Coxiella burnetii is a global, highly infectious intracellular bacterium, able to infect a wide range of hosts and to persist for months in the environment. It is the etiological agent of Q fever—a zoonosis of global priority. Currently, there are no national surveillance data on C. burnetii’s seroprevalence for any South American country, reinforcing the necessity of developing novel and inexpensive serological tools to monitor the prevalence of infections among humans and animals—especially cattle, goats, and sheep. In this study, we used immunoinformatics and computational biology tools to predict specific linear B-cell epitopes in three C. burnetii outer membrane proteins: OMP-H (CBU_0612), Com-1 (CBU_1910), and OMP-P1 (CBU_0311). Furthermore, predicted epitopes were tested by ELISA, as synthetic peptides, against samples of patients reactive to C. burnetii in indirect immunofluorescence assay, in order to evaluate their natural immunogenicity. In this way, two linear B-cell epitopes were identified in each studied protein (OMP-H(51–59), OMP-H(91–106), Com-1(57–76), Com-1(191–206), OMP-P1(197–209), and OMP-P1(215–227)); all of them were confirmed as naturally immunogenic by the presence of specific antibodies in 77% of studied patients against at least one of the identified epitopes. Remarkably, a higher frequency of endocarditis cases was observed among patients who presented an intense humoral response to OMP-H and Com-1 epitopes. These data confirm that immunoinformatics applied to the identification of specific B-cell epitopes can be an effective strategy to improve and accelerate the development of surveillance tools against neglected diseases.
Introduction: Q fever is a highly infectious but largely 'neglected zoonosis' caused by Gramnegative bacterium, Coxiella burnetii, which is listed as a biological warfare agent and infects a wide range of domestic and wild animals as well as humans. The disease in animals is associated to late abortions, stillbirths and other reproductive disorders. In humans, acute Q fever has a wide clinical spectrum, ranging from asymptomatic to influenza-like or pneumonia cases, and may progress to chronic diseases complicated by endocarditis, meningoencephalitis and/or osteomyelitis. Coxiella burnetii survive in environment for months and can spread by aerosolization or ingestion of contaminated milk and derivatives. Considering its severity, Q fever may have a huge impact on livestock and public health, especially in developing countries like Brazil, where meat of cattle, milk and its derivatives are important economic issues and largely consumed. Besides, the true picture of infection remains obscure due to limited diagnostic and surveillance strategies. On this context, outer-membrane proteins (OMPs) emerge as targets to novel diagnosis reactive, once previous studies showed the reactivity of members of these proteins against serum of humans and animals infected by C. burnetii. However, in order to avoid crossreactions with other bacterial pathogens, in silico analysis of OMPs rise as a promising approach to identification of highly specific immunogenic regions on Coxiella burnetii targets. Objective: This study aimed to identify potential B-cell epitopes on OMPs (Omp-H, Omp-P1, Omp-Com1) of Coxiella burnetii. Methodology: To predict linear B-cell epitopes, sequences of OMP-H, Omp-Com1 and Omp-P1 were obtained from Uniprot and explored by three prediction algorithms (BCpred,BepiPred-1.0,EMINI-Surface-Accessibility-Prediction). All sequences predicted by at least two algorithms and with more than 9 mers were analyzed by VaxiJen, to predict its immunogenicity. Moreover, the conservation degree of predicted epitopes was evaluated by comparison (BLASTp) between each sequences and correlated bacteria (Francisella tularensis, Legionella pneumophila, Escherichia coli, Pseudomonas aeruginosa, Rickettsia rickettsia, Ehrlichia chaffeensis,Bartonella henselae, Brucella melitensis, Afipia felis, Campylobacter jejuni). Finally, the 3D structures of OMP-H, OMP-P1 and OMP-Com1 were predicted by Robetta algorithm and its oligomeric structures predicted by Galaxy Protein Modelling Program. Results: Firstly, in each studied OMP, two sequences were predicted as linear B-cell epitope (OmpH-E1, OmpH-E2, OmpC-E1, OmpC-E2, OmpP-E1, and OmpP-E2). Among these sequences, OmpH-E2, OmpC-E1, OmpP-E1 and OmpP-E2 were predicted as immunogenic epitopes and evaluated for its conservation degree. Remarkably, all sequences predicted as immunogenic linear B-cell epitopes were specific of Coxiella burnetii, once that presented a low conservation degree in comparison with correlated bacteria. Moreover, all immunogenic predicted epitopes are exposed on surface...
