Vector-Borne Viral Infections
Malaria is by far the most important tropical parasitic disease, killing two children aged less than 5 years every minute. Most of these deaths occur in sub-Saharan Africa 
. It is estimated that around 900 000 people die each year from malaria and that more than 3 billion people are exposed to the risk of acquiring the disease worldwide, especially in sub-Saharan Africa, India and South East Asia
. Deployment of an effective vaccine could save countless lives and improve the overall quality of life in the tropics and subtropics.
Malaria is caused by infection of red blood cells with protozoan parasites of the genus Plasmodium, which are transmitted by the bite of a feeding female mosquito of any one of the 50 species of Anopheles mosquitoes, of which the best known is A gambiae. Four Plasmodium species infect humans, P. falciparum, P. vivax, P. ovale and P. malariae, causing a spectrum of clinical disease ranging from moderate flu-like symptoms to severe malaria disease characterized by respiratory distress, coma, severe anaemia, hypoglycaemia, generalized convulsions and high mortality.
Uncomplicated malaria, with no evidence of vital organ dysfunction, has a relatively low case-fatality rate provided there is early case detection and appropriate management, including effective drug therapy. However, in areas of high disease burden and lack of prompt access to health care and accurate diagnostics, delay in treatment or use of ineffective drugs, uncomplicated malaria can rapidly progress to severe disease with case fatality rates in people receiving treatment of up to 15-20%. Untreated severe malaria is almost always fatal. Most at risk are those who have not acquired immunity to malaria, such as young children, travelers, and displaced persons. Malaria contributes significantly to anaemia in children living in endemic countries. In those who survive, profound sequelae may affect physical and mental development    . Malaria in pregnant women is a cause of adverse birth outcomes such as spontaneous abortion, stillbirth, premature delivery and low birth weight  as well as profound anaemia. Malaria transmission was successfully reduced or eliminated between 1957 and 1972 from areas where it had occurred at low intensities in the Americas, Asia, Europe and Transcaucasia. This was achieved through vector control - DDT spraying - combined with improved access to treatment. In contrast, most of sub-Saharan Africa and some foci elsewhere continued to suffer high intensity malaria transmission. In recent years, malaria has reemerged in several areas after interruption of malaria control efforts that were not sustainable  and in the face of increasing drug and insecticide resistance. The combination of tools and methods to combat malaria now includes long-lasting insecticidal bed nets (LLIN) and artemisinin-based combination therapy (ACT), supported by indoor residual spraying of insecticide (IRS) and intermittent preventive treatment in pregnancy (IPTp)   .
There were an estimated 247 million malaria cases among 3.3 billion people at risk in 2006, causing nearly a million deaths, mostly of children under 5 years of age. In 2008, 109 countries were endemic for malaria, 45 of which within the WHO African region. Provisional country-level estimates continue to be refined based on efforts to improve the global malaria endemicity map. As of January 2005, estimates of rates of total clinical malaria incidence indicated that around 59% of the world's clinical malaria cases occurred in Africa, around 38% in Asia and around 3% in the Americas. Over 80% of the deaths from malaria occurred in sub-Saharan Africa. In Mali alone, malaria kills close to 70 000 children under 5 years of age every year and contributes to substantial yearly economic losses. Malaria is estimated to be responsible for an estimated average annual reduction of 1.3% in economic growth for those countries with the highest burden 
Parasite-vector-human transmission dynamics such as transmission potential of the different anopheline mosquitoes and climatic conditions greatly influence the variation in disease burden in different regions of the world. In addition, socioeconomic factors such as degree of poverty, quality of housing and access to health care and health education, as well as the existence of active malaria control programs providing access to malaria prevention and treatment measures also greatly affect the disease burden variability. The most efficient malaria vector, Anopheles gambiae, occurs exclusively in Africa and also is one of the most difficult to control. Tropical areas of the world have the best combination of adequate rainfall, temperature and humidity allowing for breeding and survival of anophelines. A recent study shows that a substantial proportion of people originating from malaria endemic countries and not showing any sign of malaria harbored Plasmodium parasites in their blood and could potentially be a reservoir  .
