses by protected and unprotected volunteers, when plas- mid-transfected or protein fragment-transduced or peptide-stimulated APCs are co-cultured with lym- phocytes derived from the volunteers, followed by quantification of interferon-gamma or other cyto- kine induction using the ELISPOT assay or other cellular assays such as intracellular cytokine stain- ing by fluorescence activated cell sorting, or lymphoproliferation. Similarly, the protective potential of a candidate blood stage antigen or epitope can be demonstrated by differential bind- ing of antibodies in any suitable high-throughput assay, such as protein arrays produced by cell-free expressions, again comparing protected to unprotected volunteers. In a parallel process to that using reagents derived from irradiated spor- ozoite-immunized volunteers, sera and cells can were tested over many years in humans without success. The Walter Reed Army Institute of Research Malaria Vaccine Program records over a dozen clinical trials utilizing this antigen without achieving significant levels of protection. It was not until the CSP was combined with hepatitis B surface antigen, expressed in yeast as a fusion protein and formulated in the experimental oil-in-water adjuvant ASO2A containing the immunostimulants monophosphoryl lipid A and the saponin QS21, that consistent protection was achieved against experimental sporozoite challenge in humans (RTS,S/AS02A vaccine). Experience has been similar with genetic (DNA-based) vaccines, in which the genetic material encoding the malarial antigen is injected, the genes are taken up by host cells, the antigen is expressed by host ribosomes and an immune response induced. While it was relatively easy to protect mice against malaria using DNA plasmids64 or recombinant poxvirus,65 no protection mic areas who possess clinical immunity to blood stage infection for use in humoral or cellular immunoscreening of blood stage antigens to ident- ify those associated with protection. Data from a functional immunoscreen of pre- erythrocytic stage candidate antigens using PBMCs from irradiated sporozoite-immunized volunteers are presented in Fig. 6. It is notable that several of the new antigens tested by Doolan et al. stimulated levels of interferon-gamma production greatly exceeding those induced by traditional antigens such as CSP.61 This lends credence to the claim that the genomes-to-vaccines approach will yield superior antigens. In summary, the promise of the genomes-to-vaccine effort lies in the potential to identify the most immunogenic and protective antigens and epitopes in the P. falciparum genome, rather than relying on the small and possibly inferior subset that have been identified during the past 30 years using traditional approaches. Vaccine delivery systems Identifying the best antigens is only half of the challenge for vaccine developers; a delivery system must also be identified that can induce the desired immune responses in humans. Historically, this has been a frustrating endeavor: delivery systems that are protective in animal models have generally not been protective when tested in humans. For Malaria vaccines for travelers 203 was seen in humans immunized with a five-plasmid cocktail of pre-erythrocytic stage antigens (T. Richie, unpublished data). Thus a major effort in the realm of malaria vaccine development has been to improve vaccine delivery systems to achieve greater potency and protection in humans. For protein-based technologies, this has included embedding antigens in virus-like particles66 or PLGA microparticles,67 conjugating them with proteins such as tetanus toxoid to provide T cell help,68 assessing new adjuvants designed to favor antigen- specific Th-1 cellular responses (i.e. production of interferon-gamma),69,70 modifying codon usage and manufacturing processes to optimize folding and tertiary structure,71,72 combining epitopes into multiple antigen peptide constructs73 or epitope Figure 6 ImmunoSense algorithms* were employed to gen CD4C T cell epitopes derived from a number of putative P. f characterized P. falciparum candidate antigens previou PfTRAP/SSP2, PfLSA1, PfEXP). Peptide pools from each an recall interferon-gamma responses from peripheral blood sporozoite-immunized human volunteers at the time of ch responses of protected and unprotected volunteers are com induced a more robust interferon-gamma response in pr characteristic was not shown by any of the four established represent superior candidates for inclusion in a malaria vaccin of Science, USA) (*The ImmunoSense approach