Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) Elicits Detectable Levels of Invasion-Inhibitory Antibodies during Natural Infection in Humans.

Plasmodium falciparum cysteine-rich protective antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite-neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria-infected plasma obtained from patients in central India and analyzed for their parasite neutralizing activity via in vitro growth inhibition assays (GIA). We report that, despite being susceptible to antibodies, CyRPA is a highly conserved antigen that does not appear to be under substantial immune selection pressure, as a very low acquisition rate for anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the small amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA-based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine-induced CyRPA antibodies exhibited a robust 50% inhibitory concentration (IC50) of 21.96 µg/ml, which is comparable to the IC50 of antibodies against the leading blood-stage vaccine candidate, reticulocyte-binding-like homologous protein 5 (RH5). Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but is highly conserved compared to other leading candidates such as MSP-1 and AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

Gravidity and malaria trends interact to modify P. falciparum densities and detectability in pregnancy: a 3-year prospective multi-site observational study.

BACKGROUND: Low-density Plasmodium falciparum infections prevail in low transmission settings, where immunity is expected to be minimal, suggesting an immune-independent effect on parasite densities. We aimed to describe parasite densities in pregnancy, and determine how gravidity and antibody-mediated immunity affect these, during a period of declining malaria transmission in southern Mozambique. METHODS: We documented P. falciparum infections at first antenatal care visits (n = 6471) between November 2016 and October 2019 in Ilha Josina (high-to-moderate transmission area), Manhiça (low transmission area), and Magude (pre-elimination area). Two-way interactions in mixed-effects regression models were used to assess gravidity-dependent differences in quantitative PCR-determined P. falciparum positivity rates (PfPRqPCR) and densities, in the relative proportion of detectable infections (pDi) with current diagnostic tests (≥ 100 parasites/µL) and in antimalarial antibodies. RESULTS: PfPRqPCR declined from 28 to 13% in Ilha Josina and from 5-7 to 2% in Magude and Manhiça. In primigravidae, pDi was highest in Ilha Josina at the first study year (p = 0.048), which declined with falling PfPRqPCR (relative change/year: 0.41, 95% CI [0.08; 0.73], p = 0.029), with no differences in antibody levels. Higher parasite densities in primigravidae from Ilha Josina during the first year were accompanied by a larger reduction of maternal hemoglobin levels (- 1.60, 95% CI [- 2.49; - 0.72; p < 0.001), than in Magude (- 0.76, 95% CI [- 1.51; - 0.01]; p = 0.047) and Manhiça (- 0.44, 95% CI [- 0.99; 0.10; p = 0.112). In contrast, multigravidae during the transmission peak in Ilha Josina carried the lowest pDi (p = 0.049). As PfPRqPCR declined, geometric mean of parasite densities increased (4.63, 95% CI [1.28; 16.82], p = 0.020), and antibody levels declined among secundigravidae from Ilha Josina. CONCLUSIONS: The proportion of detectable and clinically relevant infections is the highest in primigravid women from high-to-moderate transmission settings and decreases with declining malaria. In contrast, the falling malaria trends are accompanied by increased parasite densities and reduced humoral immunity among secundigravidae. Factors other than acquired immunity thus emerge as potentially important for producing less detectable infections among primigravidae during marked declines in malaria transmission.

Antibody Combinations Targeting the Essential Antigens CyRPA, RH5, and MSP-119 Potently Neutralize Plasmodium falciparum Clinical Isolates From India and Africa.

BACKGROUND: Targeting multiple key antigens that mediate distinct Plasmodium falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low immunoglobulin G concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency. METHODS: We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from Central India and Mozambique, Africa, and characterized for their invasion properties and genetic diversity of invasion ligands. RESULTS: In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation. CONCLUSIONS: The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite neutralization against P. falciparum clones and clinical isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.

Delivery of a Cancer-Testis Antigen-Derived Peptide Using Conformationally Restricted Dipeptide-Based Self-Assembled Nanotubes.

