Effect of Heat Treatment on Protein Self-Digestion in Ruminants' Milk.

This study investigated whether heat treatments (raw, 63 °C for 30 min, and 85 °C for 5 min) affect protein hydrolysis by endogenous enzymes in the milk of ruminants (bovine, ovine, and caprine) using a self-digestion model. Self-digestion consisted of the incubation for six hours at 37 °C of the ruminants' milk. Free amino group concentration was measured by the o-phthaldialdehyde method, and peptide sequences were identified by chromatography-mass spectrometry. Results showed that heat treatments prior to self-digestion decreased the free NH2 by 59% in bovine milk heated at 85 °C/5 min, and by 44 and 53% in caprine milk heated at 63 °C/30 min and 85 °C/5 min, respectively. However, after self-digestion, only new free amino groups were observed for the raw and heated at 63 °C/30 min milk. ß-Casein was the most cleaved protein in the raw and heated at 63 °C/30 min bovine milk. A similar trend was observed in raw ovine and caprine milk. Self-digestion increased 6.8-fold the potential antithrombin peptides in the bovine milk heated at 63 °C/30 min. Enhancing bioactive peptide abundance through self-digestion has potential applications in the industry for functional products. Overall, heat treatments affected the free amino groups according to the species and heat treatment applied, which was reflected in the varying degrees of cleaved peptide bonds and peptides released during self-digestion.

The impact of heating and drying on protease activities of ruminant milk before and after in vitro infant digestion.

This study investigated the effect of heating (63°C/30 min or 75°C/15 s) and drying (spray-drying or freeze-drying) on plasmin, cathepsin D, and elastase activities in bovine, ovine, and caprine milk, compared to non-dried raw milk counterparts. Protease activities and protein hydrolysis were assessed before and after in vitro infant digestion with or without gastric and pancreatic enzymes. At 75°C/15 s, plasmin activity in caprine and ovine milk decreased (69-75%, p<0.05), while cathepsin D activity in spray-dried bovine milk heated increased (2.8-fold, p<0.05). Plasmin and cathepsin D activities increased (<1.2-fold, p<0.05) after in vitro digestion with pancreatin, regardless of milk species. Endogenous milk enzymes hydrolyzed more proteins than gastric enzymes during gastric digestion and contributed to small intestinal digestion. In summary, milk proteases remained active after processing with effects dependent on the species of milk, and they contributed to in vitro protein hydrolysis in the stomach and small intestine.

Ruminant Milk-Derived Extracellular Vesicles: A Nutritional and Therapeutic Opportunity?

Milk has been shown to contain a specific fraction of extracellular particles that are reported to resist digestion and are purposefully packaged with lipids, proteins, and nucleic acids to exert specific biological effects. These findings suggest that these particles may have a role in the quality of infant nutrition, particularly in the early phase of life when many of the foundations of an infant's potential for health and overall wellness are established. However, much of the current research focuses on human or cow milk only, and there is a knowledge gap in how milk from other species, which may be more commonly consumed in different regions, could also have these reported biological effects. Our review provides a summary of the studies into the extracellular particle fraction of milk from a wider range of ruminants and pseudo-ruminants, focusing on how this fraction is isolated and characterised, the stability and uptake of the fraction, and the reported biological effects of these fractions in a range of model systems. As the individual composition of milk from different species is known to differ, we propose that the extracellular particle fraction of milk from non-traditional and minority species may also have important and distinct biological properties that warrant further study.

Heat-Treatments Affect Protease Activities and Peptide Profiles of Ruminants' Milk.

