Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans.

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

Progression of Plasmodium berghei through Anopheles stephensi is density-dependent.

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.

An ontology for major histocompatibility restriction.

BACKGROUND: MHC molecules are a highly diverse family of proteins that play a key role in cellular immune recognition. Over time, different techniques and terminologies have been developed to identify the specific type(s) of MHC molecule involved in a specific immune recognition context. No consistent nomenclature exists across different vertebrate species. PURPOSE: To correctly represent MHC related data in The Immune Epitope Database (IEDB), we built upon a previously established MHC ontology and created an ontology to represent MHC molecules as they relate to immunological experiments. DESCRIPTION: This ontology models MHC protein chains from 16 species, deals with different approaches used to identify MHC, such as direct sequencing verses serotyping, relates engineered MHC molecules to naturally occurring ones, connects genetic loci, alleles, protein chains and multi-chain proteins, and establishes evidence codes for MHC restriction. Where available, this work is based on existing ontologies from the OBO foundry. CONCLUSIONS: Overall, representing MHC molecules provides a challenging and practically important test case for ontology building, and could serve as an example of how to integrate other ontology building efforts into web resources.

Persistence of atovaquone in human sera following treatment: inhibition of Plasmodium falciparum development in vivo and in vitro.

Published pharmacokinetic data indicate that after treatment of patients with therapeutic doses of atovaquone/proguanil hydrochloride (Malarone, GlaxoSmithKline Research Triangle Park, NC), the plasma half-lives of these drugs are 70h and 15h, respectively. However, using two biologic assays (mosquito transmission and in vitro asexual stage development), we demonstrate here that sera from volunteers treated with atovaquone/proguanil retained activity against Plasmodium falciparum up to 6 weeks after such treatment. This activity was due to atovaquone, as administration of this drug alone replicated the data obtained with the combination. Most notably, asexual stage development of an atovaquone-resistant strain (NGATV01) of P. falciparum was not inhibited by sera taken after atovaquone treatment. These data indicate that for atovaquone, biologic assays, though not quantitative, are more sensitive than the usual physicochemical assays. Also, persistence of atovaquone in plasma at low concentrations for long periods may increase the risk of resistant parasites arising.

Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara.

Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.

Complete development of mosquito phases of the malaria parasite in vitro.

Methods for reproducible in vitro development of the mosquito stages of malaria parasites to produce infective sporozoites have been elusive for over 40 years. We have cultured gametocytes of Plasmodium berghei through to infectious sporozoites with efficiencies similar to those recorded in vivo and without the need for salivary gland invasion. Oocysts developed extracellularly in a system whose essential elements include co-cultured Drosophila S2 cells, basement membrane matrix, and insect tissue culture medium. Sporozoite production required the presence of para-aminobenzoic acid. The entire life cycle of P. berghei, a useful model malaria parasite, can now be achieved in vitro.

A malaria scavenger receptor-like protein essential for parasite development.

Malaria parasites suffer severe losses in the mosquito as they cross the midgut, haemolymph and salivary gland tissues, in part caused by immune responses of the insect. The parasite compensates for these losses by multiplying during the oocyst stage to form the infectious sporozoites. Upon human infection, malaria parasites are again attenuated by sustained immune attack. Here, we report a single copy gene that is highly conserved amongst Plasmodium species that encodes a secreted protein named PxSR. The predicted protein is composed of a unique combination of metazoan protein domains that have been previously associated with immune recognition/activation and lipid/protein adhesion interactions at the cell surface, namely: (i) scavenger receptor cysteine rich (SRCR); (ii) pentraxin (PTX); (iii) polycystine-1, lipoxygenase, alpha toxin (LH2/PLAT); (iv) Limulus clotting factor C, Coch-5b2 and Lgl1 (LCCL). In our assessment the PxSR molecule is completely novel in biology and is only found in Apicomplexa parasites. We show that PxSR is expressed in sporozoites of both human and rodent malaria species. Disruption of the PbSR gene in the rodent malaria parasite P. berghei results in parasites that form normal numbers of oocysts, but fail to produce any sporozoites. We suggest that, in addition to a role in sporogonic development, PxSR may have a multiplicity of functions.