Use of tumor-associated antigens for cancer immunotherapy is limited due to their poor in vivo stability and low cellular uptake. Delivery of antigenic peptides using synthetic polymer-based nanostructures has been actively pursued but with limited success. Peptide-based nanostructures hold much promise as delivery vehicles due to their easy design and synthesis and inherent biocompatibility. Here, we report self-assembly of a dipeptide containing a non-natural amino acid, α,ß-dehydrophenylalanine (ΔF), into nanotubes, which efficiently entrapped a MAGE-3-derived peptide (M3). M3 entrapped in F-ΔF nanotubes was more stable to a nonspecific protease treatment and both F-ΔF and F-ΔF-M3 showed no cellular toxicity for four cancerous and noncancerous cell lines used. F-ΔF-M3 showed significantly higher cellular uptake in RAW 267.4 macrophage cells compared to M3 alone and also induced in vitro maturation of dendritic cells (DCs). Immunization of mice with F-ΔF-M3 selected a higher number of IFN-γ secreting CD8+ T cells and CD4+ T compared to M3 alone. On day 21, a tumor growth inhibition ratio (TGI, %) of 41% was observed in a murine melanoma model. These results indicate that F-ΔF nanotubes are highly biocompatible, efficiently delivered M3 to generate cytotoxic T lymphocytes responses, and able to protect M3 from degradation under in vivo conditions. The F-ΔF dipeptide-based nanotubes may be considered as a good platform for further development as delivery agents.

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study.

BACKGROUND: Vaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity. METHODS: We evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection. RESULTS: RTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3-0.93], p = 0.03, for MSP1; 0.52 [0.26-0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses. CONCLUSIONS: Increased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

Targeted delivery of microRNA-199a-3p using self-assembled dipeptide nanoparticles efficiently reduces hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is an aggressive tumor with limited systemic and locoregional modalities of treatment. Although microRNA (miRNA) based therapies have significant potential, their targeted delivery remains a major challenge. miR-199a-3p functions as an important tumor suppressor in HCC, which regulates various cellular processes. Recently, peptide-based nanoparticles (NPs) have been developed to deliver oligonucleotides including miRNA. Here, we describe the synthesis and characterization of arginine α,ß-dehydrophenylalanine (RΔF) nanoparticles for the selective delivery of miR-199a-3p to restore dysregulated gene expression in HCC. Targeted delivery was achieved by conjugating lactobionic acid (LA) with RΔF NPs (RΔF-LA NPs), a ligand for the asialoglycoprotein receptor known to be overexpressed in HCC cell lines. RΔF-LA NPs condensed miR-199a-3p had an average size of ∼60nm and a zeta potential of ∼+2.54 mV. RΔF-LA/miR NPs were found to be stable in serum as well as against RNase attack. RΔF-LA/miR NPs showed an enhanced cellular uptake and an efficient delivery of miR-199a-3p leading to a significant increase in miR-199a-3p levels (over 500 fold). The increased miR-199a-3p levels remarkably suppressed cell proliferation and migration as well as induced cellular apoptosis and downregulation of the specific target gene (mTOR) in vitro. RΔF-LA/miR NPs showed high tumor/ low organ ratios after intravenous injection into HCC tumor bearing nude mice. RΔF-LA/miR NPs treated mice demonstrated>50% decline in tumor growth, which also corresponded well with suppression of mTOR protein expression, tumor cell proliferation and increased survival rate (P < 0.05). CONCLUSION: RΔF-LA/miR NPs showed significantly enhanced delivery of the miRNA which underscores their potential for further development as a therapeutic approach for HCC. (Hepatology 2018;67:1392-1407).

Comparative evaluation of the aqueous humor proteome of primary angle closure and primary open angle glaucomas and age-related cataract eyes.