The pattern and intensity of malaria transmission determines the degree of protective immunity acquired by the residents of affected areas and the nature of the clinical disease profile. The majority of deaths in tropical Africa occur in areas of high transmission of P falciparum malaria. In much of sub-Saharan Africa, populations are continuously exposed to a fairly constant rate of malarial inoculations, and if the inoculation rates are high (annual entomological inoculation rate (EIR) >10 bites), then partial immunity to the clinical disease and to its severe manifestations is acquired early in childhood. In such areas, acute clinical disease is almost always confined to young children who have not yet acquired clinical immunity, and to pregnant women, whose immunity to malaria is temporarily impaired. In these 'stable' and high-transmission areas, adolescents and adults are partially immune and rarely suffer clinical disease, although they continue to harbour low blood-parasite densities. Immunity appears to be lost when individuals move out of the transmission zone, although the time course of this waning of immunity is not well documented.
In areas of low or highly seasonal P falciparum malaria transmission, where annual EIRs are usually <5 bites and may be <1, acquisition of immunity is slower. As a result, people of all ages may be at risk of suffering acute clinical malaria episodes, with a high risk of progression to life-threatening severe disease if not appropriately treated. This situation is seen in much of Asia and Latin America and the remaining parts of the world where malaria is endemic. In these areas, malaria epidemics may occur when inoculation rates increase rapidly, leading to a high incidence of malaria in all age groups.
The estimated costs of malaria in terms of strain on the health systems are enormous: in endemic countries, as many as 3 out of 10 hospital beds can be occupied by victims of the disease. A study in 2002 in Mozambique showed that 40% of outpatients and 60% of children admitted to hospitals suffered from malaria. The cost of malaria in sub-Saharan Africa is estimated to represent 1%-5% of GDP, a cost of about $12 billion a year  .
The agents of human malaria are four species of Plasmodium protozoa: P vivax, P falciparum, P ovale, and P malariae. All are transmitted by Anopheles mosquitoes. The majority of malaria cases are caused by P. falciparum and P. vivax.
. Human cases of P. knowlesi malaria also occur in Malaysia, Thailand, Myanmar and parts of the Philippines as a zoonotic disease
. About 86% of all clinical malaria cases occur in Africa. Among the cases that occur outside the African Region, 80% are in India, Sudan, Myanmar, Bangladesh, Indonesia, Papua New Guinea and Pakistan. The second most common malaria parasite, P vivax, accounts for a higher proportion of the total malaria disease burden in Asia and in parts of the Americas, Europe and North Africa.
Plasmodium parasites have a complex life cycle that begins when an infected female mosquito injects sporozoites into a human when taking a blood meal    . The sporozoites invade the blood stream and within about 30 minutes migrate to the liver and invade hepatocytes. Sporozoites mature in the liver where they give rise to tens of thousands of merozoites over a period of 6-16 days. The sporozoite and liver stages are known together as the pre-erythrocytic stage of the life cycle. This initial phase is followed by the erythrocytic or asexual blood stage, when merozoites erupt into the blood stream, invade red blood cells (RBCs), multiply and mature over a few days and then are released by lysis of the infected RBCs. The RBC invasion-multiplication cycle repeats itself time after time, giving rise to the classical malaria acute febrile episodes and rigors that occur every 48-72 hours due to the lysis of infected RBCs, which leads to the release of new mature merozoites in the blood. The third stage of the parasite is the sexual stage, when some merozoites differentiate into gametocytes, which, after being taken up by an anopheline mosquito, will sexually combine in the insect host to generate a zygote, from which new sporozoites will eventually emerge, ready to reinitiate the cycle.
Both the 23.3 megabase P falciparum and the 26.8 megabase P vivax genome sequences have been reported: they each carry more than 5200 genes distributed over 14 chromosomes   .
