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As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)–encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8 + T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

O presente trabalho apresenta o projeto de um dispositivo e os testes realizados em um sistema de auxílio na prevenção de contaminações pelo Sars-Cov2 com a desinfecção do ar pela utilização de UVC como ação germicida. O sistema foi testado com E.colis e proporcionou no ar tratado uma visível taxa de inativação das colônias de bactérias aspergidas em sua entrada.

During the pandemic period of COVID-19 in 2020, suspension of face-to-face activities, a research group was formed at IFSP, campus São Paulo, for the development of systems with special lamps using UV radiation technology as an agent with germicidal action in order to help reduce the spread of SARS-CoV-2 in different environments. Key words. Air disinfection, UVC radiation, Sars-CoV2.

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 45–90 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence in CD36 adherent parasites. AP2-O-knocked-down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is, beside its role in transmission stages, also one of the virulence gene transcriptional regulators and may therefore be exploited as an important target to disrupt severe malaria and block parasite transmission.

Chikungunya virus (CHIKV) is responsible for a self-limited illness that can evolve into long-lasting painful joint inflammation. In this study, we report a novel experimental CHIKV vaccine formulation of lipid nanoparticles loaded with a recombinant protein derived from the E2 structural protein. This antigen fragment, designated ∆E2.1, maintained the antigenicity of the native viral protein and was specifically recognized by antibodies induced in CHIKV-infected patients. The antigen has been formulated into nanoparticles consisting of nano-multilamellar vesicles (NMVs) combined with the adjuvant monophosphoryl lipid A (MPLA). The vaccine formulation demonstrated a depot effect, leading to controlled antigen release, and induced strong antibody responses significantly higher than in mice immunized with the purified protein combined with the adjuvant. More relevantly, E2-specific antibodies raised in mice immunized with ∆E2.1-loaded NMV-MPLA neutralized CHIKV under in vitro conditions. Taken together, the results demonstrated that the new nanoparticle-based vaccine formulation represents a promising approach for the development of effective anti-CHIKV vaccines.

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 50-60 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes and other multigenic families. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence. AP2-O-knocked down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is one of the major virulence gene transcriptional regulators and may, therefore, be exploited as an important target to disrupt severe malaria and block parasite transmission.

Background: Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist. Methods: We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested. Results: Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions: Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.

Air pollution represents a considerable threat to health worldwide. The Sao Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1β, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

BackgroundPlasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1.MethodsFive regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry.ResultsWe found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins.ConclusionRecombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.

Amphotericin B (Amph-B) is an antifungal drug used intravenously for the treatment of leishmaniasis. Side-effects from Amph-B treatment can arise such as cardiac arrhythmia and renal dysfunctions, which will lead to discontinuation of treatment. Unfortunately, patients in endemic countries do not have access to alternative therapies. The objective of this study was to analyze the effects of Cobalt-60 gamma irradiation on crosslinking polymeric hydrogels (Hydg) and the incorporation of Amph-B into the gel as a controlled-release drug delivery alternative. Polyvinylpyrrolidone (PVP)/Amph-B solutions were irradiated with 15 kGy at 0 °C and 25 °C. The drug's stability was ascertained by UV–visible spectrometry, liquid chromatography/mass spectrometry and proton nuclear magnetic resonance. Irradiated Hydg/Amph-B achieved similar stability to the standard Amph-B solution and was enough to promote hydrogel crosslinking. In vitro trials were carried out to ensure Amph-B was still biologically active after irradiation. The results from flow cytometry and MTT assay show that Amph-B had an IC50 = 16.7 nM. A combination of Hydg at 1.324 gmL−1 and Amph-B at 25.1 nM for 24 h lead to the greatest inhibition of L. amazonensis promastigotes, and could be used as an alternative treatment method for cutaneous leishmaniosis.

Liposomes loaded with GPI-anchored proteins induce a strong and effective response against protozoan pathogens when immunized in mice. On the basis of results using Plasmodium falciparum merozoite proteins loaded on liposomes, we tested whether a similar approach would be protective in the P. yoelii XL and P. berghei NK65 models of lethal murine malaria infections. We also analyzed the enhancing capacity of additional adjuvants. As expected, merozoite protein loaded liposomes provided strong humoral responses (IgG titers of 105 against MSP119) in BALB/c and C57BL/6 host backgrounds and showed total protection in lethal challenges (100% survival) with P. yoelii, while protection against lethal challenge with P. berghei NK was at least partial (11%–20% in C57BL/6 mice and no protection in BALB/c). Thus, immunization with proteoliposomes is not only highly immunogenic but also provides a protective response in widely used murine models of malaria.