PURPOSE: To analyze and compare the total proteome of aqueous humor (AH) from patients having primary angle closure glaucoma (PACG), primary open angle glaucoma (POAG) and age-related cataract. MATERIALS AND METHODOLOGY: Aqueous humor was collected from age-matched PACG, POAG and cataract patients who underwent surgery, and it was immediately stored at - 80 °C until analysis. From each sample, 25 µg of total protein was subjected to trypsin digestion and subsequently LC-MS/MS analysis was performed for the deep proteome analysis. The data acquired after the LC-MS/MS analysis were analyzed using Proteome Discoverer 1.4. The identified peptide matches were validated using percolator, at less than 1% false discovery rates. RESULTS: A total of 625, 594 and 636 proteins were identified in PACG, POAG and cataract groups, respectively (n = 9 in each group). The inter-group comparison among all these groups showed that 246 proteins were identified in all the three groups. An average of 236 ± 42, 218 ± 40 and 214 ± 62 proteins from each AH sample of PACG, POAG and cataract, respectively, was identified. There were 53 proteins commonly found in all 9 PACG AH, 59 proteins in POAG AH and 42 proteins in 9 cataracts AH samples. In the individual analysis, there were 28 proteins found in all the samples analyzed representing the "constitutive AH proteome." Spectral counting analysis of 246 proteins identified in all three group types showed significant differences in protein abundance. In proteins unique to PACG AH, 7 proteins viz. ARHGEF12, APC2, WAS, PIK3CG, ITGB1, MSN and PFN1 out of 226 were found in "Regulation of Actin Cytoskeleton" pathway, whereas in POAG 5 out of 206 proteins viz. ADCY2, ITPR1, MAPK3, MAP3K2 and TUBB1 were found in "Gap Junction" pathway. CONCLUSIONS: A qualitative as well as a quantitative comparison of proteomes of AH from PACG, POAG and age-related cataract eyes showed significant differences, thus providing clues to the disease pathophysiology.

Identifying Immune Correlates of Protection Against Plasmodium falciparum Through a Novel Approach to Account for Heterogeneity in Malaria Exposure.

Background: A main criterion to identify malaria vaccine candidates is the proof that acquired immunity against them is associated with protection from disease. The age of the studied individuals, heterogeneous malaria exposure, and assumption of the maintenance of a baseline immune response can confound these associations. Methods: Immunoglobulin G/immunoglobulin M (IgG/ IgM) levels were measured by Luminex® in Mozambican children monitored for clinical malaria from birth until 3 years of age, together with functional antibodies. Studied candidates were pre-erythrocytic and erythrocytic antigens, including EBAs/PfRhs, MSPs, DBLs, and novel antigens merely or not previously studied in malaria-exposed populations. Cox regression models were estimated at 9 and 24 months of age, accounting for heterogeneous malaria exposure or limiting follow-up according to the antibody's decay. Results: Associations of antibody responses with higher clinical malaria risk were avoided when accounting for heterogeneous malaria exposure or when limiting the follow-up time in the analyses. Associations with reduced risk of clinical malaria were found only at 24 months old, but not younger children, for IgG breadth and levels of IgG targeting EBA140III-V, CyRPA, DBL5ε and DBL3x, together with C1q-fixation activity by antibodies targeting MSP119. Conclusions: Malaria protection correlates were identified, only in children aged 24 months old when accounting for heterogeneous malaria exposure. These results highlight the relevance of considering age and malaria exposure, as well as the importance of not assuming the maintenance of a baseline immune response throughout the follow-up. Results may be misleading if these factors are not considered.

Optimization of incubation conditions of Plasmodium falciparum antibody multiplex assays to measure IgG, IgG1-4, IgM and IgE using standard and customized reference pools for sero-epidemiological and vaccine studies.

BACKGROUND: The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1-4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different incubation times and temperatures (4 °C overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions. RESULTS: Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels, thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG1-4 responses were highest when the WHO reference reagent was incubated with antigen-coupled beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h and 4 °C overnight, indicating no difference in unspecific binding. CONCLUSIONS: With this study, the immunogenicity profile of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity) and isotypes/subclasses (e.g. IgM) under study.

