Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as <it>Pf</it>RONs, <it>Pf</it>92, <it>Pf</it>38, <it>Pf</it>12 and <it>Pf</it>34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the <it>P. vivax </it>rhoptry neck protein 1 (<it>Pv</it>RON1) and examine its antigenicity in natural <it>P. vivax </it>infections.</p> <p>Methods</p> <p>The <it>Pv</it>RON1 encoding gene, which is homologous to that encoding the <it>P. falciparum </it>apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The <it>pvron1 </it>gene transcription was evaluated by RT-PCR using RNA obtained from the <it>P. vivax </it>VCG-1 strain. Two peptides derived from the deduced <it>P. vivax </it>Sal-I <it>Pv</it>RON1 sequence were synthesized and inoculated in rabbits for obtaining anti-<it>Pv</it>RON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the <it>Pv</it>RON1 protein was assessed using human sera from individuals previously exposed to <it>P. vivax </it>malaria by ELISA.</p> <p>Results</p> <p>In the <it>P. vivax </it>VCG-1 strain, RON1 is a 764 amino acid-long protein. <it>In silico </it>analysis has revealed that <it>Pv</it>RON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The <it>Pv</it>RON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with <it>P. vivax</it>.</p> <p>Conclusions</p> <p>This study shows the identification and characterization of the <it>P. vivax </it>rhoptry neck protein 1 in the VCG-1 strain. Taking into account that <it>Pv</it>RON1 shares several important characteristics with other <it>Plasmodium </it>antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the <it>Aotus </it>monkey model are thus recommended.</p

Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein

<p>Abstract</p> <p>Background</p> <p>Malaria caused by <it>Plasmodium vivax </it>is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to <it>Plasmodium falciparum</it>, due in part to the difficulties of maintaining an <it>in vitro </it>culture of <it>P. vivax</it>. This study describes the identification of the <it>P. falciparum </it>thrombospondin-related apical merozoite protein homologue in <it>P. vivax </it>(PvTRAMP) and examines its potential to be further evaluated as vaccine candidate.</p> <p>Methods</p> <p>The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of <it>P. vivax</it>. Genes adjacent to <it>pvtramp </it>were identified <it>in silico </it>to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in <it>P. falciparum </it>and <it>Plasmodium knowlesi</it>. The <it>pvtramp </it>gene was amplified from cDNA of <it>P. vivax </it>schizont stages, cloned and expressed in <it>Escherichia coli</it>. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from <it>P. vivax-</it>infected individuals living in endemic areas was also assessed by ELISA.</p> <p>Results</p> <p>The PfTRAMP homologue in <it>P. vivax</it>, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with <it>P. vivax.</it></p> <p>Conclusions</p> <p>The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the <it>Aotus </it>animal model.</p

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p &lt; 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

PvGAMA reticulocyte binding activity: predicting conserved functional regions by natural selection analysis

Background: Adhesin proteins are used by Plasmodium parasites to bind and invade target cells. Hence, characterising molecules that participate in reticulocyte interaction is key to understanding the molecular basis of Plasmodium vivax invasion. This study focused on predicting functionally restricted regions of the P. vivax GPI-anchored micronemal antigen (PvGAMA) and characterising their reticulocyte binding activity. Results: The pvgama gene was initially found in P. vivax VCG-I strain schizonts. According to the genetic diversity analysis, PvGAMA displayed a size polymorphism very common for antigenic P. vivax proteins. Two regions along the antigen sequence were highly conserved among species, having a negative natural selection signal. Interestingly, these regions revealed a functional role regarding preferential target cell adhesion. Conclusions: To our knowledge, this study describes PvGAMA reticulocyte binding properties for the first time. Conserved functional regions were predicted according to natural selection analysis and their binding ability was confirmed. These findings support the notion that PvGAMA may have an important role in P. vivax merozoite adhesion to its target cells. © 2017 The Author(s)

Identificar las moléculas HLA DRB1-DQB1 y predecir los epítopos asociados con la eliminación y redetección de la infección por VPH de alto riesgo.

Several determining factors are involved in HPV infection outcomes; human leukocyte antigen (HLA) polymorphisms have been described as related factors. This study has ascertained the effect of genetic variation on HLA-DRB1 and DQB1 genes on HPV-16/-18/-31/-33/-45 and -58 clearance and redetection in Colombian women. PCR and qPCR were used for viral identification and the Illumina MiSeq system was used for HLA-typing of cervical samples (n=276). Survival models were adjusted for identifying alleles/haplotypes related to HPV clearance/redetection; L1/L2 protein-epitope binding to MHC-II molecules was also predicted. Significant associations suggested effects favouring or hampering clearance/redetection events depending on the viral type involved in infection, e.g. just DRB1*12:01:01G favoured HPV-16 (coeff: 4.8) and HPV-45 clearance (coeff: 12.65) whilst HPV-18 (coeff: 2E-15), HPV-31 (coeff: 8E-17) and HPV-58 hindered elimination (coeff: 1E-14). An effect was only observed for some alelles when configured as haplotypes, e.g. DRB1*04:07:01G (having the greatest frequency in the target population) was associated with DQB1*02:01:1G or *03:02:03. Epitope prediction identified 23 clearance-related peptides and 29 were redetection-related; eight might have been related to HPV-16/-18 and -58 persistence and one to HPV-18 elimination. HLA allele/haplotype relationship with the course of HPV infection (clearance/redetection) depended on the infecting HPV type, in line with the specific viral epitopes displayed