was developed Diego, in collaboration with Denise Doolan at the Naval Medi protective epitopes directly using propriety computer algorith amino acid motifs predicted to bind strongly to HLA Cla supertype).110 This strategy results in the a priori eliminatio candidate genes, without the need to clone, express or identification of target antigens as well as the target epitop sequences representing those peptide epitopes associated wi be strung together with appropriate spacers to form multi-epi to construct ‘high impact’ recombinant plasmid or virally vec strings74 or combining recombinant proteins or peptides that independently induce protective responses, in the hope of achieving synergy.75 For DNA-based vaccines, this has included replacing native genes with synthetic genes using codon frequencies optimized for host expression,76 adding sequences such as the Fc region of immunoglobulin that increase antigen uptake by dendritic cells,77 complexing plasmids with cationic lipids such as Vaxfectin,78 particle-mediated gene transfer79 or jet-injection80 to enhance plasmid delivery, co- stimulation with cytokines or plasmids encoding cytokines81,82 or immunostimulatory molecules such as CpG DNA,83 vectoring genes with replica- tion-incompetent poxviruses,65 alphaviruses84 or adenoviruses,85 or, as with protein-based vaccines, erate pools of peptide representing predicted CD8C and alciparum proteins and from four ‘established’ and well- sly discovered using traditional approaches (PfCSP, tigen were tested separately for their ability to induce mononuclear cells (PBMC’s) collected from irradiated allenge with infectious P. falciparum sporozoites. The pared. Some of these antigens (for example, 2 and 13) otected as compared to unprotected volunteers. This antigens and may indicate that the new antigens may e. Data are from reference 61. (q 2003. National Academy by Alessandro Sette and colleagues at Epimmune Inc., San cal Research Center. This approach identifies potentially ms to search the sequences of candidate antigens for the ss I or II supertypes (e.g. HLA-A2 supertype, HLA-DR n of the majority of genetic sequence contained by the evaluate this material, and additionally results in the es on those antigens (see Fig. 5). The DNA or amino acid th strong recall responses from protected individuals can tope vaccine constructs,111 which may be an efficient way tored vaccines.) combining antigens to achieve synergy.86 While responses typically induced by RTS,S as well as T.L. Richie204 many of these approaches have worked in animal models, none of those tested in humans with the possible exception of the novel adjuvant ASO2A in combination with RTS,S, has yet shown comparable success. One of the most promising vaccine delivery approaches is to combine technologies in heter- ologous, ‘prime-boost’ vaccination regimens. In this approach, one delivery system, such as a DNA plasmid, is used to prime malaria-specific immune responses, and then a second delivery system, such as a recombinant protein or a recombinant poxvirus or adenovirus encoding the identical antigen, is used to boost these responses. Priming appears to induce long-lasting memory cells that serve to focus the immune response to the subsequent heterologous boost, which otherwise would be dominated by vector-specific responses directed against pox or adenovirus proteins, for example. Natural patho- gens such as adenoviruses or poxviruses may also present the immune systems with ‘danger signals,’ further improving immunogenicity. Such prime- boost approaches have achieved significant levels of protection in animal models.87–90 A particularly encouraging outcome was achieved in rhesus mon- keys by a series of priming immunizations with four plasmids each encoding a different pre-erythrocytic or erythrocytic stage antigen from the simian malaria P. knowlesi, followed by a boosting immu- nization with four recombinant poxviruses encoding the same four antigens. Eighty percent of the monkeys were clinically protected against the usually lethal P. knowlesi sporozoite challenge, and a quarter of those protected (20% overall) were sterilely protected against parasitemia.91 Using the P. falciparum orthologue of one of these four antigens (TRAP/SSP2), coupled with a string of epitopes derived from several additional pre-ery- throcytic stage antigens, partial protection has been achieved in humans with a similar prime-boost approach using fowlpox priming and mammalian pox boosting.92 Further improvements may be possible with a triple heterologous regimen.93 One of