Arginine-α, ß-dehydrophenylalanine Dipeptide Nanoparticles for pH-Responsive Drug Delivery.

PURPOSE: Nanoparticles (NPs) exhibiting responsiveness towards pH variations in organs, tissue microenvironments and cellular compartments can significantly add on to the drug delivery potential. Here, we have developed NPs from an amphipathic dipeptide, Arginine-α, ß-dehydrophenylalanine (RΔF), and tried to explore their pH responsive drug delivery potential in various cancer cells. METHODS: RΔF-NPs were architectured by harnessing the process of molecular self-assembly followed by the assessment of effect of pH on NPs morphology using zetasizer, SEM and CD. FTIR and PXRD analysis of the dipeptide and doxorubicin (Dox) were carried out for compatibility assessment followed by encapsulation of Dox in RΔF-NPs. RΔF-Dox-NPs were evaluated for pH dependent release as well as for in-vitro cellular internalization and efficacy in cancer cells. RESULTS: RΔF self-assembled to form monodispersed particles at pH 7. SEM analysis revealed a loss of overall particle morphology along with particle aggregation at highly acidic and basic pH respectively. The NPs demonstrated a slow and sustained release behaviour at pH 7 (97.64 ± 4.71% after 36 h) in comparison to pH 2 (90.27 ± 1.45% after 8 h) and pH 10 (96.39 ± 3.87% after 12 h). In-vitro efficacy studies carried-out in various cancer cells revealed that RΔF-Dox-NPs exhibited higher efficacy with 1.65, 1.95 and 13.34 fold lower IC50 values in comparison to Dox in C6, HCT-116 and AGS cell lines. CONCLUSIONS: RΔF-Dox-NPs with higher drug release at acidic pH, enhanced internalization in cancer cells along with higher cytotoxic potential can act as effective pH responsive drug delivery systems.

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion.

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.

Efficacy of Dipeptide-Coated Magnetic Nanoparticles in Lung Cancer Models Under Pulsed Electromagnetic Field.

Lung cancer is the leading cause of cancer deaths and the overall 5-year survival rate is less than 17%. Hyperthermia is an alternative approach for the treatment of lung cancer and is associated with fewer side effects. We employed ironoxide nanoparticles in inducing localized hyperthermia in lung cancer cells using a pulsed electromagnetic field (PEMF). We synthesized, characterized and determined the uptake of dipeptide-coated iron oxide nanoparticles. Further, their ability in inducing localized hyperthermia in PEMF on lung cancer cells was assessed. Results showed nanoparticles are non-cytotoxic and showed enhanced cellular uptake in lung cancer cells. In vivo studies in nude mice lung tumor xenografts confirmed the presence in the tumors. Lung cancer cells pretreated with dipeptide-coated magnetic nanoparticles upon PEMF exposure induced cell death.

Prevalence of afebrile parasitaemia due to Plasmodium falciparum & P. vivax in district Balaghat (Madhya Pradesh): Implication for malaria control.

BACKGROUND & OBJECTIVES: Balaghat district in Central India is a highly malarious district where both Plasmodium falciparum and P. vivax are prevalent. In this district, the persistence of malaria was on an increase and not responsive to intervention measures even though there was no drug resistance. This study was undertaken by conducting mass screening to determine the prevalence of malaria among particularly vulnerable tribe of Balaghat, for developing evidence-based intervention measures for malaria control in hard to reach areas. METHODS: This prospective study was carried out during 2013-2014 by conducting mass survey of the population in 10 villages of Birsa community health centre (CHC) and 12 villages of Baihar CHC. Finger-pricked blood smears were collected from all consenting individuals with or without fever for microscopic examination. RESULTS: In the febrile group, the slide positivity rate (SPR) and slide falciparum rate (SFR) were 32.4 and 28.9 per cent, respectively, with 89.4 per cent P. falciparum, while in the afebrile individuals also, the SPR and SFR were high (29 and 26%, respectively), but these were significantly lower than that of febrile group. The gametocyte carriers were significantly higher (odds ratio 1.67, 95% confidence interval 1.25-2.25, P=0.0004) in afebrile patients when compared with febrile group. Vector incrimination showed the presence of four sporozoite-positive Anopheles culicifacies out of 1953 assayed. INTERPRETATION & CONCLUSIONS: Plasmodium falciparum malaria was high in young children (up to 8 years) as compared to the adult in both afebrile and febrile group in Balaghat district. High prevalence of gametocyte was observed in all age groups among the afebrile cases. The identification of afebrile malaria parasitaemia is an important challenge for the malaria elimination initiatives. A strong malaria surveillance system is fundamental to both programme design and implementation.