Several lines of evidence suggest that a prophylactic malaria vaccine for humans is feasible. Firstly, naturally acquired immunity builds up during the first two decades of life in people living in malaria-endemic countries. This naturally acquired immunity is however partial and short-lived, and appears to depend on continuous antigenic stimulation, waning when antigen exposure ceases. Protection has been elicited by passive transfer of immunoglobulins from malaria-immune adults to malaria-naïve human volunteers. There also is experimental evidence that immunization of humans and animals with irradiated sporozoites results in partial or complete protection from an experimental infection with viable sporozoites
, which might pave the way to a genetically attenuated live sporozoite vaccine
However, multiple obstacles to the development of a malaria vaccine remain, that include the lack of general agreement on possible immune correlates of protection, the lack of predictive animal models and assays, and the multiple stages and antigenic diversity and variability of the parasite. Different antigens often are expressed at different stages of the parasite life cycle, and most show considerable polymorphism  . The genetic complexity of the parasite remains a significant challenge. Plasmodia have more than 5000 genes, and finding which ones code for appropriate candidate vaccine antigens may be a quagmire   .
Different vaccines are being developed that target the different stages of the parasite life cycle: pre-erythrocytic (sporozoite and liver stage), erythrocytic (blood stage) and sexual stages (for reviews, see       ). Pre-erythrocytic stage vaccine strategies aim to generate an antibody response that will prevent sporozoites from invading hepatocytes as well as to elicit a cell-mediated immune response that will inhibit intra-hepatic parasite development. This type of vaccine would be ideal for travelers because it would prevent infection and the advent of clinical disease. Erythrocytic stage vaccine strategies aim to elicit antibodies that will target merozoite antigens and/or antigens expressed on the surface of infected RBCs. These vaccines ideally should be able to induce antibody-mediated cellular toxicity and/or complement lysis, as well as T-cell responses that will inhibit the development of merozoites in RBCs. This type of vaccine is hypothesized to allow parasite densities to be controlled at levels which would minimise morbidity and would therefore be suitable for residents of endemic countries for morbidity reduction, but not for prevention of infection. Finally, vaccines targeting the sexual stage of the parasite aim not to prevent infection or disease but to prevent the transmission of the parasite to new hosts. Efficacious transmission blocking vaccines are thought to be highly desirable in pre elimination settings where interruption of transmission becomes a key aim of an immunization programme.
Almost all of the vaccines under development are directed at P falciparum, which is responsible for the vast majority of severe malaria disease and deaths  .
The most advanced malaria vaccine candidate at this time is a pre-erythrocytic vaccine based on the circum-sporozoite protein (CSP) that is the predominant surface antigen of the sporozoites and is expressed early on infected hepatocytes. This vaccine, RTS,S/AS01, is made of recombinant chimeric virus-like particles (VLPs) produced in Saccharomyces cerevisiae combining the Hepatitis B surface antigen (HBsAg) with the C-terminus portion (aa 207-395) of P falciparum CSP. The candidate vaccine has shown 40% protective efficacy in multiple clinical challenge studies conducted in partnership with the US Military Malaria Vaccine Program. A randomized controlled field efficacy trials in Gambian adults demonstrated 71% efficacy against time to infection for the first 9 weeks after vaccination, but low efficacy for the subsequent 6 weeks. Efficacy over the entire 15 week period was 34% and it has been proposed that heterogeneity of risk may partly or wholly account for the apparent waning seen in this study. The vaccine was found to be safe and well tolerated in adults in a hyperendemic region of western Kenya  as well as in 1-4 years-old children in Mozambique in whom it induced a strong antibody response to both the CSP and to HBsAg  and a TH1 CD4+ T cell response. The paediatric clinical development of RTS,S has occurred as a partnership between the PATH Malaria Vaccine Initiative and GSK.
A randomized phase IIb trial in 2022 Mozambiquan children aged 1 to 4 years showed 30% efficacy (95%CI 11-45%) over six months against time to first or only clinical malaria episode and 57% efficacy (95%CI 16-80%) against severe malaria  . Vaccine efficacy over an extended 18-month follow-up period was 35.3% against time to first or only episode of malaria and 48.6% against severe disease   . In a study in Bagamoyo, Tanzania, 170 infants were immunized at 8, 12 and 16 weeks of age with RTS,S in co-administration with a DTPw/Hib vaccine. Seroconversion to CSP was 98.6%, and the efficacy of the RTS,S vaccine against first infection with P falciparum at 6 month after vaccination was 65.2%, but GMT to diphtheria and tetanus vaccines were lower in co-administration with RTS,S  .
