Publications by authors named "Rafael Dhalia"

24 Publications

  • Page 1 of 1

Are Zika virus cross-reactive antibodies against aquaporin-4 associated to Neuromyelitis Optica Spectrum Disorder?

J Neuroimmunol 2021 Aug 20;360:577697. Epub 2021 Aug 20.

Department of Virology, Aggeu Magalhães Institute, Oswaldo Cruz Foundation, Recife, PE 50740-465, Brazil. Electronic address:

Zika virus (ZIKV) infection has been associated with the development of Neuromyelitis Optica Spectrum Disorder (NMOSD). ZIKV-induced antibodies that putatively cross-react to aquaporin-4 (AQP4) protein are suggested to cause inflammation of the optic nerve. A region of similarity between AQP4 and the ZIKV NS2B protein was identified. Our data showed that ZIKV-associated NMOSD patients develop anti-AQP4 antibodies, but not anti-ZIKV NS2B antibodies, revealing that cross-reacting antibodies are not the underlying cause of this phenotype. ZIKV infection in mice showed persistent viral replication in the eye tissue, suggesting that NMOSD symptoms are consequence of viral infection of the optic nerve cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jneuroim.2021.577697DOI Listing
August 2021

Zika-related adverse outcomes in a cohort of pregnant women with rash in Pernambuco, Brazil.

PLoS Negl Trop Dis 2021 03 8;15(3):e0009216. Epub 2021 Mar 8.

Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, United Kingdom.

Background: While Zika virus (ZIKV) is now widely recognized as a teratogen, the frequency and full spectrum of adverse outcomes of congenital ZIKV infection remains incompletely understood.

Methods: Participants in the MERG cohort of pregnant women with rash, recruited from the surveillance system from December/2015-June/2017. Exposure definition was based on a combination of longitudinal data from molecular, serologic (IgM and IgG3) and plaque reduction neutralization tests for ZIKV. Children were evaluated by a team of clinical specialists and by transfontanelle ultrasound and were classified as having microcephaly and/or other signs/symptoms consistent with congenital Zika syndrome (CZS). Risks of adverse outcomes were quantified according to the relative evidence of a ZIKV infection in pregnancy.

Findings: 376 women had confirmed and suspected exposure to ZIKV. Among evaluable children born to these mothers, 20% presented with an adverse outcome compatible with exposure to ZIKV during pregnancy. The absolute risk of microcephaly was 2.9% (11/376), of calcifications and/or ventriculomegaly was 7.2% (13/180), of additional neurologic alterations was 5.3% (13/245), of ophthalmologic abnormalities was 7% (15/214), and of dysphagia was 1.8% (4/226). Less than 1% of the children experienced abnormalities across all of the domains simultaneously. Interpretation: Although approximately one-fifth of children with confirmed and suspected exposure to ZIKV in pregnancy presented with at least one abnormality compatible with CZS, the manifestations presented more frequently in isolation than in combination. Due to the rare nature of some outcomes and the possibility of later manifestations, large scale individual participant data meta-analysis and the long-term evaluation of children are imperative to identify the full spectrum of this syndrome and to plan actions to reduce damages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0009216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971861PMC
March 2021

Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis.

PLoS Negl Trop Dis 2020 07 27;14(7):e0008488. Epub 2020 Jul 27.

Instituto Aggeu Magalhães (IAM)-Fundação Oswaldo Cruz (Fiocruz), Recife, Pernambuco, Brazil.

Background: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL.

Methodology/principal Findings: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis.

Conclusion: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410341PMC
July 2020

Zika virus infection in pregnancy: Establishing a case definition for clinical research on pregnant women with rash in an active transmission setting.

PLoS Negl Trop Dis 2019 10 7;13(10):e0007763. Epub 2019 Oct 7.

Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, PE, Brasil.

