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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.
Assuntos
Vacinas Anticâncer , Neoplasias , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Vacinas de DNA , Animais , Feminino , Camundongos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Imunização , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes , RNA Mensageiro/genéticaRESUMO
Background: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. Methods: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. Results: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. Conclusion: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.
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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.
Assuntos
Malária Falciparum , Plasmodium falciparum , Animais , Eritrócitos , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Desenvolvimento Sexual , Fatores de Transcrição/genética , Transcrição Gênica , Virulência/genéticaRESUMO
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 µg 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.
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Studies on canine babesiosis in northeastern Brazil are scarce, although the weather conditions in this region are favorable for the development of the tick vector. This study determined the prevalence of Babesia vogeli in dogs sampled in Teresina, state of Piauí, northeast Brazil, using direct and indirect diagnostic methods and performed a phylogenetic analysis of 18S rRNA sequences. A total of 315 dogs were screened during routine care regardless of clinical suspicion. Blood was collected by jugular venipuncture to perform indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR) and for parasite screening in peripheral blood smears. Positivity was 2.2% (7/315) by microscopy, 4.8% (15/315) by PCR, and 48.6% (153/315) by IFA. PCR amplified a 602-bp fragment of the piroplasmid 18S rRNA gene, and sequence alignment and analysis revealed 99% homology with B. vogeli isolates from other regions of Brazil and other countries. In addition, there was high variability among sequences from other northeast states of Brazil. This study is the first to perform the molecular analysis of B. vogeli in Piauí. The results demonstrate that canine babesiosis is endemic in dogs sampled in Teresina and that PCR may be the method of choice to perform parasite screening in this region.
Estudos sobre a babesiose canina são escassos no Nordeste do Brasil, apesar das condições climáticas favoráveis ao desenvolvimento do carrapato vetor. Esta pesquisa objetivou determinar a ocorrência de Babesia vogeli em cães amostrados em Teresina, estado do Piauí, região Meio Norte do Brasil, através de métodos diretos e indiretos de diagnóstico, além de realizar análise filogenética das sequências 18S rRNA de piroplasmídeos obtidas no estudo. Foram avaliados 315 cães atendidos em clínicas veterinárias, sob qualquer suspeita clínica. Desses animais, foi colhido sangue por venopunção jugular para Reação de Imunofluorescência Indireta (RIFI) e Reação em Cadeia pela Polimerase (PCR). Além disso, esfregaços de sangue periférico foram realizados para pesquisa direta do parasita. A positividade dos animais foi de 2,2% (7/315) ao esfregaço sanguíneo, 4,8% (15/315) à PCR e 48,6% (153/315) à RIFI. O sequenciamento de amostras positivas à PCR resultou em um fragmento de 602 pb do gene 18S rRNA de piroplasmídeos, cujo alinhamento e análise da sequência revelaram 99% de homologia com isolados de B. vogeli de outras regiões do Brasil, além de outros países. É interessante ressaltar que, comparando isolados em diferentes estados do Nordeste, a homologia pode ser bastante variável. Esses são os primeiros resultados sobre a análise molecular de B. vogeli no Estado do Piauí. Além disso, este estudo demonstra que a babesiose canina é endêmica em cães de Teresina, Nordeste do Brasil, e que a PCR pode ser o método de escolha para diagnóstico da doença nessas áreas.
