Resposta de anticorpos IgG contra a região C-terminal da Proteína 1 da Superfície de Merozóitos de Plasmodium vivax em indivíduos que residem em áreas de transmissão de malária no Estado do Pará

Neste estudo avaliamos o potencial antigênico da proteína recombinante His₆-P1₁₉ contendo a região C-terminal da Proteína 1 da Superfície de Merozoítos de Plasmodium vivax. Analisamos a aquisição e os níveis de anticorpos IgG, e comparamos duas áreas com transmissão de malária, localizadas nos municípios de Trairão e Itaituba, Pará. Foram analisadas 391 amostras, sendo 208 amostras coletadas em Três Boeiras (TB) e 183 em São Luiz do Tapajós (SLT). No momento da coleta, foi realizado o exame da gota espessa e foram obtidos dados como idade, número de episódios prévios de malária e tempo decorrido desde o último episódio. As amostras foram analisadas por (ELISA) e os aspectos imunoepidemiológicos foram descritos. A comparação entre as duas áreas mostrou que tanto a freqüência de soros que reconheceram a His₆-MSP1₁₉ como a concentração dos anticorpos IgG foi maior na população de TB, quando comparado com SLT. A freqüência de soros positivos foi 64,42% e 20,22% e a média dos índices de reatividade foi 6,11 ± 4,58 e 2,56 ± 1,96, respectivamente. A idade não influenciou a aquisição de anticorpos IgG na população mais expostas (TB), mas na população menos exposta (SLT) a percentagem de positivos aumentou entre os adultos. Nos grupos de indivíduos que relataram nunca terem tido malária, a freqüência de positivos foi semelhante nas duas localidades. No grupo que relatou ter tido malária, a percentagem de positivos foi maior em TB. Nesses dois grupos, a concentração de anticorpos IgG foi maior na população de TB. Nas duas áreas, o número de episódio influenciou a resposta de anticorpos específicos, sendo que em TB contribuiu para o aumento da percentagem de positivos e da concentração de anticorpos, mas em SLT influenciou apenas na percentagem de positivos. Entretanto, nas duas áreas, não foi possível associar a resposta de anticorpo com proteção, uma vez que os mais expostos estavam tendo malária recentemente. As áreas de estudo apresentaram perfil diferente quanto à intensidade da transmissão de malária e a aquisição natural de anticorpos. Os aspectos imunoepidemiológicos mostraram que a exposição contínua ao parasito contribuiu para a aquisição de anticorpos IgG específicos contra antígeno da fase sangüínea de P. vivax. No entanto, esta resposta não foi efetiva para conferir proteção.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP119, PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP119. Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Early skin immunological disturbance after Plasmodium-infected mosquito bites

Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or C086 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host. (C) 2012 Elsevier Inc. All rights reserved.

Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Hepatocyte determinants of susceptibility to pre-erythrocytic Plasmodium infection

Thesis (Ph.D.)--University of Washington, 2016-08

Serological and parasitological study and report of the first case of human babesiosis in Colombia

A study on the presence of Babesia in humans was performed in Puerto Berrío (Latitude 6.50deg. Longitude: -74.38deg. River: Magdalena. Area: 74.410km², Colombia-South America). Indirect immunofluorescence, thin and thick blood smears were used to study 194 individuals. Patients were grouped according to their risk-factors for Babesia infection: (group 1) individuals with fever, chills, sweating and other malaria-type symptoms; (group 2) symptomatic and asymptomatic individuals from local cattle ranches, which were enrolled in an active form, and (group 3) workers from the local slaughterhouse. Seven individuals were serologically positive for Babesia: Three individuals presented IgM antibodies against B. bovis, while one had IgG against this species; one individual had IgM against B. bigemina, another had IgG and a third both IgM and IgG against this species. Only one individual was parasitologically positive for Babesiaand serologically positive for Babesia bovis (IgM 1:64)

Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Natural antibody response to Plasmodium falciparum merozoite antigens MSP5, MSP9 and EBA175 is associated to clinical protection in the Brazilian Amazon

BACKGROUND: Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. METHODS: Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients' plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. RESULTS: 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2(DF=1) = 9.26/p = 0.0047) and MSP5 (X2(DF=1) = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2(DF=1) = 6.41/p = 0.0206, Fisher's exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p < 0.03), MSP5 (U = 212, p < 0.004), MSP9 (U = 189.5, p < 0.002) and EBA175 (U = 197, p < 0.014, Mann-Whitney's U test). IgG1 and IgG3 were predominant for all antigens, but some patients also presented with IgG2 and IgG4. The recognition of MSP5 (OR = 0.112, IC95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. CONCLUSIONS: Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Functional characterisation of Schistosoma japonicum acetylcholinesterase

BACKGROUND: Acetylcholinesterase (AChE) is an important metabolic enzyme of schistosomes present in the musculature and on the surface of the blood stage where it has been implicated in the modulation of glucose scavenging from mammalian host blood. As both a target for the antischistosomal drug metrifonate and as a potential vaccine candidate, AChE has been characterised in the schistosome species Schistosoma mansoni, S. haematobium and S. bovis, but not in S. japonicum. Recently, using a schistosome protein microarray, a predicted S. japonicum acetylcholinesterase precursor was significantly targeted by protective IgG1 immune responses in S. haematobium-exposed individuals that had acquired drug-induced resistance to schistosomiasis after praziquantel treatment.

RESULTS: We report the full-length cDNA sequence and describe phylogenetic and molecular structural analysis to facilitate understanding of the biological function of AChE (SjAChE) in S. japonicum. The protein has high sequence identity (88 %) with the AChEs in S. mansoni, S. haematobium and S. bovis and has 25 % sequence similarity with human AChE, suggestive of a highly specialised role for the enzyme in both parasite and host. We immunolocalized SjAChE and demonstrated its presence on the surface of adult worms and schistosomula, as well as its lower expression in parenchymal regions. The relatively abundance of AChE activity (90 %) present on the surface of adult S. japonicum when compared with that reported in other schistosomes suggests SjAChE may be a more effective drug or immunological target against this species. We also demonstrate that the classical inhibitor of AChE, BW285c51, inhibited AChE activity in tegumental extracts of paired worms, single males and single females by 59, 22 and 50 %, respectively, after 24 h incubation with 200 μM BW284c51.

CONCLUSIONS: These results build on previous studies in other schistosome species indicating major differences in the enzyme between parasite and mammalian host, and provide further support for the design of an anti-schistosome intervention targeting AChE.

Malaria transmission blocking immunity and sexual stage vaccines for interrupting malaria transmission in Latin America

Malaria is a vector-borne disease that is considered to be one of the most serious public health problems due to its high global mortality and morbidity rates. Although multiple strategies for controlling malaria have been used, many have had limited impact due to the appearance and rapid dissemination of mosquito resistance to insecticides, parasite resistance to multiple antimalarial drug, and the lack of sustainability. Individuals in endemic areas that have been permanently exposed to the parasite develop specific immune responses capable of diminishing parasite burden and the clinical manifestations of the disease, including blocking of parasite transmission to the mosquito vector. This is referred to as transmission blocking (TB) immunity (TBI) and is mediated by specific antibodies and other factors ingested during the blood meal that inhibit parasite development in the mosquito. These antibodies recognize proteins expressed on either gametocytes or parasite stages that develop in the mosquito midgut and are considered to be potential malaria vaccine candidates. Although these candidates, collectively called TB vaccines (TBV), would not directly stop malaria from infecting individuals, but would stop transmission from infected person to non-infected person. Here, we review the progress that has been achieved in TBI studies and the development of TBV and we highlight their potential usefulness in areas of low endemicity such as Latin America.

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis.