Rice‐produced MSP142 of Plasmodium falciparum elicits antibodies that inhibit parasite growth in vitro

Many malaria antigens contain multiple disulphide bonds involved in the formation of inhibitory B‐cell epitopes. Producing properly folded malaria antigens in sufficient quantities for vaccination is often a challenge. The 42‐kDa fragment of Plasmodium falciparum merozoite surface protein 1 (MSP1₄₂) is such a kind of malaria antigen. In this study, we investigated the expression of MSP1₄₂ in a rice system (9522, a cultivar of Oryza sativa ssp. japonica), which was used as a bioreactor for protein production. The MSP1₄₂ gene was synthesized according to rice‐preferred codons and transformed into rice plants via an Agrobacterium‐mediated method. The recombinant antigen was efficiently expressed in rice seeds with a level up to 1.56% of total soluble protein and was recognized by both the conformational monoclonal antibody 5.2 (mAb5.2) and the pooled sera of P. falciparum malaria patients. Rabbits were immunized intramuscularly with the purified MSP1₄₂ formulated with Freund's adjuvant. High antibody titres against MSP1₄₂ were elicited. The rabbit immune sera reacted well with the native protein of P. falciparum parasite and strongly inhibited the in vitro growth of blood‐stage P. falciparum parasites, demonstrating that transgenic rice can become an efficient bioreactor for the production of malaria vaccine antigens.     

Th1 immune response to Plasmodium falciparum recombinant thrombospondin‐related adhesive protein (TRAP) antigen is enhanced by TLR3‐specific adjuvant,poly(I:C) in BALB/c mice

Sporozoite‐based malaria vaccines have provided a gold standard for malaria vaccine development, and thrombospondin‐related adhesive protein (TRAP) serves as the main vaccine candidate antigen on sporozoites. As recombinant malaria vaccine candidate antigens are poorly immunogenic, additional appropriate immunostimulants, such as an efficient adjuvant, are highly essential to modulate Th1‐cell predominance and also to induce a protective and long‐lived immune response. In this study, polyinosinic:polycytidylic acid [poly(I:C)], the ligand of TLR3, was considered as the potential adjuvant for vaccines targeting stronger Th1‐based immune responses. For this purpose, BALB/c mice were immunized with rPfTRAP delivered in putative poly(I:C) adjuvant, and humoural and cellular immune responses were determined in different immunized mouse groups. Delivery of rPfTRAP with poly(I:C) induced high levels and titres of persisted and also high‐avidity anti‐rPfTRAP IgG antibodies comparable to complete Freund's adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) adjuvant after the second boost. In addition, rPfTRAP formulated with poly(I:C) elicited a higher ratio of IFN‐γ/IL‐5, IgG2a/IgG1, and IgG2b/IgG1 than with CFA/IFA, indicating that poly(I:C) supports the induction of a stronger Th1‐based immune response. This is a first time study which reveals the potential of rPfTRAP delivery in poly(I:C) to increase the level, avidity and durability of both anti‐PfTRAP cytophilic antibodies and Th1 cytokines.     

Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname

Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether–lumefantrine in South‐East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America.     

Immunization against Plasmodium falciparum with recombinant polypeptides produced in Escherichia coli

Two proteins produced in recombinant Escherichia coli and containing amino acid sequences from the Plasmodium falciparum precursor to major merozoite surface antigens (PMMSA) have been partially purified. These proteins, together with a preparation of merozoites, have been used to immunize animals. The antibody response and the degree of protection were compared. Animals immunized with merozoites produced antibodies reacting with many P. falciparum proteins, whereas a response specific for PMMSA was detected in those receiving the recombinant material. Incomplete protection was conferred to both groups and there was no apparent correlation between antibody levels and protection.     

Severity of anaemia is associated with bone marrow haemozoin in children exposed to Plasmodium falciparum

There are no large‐scale ex vivo studies addressing the contribution of Plasmodium falciparum in the bone marrow to anaemia. The presence of malaria parasites and haemozoin were studied in bone marrows from 290 anaemic children attending a rural hospital in Mozambique. Peripheral blood infections were determined by microscopy and polymerase chain reactions. Bone marrow parasitaemia, haemozoin and dyserythropoiesis were microscopically assessed. Forty‐two percent (123/290) of children had parasites in the bone marrow and 49% (111/226) had haemozoin, overlapping with parasitaemia in 83% (92/111) of cases. Sexual and mature asexual parasites were highly prevalent (62% gametocytes, 71% trophozoites, 23% schizonts) suggesting their sequestration in this tissue. Sixteen percent (19/120) of children without peripheral infection had haemozoin in the bone marrow. Haemozoin in the bone marrow was independently associated with decreased Hb concentration (P = 0·005) and was more common in dyserythropoietic bone marrows (P = 0·010). The results of this ex vivo study suggest that haemozoin in the bone marrow has a role in the pathogenesis of malarial‐anaemia through ineffective erythropoiesis. This finding may have clinical implications for the development of drugs targeted to prevent and treat malarial‐anaemia.     

Melatonin activates FIS1, DYN1, and DYN2 Plasmodium falciparum related‐genes for mitochondria fission: Mitoemerald‐GFP as a tool to visualize mitochondria structure

Malaria causes millions of deaths worldwide and is considered a huge burden to underdeveloped countries. The number of cases with resistance to all antimalarials is continuously increasing, making the identification of novel drugs a very urgent necessity. A potentially very interesting target for novel therapeutic intervention is the parasite mitochondrion. In this work, we studied in Plasmodium falciparum 3 genes coding for proteins homologues of the mammalian FIS1 (Mitochondrial Fission Protein 1) and DRP1 (Dynamin Related Protein 1) involved in mitochondrial fission. We studied the expression of P. falciparum genes that show ample sequence and structural homologies with the mammalian counterparts, namely FIS1, DYN1, and DYN2. The encoded proteins are characterized by a distinct pattern of expression throughout the erythrocytic cycle of P. falciparum, and their mRNAs are modulated by treating the parasite with the host hormone melatonin. We have previously reported that the knockout of the Plasmodium gene that codes for protein kinase 7 is essential for melatonin sensing. We here show that PfPk7 knockout results in major alterations of mitochondrial fission genes expression when compared to wild‐type parasites, and no change in fission proteins expression upon treatment with the host hormone. Finally, we have compared the morphological characteristics (using MitoTracker Red CMX Ros) and oxygen consumption properties of P. falciparum mitochondria in wild‐type parasites and PfPk7 Knockout strains. A novel GFP construct targeted to the mitochondrial matrix to wild‐type parasites was also developed to visualize P. falciparum mitochondria. We here show that, the functional characteristics of P. falciparum are profoundly altered in cells lacking protein kinase 7, suggesting that this enzyme plays a major role in the control of mitochondrial morphogenesis and maturation during the intra‐erythrocyte cell cycle progression.     

Immunization with Brugia malayi Hsp70 protects mice against Litomosoides sigmodontis challenge infection

More than 1·5 billion people are at risk of being infected with filarial nematodes worldwide. Therapy and control of transmission are mainly based on mass drug distribution. As these drugs have to be administered annually or biannually and might be loosing their efficacy, a vaccine against filariae is an alternative approach to chemotherapy. In the current study, we have analysed the potential of Brugia malayi heat shock protein 70 (BmHsp70) as a vaccine candidate in a murine helminth infection. Immunization of BALB/c mice with alum‐precipitated recombinant BmHsp70 conferred partial protection against subsequent challenge infection with the rodent parasite Litomosoides sigmodontis. Immunization resulted in reduced numbers of larvae in the pleural cavity as well as reduced numbers of circulating microfilariae. Reduced parasite burden was associated with high titres of BmHsp70‐specific antibodies and increased production of type I and II cytokines in response to L. sigmodontis antigen and BmHsp70. In summary, the immunization with BmHsp70 induced cellular and humoral immune responses and partially protected against L. sigmodontis in a challenge infection. Therefore, we hypothesize that BmHsp70 might be considered as a potential vaccine candidate for reduction in the incidence of B. malayi infections in future studies.     

Differential T‐cell responses to a chimeric Plasmodium falciparum antigen; UB05‐09, correlates with acquired immunity to malaria

The development of a sterilizing and cost‐effective vaccine against malaria remains a major problem despite recent advances. In this study, it is demonstrated that two antigens of P. falciparum UB05, UB09 and their chimera UB05‐09 can serve as protective immunity markers by eliciting higher T‐cell responses in malaria semi‐immune subjects (SIS) than in frequently sick subjects (FSS) and could be used to distinguish these two groups. UB05, UB09 and UB05‐09 were cloned, expressed in E. coli, purified and used to stimulate PBMCs isolated from 63 subjects in a malaria endemic area, for IFN‐γ production, which was measured by the ELISpot assay. The polymorphism of UB09 gene in the malaria infected population was also studied by PCR/sequencing of the gene in P. falciparum field isolates. All three antigens were preferentially recognized by PBMCs from SIS. IFN‐γ production induced by these antigens correlated with the absence of fever and parasitaemia. UB09 was shown to be relatively well‐conserved in nature. It is concluded that UB05, UB09 and the chimera UB05‐09 posses T‐cell epitopes that are associated with protection against malaria and could thus be used to distinguish SIS from FSS eventhough acute infection with malaria has been shown to reduce cytokine production in some studies. Further investigations of these antigens as potential diagnostic and/or vaccine candidates for malaria are indicated.     

C2株蓝氏贾第鞭毛虫TCTP基因的克隆、表达和序列分析

目的克隆C2株蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)的翻译调控肿瘤蛋白(Translationally controlled tumor protein,TCTP)编码区,对其进行生物信息学分析和原核表达。方法根据WB株贾第虫TCTP编码区序列设计引物,以C2株贾第虫基因组DNA为模板扩增获得TCTP编码区片段,双酶切连入原核表达载体pET-28a(+),将酶切和测序验证正确的重组质粒转化E. coli Rosetta(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE及Western blot鉴定蛋白产物,采用Ni-NTA经亲和层析纯化重组蛋白。同时根据测序结果分析C2株贾第虫TCTP蛋白结构特征和进化关系。结果克隆了包含酶切位点全长470 bp的C2株贾第虫TCTP编码区,构建了原核表达载体pET-28a(+)-TCTP,在Rosetta(DE3)中表达、纯化得到了相对分子量约18.5 kDa的融合蛋白,与预期相符;测序结果显示C2株贾第虫TCTP序列与WB株相同,生物信息学分析显示贾第虫TCTP蛋白虽然序列长度较其它物种更短但与裂殖酵母TCTP空间结构相似,均由4个β折叠和3个主要的α螺旋组成,位于序列中部的不规则环状结构更小,仅9个氨基酸残基组成,在进化上与其它真核生物亲缘关系较远。结论成功克隆表达了贾第虫TCTP,为TCTP功能及贾第虫致病机制的研究提供了实验依据。     

恶性疟原虫裂殖子表面蛋白2融合HBS基因重组质粒的免疫原性研究

目的　构建恶性疟原虫海南分离株 (FCC1/HN)裂殖子表面蛋白 2 (MSP2 )融合HBsAg基因片断真核重组表达质粒 pVXORF1-PfMSP2 -HBS ,观察该DNA疫苗的免疫特性。方法　 (1)以恶性疟原虫海南分离株 (FCC1/HN)基因组为模板 ,扩增出pfMSP2 DNA片断 ,将该片段克隆入质粒pVXORF1-HBS中HBS的上游并与之紧密相连 ,构建质粒 pVXORF1-PfMSP2 -HBS ;构建真核重组表达质粒 pVXORF1-PfMSP2 以作为对照。 (2 )用上述构建的质粒及空质粒 pVXORF1免疫KM小鼠 ,以ELISA检测血清IgG抗体。 结果　 (1)筛选出的重组子为编码FCC1/HNMSP2 基因片断的重组质粒 pVXORF1-PfMSP2 -HBS及 pVXORF1-PfMSP2 。 (2 )裸DNA尾根多点注射KM小鼠 ,显示 pVXORF1-PfMSP2 -HBS组小鼠较pVXORF1-PfMSP2 组小鼠产生抗PfMSP2 蛋白的抗体效价明显增高 ,二者比较 ,差异有非常显著意义 (P <0 0 1)。结论　PfMSP2 融合HBsAg基因片段激发机体产生抗PfMSP2 抗体的能力显著增强 ,提示PfMSP2 融合HBsAg基因有可能作为高效抗红内期恶性疟原虫复合多价基因疫苗的有效组成部分     

Neutrophil alterations in pregnancy‐associated malaria and induction of neutrophil chemotaxis by Plasmodium falciparum