All studies cited to date had used AS02 adjuvant, an oil-in-water emulsion with added MPL and QS21. Paediatric development has since shifted to AS01, which consists of a liposomal preparation with MPL and QS21. This adjuvant is more immunogenic for both IgG and CD4+ T cell responses. A randomised controlled study with about 850 children aged 5 to 17 months who were followed for 8 months in Kilifi, Kenya and Korogwe, Tanzania, showed an efficacy of 55% in terms of the rate of all episodes of malaria for RTS,S/AS01  . The magnitude of the anti-CSP antibody responses in that study was substantially higher than in children that had received the RTS, S/AS02 vaccine previously. Whether the higher antibody titers associated with the use of AS01 will translate into a longer duration of protective efficacy for the RTS, S vaccine remains to be demonstrated.
Other clinical trials of the RTS,S vaccine are under way in combination with other candidate malaria vaccines    . A pivotal phase III trial design was planned to start in the second quarter of 2009 and is intended to enrol up to 16,000 children in 11 different sites in Burkina Faso, Ghana, Gabon, Malawi, Mozambique, Kenya and Tanzania, covering different transmission patterns.
Another CSP-based candidate vaccine was developed by Dictagen, Inc, in collaboration with the University of Lausanne, Switzerland, that contained a 102 aa synthetic peptide representing the C terminal portion of the CSP antigen formulated with Montanide ISA 720. The formulation was found to be safe in human volunteers and to elicit both an antibody response and a cellular immune response. The vaccine currently is in Phase IIa clinical trials.
The US Military Malaria Vaccine Program (USMMVP), in collaboration with Vical, Inc, developed a candidate DNA vaccine for malaria by mixing five plasmids that encoded five different Plasmodium falciparum antigens, including CSP, liver stage antigens 1 and 3 (LSA-1 and -3), exported protein 1 (EXP-1) and the sporozoite surface protein 2 (SSP-2, also known as thrombospondin-related adhesive protein, TRAP). The vaccine however only showed modest immunogenicity in nonhuman primates and elicited little protection against sporozoite challenge in human volunteers. More recently, USMMVP has conducted Phase I trials with Adenovirus 5 vectors that expressed CSP and the merozoite antigen AMA1. In addition, Crucell has been conducting a Phase I trial with adenovirus 35 recombinant for CSP in the USA in conjunction with NIAID.
The vaccine potential of P falciparum LSA-3 was further investigated in nonhuman primates: both a DNA vaccine and a long LSA-3 synthetic peptide vaccine were able to elicit sterilizing immunity against sporozoite challenges in chimpanzees and Aotus monkeys, respectively   .
Another liver stage antigen, the TRAP antigen, was developed as a candidate vaccine by the Oxford University Malaria Vaccine Clinical Trials Group, which conducted studies of a DNA, a fowlpoxvirus (FPV) and a MVA-based vaccines expressing the TRAP antigen. The recombinant FPV and MVA vaccines were tested independently or in prime-boost combinations with or without the DNA vaccine and found to be well tolerated and highly immunogenic in human volunteers in the UK in terms of induction of specific CD4+ T cells   . Trials in Gambia and in Kenya (DNA/MVA prime-boost and FPV/MVA prime-boost) failed however to demonstrate protective efficacy of these approaches  .
Live recombinant vaccines expressing CSP determinants also were developed by Oxford University using a chimpanzee Adenovirus (AdCh63), MVA or FPV as vectors. These vaccines are in early clinical development. The first clinical trials of AdCh63/MVA ME-TRAP were begun in 2008 with a challenge trial planned for 2009. The prime-boost combination of an Ad35-CSP vaccine with the RTS,S/AS01 vaccine was found to be very immunogenic and to elicit robust protection against challenge in rhesus macaques . Results were less clear-cut for prime-boost immunization with RTS,S and the MVA-CSP recombinant vaccine, whose combination only elicited a modest 33% protection against sporozoite challenge in volunteers  .
The choice of clinical case definitions and end-points in efficacy trials of erythrocytics vaccines, which aim to decrease morbidity by reducing parasitemia, still remains a difficult issue   . Various groups, focusing on different merozoite antigens and different regions of the proteins, and using different antigen expression systems and formulations are developing a variety of erythrocytic stage malaria vaccine candidates. Results of preliminary efficacy trials involving some of these candidates should be made available over the next couple of years.
Blood stage vaccine candidates currently in early clinical trials are the merozoite surface proteins 1 (MSP-1), 2 (MSP-2), and 3 (MSP-3), apical membrane antigen 1 (AMA-1), the glutamate-rich protein (GLURP) and the serine repeat antigen protein (SERA)  . Anti-MSP-1 antibodies have been reported to strongly correlate with reduced risk of clinical malaria in Ghanaian children  . The two allelic forms of the C-terminal 42 kD region of MSP-1 were formulated with Alhydrogel and used to immunize 60 malaria naïve individuals, the large majority of whom developed anti-MSP-1 antibodies. However, these antibodies only showed minimal growth inhibition of P falciparum in vitro  . A candidate vaccine containing MSP-1 adjuvanted with AS02, which had shown promising protective efficacy in nonhuman primate models, also failed to induce protection or to decrease parasitemia in 1-4 years old children in Kenya in spite of good immunogenicity results  .
The Combination B vaccine, which was developed in collaboration between the Walter and Eliza Hall Research Institute in Australia, the Swiss Tropical Institute in Lausanne and the Papua New Guinea Institute for Medical Research. combined the merozoite surface proteins MSP-1 and MSP-2 with the ring-stage infected erythrocyte surface antigen RESA and induced a 62% reduction in parasitemia in a Phase II trial on 5-9 years old children in Papua New Guinea  , but this effect was restricted to parasites expressing one of the two allelic forms of MSP-2  . A bi-allele MSP2 vaccine is now under development and a phase 1 trial of this vaccine recently took place in Australia.
Another promising vaccine in clinical development is an MSP-3 -based vaccine. The vaccine was tested in Phase I trials   and found to elicit antibodies able to block multiplication of P falciparum in red blood cells in vitro in a monocyte-dependent manner, a property of natural cyclophilic IgG1 and IgG3 antibodies from malaria-immune African adults  . Passive transfer of the vaccinated volunteers' sera into P falciparum-infected humanized SCID mice reduced or abrogated parasitemia  . The levels of cytophilic IgG3 antibodies against conserved regions of MSP-3 and the 24 kD glutamate-rich protein (GLURP) both significantly correlated with protection against clinical P falciparum malaria in naturally exposed individuals in an area of hyperendemicity in Myanmar  . A GLURP-based candidate vaccine formulated with either alum or montanide ISA 720 similarly elicited dose-dependent cellular and humoral immune responses with high levels of cytophilic IgG1 antibodies that inhibited P falciparum growth in vitro in the presence of monocytes  . The MSP-3-based vaccine is currently in Phase II clinical trials in Mali. A MSP3-GLURP fusion protein termed GMZ2 has progressed to the stage of phase 1 trials in sub-Saharan Africa with plans to presently progress to Phase 2 trials.
Another antigen of interest that was tested in Phase I trials is the AMA-1 antigen, which is known to be expressed at both the hepatic and erythrocytic stages. The AMA-1 vaccine which was developed by the Walter Reed Army Institute of Research using AS02 as the adjuvant induced a robust humoral immune response that lasted for more than one year. Field trials of that formulation have been initiated in Mali  . Phase I trials of an alhydrogel AMA-1 formulation were also conducted in Mali: the vaccine was found to be well tolerated   . AMA-1 was also expressed in combination with MSP-1 as a fusion protein (PfCP2.9) that was formulated with montanide ISA 720. The resulting candidate vaccine was tested in Phase I trials in Chinese volunteers   and was found to be well tolerated and to elicit high anti-PfCP2.9 antibody titers. The resulting antibodies, however, failed to show much effect on the growth of the parasite in vitro. The ISA720 formulation has been found to introduce several challenges for clinical vaccine development and is not now recommended for use by IVR.