Vaccines are the primary means of controlling and preventing pandemics and outbreaks of pathogens such as bacteria, viruses, and parasites. However, a major drawback of naked DNA-based vaccines is their low immunogenicity and the amount of plasmid DNA necessary to elicit a response. Nano-sized liposomes can overcome this limitation, enhancing both nucleic acid stability and targeting to cells after administration. We tested two different DNA vaccines in cationic liposomes to improve the immunogenic properties. For this, we cloned the coding sequences of the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5) either alone or fused with small the small hepatitis virus (HBV) envelope antigen (HBsAg) encoding sequences, potentially resulting in HBsAg particles displaying PfRH5 on their outside. Instead of invasive intraperitoneal or intramuscular immunization, we employed intradermal immunization by tattooing nano-encapsulated DNA. Mice were immunized with 10 &mu, g encapsulated DNA encoding PfRH5 alone or in fusion with HBsAg and this elicited antibodies against schizont extracts (titer of 104). Importantly, only IgG from animals immunized with PfRH5-HBs demonstrated sustained IgG-mediated inhibition in in vitro growth assays showing 58% and 39% blocking activity after 24 and 48 h, respectively. Intradermal tattoo-vaccination of encapsulated PfRH5-HBsAg coding plasmid DNA is effective and superior compared with an unfused PfRH5-DNA vaccine, suggesting that the HBsAg fusion may be advantageous with other vaccine antigens.

Microlipid vesicles (MLV) have a broad spectrum of applications for the delivery of molecules, ranging from chemical compounds to proteins, in both in vitro and in vivo conditions. In the present study, we developed a new set of nanosize multilayer lipid vesicles (NMVs) containing a unique combination of lipids. The NMVs enable the adsorption of histidine-tagged proteins at the vesicle surface and were demonstrated to be suitable for the in vivo delivery of antigens. The NMVs contained a combination of neutral (DOPC) and anionic (DPPG) lipids in the inner membrane and an external layer composed of DOPC, cholesterol, and a nickel-containing lipid (DGS-NTA [Ni]). NMVs combined with a recombinant form of the B subunit of the Shiga toxin (rStx2B) produced by certain enterohemorragic Escherichia coli (EHEC) strains enhanced the immunogenicity of the antigen after parenteral administration to mice. Mice immunized with rStx2B-loaded NMVs elicited serum antibodies capable of neutralizing the toxic activities of the native toxin; this result was demonstrated both in vitro and in vivo. Taken together, these results demonstrated that the proposed NMVs represent an alternative for the delivery of antigens, including recombinant proteins, generated in different expression systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-018-0035-0) contains supplementary material, which is available to authorized users.

Increased resistance to the first-line treatment against P. falciparum malaria, artemisinin-based combination therapies, has been reported. Here, we tested the effect of crude ethanolic extract of the fungus Trichoderma stromaticum (Ext-Ts) on the growth of P. falciparum NF54 in infected human red blood cells (ihRBCs) and its anti-malarial and anti-inflammatory properties in a mouse model of experimental cerebral malaria. For this purpose, ihRBCs were treated with Ext-Ts and analysed for parasitaemia; C57BL/6 mice were infected with P. berghei ANKA (PbA), treated daily with Ext-Ts, and clinical, biochemical, histological and immunological features of the disease were monitored. It was observed that Ext-Ts presented a dose-dependent ability to control P. falciparum in ihRBCs. In addition, it was demonstrated that Ext-Ts treatment of PbA-infected mice was able to increase survival, prevent neurological signs and decrease parasitaemia at the beginning of infection. These effects were associated with systemically decreased levels of lipids and IFN-γ, ICAM-1, VCAM-1 and CCR5 cerebral expression, preserving blood brain barrier integrity and attenuating the inflammatory lesions in the brain, liver and lungs. These results suggest that Ext-Ts could be a source of immunomodulatory and antimalarial compounds that could improve the treatment of cerebral malaria.

The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors.