the advantages of heterologous vacci- nation is that it may broaden the immune responses to include both strong antibody responses tradition- ally observed with protein-based vaccines (needed for protection against sporozoites and blood stage parasites) and the T cell responses observed with DNA-based vaccines (needed for protection against liver stage parasites). In a recent study, volunteers immunized with the RTS,S/AS02A vaccine up to 15 months following three priming immunizations with a DNA vaccine encoding the P. falciparum CSP showed both the strong antibody and CD4C T cell Development efforts Many malaria vaccines have been tested in humans during the past 20 years. It was estimated that by 1998 roughly 40 trials had been carried out.97 A review by Ballou et al. lists 35 candidate malaria vaccines currently in pre-clinical or clinical testing, five of which have been evaluated in endemic areas.98 The majority (25/35 or 71%) are recombi- nant proteins or long synthetic peptides, four are vectored by recombinant poxviruses, two are prime-boost combinations of poxviruses and recom- binant proteins, two are DNA vaccines, one is a lipopeptide and one is a virus-like particle. About half are directed against sporozoite and liver stages, half against blood stages, two against gametocyte stages and two include antigens from more than one parasite stage. These many vaccines are supported by a surprising diversity of insti- tutions and partnerships representing both public and private sectors and both developed and devel- oping nations. Historically, large pharmaceutical companies have been reluctant to undertake the major investment required to develop a malaria vaccine due to perception that the markets provided by the CD8C T cell-mediated cytotoxic T cell responses (CTLs) and CD8C T cell-mediated, short-term, interferon-gamma responses typically induced by the CSP DNA vaccine.94,95 Although the protective efficacy induced by this DNA prime, protein boost approach was not tested in this trial, it is hoped that as heterologous approaches are systematically evaluated in the clinic in the coming years, significant protection against sporozoites challenge will be observed. One of the drawbacks of utilizing viral vectors in prime-boost approaches is that the anti-viral immune response engendered by immunization or alternatively by prior, natural exposure to a wild type infection (e.g. adenovirus respiratory infec- tion) can dampen the immunogenicity of sub- sequent immunizations with any vaccine vectored by the same virus.96 It is hoped that it will be possible to circumvent pre-existing anti-vector immunity by priming with DNA plasmids, by choos- ing viral vectors representing rare serotypes, by alternating vectors, or by mutating the surface proteins of the vector so that they are no longer immunologically cross-reactive with naturally occurring or vaccine-induced anti-vector responses. travelers and by endemic countries are limited and protein-based vaccines such as RTS,S/AS02A, which Malaria vaccines for travelers 205 that the scientific and technical feasibility of such a vaccine has yet to be unambiguously demonstrated. As described earlier, various chemoprophylaxis regimens demonstrate efficacies approaching 100% in controlled field trials, and even if the effective- ness of drugs is reduced to 60 or 70% by problems of access, compliance, side effects, etc. there is still no protein-based or nucleic-acid based sub-unit vaccine that attains this level of protection in malaria-naı¨ve individuals. The level of protection against infection afforded by the best current vaccine, RTS,S/AS02A, is not strong enough nor sufficiently long-lasting for it to replace chemopro- phylaxis. However, the development of RTS,S/AS02A by GlaxoSmithKline Biologicals (GSKBio, Rixensart, Belgium) does represent an important exception to the relative lack of interest shown by ‘big pharma,’ and illustrates how public– private partnerships, in this case including the US Army, the US Agency for International Develop- ment, and the Malaria Vaccine Initiative (MVI) (funded by the Bill and Melinda Gates Foundation) can propel vaccine development efforts forward. Humanitarian organizations, such as MVI, the European Malaria Vaccine Initiative (EMVI), the Wellcome Trust and US AID all aim to alleviate the suffering and economic loss inflicted by malaria on tropical countries, and their support will clearly be needed to facilitate what must ulti- mately be a commercial effort to manufacture and market a vaccine. Equally important to the effort are ‘north–south’ partnerships: developing countries are making an increasing contribution despite significant limi- tations in budget, both in terms of basic science research and in terms of developing high quality test sites in endemic areas where vaccines will eventually be deployed. To cite just three among many examples: the Centro de Investigac¸ao em Saude, in Manhic¸a, Mozambique, is currently assessing RTS,S/AS02A efficacy in young children; the Malaria Vaccine and Drug Testing Center in Cali, Colombia, is testing P. vivax vaccines; and the Noguchi Memorial Institute for Medical Research and the Navrongo Health Research Centre in Ghana has just begun a 7-year effort to test a variety of vaccines under a contract with the US National Institutes of Health extramural program. As mentioned, the majority of development efforts are relying on protein-based technologies due to the ability of recombinant proteins and synthetic peptides to induce strong antibody responses, the fact that antibody responses appear to underlie the clinical protection of NAI, and the encouraging results of early field efficacy trials of is now entering phase 2b testing in 1 to 4 year-old children in Mozambique. The protein-based approach is particularly attractive to anti-disease vaccine developers utilizing blood stage antigens (described in detail in the reviews by Ballou98 and Mahanty).99 More recently, however, there has been a trend toward other technologies which may be better suited to inducing the cell-mediated immunity that is likely key to halting parasite development early in infection.100 This is particu- larly true for the pre-erythrocytic stage vaccines, where protection, including that afforded by pro- tein-based vaccines such as RTS,S,101,102 appears to be primarily cell-mediated.103 A leader in this area is Adrian Hill’s group at Oxford University in the UK, which has demonstrated significant reductions in liver stage parasite burden by priming human volunteers with a recombinant DNA plasmid or a recombinant fowl pox 9, followed by boosting with modified Vaccinia Ankara (MVA), another recombi- nant pox vector. These vaccines each contain the genetic sequence for the pre-erythrocytic stage antigen thrombospondin-related adhesive protein (TRAP/SSP2), fused to a string of CD4C and CD8C epitopes derived from six different pre-erythrocytic stage antigens. Although only a handful of volun- teers immunized with this heterologous approach has been completely protected against parasitemia following experimental sporozoite challenge, many have experience a delay in the onset of parasitemia indicating a significant reduction in liver stage burden.92,104 More recently, the Oxford group has constructed a six antigen polyprotein, also com- posed of pre-erythrocytic stage antigens.105 These various candidate vaccines are being developed with support from the Wellcome Trust, MVI and EMVI. The US military is another group increasing its focus on alternative technologies. The Walter Reed Army Institute of Research (WRAIR) Malaria Vaccine Program, a co-developer of RTS,S/AS02A, hopes to combine adenovirus serotype 35-vectored and protein-based vaccines in prime-boost regimens, in order to achieve higher levels of efficacy than currently exhibited by RTS,S/AS02A alone. The WRAIR development strategy includes the combi- nation of two or more pre-erythrocytic stage proteins, the combination of pre-erythrocytic stage and erythrocytic stage proteins and the testing of alternative adjuvants to AS02A to enhance cell-mediated immunity. As another arm of the US Department of Defense effort, the Naval Medical Research Center Malaria Vaccine Program is utilizing a genomes-to-vaccines approach to ident- ify superior candidate antigens, and is designing and testing multi-antigen, multi-stage vaccines containing half a dozen down-selected antigens. with dwindling efficacy secondary to the evolution vaccines by preventing the transmission of vaccine-resistant genotypes. Wild cards in anticipating the future are the T.L. Richie206 The future The first achievement for malaria vaccine devel- opers may be a reduction in morbidity and mortality of childhood malaria using a simple, possibly single antigen, protein-based, blood stage vaccine. This is likely the easiest goal to achieve, because clinical immunity occurs naturally and the development of an anti-disease vaccine aims to accelerate the transition from clinically naı¨ve to clinically immune rather than prevent infection altogether. The problem that may plague such a success, however, is