Defining cases of Zika virus (ZIKV) infection is a critical challenge for epidemiological research. Due to ZIKV's overlapping clinical features and potential immunologic cross-reactivity with other flaviviruses and the current lack of an optimal ZIKV-specific diagnostic assay, varying approaches for identifying ZIKV infections have been employed to date. This paper presents the laboratory results and diagnostic criteria developed by the Microcephaly Epidemic Research Group for defining cases of maternal ZIKV infection in a cohort of pregnant women with rash (N = 694) recruited during the declining 2015-2017 epidemic in northeast Brazil. For this investigation, we tested maternal sera for ZIKV by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Immunoglobulin (Ig) M and IgG3 enzyme-linked immunosorbent assays (ELISAs), and Plaque Reduction Neutralization Test (PRNT50). Overall, 23.8% of participants tested positive by qRT-PCR during pregnancy (range of detection: 0-72 days after rash onset). However, the inter-assay concordance was lower than expected. Among women with qRT-PCR-confirmed ZIKV and further testing, only 10.1% had positive IgM tests within 90 days of rash, and only 48.5% had ZIKV-specific PRNT50 titers ≥20 within 1 year of rash. Given the complexity of these data, we convened a panel of experts to propose an algorithm for identifying ZIKV infections in pregnancy based on all available lines of evidence. When the diagnostic algorithm was applied to the cohort, 26.9% of participants were classified as having robust evidence of a ZIKV infection during pregnancy, 4.0% as having moderate evidence, 13.3% as having limited evidence of a ZIKV infection but with uncertain timing, and 19.5% as having evidence of an unspecified flavivirus infection before or during pregnancy. Our findings suggest that integrating longitudinal data from nucleic acid and serologic testing may enhance diagnostic sensitivity and underscore the need for an on-going dialogue regarding the optimization of strategies for defining cases of ZIKV in research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0007763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797234PMC
October 2019

Perinatal analyses of Zika- and dengue virus-specific neutralizing antibodies: A microcephaly case-control study in an area of high dengue endemicity in Brazil.

PLoS Negl Trop Dis 2019 03 11;13(3):e0007246. Epub 2019 Mar 11.

Aggeu Magalhães Institute, Oswaldo Cruz Foundation (FIOCRUZ), Recife, Pernambuco, Brazil.

Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0007246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428350PMC
March 2019

Evaluation of the recombinant antigens Wb14 and WbT for the capture antibody diagnosis of lymphatic filariasis.

Mem Inst Oswaldo Cruz 2018 Mar 26;113(5):e170435. Epub 2018 Mar 26.

Fundação Oswaldo Cruz-Fiocruz, Instituto Aggeu Magalhães, Recife, PE, Brasil.

Background: Lymphatic filariasis (LF) is a parasitic disease caused mainly by the Wuchereria bancrofti worm and that affects up to 120 million people worldwide. LF is the second cause of chronic global deformity, responsible for 15 million people with lymphedema (elephantiasis) and 25 million men with scrotal hydrocele. Its diagnosis is still associated with numerous difficulties, such as the sample collection periods (microfilaria nocturnal periodicity) and limited diagnostic kits.

Objectives: The aim of this work was to evaluate two recombinant antigens (Wb14 and WbT) as part of an enzyme-linked immunosorbent assay (ELISA) based antibody capture tests for LF.

Methods: The recombinant antigens rWb14 and rWbT were expressed in Escherichia coli BL21 and an antibody capture ELISA was performed. For this, sera were used from microfilaremic individuals with W. bancrofti (MF), chronic pathology (CP), individuals infected with Strongyloides (SP) and healthy controls from endemic (EN) and non-endemic (NE) areas.

Findings: Both tests showed similar results, with 90% sensitivity and 96.6% specificity. In comparison with the BM14 ELISA commercial test, the Wb14 and WbT antigens performed with identical sensitivity but greater specificity. Reduced positivity with the CP suggested a potential to monitor cure. This was not confirmed, however, when sera from individuals up to seven years after treatment were assayed.

Main Conclusions: The Wb14 and WbT ELISAs were considered efficient and promising diagnostic tests. Due to the importance of antibody capture analysis to evaluate the Global Program to Eliminate Lymphatic Filariasis (GPELF), the tests proposed here appear as great alternatives to the available commercial system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/0074-02760170435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868868PMC
March 2018

Association between microcephaly, Zika virus infection, and other risk factors in Brazil: final report of a case-control study.

Lancet Infect Dis 2018 03 11;18(3):328-336. Epub 2017 Dec 11.

Department of Infectious Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK.

Background: A Zika virus epidemic emerged in northeast Brazil in 2015 and was followed by a striking increase in congenital microcephaly cases, triggering a declaration of an international public health emergency. This is the final report of the first case-control study evaluating the potential causes of microcephaly: congenital Zika virus infection, vaccines, and larvicides. The published preliminary report suggested a strong association between microcephaly and congenital Zika virus infection.