Proteases present in milk are heat-sensitive, and their activities increase or decrease depending on the intensity of the thermal treatment applied. The thermal effects on the protease activity are well-known for bovine milk but poorly understood for ovine and caprine milk. This study aimed to determine the non-specific and specific protease activities in casein and whey fractions isolated from raw bovine, ovine, and caprine milk collected in early lactation, and to determine the effects of low-temperature, long-time (63°C for 30 min) and high-temperature, short-time (85°C for 5 min) treatments on protease activities within each milk fraction. The non-specific protease activities in raw and heat-treated milk samples were determined using the substrate azocasein. Plasmin (the main protease in milk) and plasminogen-derived activities were determined using the chromogenic substrate S-2251 (D-Val-Leu-Lys-pNA dihydrochloride). Peptides were characterized using high-resolution liquid chromatography coupled with tandem mass spectrometry. The activity of all native proteases, shown as non-specific proteases, was similar between raw bovine and caprine milk samples, but lower (P < 0.05) than raw ovine milk in the whey fraction. There was no difference (P > 0.05) between the non-specific protease activity of the casein fraction of raw bovine and caprine milk samples; both had higher activity than ovine milk. After 63°C/30 min, the non-specific protease activity decreased (44%; P > 0.05) for the bovine casein fraction only. In contrast, the protease activity of the milk heated at 85°C/5 min changed depending on the species and fraction. For instance, the activity decreased by 49% for ovine whey fraction, but it increased by 68% for ovine casein fraction. Plasmin and plasminogen were in general inactivated (P > 0.05) when all milk fractions were heated at 85°C/5 min. Most of the peptides present in heat-treated milk were derived from ß-casein and αS1-casein, and they matched the hydrolysis profile of cathepsin D and plasmin. Identified peptides in ruminant milk samples had purported immunomodulatory and inhibitory functions. These findings indicate that the non-specific protease activity in whey and casein fractions differed between ruminant milk species, and specific thermal treatments could be used to retain better protease activity for all ruminant milk species.

A protocol for selection of Trichoderma spp. to protect grapevine pruning wounds against Lasiodiplodia theobromae.

There is no standardized protocol to select Trichoderma against trunk disease pathogens in grapevine. This study describes a protocol to select Trichoderma isolates for protecting the pruning wounds of grapevine against Lasiodiplodia theobromae. Thus, four assays were performed: (1) in vitro antagonism of Trichoderma isolates, molecularly identified, to control L. theobromae; (2) in vitro effect of grapevine fungicides on the conidia of the antagonists; (3) capability of Trichoderma to survive and endophytically penetrate in grapevine canes; and (4) efficiency of Trichoderma and fungicides to protect the pruning wounds of grapevine shoots against L. theobromae. T. asperelloides (02/03), T. koningiopsis (09/02), and T. asperellum (01/13) were the best antagonists for L. theobromae. The conidia of the three isolates were affected only by tebuconazole, with the first two fungi being the most tolerant to the fungicides. The Trichoderma isolates survived on the cane tip for 15 days, but only T. asperelloides (02/03) penetrated endophytically 4 cm in the cane and showed preventive and curative capability to control L. theobromae, with similar efficiency as that of the fungicides in both the periods tested post inoculation. These four assays are suggested to select Trichoderma against L. theobromae or other trunk disease pathogens.

Genetic sequence characterization and naturally acquired immune response to Plasmodium vivax Rhoptry Neck Protein 2 (PvRON2).

BACKGROUND: The genetic diversity of malaria antigens often results in allele variant-specific immunity, imposing a great challenge to vaccine development. Rhoptry Neck Protein 2 (PvRON2) is a blood-stage antigen that plays a key role during the erythrocyte invasion of Plasmodium vivax. This study investigates the genetic diversity of PvRON2 and the naturally acquired immune response to P. vivax isolates. RESULTS: Here, the genetic diversity of PvRON21828-2080 and the naturally acquired humoral immune response against PvRON21828-2080 in infected and non-infected individuals from a vivax malaria endemic area in Brazil was reported. The diversity analysis of PvRON21828-2080 revealed that the protein is conserved in isolates in Brazil and worldwide. A total of 18 (19%) patients had IgG antibodies to PvRON21828-2080. Additionally, the analysis of the antibody response in individuals who were not acutely infected with malaria, but had been infected with malaria in the past indicated that 32 patients (33%) exhibited an IgG immune response against PvRON2. CONCLUSIONS: PvRON2 was conserved among the studied isolates. The presence of naturally acquired antibodies to this protein in the absence of the disease suggests that PvRON2 induces a long-term antibody response. These results indicate that PvRON2 is a potential malaria vaccine candidate.

Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate.

BACKGROUND: Technical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated. RESULTS: Detailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity. CONCLUSIONS: The MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection.

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

Significant biochemical, biophysical and metabolic diversity in circulating human cord blood reticulocytes.

