Structural and immunogenicity analysis of reconstructed ancestral and consensus P48/45 for cross-species anti malaria transmission-blocking vaccine.

The development of the anti-malaria vaccine holds a promising future in malaria control. One of the anti-malaria vaccine strategies known as the transmission-blocking vaccine (TBV) is to inhibit the parasite transmission between humans and mosquitoes by targeting the parasite gametocyte. Previously, we found that P48/45 included in the 6-Cysteine protein family shared by Plasmodium sp. We also detected vaccine properties possessed by all human-infecting Plasmodium and could be used as a cross-species anti-malaria vaccine. In this study, we investigated the efficacy of P48/45 through the ancestral and consensus reconstruction approach. P48/45 phylogenetic and time tree analysis was done by RAXML and BEAST2. GRASP server and Ugene software were used to reconstruct ancestral and consensus sequences, respectively. The protein structural prediction was made by using a psipred and Rosetta program. Each protein characteristic of P48/45 was analyzed by assessing hydrophobicity and Post-Translational Modification sites. Meanwhile, the Epitope sequence for B-cell, T-cell, and HLA was determined using an immunoinformatics approach. Lastly, molecular docking simulation was done to determine native binding interactions of P48/45-P230. The result showed a distinct protein characteristic of ancestral and consensus sequences. The immunogenicity analysis revealed the number of epitopes in the ancestral sequence is greater than the consensus sequence. The study also found a conserved epitope located in the binding site and consists of specific Post-Translational Modification sites. Hence, our research provides detailed insight into ancestral and consensus P48/45 efficacy for the cross-species anti-malaria vaccine.

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

The Toll-Like Receptor 2 agonist PEG-Pam2Cys as an immunochemoprophylactic and immunochemotherapeutic against the liver and transmission stages of malaria parasites.

Both vaccine and therapeutic approaches to malaria are based on conventional paradigms; whole organism or single antigen epitope-based vaccines administered with or without an adjuvant, and chemotherapeutics (anti-malaria drugs) that are toxic to the parasite. Two major problems that limit the effectiveness of these approaches are i) high levels of antigenic variation within parasite populations rendering vaccination efficacy against all variants difficult, and ii) the capacity of the parasite to quickly evolve resistance to drugs. We describe a new approach to both protection from and treatment of malaria parasites that involves the direct stimulation of the host innate immune response through the administration of a Toll-Like Receptor-2 (TLR2) agonist. The activity of PEG-Pam2Cys against the hepatocytic stages, erythrocytic stages and gametocytes of the rodent malaria parasite Plasmodium yoelii was investigated in laboratory mice. We show that administration of PEG-Pam2Cys, a soluble form of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (Pam2Cys), significantly and dramatically reduces the numbers of malaria parasites that grow in the livers of mice following subsequent challenge with sporozoites. We also show that treatment can also clear parasites from the liver when administered subsequent to the establishment of infection. Finally, PEG-Pam2Cys can reduce the numbers of mosquitoes that are infected, and the intensity of their infection, following blood feeding on gametocytaemic mice. These results suggest that this compound could represent a novel liver stage anti-malarial that can be used both for the clearance of parasites following exposure and for the prevention of the establishment of infection.

Opportunities for Host-targeted Therapies for Malaria.

Despite the recent successes of artemisinin-based antimalarial drugs, many still die from severe malaria, and eradication efforts are hindered by the limited drugs currently available to target transmissible gametocyte parasites and liver-resident dormant Plasmodium vivax hypnozoites. Host-targeted therapy is a new direction for infectious disease drug development and aims to interfere with host molecules, pathways, or networks that are required for infection or that contribute to disease. Recent advances in our understanding of host pathways involved in parasite development and pathogenic mechanisms in severe malaria could facilitate the development of host-targeted interventions against Plasmodium infection and malaria disease. This review discusses new opportunities for host-targeted therapeutics for malaria and the potential to harness drug polypharmacology to simultaneously target multiple host pathways using a single drug intervention.

Blood-stage immunity to Plasmodium chabaudi malaria following chemoprophylaxis and sporozoite immunization.

Protection against malaria in humans can be achieved by repeated exposure to infected mosquito bites during prophylactic chloroquine treatment (chemoprophylaxis and sporozoites (CPS)). We established a new mouse model of CPS immunization to investigate the stage and strain-specificity of malaria immunity. Immunization with Plasmodium chabaudi by mosquito bite under chloroquine cover does not generate pre-erythrocytic immunity, which is acquired only after immunization with high sporozoite doses. Instead, CPS immunization by bite elicits long-lived protection against blood-stage parasites. Blood-stage immunity is effective against a virulent, genetically distinct strain of P. chabaudi. Importantly, if exposure to blood-stage parasitemia is extended, blood-stage parasites induce cross-stage immunity targeting pre-erythrocytic stages. We therefore show that CPS immunization can induce robust, long-lived heterologous blood-stage immunity, in addition to protection against pre-erythrocytic parasites following high dose sporozoite immunization. Cross-stage immunity elicited by blood-stage parasites may further enhance efficacy of this immunization regimen.