The formulation of an effective vaccine against malaria is still a significant challenge and the induction of high anti-parasite antibody titers plus a sustained T cell response is mandatory for the success of such a vaccine. We have developed a nanoliposome-based structure which contains plasma membrane-associated proteins (PfMNP) of Plasmodium falciparum merozoites on its surface. Incorporation of parasite-derived proteins led to a significant increase in the size and dispersity of particles. Immunization of particles in BalbC and C57BL/6 mice led to high anti-MSP119 IgG titers (10(4)) after the first dose and reached a plateau (>10(6)) after the third dose. While very high titers were observed against the C-terminal domain of the vaccine candidate MSP1, only modest titers (≤10(3)) were detected against MSP2. The induced antibodies showed also a strong growth-inhibiting effect in reinvasion assays. In addition, PfMNP immunization generated antibodies which partially blocked the inflammatory response, probably by blocking TLR-induced activation of macrophages by malarial toxins such as GPI anchors. The results underline the potential of nanoliposome-based formulations as anti-malarial vaccines.

Background Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite’s redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. Results Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. Conclusions Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs. Electronic supplementary material The online version of this article (10.1186/s13071-017-2402-3) contains supplementary material, which is available to authorized users.

Malaria-associate pregnancy has a significant impact on infant morbidity and mortality. The detrimental effects of malaria infection during pregnancy have been shown to correlate with immune activation in the placental tissue. Herein we sought to evaluate the effect of Toll-like receptors (TLRs) activation on placental malaria (PM) development by using the Plasmodium berghei NK65GFP infection model. We observed that activation of the innate immune system by parasites leads to PM due to local inflammation. We identified TLR4 activation as the main pathway involved in the inflammatory process in the placental tissue since the absence of functional TLR4 in mice leads to a decrease in the pro-inflammatory responses, which resulted in an improved pregnancy outcome. Additionally, a similar result was obtained when infected pregnant mice were treated with IAXO-101, a TLR4/CD14 blocker. Together, this study illustrates the importance of TLR4 signalling for the generation of the severe inflammatory response involved in PM pathogenesis. Therefore, our results implicate that TLR4 blockage could be a potential candidate for therapeutic interventions to reduce malaria-induced pathology both in the mother and the fetus.

Producing recombinant antigens in GMP grade for vaccine purposes can be very difficult and in general, a time-consuming process [1]. Overcoming these two problems was one of the most important goals of DNA vaccines [2]. Producing and purifying plasmids that encode antigens has many advantages, namely easy storage and high stability [3]. However, there are different roadblocks concerning DNA for use in vaccines.

A relação evolutiva entre mamíferos e Plasmodium compreende infecções com reflexos adaptativos de ambos os lados. A evasão imune pelo parasita e a aquisição imune do hospedeiro contra antígenos importantes são extremos de um longo processo. Genes e antígenos variantes do parasita funcionam nesse processo desempenhando papel evasivo, de citoaderência e manutenção do processo infeccioso. Hospedeiros podem adquirir imunidade efetiva por vacinas contra antígenos variantes e antígenos de caráter reinvasivo. Testamos aqui três desenhos experimentais visando: 1- Definir se antigenos variantes BIR estão associados a citoadesão em infecção murina e se sua localização é em eritrócitos infectados. 2- O papel de um transgene controlado por um promotor de genes variantes no processo adaptativo infeccioso, frente a hospedeiros que diferem em apenas um único gene. 3- Criação de um modelo vacinal aplicável a qualquer antígeno relevante em infecções pelo gênero Plasmodium. Como resultados obtivemos que: 1- Antigenos BIR não parecem estar relacionados na cito adesão em modelo murino. Identificamos um outro grupo de antígenos com domínio LCCL que talvez desempenhe um papel de ligante. 2- promotores bir podem sofrer modulação em uma única infecção que difiere em somente um gene em hospedeiros. Os efeitos desencadeados foram maior parasitemia, anergia e tolerância imune sem afetar a morbidade da infecção de maneira nociva ao hospedeiro. Esse efeito parece ser mediado por subpopulações parasitarias usando exossomos entre parasita e hospedeiro. 3- Produzimos um sistema funcional de produção de proteínas recombinantes fusionadas a GPI que permite integração em lipossomos para usos vacinais. A prova de princípio foi o uso de antígenos PfRH5-GPI recombinantes em teste vacinal que conseguiram gerar anticorpos com alta atividade inibitória em cultivos de P. falciparum. Tomados em conjunto mostramos que o hospedeiro é capaz influenciar a expressão de transgenes controlados por promotores bir e que proteolipossomos contendo antígenos relevantes, como PfRH5, possuem potencial protetor quando vacinados contra malaria. The relation between mammals and Plasmodium comprise infections with adaptive reflections for both sides. The immune evasion by parasites and immune acquisition by the host against important antigens are end points of a long process. Variant genes and proteins of the parasite can exert a role in this process by enbling immune evasion, cytoadherence resulting in the maintainence of the infective process. We tested three experimental approaches focusing on the following points:1-Show if variant BIR antigens are associated with cytoadherence during murine infections 2- The role of a transgene under the control of a variant gene promoter in adaptation to infection in hosts which express or not the transgene 3- Creation of a vaccine model applicable to any relevant antigen in infections with Plasmodium. As results we showed that: 1-BIR antigenes are likely not related to cito adhesion in the murine model. Cytoadherence in this model is probably related to exported parasite proteins with LCCL domains 2-bir promoters can be modulated during infection in hosts which which differ in one unique gene.The effects observed in this case was an increase in parasitemia, anergy and immune tolerance without affecting the morbidity of infection in the host. These effects are apparently mediated by parasite subpopulations producing exosomes that signal from the parasite to the host.3- We generated a system for recombinant protein production where antigens are fused to GPI and then integrated onto liposomes for vaccine usage. The proof of principle was the use of recombinant PfRH5-GPI as vaccine which elicited antibodies with strong blocking activity in P. falciparum cultures. Together we have shown that the host environment is capable of modulating the activity of variant bir gene promoters and that proteoliposomes loaded with relevant malarial antigens such as PfRH5, are potentially protective when used as malaria vaccine.