Pregnancy‐associated malaria (PAM) is a severe form of the disease caused by sequestration of Plasmodium falciparum‐infected red blood cells (iRBCs) in the developing placenta. Pathogenesis of PAM is partially based on immunopathology, with frequent monocyte infiltration into the placenta. Neutrophils are abundant blood cells that are essential for immune defence but may also cause inflammatory pathology. Their role in PAM remains unclear. We analysed neutrophil alterations in the context of PAM to better understand their contribution to disease development. Pregnant women exposed to Plasmodium falciparum had decreased numbers of circulating neutrophils. Placental‐like BeWo cells stimulated with malaria parasites produced the neutrophil chemoattractant IL‐8 and recruited neutrophils in a trans‐well assay. Finally, immunostaining of a PAM placenta confirmed neutrophil accumulation in the intervillous space. Our data indicate neutrophils may play a role in placental malaria and should be more closely examined as an etiological agent in the pathophysiology of disease.     

Plasmodium falciparum infection transmitted by transfusion: A cause of hemophagocytic syndrome after bone marrow tranplantation in a non‐endemic country

A 27‐year‐old man with severe aplastic anemia underwent bone marrow transplantation from his HLA identical brother in July 2016. Conditioning included ATGAM 30 mg/kg for 3 days and Cyclophosphamide 50 mg/kg for 4 days. The patient received several platelet and red blood cell transfusions before and after the conditioning. The patient received broad spectrum antibiotics and caspofungin because persistant febrile neutropenia without bacteriological or mycological documentation. Hemophagocytic syndrome was diagnosed on day +12. Steroids at 1 mg/kg were started on day +12. Fever resolved the same day but resumed 3 days later associated to intravascular hemolysis with no schizocytes on blood smears and negative DAT. Thick blood film smears performed on day +26 revealed Plasmodium falciparum parasites (parasitemia = 20%). Except the level of parasitemia, there were no signs of gravity. Quinine was started on day 26 at a loading dose of 15 mg/kg followed by 8 mg/kg three times a day for 20 doses. Fever vanished after 2 days. Parasitemia cleared in 3 days and remained negative thereafter. Investigations revealed that the patient was transfused by a red cell unit harvested in a voluntary donor native of a malaria endemic country. PCR for P. falciparum performed in this donor in the frame of investigations was positive. The patient is alive with a normal blood count 1 year after BMT.     

Antibody responses to surface antigens of Plasmodium falciparum gametocyte‐infected erythrocytes and their relation to gametocytaemia

An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra‐erythrocytic gametocytes produced either by a laboratory‐adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti‐gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti‐GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06–1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte‐infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti‐gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.     

Immunization against malaria with a recombinant protein

We have expressed in bacteria the C-terminal part of Plasmodium yoelii merozoite surface protein-1 (MSP1) containing the two epidermal growth factor-like domains. The protein, either alone or fused to glutathione Stransferase, was highly effective as a vaccine and protected mice against challenge infection. Reduction and alkylation abolished the protection obtained with the protein. This shows for the first time the absolute requirement of the disulphide-bonded conformation for immunogenicity. In a short term experiment, mice were protected against a massive challenge. The immunity was effective at the time of merozoite release/reinvasion. Recombinant protein based on this part of MSP1 may be suitable as a vaccine against malaria.     

The role of regulatory T cells during Plasmodium chabaudi chabaudi AS infection in BALB/c mice

An inappropriate immune response to parasite infection is one of the primary drivers of malaria pathogenesis. Regulatory T cells (Tregs), an important subset of CD4⁺ T cells, can maintain self‐tolerance and prevent autoimmune diseases. However, there is little consensus about their role in malaria pathogenesis. In this study, we transiently depleted Tregs (CD25⁺ T cells) using an anti‐CD25 mAb (7D4 clone) at different time points following Plasmodium chabaudi chabaudi AS infection in BALB/c mice and investigated the effect of depletion of Tregs in this model. In control mice, Tregs proliferated significantly and their suppressive function was enhanced after infection. IL‐10 was increased drastically during infection. Depletion of Tregs at various time points can lead to divergent outcomes. When Tregs were depleted prior to or during the early phase of infection, most mice survived and had a robust Th1 immune response. In contrast, when Tregs were depleted close to peak parasitemia, all mice died as a result of inflammation. Taken together, these data suggest that in P. c. chabaudi AS‐infected BALB/c mice, Tregs inhibit the Th1 response and macrophage activation, leading to increased parasite load; however, they also control inflammation‐mediated pathology by secreting high levels of IL‐10.