Despite encouraging progress, the lack of immune correlates of protection together with the high polymorphism of many of the erythrocytic stage antigens constitute major obstacles to the development of vaccines that target the blood stage of the parasite cycle. In contrast with pre-erythrocytic stage vaccine candidates, erythrocytic vaccine candidates lack an appropriate human artificial challenge model and have had to rely on natural transmission in the field to provide a proof-of-concept of their efficacy. Their development is therefore slower and necessitates major commitment, intensive collaboration as well as high-level coordination supported by adequate funding. A recent meeting stressed the potential of the use of optimised clinical challenge models for screening blood stage vaccines in the future.
Transmission-blocking vaccines aim to prevent onward transmission to humans by targeting the sexual stages of P. falciparum and blocking sexual mating so as to prevent the development of sporozoites in Anopheles mosquitoes. Antibodies against gametocytes could act directly in humans, or at a later stage in mosquitoes. This approach has the advantage of having robust in vitro assays that could be used to demonstrate proof-of-concept, as well as a relatively clear effector immune response. Several candidates are in clinical development, including vaccines that target the Pfs25 or Pvs25 and Pvs 28 surface proteins, but the ISA51 formulation of these vaccines turned out to be unacceptably reactogenic  . Also, a major challenge for this vaccine approach is proving true field efficacy   .
Pregnancy malaria vaccines
In Africa, where an estimated 50 million women become pregnant each year, maternal malaria causes untold numbers of abortions, stillbirths and over 10 000 maternal deaths. In addition, malaria infection causes more than 200 000 low-birthweight babies to die within their first year of life. The possibility of developing a placental malaria vaccine by targeting Plasmodium antigens expressed on the surface of infected erythrocytes that attach to placental proteins is the subject of promising R&D efforts .
P. vivax vaccines
If overall malaria disease burden continues to decrease then it is likely that P. vivax control will become a high priority within the malaria community. P. vivax disease already accounts for a rising proportion of cases in co-endemic areas. The WHO has taken a leading role in providing guidance on the P. vivax vaccine R&D agenda. Phase 1 trials of Pvs25 and P. vivax CSP-based peptide vaccines have been the only P. vivax clinical trials in recent years. A potentially promising approach based on P. vivax Duffy Binding Protein is under development by a group in India.
Given the complexity of the parasite and the slow development of naturally acquired immunity, it is increasingly thought by many experts that an effective malaria vaccine may have to contain antigens from multiple stages of the parasite. Several groups are working on such combination approaches for which numerous challenges have to be overcome including possible competition between the different antigens, compatibility of antigen presentation systems, and cost and complexity of evaluating incremental improvements.
International efforts to combat malaria have been scaled up in recent years, including among its many actors: the WHO, UNICEF, UNDP, the World Bank, the NIH in the USA, the Wellcome Trust in the UK, USAID, the European Malaria Vaccine Initiative (EMVI), the Malaria Vaccine Initiative (MVI) of PATH, The European Commission, EDCTP, and the Bill and Melinda Gates Foundation. The Roll Back Malaria Partnership plays a key role in coordinating malaria control strategy http://rbm.who.int/aboutus.html   .
It has been estimated that the cost for supporting the minimal set of malaria interventions required to effectively control malaria is around US$ 3.2 billion per year for the 82 countries with the highest burden of disease (US$ 1.9 billion for Africa alone). Increased commitment and financial support, through programmes such as the Global Fund to Fight AIDS, Tuberculosis and Malaria which disbursed more than US$ 200 million in 2003-2004 to 28 countries, will be needed to support control strategies in an effective and sustainable way. The next 5 years is likely for the first time to witness the submission of a malaria vaccine for possible registration (or "positive scientific opinion" - the EMEA article 58 equivalent). The malaria community will then need to consider the role of a partially efficacious pre-erythrocytic malaria vaccine as an addition to the current complement of malaria control measures. Generation of appropriate clinical trial data to allow assessment of public health impact of vaccination in the context of existing control measures will be a crucial component of this process.