The delivery of antigens as DNA vaccines is an efficient alternative to induce immune responses against antigens, which are difficult to produce in recombinant form. However, the delivery of naked DNA is ineffective or relies on sophisticated ballistic devices. Here, we show a combination of liposome application and naked DNA vaccine that successfully overcomes these problems. Upon entrapment of plasmids encoding different antigens in cationic particles, transfection efficiencies similar to commercial kits were achieved in in vitro cell cultures. The liposome-based approach provided strong humoral responses against three malarial antigens, namely the Circumsporozoite protein and the C terminus of merozoite surface protein 1 from Plasmodium vivax (titers 104 or 103–104, respectively) and P. falciparum Rhoptry antigen 5 from Plasmodium falciparum (titers 103–104). When employed in P. falciparum growth-inhibition assays, antibodies demonstrated consistent reinvasion-blocking activities that were dose dependent. Liposome-formulated DNA vaccines may prove useful when targets cannot be produced as recombinant proteins and when conformation-dependent and highly specific antibodies are mandatory., Graphical Abstract

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Lipid particles for drug delivery can be modified to create multilayer vesicles with higher stability and improved cargo interaction. Here, we used lipids capable of forming hydrogen bonds instead of covalent bonds and designed stable vesicles-inside-vesicles with a high capacity of entrapping antimalarial drugs such as chloroquine (hydrophilic) and Artemisinin (lipophilic). In vitro treatment of the drug-sensitive P. falciparum strain NF54 showed that encapsulated drugs resulted in 72% and 60% lower IC50 values for each drug, respectively. Fluorochrome-labeling of a cargo-peptide or of membrane-resident lipids indicated that vesicles interacted more specifically with parasite-infected erythrocytes than with normal red blood cells. Accordingly, vesicle-confined chloroquine was able to elicit a stronger antiparasitic effect than free chloroquine in a lethal murine model of infection. Being permissive not only to small molecules but also to larger peptides, hydrogen-bond linked multilamellar liposomes are a very promising approach for enhanced drug delivery.

Made available in DSpace on 2018-11-26T15:37:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1 alpha domain diversity. Among the most commonly expressed DBL1 alpha types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1 alpha types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. (C) 2016 Elsevier Inc. All rights reserved. Univ Sao Paulo, Inst Biomed Sci, Av Prof Lineu Prestes 1374, BR-05508900 Sao Paulo, Brazil State Univ Sao Paulo, Inst Biotechnol, Al Tecomarias S-N, BR-18607440 Botucatu, SP, Brazil Univ Estadual Campinas, Inst Biol, R Monteiro Lobato 255,Cidade Univ, BR-13083862 Campinas, SP, Brazil Univ Fed Bahia, Hosp Prof Edgard Santos, Med Genet Serv, R Dr Augusto Viana S-N, BR-40110060 Salvador, BA, Brazil State Univ Sao Paulo, Inst Biotechnol, Al Tecomarias S-N, BR-18607440 Botucatu, SP, Brazil FAPESP: 2009/17114-3 FAPESP: 2010/50333-8 CNPq: 576128/2008-2 FAPESP: 2009/53702-7