Methods: We did a case-control study in eight public maternity hospitals in Recife, Brazil. Cases were neonates born with microcephaly, defined as a head circumference of 2 SD below the mean. Two controls without microcephaly were matched to each case by expected date of delivery and area of residence. We tested the serum of cases and controls and the CSF of cases for detection of Zika virus genomes with quantitative RT-PCR and for detection of IgM antibodies with capture-IgM ELISA. We also tested maternal serum with plaque reduction neutralisation assays for Zika and dengue viruses. We estimated matched crude and adjusted odds ratios with exact conditional logistic regression to determine the association between microcephaly and Zika virus infection.

Findings: We screened neonates born between Jan 15 and Nov 30, 2016, and prospectively recruited 91 cases and 173 controls. In 32 (35%) cases, congenital Zika virus infection was confirmed by laboratory tests and no controls had confirmed Zika virus infections. 69 (83%) of 83 cases with known birthweight were small for gestational age, compared with eight (5%) of 173 controls. The overall matched odds ratio was 73·1 (95% CI 13·0-∞) for microcephaly and Zika virus infection after adjustments. Neither vaccination during pregnancy or use of the larvicide pyriproxyfen was associated with microcephaly. Results of laboratory tests for Zika virus and brain imaging results were available for 79 (87%) cases; within these cases, ten were positive for Zika virus and had cerebral abnormalities, 13 were positive for Zika infection but had no cerebral abnormalities, and 11 were negative for Zika virus but had cerebral abnormalities.

Interpretation: The association between microcephaly and congenital Zika virus infection was confirmed. We provide evidence of the absence of an effect of other potential factors, such as exposure to pyriproxyfen or vaccines (tetanus, diphtheria, and acellular pertussis, measles and rubella, or measles, mumps, and rubella) during pregnancy, confirming the findings of an ecological study of pyriproxyfen in Pernambuco and previous studies on the safety of Tdap vaccine administration during pregnancy.

Funding: Brazilian Ministry of Health, Pan American Health Organization, and Enhancing Research Activity in Epidemic Situations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1473-3099(17)30727-2DOI Listing
March 2018

The phenotypic spectrum of congenital Zika syndrome.

Am J Med Genet A 2017 Apr;173(4):841-857

Departamento de Genetica, Universidade Federal de Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.

In October 2015, Zika virus (ZIKV) outbreak the Brazilian Ministry of Health (MoH). In response, the Brazilian Society of Medical Genetics established a task force (SBGM-ZETF) to study the phenotype of infants born with microcephaly due to ZIKV congenital infection and delineate the phenotypic spectrum of this newly recognized teratogen. This study was based on the clinical evaluation and neuroimaging of 83 infants born during the period from July, 2015 to March, 2016 and registered by the SBGM-ZETF. All 83 infants had significant findings on neuroimaging consistent with ZIKV congenital infection and 12 had confirmed ZIKV IgM in CSF. A recognizable phenotype of microcephaly, anomalies of the shape of skull and redundancy of the scalp consistent with the Fetal Brain Disruption Sequence (FBDS) was present in 70% of infants, but was most often subtle. In addition, features consistent with fetal immobility, ranging from dimples (30.1%), distal hand/finger contractures (20.5%), and feet malpositions (15.7%), to generalized arthrogryposis (9.6%), were present in these infants. Some cases had milder microcephaly or even a normal head circumference (HC), and other less distinctive findings. The detailed observation of the dysmorphic and neurologic features in these infants provides insight into the mechanisms and timings of the brain disruption and the sequence of developmental anomalies that may occur after prenatal infection by the ZIKV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajmg.a.38170DOI Listing
April 2017

Dengue Virus-Specific Antibodies Enhance Brazilian Zika Virus Infection.

J Infect Dis 2017 03;215(5):781-785

Aggeu Magalhães Research Center, Oswaldo Cruz Foundation (FIOCRUZ), Recife, Brazil.