Protective hinge in insulin opens to enable its receptor engagement.

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in ß cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 ß-turn, coupling reorientation of Phe(B24) to a 60° rotation of the B25-B28 ß-strand away from the hormone core to lie antiparallel to the receptor's L1-ß2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile(A2), Val(A3), Val(B12), Phe(B24), and Phe(B25)) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.

Characterization of fine specificity of the immune response to a Plasmodium falciparum rhoptry neck protein, PfAARP.

BACKGROUND: Immunological characterization of potential blood-stage malaria antigens would be a valuable strategy in the development of an effective vaccine. Identifying B and CD4(+) T cell epitopes will be important in understanding the nature of immune response. A previous study has shown that Plasmodium falciparum apical asparagine-rich protein (PfAARP) stimulates immune response and induces potent invasion-inhibitory antibodies. Antibodies to PfAARP provide synergistic effects in inhibition of parasite invasion when used in combination with antibodies to other antigens. In the present study, an attempt was made to identify B cell and CD4(+) T cell epitopes of PfAARP. METHODS: Balb/c mice were immunized with recombinant PfAARP and both cellular and humoral responses were analysed at various time points. Computerized databases [immune epitope database (IEDB) and B cell epitope prediction (BCEPred)] were used to predict epitope sequences within PfAARP and predicted peptides were synthesized. In addition, nine 18 amino acid, long-overlapping peptides spanning the entire length of PfAARP were synthesized. Using these peptides, B cell and CD4(+) T cell responses in PfAARP immunized mice were measured by ELISA and ELISPOT assays. RESULTS: Here, it is demonstrated that immunization of mice with PfAARP induced long-lasting, high-titre antibodies (4 months post immunization). Also, the recombinant protein was effective in inducing a pronounced Th1 type of immune response quantified by IFN-γ ELISA and ELISPOT. It was found that the predicted peptides did not represent the immunogenic regions of PfAARP. However, of the nine overlapping peptides, three peptides (peptides 3, 5 and 7) were strongly recognized by PfAARP-immunized sera and represented B cell epitopes. Also, peptide 3 elicited IFN- γ response, suggesting it to be a T-cell epitope. CONCLUSIONS: Induction of long-lasting humoral and cellular response on PfAARP immunization in mice underscores its possible use as a blood-stage malaria vaccine candidate. Mapping of immunogenic regions may help in designing fusion chimera containing immunologically relevant regions of other vaccine target antigens and/or for multi-component vaccine candidates.

Short peptide based nanotubes capable of effective curcumin delivery for treating drug resistant malaria.