BACKGROUND: The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these 'sub-vital' stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies. DESIGN AND METHODS: Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile. RESULTS: Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes. CONCLUSIONS: Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.

Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3.

A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3ß of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3ß (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3ß, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3ß elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

On cytoadhesion of Plasmodium vivax: raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

Gestational malaria associated to Plasmodium vivax and Plasmodium falciparum placental mixed-infection followed by foetal loss: a case report from an unstable transmission area in Brazil.

Gestational malaria is a multi-factorial syndrome leading to poor outcomes for both the mother and foetus. Although an unusual increasing in the number of hospitalizations caused by Plasmodium vivax has been reported in Brazil, mortality is rarely observed. This is a report of a gestational malaria case that occurred in the city of Manaus (Amazonas State, Brazil) and resulted in foetal loss. The patient presented placental mixed-infection by Plasmodium vivax and Plasmodium falciparum after diagnosis by nested-PCR, however microscopic analysis failed to detect P. falciparum in the peripheral blood. Furthermore, as the patient did not receive proper treatment for P. falciparum and hospitalization occurred soon after drug treatment, it seems that P. falciparum pathology was modulated by the concurrent presence of P. vivax. Collectively, this case confirms the tropism towards the placenta by both of these species of parasites, reinforces the notion that co-existence of distinct malaria parasites interferes on diseases' outcomes, and opens discussions regarding diagnostic methods, malaria treatment during pregnancy and prenatal care for women living in unstable transmission areas of malaria, such as the Brazilian Amazon.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice.

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund's Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria.

On the cytoadhesion of Plasmodium vivax-infected erythrocytes.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Human Vaccinia virus and Pseudocowpox virus co-infection: clinical description and phylogenetic characterization.

BACKGROUND: Occupational exanthematic diseases represent an important cause of public health impact and economical losses. Among the viral exanthematic diseases, two caused by poxviruses are noteworthy: the bovine vaccinia (BV), caused by the Vaccinia virus (VACV); and the milker's nodule, in which the agent is the Pseudocowpox virus (PCPV). Both agents are zoonotic and have been associated with several cases of bovine infection. In Brazilian rural areas BV has been highly prevalent, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by several systemic symptoms. Although VACV and PCPV present with similar epidemiological and transmission patterns, no VACV and PCPV co-infection cases have to date been described. OBJECTIVES: To describe the first case of zoonotic VACV and PCVP co-infection, based on serological and molecular methods. STUDY DESIGN AND RESULTS: In this work we report a case of a Brazilian rural worker who presented with a large severely ulcerated-pustule skin lesion, associated with fever, headache, malaise, myalgia and axillary, inguinal and cervical limphadenopathy. The worker declared occupational contact with cattle that had notable injuries on their teats. Human and bovine clinical samples were collected and submitted to serological and molecular tests. PCR and phylogenetic analysis revealed the presence of VACV DNA and PCPV DNA in the patient's lesion. Serological tests indicated anti-VACV neutralizing antibodies and molecular assays showed the presence of VACV and PCPV DNA in the patient sera. VACV and PCPV also were detected in dairy cattle. CONCLUSION: Together, these results indicate a case of zoonotic VACV/PCPV co-infection. Epidemiological surveillance and appropriate medical treatment are essential for the control of both diseases, especially in the most severe cases, as described in the present study.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella typhimurium.

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His(6)FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP1(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.

Immunogenic properties of a recombinant fusion protein containing theC-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptidebased on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP119) from Plasmodiumvivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC).Herein, we tested whether the same strategy, based on the MSP119 component of the deadly malariaparasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity.The His6FliC-MSP119 fusion protein was expressed from a recombinant Escherichia coli and showedpreserved in vitro TLR5-binding activity. In contrast to animals injected with His6MSP119, mice subcutaneouslyimmunised with the recombinant His6FliC-MSP119 developed strong MSP119-specific systemicantibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN1826, complete and incomplete Freund’s adjuvants or Quil-A, improved the IgG responses after the second,but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, asevaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by thedetection of antigen-specific interferon- secretion by immune spleen cells. MSP119-specific antibodiesrecognised not only the recombinant protein, but also the native protein expressed on the surface of P.falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the invitro growth of three different P. falciparum strains. In summary, these results extend our previous observationsand further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigensis a promising alternative to improve their immunogenicity