Anti-Flavivirus antibodies are highly cross-reactive and may facilitate Zika virus (ZIKV) infection through the antibody-dependent enhancement (ADE) mechanism. We demonstrate that dengue-specific antibodies enhance the infection of a primary Brazilian ZIKV isolate in a FcγRII-expressing K562 cell line. In addition, we demonstrate that serum samples from dengue-immune pregnant women enhanced ZIKV infection. These findings highlight the need for epidemiological studies and animal models to further confirm the role of ADE in the development of congenital and neurological complications associated with ZIKV infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiw638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854042PMC
March 2017

Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

J Infect Dis 2016 Dec 5;214(12):1897-1904. Epub 2016 Oct 5.

Department of Virology, Aggeu Magalhães Research Center, Fundação Oswaldo Cruz.

Background:  Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates.

Methods:  We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated.

Results:  Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001).

Conclusions:  Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiw477DOI Listing
December 2016

Association between Zika virus infection and microcephaly in Brazil, January to May, 2016: preliminary report of a case-control study.

Lancet Infect Dis 2016 Dec 16;16(12):1356-1363. Epub 2016 Sep 16.

The Research Center Aggeu Magalhães (CPqAM) and Oswaldo Cruz Foundation (Fiocruz), Recife, Brazil; Department of Community Health, Federal University of Goiás, Goiânia, Brazil.

Background: The microcephaly epidemic, which started in Brazil in 2015, was declared a Public Health Emergency of International Concern by WHO in 2016. We report the preliminary results of a case-control study investigating the association between microcephaly and Zika virus infection during pregnancy.

Methods: We did this case-control study in eight public hospitals in Recife, Brazil. Cases were neonates with microcephaly. Two controls (neonates without microcephaly), matched by expected date of delivery and area of residence, were selected for each case. Serum samples of cases and controls and cerebrospinal fluid samples of cases were tested for Zika virus-specific IgM and by quantitative RT-PCR. Laboratory-confirmed Zika virus infection during pregnancy was defined as detection of Zika virus-specific IgM or a positive RT-PCR result in neonates. Maternal serum samples were tested by plaque reduction neutralisation assay for Zika virus and dengue virus. We estimated crude odds ratios (ORs) and 95% CIs using a median unbiased estimator for binary data in an unconditional logistic regression model. We estimated ORs separately for cases with and without radiological evidence of brain abnormalities.

Findings: Between Jan 15, 2016, and May 2, 2016, we prospectively recruited 32 cases and 62 controls. 24 (80%) of 30 mothers of cases had Zika virus infection compared with 39 (64%) of 61 mothers of controls (p=0·12). 13 (41%) of 32 cases and none of 62 controls had laboratory-confirmed Zika virus infection; crude overall OR 55·5 (95% CI 8·6-∞); OR 113·3 (95% CI 14·5-∞) for seven cases with brain abnormalities; and OR 24·7 (95% CI 2·9-∞) for four cases without brain abnormalities.

Interpretation: Our data suggest that the microcephaly epidemic is a result of congenital Zika virus infection. We await further data from this ongoing study to assess other potential risk factors and to confirm the strength of association in a larger sample size.

Funding: Brazilian Ministry of Health, Pan American Health Organization, and Enhancing Research Activity in Epidemic Situations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1473-3099(16)30318-8DOI Listing
December 2016

Initial Description of the Presumed Congenital Zika Syndrome.

Am J Public Health 2016 Apr;106(4):598-600

Demócrito de Barros Miranda-Filho, Ricardo Arraes de Alencar Ximenes, Maria Angela Wanderley Rocha, and Regina Coeli Ferreira Ramos are with the University of Pernambuco, Recife, Brazil. Celina Maria Turchi Martelli, Rafael Dhalia, Rafael Freitas de Oliveira França, and Ernesto T. A. Marques Júnior are with The Research Center Aggeu Magalhães (CPqAM)/Oswaldo Cruz Foundation (Fiocruz), Recife. Thalia Velho Barreto Araújo and Ricardo Arraes de Alencar Ximenes are with the Federal University of Pernambuco, Recife. Laura Cunha Rodrigues is with the London School of Hygiene and Tropical Medicine, London, UK.

Objectives: To provide an initial description of the congenital syndrome presumably associated with infection by Zika virus compared with other syndromes including congenital infections of established etiologies.

Methods: We provide an overview of a published case series of 35 cases, a clinical series of 104 cases, and published and unpublished reports of clinical and laboratory findings describing cases diagnosed since the beginning of the epidemic of microcephaly in Brazil.