BACKGROUND: Curcumin (Ccm) has shown immense potential as an antimalarial agent; however its low solubility and less bioavailability attenuate the in vivo efficacy of this potent compound. In order to increase Ccm's bioavailability, a number of organic/inorganic polymer based nanoparticles have been investigated. However, most of the present day nano based delivery systems pose a conundrum with respect to their complex synthesis procedures, poor in vivo stability and toxicity issues. Peptides due to their high biocompatibility could act as excellent materials for the synthesis of nanoparticulate drug delivery systems. Here, we have investigated dehydrophenylalanine (ΔPhe) di-peptide based self-assembled nanoparticles for the efficient delivery of Ccm as an antimalarial agent. The self-assembly and curcumin loading capacity of different ΔPhe dipeptides, phenylalanine-α,ß-dehydrophenylalanine (FΔF), arginine-α,ß-dehydrophenylalanine (RΔF), valine-α,ß-dehydrophenylalanine (VΔF) and methonine-α,ß-dehydrophenylalanine (MΔF) were investigated for achieving enhanced and effective delivery of the compound for potential anti-malarial therapy. RESULTS: FΔF, RΔF, VΔF and MΔF peptides formed different types of nanoparticles like nanotubes and nanovesicles under similar assembling conditions. Out of these, F∆F nanotubes showed maximum curcumin loading capacity of almost 68 % W/W. Ccm loaded F∆F nanotubes (Ccm-F∆F) showed comparatively higher (IC50, 3.0 µM) inhibition of Plasmodium falciparum (Indo strain) as compared to free Ccm (IC50, 13 µM). Ccm-F∆F nano formulation further demonstrated higher inhibition of parasite growth in malaria infected mice as compared to free Ccm. The dipeptide nanoparticles were highly biocompatible and didn't show any toxic effect on mammalian cell lines and normal blood cells. CONCLUSION: This work provides a proof of principle of using highly biocompatible short peptide based nanoparticles for entrapment and in vivo delivery of Ccm leading to an enhancement in its efficacy as an antimalarial agent.

Malaria and HIV infection in Mozambican pregnant women are associated with reduced transfer of antimalarial antibodies to their newborns.

BACKGROUND: Malaria and human immunodeficiency virus (HIV) infection during pregnancy affect the transplacental transfer of antibodies against several pathogens from mother to fetus, although the effect of malaria and HIV infection on the transfer of antimalarial antibodies remains unclear. METHODS: Levels of total immunoglobulin G (IgG), immunoglobulin M (IgM), and IgG subtypes against the following Plasmodium falciparum antigens were measured in 187 pairs of mother-cord plasma specimens from Mozambique: 19-kDa fragment of merozoite surface protein 1 (MSP119), erythrocyte binding antigen 175 (EBA175), apical membrane antigen 1 (AMA1), and parasite lysate. Placental antibody transfer was defined as the cord-to-mother ratio (CMR) of antibody levels. RESULTS: Maternal malaria was associated with reduced CMR of EBA175 IgG (P = .014) and IgG1 (P = .029), AMA1 IgG (P = .002), lysate IgG1 (P = .001), and MSP1 IgG3 (P = .01). Maternal HIV was associated with reduced CMR of MSP1 IgG1 (P = .022) and IgG3 (P = .023), lysate IgG1 (P = .027) and IgG3 (P = .025), AMA1 IgG1 (P = .001), and EBA175 IgG3 (P = .001). Decreased CMR was not associated with increased adverse pregnancy outcomes or augmented risk of malaria in the infant during the first year of life. CONCLUSIONS: Placental transfer of antimalarial antibodies is reduced in pregnant women with malaria and HIV infection. However, this decrease does not contribute to an increased risk of malaria-associated morbidity during infancy.

Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation.

Merozoite surface protein 3 of Plasmodium falciparum, a 40-kDa protein that also binds heme, has been biophysically characterized for its tendency to form highly elongated oligomers. This study aims to systematically analyze the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through size exclusion chromatography, dynamic light scattering, and transmission electron microscopy, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid-like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C terminus of MSP3 may not be the only structural element responsible for oligomerization and that other peptide segments like MSP3(192-196) (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5-15-µm-long fibrillar structures stained by anti-MSP3 antibodies were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV spectrophotometry, HPLC, and electrophoresis. This study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point to the other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.

Rationally designed transmembrane peptide mimics of the multidrug transporter protein Cdr1 act as antagonists to selectively block drug efflux and chemosensitize azole-resistant clinical isolates of Candida albicans.

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.