Extreme CD8 T cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites.

Radiation-attenuated Plasmodium sporozoites (RAS) are the only vaccine shown to induce sterilizing protection against malaria in both humans and rodents. Importantly, these "whole-parasite" vaccines are currently under evaluation in human clinical trials. Studies with inbred mice reveal that RAS-induced CD8 T cells targeting liver-stage parasites are critical for protection. However, the paucity of defined T cell epitopes for these parasites has precluded precise understanding of the specific characteristics of RAS-induced protective CD8 T cell responses. Thus, it is not known whether quantitative or qualitative differences in RAS-induced CD8 T cell responses underlie the relative resistance or susceptibility of immune inbred mice to sporozoite challenge. Moreover, whether extraordinarily large CD8 T cell responses are generated and required for protection following RAS immunization, as has been described for CD8 T cell responses following single-antigen subunit vaccination, remains unknown. Here, we used surrogate T cell activation markers to identify and track whole-parasite, RAS-vaccine-induced effector and memory CD8 T cell responses. Our data show that the differential susceptibility of RAS-immune inbred mouse strains to Plasmodium berghei or P. yoelii sporozoite challenge does not result from host- or parasite-specific decreases in the CD8 T cell response. Moreover, the surrogate activation marker approach allowed us for the first time to evaluate CD8 T cell responses and protective immunity following RAS-immunization in outbred hosts. Importantly, we show that compared to a protective subunit vaccine that elicits a CD8 T cell response to a single epitope, diversifying the targeted antigens through whole-parasite RAS immunization only minimally, if at all, reduced the numerical requirements for memory CD8 T cell-mediated protection. Thus, our studies reveal that extremely high frequencies of RAS-induced memory CD8 T cells are required, but may not suffice, for sterilizing anti-Plasmodial immunity. These data provide new insights into protective CD8 T cell responses elicited by RAS-immunization in genetically diverse hosts, information with relevance to developing attenuated whole-parasite vaccines.

A succinct review of the general and immunological pharmacologic effects of proton pump inhibitors.

Proton pump inhibitors (PPI) are a group of anti-ulcer agents. PPI have selective anti-cancer effects via apoptosis of tumour, sensitization of cancer cell to chemotherapy and radiotherapy. Also PPI have anti-malarial and anti-leishmanial activity. Rising of endosomal (P)H inhibits the presentation of antigens that enter cell through endocytosis. PPI can affect transmigration of leucocytes from vessels to inflammatory sites and also can mitigate neutrophile adherence to endothelial cell. PPI increase the intralysosomal (P)H and decrease the expression of intracellular adhesion molecules. Therefore PPI can exert immunomodulation in immunological diseases through hampering antigen processing, antigen presentation, and leucocytes transmigration.

Prevalencia de anticuerpos antimaláricos en donantes del banco de sangre del Hospital Universitario San Vicente de Paúl de Medellín, Colombia / Antimalaric antibodies prevalence in blood donors at the University Hospital San Vicent de Paul in Medellin, Colombia

Se determinó la prevalencia de anticuerpos antimaláricos, tipo IgG, mediante la técnica de inmunofluorescencia indirecta (IFI), en 392 sueros de donantes voluntarios de sangre del Hospital Universitario San Vicente de Paúl, recolectados durante los meses de septiembre y octubre del año 1995 y mantenidos en cogelación a -70ºC durante 36 meses. La prevalencia de seropositividad fue de 1,8 por ciento (7 donantes), de los cuales, tres tenían antecedentes de haber vivido en zona endémica para malaria; de éstos, dos habían sufrido malaria, por lo menos, una vez en la vida; los cuatro donantes restantes no habían vivido en zona endémica para la malaria ni tenían antecedentes de haberla padecido. Todos los donantes tuvieron gotas gruesas negativas para Plasmodium. Para tratar de reducir el riesgo de malaria transfusional, se recomienda informar en forma clara a los donantes sobre las áreas endémicas para malaria en el departamento, completar la encuesta que realizan los bancos de sangre con gota gruesa en todas la unidades de sangre que se vayan a transfundir y determinar anticuerpos antimaláricos en todos los donantes de riesgo con encuestas sospechosas y que puedan ser transmisores potenciales de Plasmodium

Efficacy of alpha,beta-arteether in acute uncomplicated P. falciparum malaria.