Results: About 60% to 70% of mothers report rash during pregnancy; mainly in the first trimester. Principal features are microcephaly, facial disproportionality, cutis girata, hypertonia/spasticity, hyperreflexia, and irritability; abnormal neuroimages include calcifications, ventriculomegaly, and lissencephaly. Hearing and visual abnormalities may be present.

Conclusions: Preliminary data suggest that severe congenital abnormalities are linked to Zika virus infection. Cases have severe abnormalities, and although sharing many characteristics with congenital abnormalities associated with other viral infections, abnormalities presumably linked to the Zika virus may have distinguishing characteristics. These severe neurologic abnormalities may result in marked mental retardation and motor disabilities for many surviving offspring.

Policy Implications: Affected nations need to prepare to provide complex and costly multidisciplinary care that children diagnosed with this new congenital syndrome will require.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2105/AJPH.2016.303115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816005PMC
April 2016

A DNA vaccine against yellow fever virus: development and evaluation.

PLoS Negl Trop Dis 2015 Apr 13;9(4):e0003693. Epub 2015 Apr 13.

Oswaldo Cruz Foundation (FIOCRUZ), Aggeu Magalhães Research Centre, Department of Virology, Laboratório de Virologia e Terapia Experimental (LAVITE), Universidade Federal de Pernambuco (UFPE), University City, Recife, Pernambuco, Brazil.

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0003693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395287PMC
April 2015

Assessing protein conformational sampling and structural stability via de novo design and molecular dynamics simulations.

Biopolymers 2015 Jun;103(6):351-61

Department of Fundamental Chemistry, Federal University of Pernambuco, Recife, PE, 50740-560, Brazil.

Molecular dynamics and de novo techniques, associated to quality parameter sets, have excelled at determining the structure of small proteins with high accuracy. To achieve a detailed description of protein conformations, these methods must critically assess the thermodynamic features of the molecular ensembles. Here, a comparison of the conformational ensemble generated by molecular dynamics and de novo techniques were carried out for six Top7-based proteins carrying gp41 HIV-1 epitopes. The native Top7, a highly stable computationally designed protein, was used as benchmark. Structural stability, flexibility, and secondary structure content were assessed. The consistency of the latter was compared to experimental circular dichroism spectra for all proteins. While both methods are capable to identify the stable from unstable chimeric proteins, the sampled conformational space and flexibility differ significantly in both methods. Molecular dynamics simulations seem to better describe secondary structure content and identify regions responsible for conformational instability. The de novo method, as implemented in Rosetta-a prime tool for protein design, overestimates secondary structure content. On the other hand, its empirical energy function is capable to predict the threshold for protein stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bip.22626DOI Listing
June 2015

T-cell memory responses elicited by yellow fever vaccine are targeted to overlapping epitopes containing multiple HLA-I and -II binding motifs.

PLoS Negl Trop Dis 2013 31;7(1):e1938. Epub 2013 Jan 31.

Virology and Experimental Therapeutics Laboratory, Aggeu Magalhães Research Center, Fiocruz, Recife, Pernambuco, Brazil.

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4(+) and CD8(+) T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, "promiscuous" T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0001938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561163PMC
June 2013

West Nile virus T-cell ligand sequences shared with other flaviviruses: a multitude of variant sequences as potential altered peptide ligands.

J Virol 2012 Jul 9;86(14):7616-24. Epub 2012 May 9.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in ∼≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00166-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416302PMC
July 2012

The four trypanosomatid eIF4E homologues fall into two separate groups, with distinct features in primary sequence and biological properties.

Mol Biochem Parasitol 2011 Mar 24;176(1):25-36. Epub 2010 Nov 24.

Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Av. Moraes Rego s/n, Campus UFPE, Recife, PE 50670-420, Brazil.

Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molbiopara.2010.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736675PMC
March 2011

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

An Acad Bras Cienc 2009 12;81(4):663-9

Fundação Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhães, Departamento de Virologia, Laboratório de Virologia e Terapia Experimental, Recife, PE, Brasil.

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/s0001-37652009000400005DOI Listing
December 2009

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model.

Virology 2008 Aug 25;378(1):105-17. Epub 2008 Jun 25.

Johns Hopkins University, School of Medicine, Pharmacology Department, Baltimore, USA.