the development of resist- ance to the vaccine by the parasite, anticipated if the vaccine reduces gametocyte output, thereby adversely affecting the fitness of the parasite and selecting for vaccine-resistance genotypes. For this reason, it could turn out that initial reductions in disease and death will be followed by deteriorating efficacy as the parasite popu- lation adjusts evolutionarily to the new immunity exhibited by its host. If this were to occur, it would require an ongoing effort to create new vaccines employing different antigens, much as anti-malarial drug developers are continually seeking new classes of drugs to replace those Candidate vaccines will be assessed in prime-boost combinations similar to those advanced by the Oxford group, utilizing vaccine delivery platforms such as DNA, adenovirus serotype 5, MVA and alphavirus replicons. This group also plans to develop epitope strings containing key protective sequences from possibly 100s of newly identified antigens, in partnership with Epimmune, San Diego. Other groups focusing on pre-erythrocytic stage antigens include Sanaria, Inc., which aims to develop the most effective current method for inducing sterile immunity against liver stage infec- tion, a non-replicating, metabolically active, radi- ation-attenuated whole sporozoite vaccine;27 Apovia, Inc. (San Diego) is developing virus-like particles utilizing hepatitis B surface antigen as the delivery platform for CSP-derived epitopes;66 the University of Lausanne is developing long synthetic peptides based on CSP from both P. falciparum106 and P. vivax;107 the Pasteur Institute in Paris is developing liver stage antigen 3,108 which is expressed in both pre-erythrocytic and erythrocytic stages. These vaccines may be particularly relevant to the needs of travelers but may also significantly benefit residents of endemic areas by reducing liver stage burden. antitoxin vaccines such as that proposed by Louis Schofield at the Walter and Eliza Hall Institute of Medical Research in Melbourne,109 which could possibly achieve an anti-disease effect without adversely affecting parasite fitness, and trans- mission-blocking vaccines such as those under development by the US National Institutes of Health, which could control or even eradicate malaria without the need to directly prevent infection or disease by inducing immune responses attacking the parasite in the human host. If transmission-blocking vaccines reduce the trans- mission rate below the threshold needed to maintain the infection in the host population, malaria will disappear. All in all, there are many foreseeable outcomes, and the difficulty in identifying the approach or approaches most likely to succeed underscores the importance of maintaining the broad-based, diverse research effort that currently characterizes malaria vaccine development. Acknowledgements The author wishes to gratefully acknowledge Denise Doolan for her comments and suggestions regarding the section on genomes-to-vaccines and Fig. 5, and for permission to use Fig. 6; Adrian Hill, Joe Cohen and Gray Heppner for their comments and sugges- tions regarding the sections on their respective vaccine programs, and Oh Dong from Giant Phar- macies for supplying the costs for anti-malarial chemoprophylaxis. The studies reported herein that were conducted by the Naval Medical Research Center Malaria Program were conducted in accordance with US Navy regulations governing of parasite resistance. More difficult to achieve will be an anti-infection vaccine for travelers. This will most likely require a multivalent, multi-stage approach (Fig. 4), and will probably depend upon both the identification of new antigens (Figs. 5 and 6) and the development of superior vaccine delivery systems designed to simultaneously induce powerful cellular and humoral immune responses. Because such a vaccine is certain to negatively impact parasite fitness, the evolution of resistance will again be a major concern. Transmission-blocking vaccine components may be integral to preserving the efficacy of both anti-infection and anti-disease the protection of human subjects in medical Organ 1993;71(3/4):385–8. 7. Sheng HF, Zhou SS, Gu ZC, Zheng X. Malaria situation in the People’s Republic of China in 2002. Zhongguo Ji Sheng 12. Hamer DH, Connor BA. Travel health knowledge, attitudes Malaria vaccines for travelers 207 Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2003;21(4):193–6 [in Chinese]. 8. Rowland M, Rab MA, Freeman T, Durrani N, Rehman N. 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