Introduction: Coxiella burnetii is the etiological agent of the zoonosis Q fever, a neglected disease which causes an acute or a chronic, life-threatening disease in humans. In its late course, Q fever can be complicated by fatal (eg, endocarditis) or debilitating (eg, chronic fatigue syndrome) disorders. Ruminants are considered as the main reservoirs for human infections but are usually asymptomatic or may manifest as late term abortions and therefore have significant economic impact. C. burnetii presents a highly stable and infectious cell form, which persists in the environment and is transmitted via inhalation of aerosols and/or consumption of contaminated milk. Q fever infection is most commonly diagnosed by serology tests but due to its impaired sensitivity, reliable clinic and veterinary diagnostic tests need to be developed. In this sense, recent immunoproteomic studies identified several proteins, which presented reactivity against sera from Q feverpatients, and are potential candidates for the development of new diagnostic tests. Objective: To identify, through a combination of prediction algorithms, immunogenic and non-conserved B-cell epitopes present on Coxiella burnetii immunoreactive proteins (outer membrane protein A, YAJc and LemA). Methodology: FASTA sequences of outer membrane protein A (ompA;CBU-1260), YAJc (yajC;CBU-1143) and LemA (lemA;CBU-0545) from C. burnetii were obtained from UNIPROT. The B-cell epitope prediction was carried out using two different algorithms, BepiPred and BCPred (threshold 0.5), whereas surface accessibility was predicted by Emini Surface Accessibility algorithm (threshold 1.0). We considered a potential B-cell epitope, sequences predicted by 2 or more algorithms that comprises, at least, 9 mers of length. Thereafter, the immunogenic potential from B-cell sequences were predicted by Vaxijen algorithm (threshold 0.4). The degree of sequence conservation between immunogenic B-cell epitopes and other C.burnetii related species (Francisella tularensis, Legionella pneumophila, Escherichia coli, Pseudomonas aeruginosa, Campylobacter jejuni, Rickettsia rickettsia, Ehrlichia chaffeensis, Bartonella henselae, Brucella melitensis, Afipia felis) were compared using protein BLAST databank. Results: Combining different prediction algorithms, we described four B-cell epitopes present on OMP-A (OMP-AE1, OMP-AE2, OMP-AE3 and OMP-AE4). OMP-AE1 and OMP-AE2 appears as two potential highly immunogenic B-cell candidates (Vaxijen score: 1.2 and 2.1, respectively), both with a low degree of sequence conservation (E-value >20), whereas OMP-AE3 and OMP-AE4 were predicted as B-cell sequences poorly immunogenic (Vaxijen score: 0.3 and 0.01, respectively). YAJc and LemA possess potential B-cell epitopes (YAJ-E01, LEM-E01 and LEM-E02; Vaxijen score: 1.2, 1.5 and 1.1, respectively), but although highly immunogenic, the predicted sequences are highly conserved (E-value < 1) among phylogenetic C. burnetii related species. Conclusion: Taken together, our in silico analysis suggests that OMP-AE1 and ...
Introduction: Zika virus (ZIKV) is an arbovirus and belongs to the Flaviviridae family, like the related dengue, yellow fever, West Nile, and Japanese encephalitis viruses. Although ZIKV usually causes an asymptomatic or mildly symptomatic disease in infected adults, it can lead to severe brain abnormalities in fetuses, who are infected in utero by vertical transmission of the virus through the placenta, and is associated with serious neurological complications such Guillain-Barre syndrome and meningoencephalitis. Despite this, there are no specific treatments or vaccines against ZIKV. On this context, considering that the Envelope protein of Flavivirus is essential to invasion of host cells and is the major target of neutralizing antibodies, the identification of its immunogenic regions is a crucial step to the development of immunotherapies based on monoclonal antibodies and novel vaccines. Objective: The present study aimed to identify non-conserved B-cell epitopes on ZIKV's Envelope protein, which could be applied in novel vaccine formulations and development of monoclonal antibodies to immunotherapies against ZIKA. Methodology: The combination of three prediction algorithms (BepiPred 1.0, BepiPred 2.0, EMINI Surface Accessibility Prediction) was used to predict linear B-cell epitopes on entire sequence of ZIKV Envelope protein. To evaluate the conservation degree of predicted epitopes, we aligned its sequences to Envelope proteins of other arbovirus and compared the similarity among them, using BioEdit Sequence Alignments Editor. Finally, for non-conserved sequences, specific epitopes were considered and synthetized as linear peptides and tested by their reactivity against samples from patients infected by ZIKV (n=21) and patients infected by Dengue virus in 2007 or 2008 (n=17), who never presented Zika episodes. Finally, to experimentally validate the predicted epitopes; we compared the magnitude of response against each epitope between ZIKV and Dengue patients. Results: Firstly, we predicted three linear B-cell epitopes on Domain-III (E4, E5, E6) of Envelope protein, the main region target of neutralizing antibodies, two epitopes on Domain-II (E1, E3) and one in Domain-I (E2). Interestingly, epitopes located on Domain II were conserved epitopes, presenting more than 50% of similarity with Flavivirus. Based on the low conservation degree (similarity <50%), the epitopes located on Domain-III (E4, E5 and E6) and epitope located on Domain-I (E2) were selected to experimental validation. Remarkably, against all selected epitopes (E2, E4, E5 and E6), we observed responder individuals, which presented reactivity values higher than double of Dengue patients value. Moreover, the optical densities against epitopes E2, E4 and E5 were statistically higher in Zika patients than in Dengue patients (p=0.037, p=0.037 and p=0.004, respectively). Conclusion: Our study allowed the identification of four promising linear B-cell epitopes on ZIKV's Envelope protein, which were non-conserved among Flavivirus and could be applied...
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