A phase-III clinical trial was conducted in 50 patients (42M + 8F) with acute uncomplicated falciparum malaria from Delhi during the period of September to November 1995. Their mean age was 27.2 years, and the mean parasitaemia on day 0 was 0.65%. Patients were hospitalized and treated with a new ethyl derivative of artemisinin developed at CDRI called alpha, beta-arteether, at the dosage of 150 mg l/M for three consecutive days. Peripheral smears were examined every day for 4 days and then weekly up to 28 days. The results of the study showed that the mean parasite and fever clearance times were respectively 19.94 +/- 6.87 and 37.81 +/- 21.67 hours. Within 48 h, 70% of the cases became afebrile and the peripheral smear was negative in 100% of the cases. The drug was well tolerated. Three cases (6%) had recrudescence within 28 days. It is concluded that alpha, beta-arteether is a safe, effective and rapidly acting antimalarial.

The influence of Maloprim chemoprophylaxis on cellular and humoral immune responses to Plasmodium falciparum asexual blood stage antigens in schoolchildren living in a malaria endemic area of Mozambique.

We examined the impact of chemoprophylaxis on the cellular and humoral immune responses to polypeptides of the asexual Plasmodium falciparum blood stage antigens, the glutamate rich protein GLURP and Pf155/RESA, both of which in previous field studies have been identified as potentially protective antigens. The study was carried out in the Escola Primária de Lingamo, a primary school in a suburban area of Maputo, Mozambique. A cohort of 392 schoolchildren (aged 7-12 years) was randomly allocated to two equal groups, one receiving chemoprophylaxis with dapsone/pyrimethamine (Maloprim), the other receiving placebo every week from December 1989 to November 1990. The groups were then followed until November 1991 without chemoprophylaxis. Cellular responses to immunodominant epitopes from Pf155/RESA and GLURP, and to non malaria antigens C. albicans and PPD, were assessed by lymphocyte proliferation assays in vitro. Anti-GLURP and anti-Pf155/RESA antibodies were detected by enzyme-linked immunosorbent assay (ELISA) and erythrocyte membrane immunofluorescence (EMIF), and total anti-P. falciparum antibodies were measured by indirect fluorescent antibody test (IFAT). Immunological reactivities were evaluated every six months, at the end of the rainy season and at the end of the dry season, both during the period of chemoprophylaxis and during the follow-up. The antibody response rate to the GLURP was lower in the Maloprim group than in the placebo group during the intervention phase. The lymphoproliferative response rate to the malaria antigens was significantly lower at the end of the rainy season than at the end of the dry season, but the difference between the experimental group and the control group of schoolchildren was not statistically significant. These results suggest that the antibody responses to the GLURP molecule and partly to the Pf155/RESA antigen in this study population were shortlived and dependent on frequent boostering, but whether these antigens play a role in the development of natural clinical immunity remains open. In the experimental group of schoolchildren weekly chemoprophylaxis successfully reduced the parasite rate during the rainy season from 43% to 4%, and during the dry season from 18% to 0%. Chemoprophylaxis may therefore have a useful role in combination with another partially effective malaria control measure such as insecticide-impregnated bed nets or a malaria vaccine.

Magainin-like immunoreactivity in human submandibular and labial salivary glands.

Magainins, antimicrobial peptides secreted by granular glands of frog skin, may be related to the high resistance to infections of this epithelial surface. The oral mucosa of healthy individuals is another tissue in which infection is not frequent, probably owing to the activity of potent salivary and mucosal defense mechanisms. To investigate if magainin-like factors are a component of these oral defense mechanisms, human and animal minor (mucosal) and major salivary glands were examined by immunohistochemistry, using a polyclonal rabbit anti-magainin antibody. Cryostat sections of (para) formaldehyde-fixed tissues were incubated with the antibody and then stained with fluorescein-complexed anti-rabbit IgG. Specific staining was observed in the apical portion of the cytoplasm of ductal epithelial cells of human submandibular and labial salivary glands. Diffuse staining was present in submandibular acinar cells. Bovine, rat, hamster, and mouse tissues were unreactive. The presence of magainin-like substances in human salivary gland duct cells is consistent with reports of the occurrence of other biologically active substances in salivary gland ducts.

Protocolo de vacunación antimalárica en fase III municipio Las Majadas Estado Bolívar Venezuela / Antimalaric vaccination protocol III municipio las Majadas Bolivar State Venezuela

El presente proyecto se realizó con el proposito de obtener información sobre la eficacia, seguridad,tolerancia y aceptabilidad de la vacuna sintética SPf66n contra plasmodium falciparum, en una área holoendémica, las majadas del estado Bolívar. La muestra estará representada por 330 individuos, femeninos y masculinos divididos en 2 grupos; uno vacunado y el otro no vacunado, mayores de 12 años y que no presenten criterios de exclusión. La vacuna se administraráen 3 dosis de 2 mgs de la proteina SPf66n por cada inmunización de 0,5 cc subcutaneo. El estudio tendrá un período de observación de un año posrerior ala tercera dosis. Los análisis y resultados se presentarán en un informe final al terminar el estudio