Geoff A. Butcher

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)-γ-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.

A DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccine Encoding Thrombospondin-Related Adhesion Protein but Not Circumsporozoite Protein Partially Protects Healthy Malaria-Naive Adults against Plasmodium falciparum Sporozoite Challenge

ABSTRACT The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-γ ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-γ ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Regulation of infectivity of Plasmodium to the mosquito vector

Publisher Summary This chapter discusses the complex factors regulating the infectivity of the gametocyte to the mosquito vector and describes the patterns of parasite biology that individually advantageous to particular host parasite combinations. The chapter discusses the gametocytogenesis, including the factors that regulate the differentiation of the erythrocytic stage parasites into sexual or asexual parasites, and the development of the gametocytes into either male or female cells. The biology of the mature gametocyte, including its cell biology, distribution and longevity/viability in the peripheral bloodstream, and the biology of the gametocyte-infected host and the regulation of mosquito infection by host factors that interact either directly with the parasite or indirectly upon the parasite following its ingestion by the mosquito vector are presented in the chapter. Gametogenesis, its molecular regulation with respect to the inducers required to trigger the gametocyte to leave its arrested state, the secondary signal pathways that regulate the constituent events of microgametogenesis (exflagellation), and the impact of external regulatory molecules (usually inhibitors) on gamete formation are described. The chapter also explains fertilization, its efficiency and role as a developmental regulator and outlines the post-fertilization development, including the mechanisms that regulate the survival or destruction of the parasite before its successful establishment under the midgut basal lamina where the oocyst then forms.

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.

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.

Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

ABSTRACT After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement onPlasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.

Autoimmunity and malaria

The recent review by Zaccone et al.[1] on the prevention of autoimmune disease by helminths demonstrated how much knowledge of this topic has advanced in recent years. However, for those unfamiliar with this field, evidence for malaria acting similarly was published 40 years ago, although it was not mentioned by these authors.

Inhibition of the mosquito transmission of Plasmodium berghei by Malarone™ (atovaquone-proguanil)

Sera from patients treated with atovaquone-proguanil (Malarone™) have previously been shown to inhibit the mosquito transmission of Plasmodium falciparum, though the inhibition was not complete and the effect declined 2 weeks after treatment. In marked contrast, the inhibition of transmission of P. berghei by human sera (fed to mosquitoes, with P. berghei gametocytes, via membrane feeders) from volunteers treated with atovaquone-proguanil was total up to day 28 post-treatment and still very significant at day 56. In view of the short half-lives of atovaquone and proguanil, this was unexpected, and further experiments, reported here, were undertaken. In contrast to the incomplete blockade of infectivity of P. falciparum by serum taken 4 days post-treatment, such serum was totally inhibitory against P. berghei at a 1:10 000 dilution, indicating a remarkable sensitivity of P. berghei and demonstrating an unusual difference between the two Plasmodium species in response to a drug. The inhibitory effect on P. berghei after day 4 was caused by atovaquone and mainly through blockade of development from ookinete to oocyst. Despite previous information on the rapid elimination of atovaquone by patients, the present data indicate that low concentrations of this drug may persist in the plasma for some weeks after treatment.