Robert W. Gwadz

Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi.

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man1. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species2–4. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a β-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.

An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross.

We report a genetic linkage map of the Plasmodium falciparum genome, using the inheritance patterns of nearly 90 RFLP markers in a genetic cross. Markers were assigned to polymorphic loci on all 14 nuclear chromosomes. Genetic recombination between parental markers was detected in each of the progeny, indicating that progeny from cross-fertilization events were favored over progeny from self-fertilization of either parent alone. Inheritance patterns among the markers suggested that certain parental linkage groups on chromosomes 2, 3, 12 and 13 were favored in the cross. Recombination frequencies on five chromosomes indicated an approximate map unit size of 15-30 kb per centiMorgan for P. falciparum.

Multistage Multiantigen Heterologous Prime Boost Vaccine for Plasmodium knowlesi Malaria Provides Partial Protection in Rhesus Macaques

ABSTRACT A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.

Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae.

Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen

ABSTRACT We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-γ) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/106 PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-γ responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.

Plasmodium gallinaceum: transmission-blocking immunity in chickens. I. Comparative immunogenicity of gametocyte- and gamete-containing preparations.

Abstract Immunization of chickens by intravenous inoculation of preparations derived from blood infected with Plasmodium galinaceum led to reduced infectivity to mosquitoes ( Aedes aegypti ) during subsequently induced blood infection but had little effect on the course of asexual parasitemia. Immunization with preparations containing intracellular gametocytes was much less effective than immunization with preparations in which the extracellular gametes had been released during gametogenesis (emergence and exflagellation) prior to inoculation. By far the most effective material were preparations of partially purified gametes of both sexes. Three weekly inoculations of this material resulted in 99.99% suppression of infectivity to mosquitoes during subsequently induced blood infection. Preparations of purified gamete material from which gametes of one or another sex were absent were considerably less effective as transmission-blocking immunogens than the mixed gamete preparation. It is possible that the two sexes of gamete act synergistically to induce transmission-blocking immunity.

Plasmodium gallinaceum: Transmission-blocking immunity in chickens: II. The effect of antigamete antibodies in vitro and in vivo and their elaboration during infection

Abstract Heat-inactivated serum from chickens with transmission-blocking immunity to Plasmodium gallinaceum prevented the in vitro development of ookinetes from gametocytes of P. gallinaceum only when present during the period between the initiation of gametogeriesis and the release of the microgametes. When added after this time immune serum failed to suppress ookinete development. Immune serum did not prevent the formation of gametes from gametocytes. These results are interpreted to indicate that immune serum contains factors which prevent fertilization of the malarial gametes but which do not affect the development of the zygote once fertilization has taken place. Two distinct reactions of malarial gametes with serum from chickens with transmission-suppressing immunity are described—the gamete-agglutination (AG) reaction and the microgamete surface-fixation (SF) reaction. Both reactions were associated with the immunoglobulin fraction of immune serum. The presence of SF antibodies during a blood infection correlated closely with effective transmission-blocking immunity in vivo ; AG antibodies, on the other hand, were present in various circumstances in the absence of transmission-blocking immunity. AG and SF antibodies occurred not only in birds immunized with P. gallinaceum -gamete preparations but also during or following infections in unimmunized birds; SF antibodies appeared only following the peak of asexual infection in unimmunized birds and were of low titer. In immunized birds blood infections following live challenge invariably boosted low levels of SF antibodies. The results of immunization of chickens and Rhesus monkeys with gametes of their respective malaria parasites, P. gallinaceum and P. knowlesi , are compared.

The distribution of hatching time in Anopheles gambiae

BackgroundKnowledge of the ecological differences between the molecular forms of Anopheles gambiae and their sibling species, An. arabiensis might lead to understanding their unique contribution to disease transmission and to better vector control as well as to understanding the evolutionary forces that have separated them.MethodsThe distributions of hatching time of eggs of wild An. gambiae and An. arabiensis females were compared in different water types. Early and late hatchers of the S molecular form were compared with respect to their total protein content, sex ratio, development success, developmental time and adult body size.ResultsOverall, the distribution of hatching time was strongly skewed to the right, with 89% of the eggs hatching during the second and third day post oviposition, 10% hatching during the next four days and the remaining 1% hatching over the subsequent week. Slight, but significant differences were found between species and between the molecular forms in all water types. Differences in hatching time distribution were also found among water types (in each species and molecular form), suggesting that the eggs change their hatching time in response to chemical factors in the water. Early hatchers were similar to late hatchers except that they developed faster and produced smaller adults than late hatchers.ConclusionDifferences in hatching time and speed of development among eggs of the same batch may be adaptive if catastrophic events such as larval site desiccation are not rare and the sites quality is unpredictable. The egg is not passive and its hatching time depends on water factors. Differences in hatching time between species and molecular forms were slight, probably reflecting that conditions in their larval sites are rather similar.

Infectiousness and gamete immunization in malaria.

Publisher Summary This chapter discusses infectiousness and gamete immunization in malaria. The sources of infectiousness of a malarious individual are the gametocytes of the malaria parasite. These are the sexual stages produced under obscure circumstances during multiplication of the asexual parasites in the bloodstream. On completing their maturation, the gametocytes (male [microgametocytes] and female [macrogametocytes]) undergo no further development in the blood. Immunization with preparations containing gametes of the malaria parasite leads to highly effective suppression of infectiousness to mosquitoes during subsequent blood infection in three laboratory systems: P. gallinaceum in chickens, P. knowlesi in the rhesus monkey, and P. yoelii in the laboratory mouse. Such immunization results in the elaboration of gamete-specific antibodies. When a mosquito ingests gametocyte-carrying blood that contains such antibodies, the gametes are neutralized in the midgut of the mosquito almost immediately following their release during gametogenesis; fertilization is prevented, and the infection in the mosquito is sterilized. The immunity appears to be specific to the sexual stage. Monkeys immunized with preparations of asexual parasites alone do not produce antigamete antibodies, and their sera do not reduce the infectivity of gametocytes to mosquitoes.

Engorgement response of anopheline mosquitoes to blood fractions and artificial solutions

ABSTRACT. Anopheles stephensi Liston, Anopheles freeborni Aitken, Anopheles gambiae Giles and Anopheles dirus (Peyton & Harrison) fed equally well on whole blood, red blood cells, platelet‐rich plasma and platelet‐poor plasma. Similar feeding ability on 0.15 M NaCl containing 10‐2 M NaHCO3 was shown by the first three species, but An. dirus required an addition of albumin. The need for ATP as a phagostimulant could not be demonstrated in any of these species.