Malaria-associate pregnancy has a significant impact on infant morbidity and mortality. The detrimental effects of malaria infection during pregnancy have been shown to correlate with immune activation in the placental tissue. Herein we sought to evaluate the effect of Toll-like receptors (TLRs) activation on placental malaria (PM) development by using the Plasmodium berghei NK65

Background Antibodies have an essential role in the acquired immune response against blood stage P. falciparum infection. Although several antigens have been identified as important antibody targets, it is still elusive which antigens have to be recognized for clinical protection. Herein, we analyzed antibodies from plasmas from symptomatic or asymptomatic individuals living in the same geographic area in the Western Amazon, measuring their recognition of multiple merozoite antigens. Methods Specific fragments of genes encoding merozoite proteins AMA1 and members of MSP and EBL families from circulating P. falciparum field isolates present in asymptomatic and symptomatic patients were amplified by PCR. After cloning and expression of different versions of the antigens as recombinant GST-fusion peptides, we tested the reactivity of patients’ plasmas by ELISA and the presence of IgG subclasses in the most reactive plasmas. Results 11 out of 24 recombinant antigens were recognized by plasmas from either symptomatic or asymptomatic infections. Antibodies to MSP9 (X2 DF=1 = 9.26/p = 0.0047) and MSP5 (X2 DF=1 = 8.29/p = 0.0069) were more prevalent in asymptomatic individuals whereas the opposite was observed for MSP1 block 2-MAD20 (X2 DF=1 = 6.41/p = 0.0206, Fisher’s exact test). Plasmas from asymptomatic individuals reacted more intensely against MSP4 (U = 210.5, p p p 95% = 0.021-0.585) and MSP9 (OR = 0.125, IC95% = 0.030-0.529, cross tab analysis) predicted 8.9 and 8 times less chances, respectively, to present symptoms. Higher antibody levels against MSP5 and EBA175 were associated by odds ratios of 9.4 (IC95% = 1.29-69.25) and 5.7 (IC95% = 1.12-29.62, logistic regression), respectively, with an asymptomatic status. Conclusions Merozoite antigens were targets of cytophilic antibodies and antibodies against MSP5, MSP9 and EBA175 were independently associated with decreased symptoms.

Malária é uma doença causada por protozoários do gênero Plasmodium que causa um milhão de mortes anualmente. Quase 3 bilhões de pessoas vivem em áreas tropicais de risco de infecção com uma das 5 espécies evidenciando um problema mundial carente de solução imediata. Esse parasita apresenta um potencial para o rápido desenvolvimento de resistências contra os fármacos utilizados em seu tratamento, por isso uma das soluções preconizadas pela Organização Mundial de Saúde é o desenvolvimento de vacinas eficazes para seu controle.O objetivo desse trabalho foca-se no aperfeiçoamento de uma metodologia mais efetiva para a formulação de vacinas contra malária. Inicialmente no modelo roedor, foi utilizado o método de carregamento em nanopartículas lipossomais , com proteínas oriundas de merozoítos do gênero Plasmodium. Além da avaliação do potencial vacinal das nanopartículas (sobrevida/morbidade), avaliamos o efeito da vacina contra toxinas (por exemplo, domínios GPI). Concluímos de maneira concisa que proteolipossomos gerados com proteínas GPI ancoradas do gênero Plasmodium possuem efeitos notáveis em relação a controle de crescimento parasitológico, mostrando-se efetivo também em desafios de letalidade in vivo. No que diz respeito a aspectos que afetam humanos, os soros gerados contra proteínas do parasita são capazes de diminuir interleucinas relacionadas com sintomas graves e parecem ter grandes efeitos antiparasitários que se correlacionam diretamente com o perfil genético do hospedeiro imunizado em ensaios in vitro. Malaria is a tropical disease caused by species of the protozoan Plasmodium and around one million people die of the disease each year, while 3 billion individuals live at risk to acquire infection with one of the five species known to infect humans. Due to the parasite\'s looming resistance against most of the antimalarial compounds used in therapy, the WHO preconizes the development of effective vaccine as an important goal. The purpose of this work was to evaluate the potential of a new method of vaccine formulation against malaria. Initially tested in the rodent model, we loaded liposomal nanostructures with merozoite-derived GPI-anchored proteins. We then monitored parameters such as survival and morbidity after challenge and measured the effect against parasite derived toxines. We observed significant effects in terms of control of parasitemy and in one model complete survival of mice. We also detected the generation of antiGPI antibodies which showed to be functional in decreasing TNF-a production in an in vitro model, however, we detected that this function was dependent on the genetic background of the antibody producing immunized animal.

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.