Stephen L. Hoffman

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25–40% of the ∼515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Funding for malaria genome sequencing.

Sir — We should like to correct a statement in your Briefing on malaria about funding for the sequencing of the genome of the malaria parasite Plasmodium falciparum (Nature 386, 535–540; 1997). You report that the US Department of Defense, the US Burroughs Wellcome Fund, the UK Wellcome Trust and the US National Institutes of Health (NIH) have created a fund for this sequencing effort. In fact, no ‘fund’ has been established and there has been no pooling of funds among the donors. However, an exciting international consortium of scientists and funding agencies has been formed during the past year to provide the funds and reagents required to sequence the P. falciparum genome, and to produce, annotate and publish these sequences. We estimate that it will cost a minimum of

Protection Against Malaria by Intravenous Immunization with a Nonreplicating Sporozoite Vaccine

15 million to complete and annotate the 30-megabase P. falciparum genome. In the initial pilot phase, funding has been used to develop methods and to produce the reagents required for the high-throughput sequencing and for annotating the sequences. As a result of progress so far, the plan is to sequence the 14 chromosomes separately and to divide the work by chromosome, with about half the work being done in the United States and half in England. The work in the United States has initially been supported by grants from the NIH and the Burroughs Wellcome Fund and in England by the Wellcome Trust. Subsequent support will also be provided by the US Department of Defense. In the United States, the Naval Medical Research Institute is providing P. falciparum DNA and chromosomes, the Institute for Genomic Research will be doing a significant amount of the high-throughput sequencing and Stanford University is committed to sequencing at least one of the larger chromosomes. The donors are also supporting research on clone stability, library construction and optimization of sequencing reagents for the project at these three institutions, and at Roswell Park Cancer Institute and Harvard University. In England, the Institute of Molecular Medicine at the University of Oxford is providing P. falciparum reagents, and sequencing is being done at the Sanger Centre at Cambridge. It is expected that during the next few years a number of other laboratories may participate in the effort. The participants (scientists and donors) involved in this collaborative undertaking coordinate their activities through conference calls, e-mails and meetings. The researchers and funders met in Baltimore, Maryland, in December 1996 and will meet in Cambridge, England, on 16–17 June 1997. In addition, there has been an effort to involve the broader malaria community in this project and to facilitate widespread dissemination of the genomic information. There are substantial scientific obstacles to be overcome before the sequence of the P. falciparum genome is fully elucidated. Nonetheless, during the past year we have made great strides in identifying the funds required to pursue the project, building a strong group of scientists and institutions to execute the science, and establishing a collaborative network of scientists and donors. We believe that this project will provide a road map for malaria research in the twentyfirst century, research that will lead to improved treatment and prevention of a parasitic infection that causes hundreds of millions of illnesses, and millions of deaths annually. Stephen L. Hoffman Malaria Program, Naval Medical Research Institute, Bethesda, Maryland 20889-5607, USA e-mail: hoffmans@nmripo.nmri.nnmc.navy.mil William H. Bancroft Military Infectious Disease Research Program, US Army Medical Research and Materiel Command, Frederick, Maryland 21703, USA Michael Gottlieb Stephanie L. James Parasitology and International Programs Branch, NIAID/NIH, Bethesda, Maryland 20892, USA Enriqueta C. Bond Burroughs Wellcome Fund, Morrisville, North Carolina 27560, USA John R. Stephenson Michael J. Morgan The Wellcome Trust, London NW1 2BE, UK correspondence

Live Attenuated Malaria Vaccine Designed to Protect through Hepatic CD8+ T Cell Immunity

Malaria Sporozoite Vaccine Each year, hundreds of millions of people are infected with Plasmodium falciparum, the mosquito-borne parasite that causes malaria. A preventative vaccine is greatly needed. Seder et al. (p. 1359, published online 8 August; see the Perspective by Good) now report the results from a phase I clinical trial where subjects were immunized intravenously with a whole, attenuated sporozoite vaccine. Three of 9 subjects who received four doses and zero of 6 subjects who received five doses of the vaccine went on to develop malaria after controlled malaria infection. Both antibody titers and cellular immune responses correlated positively with the dose of vaccine received, suggesting that both arms of the adaptive immune response may have participated in the observed protection. Intravenous immunization with an attenuated whole malaria sporozoite vaccine protected volunteers in a phase I clinical trial. [Also see Perspective by Good] Consistent, high-level, vaccine-induced protection against human malaria has only been achieved by inoculation of Plasmodium falciparum (Pf) sporozoites (SPZ) by mosquito bites. We report that the PfSPZ Vaccine—composed of attenuated, aseptic, purified, cryopreserved PfSPZ—was safe and wel-tolerated when administered four to six times intravenously (IV) to 40 adults. Zero of six subjects receiving five doses and three of nine subjects receiving four doses of 1.35 × 105 PfSPZ Vaccine and five of six nonvaccinated controls developed malaria after controlled human malaria infection (P = 0.015 in the five-dose group and P = 0.028 for overall, both versus controls). PfSPZ-specific antibody and T cell responses were dose-dependent. These data indicate that there is a dose-dependent immunological threshold for establishing high-level protection against malaria that can be achieved with IV administration of a vaccine that is safe and meets regulatory standards.

Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay.

The efficacy of a sporozoite-based malaria vaccine is tested in humans, nonhuman primates, and mice. Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8+ T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8+, IFN-γ–producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

Reduction of Mortality in Chloramphenicol-Treated Severe Typhoid Fever by High-Dose Dexamethasone

Two field studies in Kenya and an experimental challenge study in the USA were done to assess the accuracy of a dipstick antigen-capture assay based on qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in peripheral blood for diagnosis of P falciparum infection. In these studies, the assay was 96.5-100% sensitive for detection of greater than 60 P falciparum asexual parasites/microL blood, 70-81% sensitive for 11-60 parasites/microL blood, and 11-67% sensitive for 10 parasites or less/microL blood. Specificity was 95% (95% CI 85-105%; n = 20) among naive American volunteers, 98% (96-101%; n = 112) among volunteers exposed to the bite of P falciparum-infected mosquitoes, and 88% (84-92%; n = 285) among Kenyans living in an area with holoendemic malaria. Our results also indicated that PfHRP-2 antigen was not detectable in blood 6 days after initiation of curative chemotherapy, and suggest that such circulating antigens rarely lead to false-positive tests. The dipstick assays sensitivity, specificity, simplicity, and speed may make it an important tool in the battle against malaria.

Induction of CD4+ T cell-dependent CD8+ type 1 responses in humans by a malaria DNA vaccine

We compared high-dose dexamethasone (initial dose, 3 mg per kilogram of body weight) with placebo in a randomized, double-blind trial involving 38 patients with culture-positive, specifically defined severe typhoid fever. The patients in the two treatment groups ranged in age from 5 to 54 and were comparable at the outset. All patients received chloramphenicol. The case-fatality rate of 10 per cent (2 of 20 patients) in the dexamethasone group was significantly lower than the fatality rate of 55.6 per cent (10 of 18) in the placebo group (P = 0.003). There was no significant difference in the incidence of complications among the survivors in either group. Delirium, obtundation, and stupor were grave prognostic signs that were useful for predicting which patients were at high risk of dying before they became comatose or went into shock. Dexamethasone is unnecessary for most patients with typhoid but is recommended for all patients with suspected typhoid fever who are delirious, obtunded, stuporous, comatose, or in shock.

Phase I/IIa Safety, Immunogenicity, and Efficacy Trial of NYVAC-Pf7, a Pox-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-γ responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8+ and CD4+ T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8+-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-γ responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasites life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data

Immunization of volunteers by the bite of mosquitoes carrying radiation-attenuated Plasmodium falciparum sporozoites protects greater than 90% of such volunteers against malaria, if adequate numbers of immunizing biting sessions and sporozoite-infected mosquitoes are used. Nonetheless, until recently it was considered impossible to develop, license and commercialize a live, whole parasite P. falciparum sporozoite (PfSPZ) vaccine. In 2003 Sanaria scientists reappraised the potential impact of a metabolically active, non-replicating PfSPZ vaccine, and outlined the challenges to producing such a vaccine. Six years later, significant progress has been made in overcoming these challenges. This progress has enabled the manufacture and release of multiple clinical lots of a 1(st) generation metabolically active, non-replicating PfSPZ vaccine, the Sanaria PfSPZ Vaccine, submission of a successful Investigational New Drug application to the US Food and Drug Administration, and initiation of safety, immunogenicity and protective efficacy studies in volunteers in MD, US. Efforts are now focused on how best to achieve submission of a successful Biologics License Application and introduce the vaccine to the primary target population of African children in the shortest possible period of time. This will require implementation of a systematic, efficient clinical development plan. Short term challenges include optimizing the (1) efficiency and scale up of the manufacturing process and quality control assays, (2) dosage regimen and method of administration, (3) potency of the vaccine, and (4) logistics of delivering the vaccine to those who need it most, and finalizing the methods for vaccine stabilization and attenuation. A medium term goal is to design and build a facility for manufacturing highly potent and stable vaccine for pivotal Phase 3 studies and commercial launch.