Assuntos
Animais , Cães , Babesia/genética , Babesia/parasitologia , Babesia/patogenicidade , Babesiose/diagnóstico , Babesiose/epidemiologia , Babesiose/genética , Babesiose/parasitologia , Babesiose/sangue , Doenças Parasitárias em Animais , ParasitosRESUMO
Studies on canine babesiosis in northeastern Brazil are scarce, although the weather conditions in this region are favorable for the development of the tick vector. This study determined the prevalence of Babesia vogeli in dogs sampled in Teresina, state of Piauí, northeast Brazil, using direct and indirect diagnostic methods and performed a phylogenetic analysis of 18S rRNA sequences. A total of 315 dogs were screened during routine care regardless of clinical suspicion. Blood was collected by jugular venipuncture to perform indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR) and for parasite screening in peripheral blood smears. Positivity was 2.2% (7/315) by microscopy, 4.8% (15/315) by PCR, and 48.6% (153/315) by IFA. PCR amplified a 602-bp fragment of the piroplasmid 18S rRNA gene, and sequence alignment and analysis revealed 99% homology with B. vogeli isolates from other regions of Brazil and other countries. In addition, there was high variability among sequences from other northeast states of Brazil. This study is the first to perform the molecular analysis of B. vogeli in Piauí. The results demonstrate that canine babesiosis is endemic in dogs sampled in Teresina and that PCR may be the method of choice to perform parasite screening in this region.(AU)
Estudos sobre a babesiose canina são escassos no Nordeste do Brasil, apesar das condições climáticas favoráveis ao desenvolvimento do carrapato vetor. Esta pesquisa objetivou determinar a ocorrência de Babesia vogeli em cães amostrados em Teresina, estado do Piauí, região Meio Norte do Brasil, através de métodos diretos e indiretos de diagnóstico, além de realizar análise filogenética das sequências 18S rRNA de piroplasmídeos obtidas no estudo. Foram avaliados 315 cães atendidos em clínicas veterinárias, sob qualquer suspeita clínica. Desses animais, foi colhido sangue por venopunção jugular para Reação de Imunofluorescência Indireta (RIFI) e Reação em Cadeia pela Polimerase (PCR). Além disso, esfregaços de sangue periférico foram realizados para pesquisa direta do parasita. A positividade dos animais foi de 2,2% (7/315) ao esfregaço sanguíneo, 4,8% (15/315) à PCR e 48,6% (153/315) à RIFI. O sequenciamento de amostras positivas à PCR resultou em um fragmento de 602 pb do gene 18S rRNA de piroplasmídeos, cujo alinhamento e análise da sequência revelaram 99% de homologia com isolados de B. vogeli de outras regiões do Brasil, além de outros países. É interessante ressaltar que, comparando isolados em diferentes estados do Nordeste, a homologia pode ser bastante variável. Esses são os primeiros resultados sobre a análise molecular de B. vogeli no Estado do Piauí. Além disso, este estudo demonstra que a babesiose canina é endêmica em cães de Teresina, Nordeste do Brasil, e que a PCR pode ser o método de escolha para diagnóstico da doença nessas áreas.(AU)
Assuntos
Animais , Cães , Parasitos , Doenças Parasitárias em Animais , Babesia/genética , Babesia/parasitologia , Babesia/patogenicidade , Babesiose/sangue , Babesiose/diagnóstico , Babesiose/epidemiologia , Babesiose/genética , Babesiose/parasitologiaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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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.
Assuntos
Anti-Inflamatórios/farmacologia , Antimaláricos/farmacologia , Misturas Complexas/farmacologia , Malária Cerebral/tratamento farmacológico , Trichoderma/química , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/isolamento & purificação , Antimaláricos/administração & dosagem , Antimaláricos/isolamento & purificação , Encéfalo/parasitologia , Encéfalo/patologia , Misturas Complexas/administração & dosagem , Misturas Complexas/isolamento & purificação , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eritrócitos/parasitologia , Histocitoquímica , Humanos , Imuno-Histoquímica , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Camundongos Endogâmicos C57BL , Parasitemia/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Análise de Sobrevida , Resultado do TratamentoRESUMO
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.
Assuntos
Proteínas de Escherichia coli/genética , Receptor 1 de Folato/genética , Peptidilprolil Isomerase/genética , Plasmídeos/química , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Receptor 1 de Folato/metabolismo , Expressão Gênica , Células HeLa , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Lipossomos/química , Lipossomos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptidilprolil Isomerase/metabolismo , Plasmídeos/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
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.