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2008.04.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615555PMC
August 2008

MBL2 gene polymorphisms protect against development of thrombocytopenia associated with severe dengue phenotype.

Hum Immunol 2008 Feb 12;69(2):122-8. Epub 2008 Feb 12.

Virology and Experimental Therapy Laboratory, Aggeu Magalhães Research Center-CPqAM/FIOCRUZ, Recife, Brazil.

Dengue disease can clinically evolve from an asymptomatic and mild disease, known as dengue fever (DF), to a severe disease known as dengue hemorrhagic fever (DHF). Recent evidence has shown how host genetic factors can be correlated with severe dengue susceptibility or protection. Many of these genes, such as CD209, TNF-a, vitamin D receptor, and FC gamma receptor IIA, are components of the innate immune system, suggesting that innate responses might have a role in dengue pathogenesis. MBL2 gene polymorphisms have been shown to modulate susceptibility or protection in many viral diseases. We investigated the involvement of MBL2 gene in the dengue clinical outcome through the analysis of MBL2 exon 1 polymorphisms (at codons 52, 54, and 57) known to be associated with reduced serum levels of the MBL protein. The genotypes of 110 well-characterized dengue-positive patients were statistically analyzed to establish possible correlations between MBL2 polymorphisms and parameters such as sex, type of infection (primary or secondary response), race/ethnicity, course of infection, and age. We found significant correlations between wild-type AA MBL2 genotype and age as associated risk factors for development of dengue-related thrombocytopenia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.humimm.2008.01.005DOI Listing
February 2008

The two eIF4A helicases in Trypanosoma brucei are functionally distinct.

Nucleic Acids Res 2006 10;34(9):2495-507. Epub 2006 May 10.

Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Avenue Moraes Rego s/n, Campus UFPE, Recife PE 50670-420, Brazil.

Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5' capped mini-exon which is linked to the 3' cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 x 10(5) molecules/cell) and depletion to approximately 10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (approximately 1-2 x 10(4) molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkl290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459412PMC
June 2006

Translation initiation in Leishmania major: characterisation of multiple eIF4F subunit homologues.

Mol Biochem Parasitol 2005 Mar;140(1):23-41

Departamento de Biologia Celular, Universidade de Brasilia, Brasilia 70910-900, D.F., Brazil.

In eukaryotes protein synthesis initiates with the binding of the multimeric translation initiation complex eIF4F - eIF4E, eIF4A and eIF4G - to the monomethylated cap present on the 5' end of mRNAs. eIF4E interacts directly with the cap nucleotide, while eIF4A is a highly conserved RNA helicase and eIF4G acts as a scaffold for the complex with binding sites for both eIF4E and eIF4A. eIF4F binding to the mRNA recruits the small ribosomal subunit to its 5' end. Little is known in detail of protein synthesis in the protozoan parasites belonging to the family Trypanosomatidae. However, the presence of the highly modified cap structure, cap4, and the spliced leader sequence on the 5' ends of all mRNAs suggests possible differences in mRNA recruitment by ribosomes. We identified several potential eIF4F homologues by searching Leishmania major databases: four eIF4Es (LmEIF4E1-4), two eIF4As (LmEIF4A1-2) and five eIF4Gs (LmEIF4G1-5). We report the initial characterisation of LmEIF4E1-3, LmEIF4A1-2 and LmEIF4G3. First, the expression of these proteins in L. major promastigotes was quantitated by Western blotting using isoform specific antibodies. LmEIF4A1 and LmEIF4E3 are very abundant, LmEIF4G3 is moderately abundant and LmEIF4E1/LmEIF4E2/LmEIF4A2 are rare or not detected. In cap-binding assays, only LmEIF4E1 bound to the 7-methyl-GTP-Sepharose resin. Molecular modelling confirmed that LmEIF4E1 has all the structural features of a cap-binding protein. Finally, pull-down assays were used to investigate the potential interaction between the eIF4A (LmEIF4A1/LmEIF4A2) and eIF4G (LmEIF4G1-3) homologues. Only LmEIF4G3, via the HEAT domain, bound specifically both to LmEIF4A1 as well as to human eIF4A. Therefore for each factor, one of the L. major forms seems to fulfil, in part at least, the expected characteristics of a translational initiation factor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molbiopara.2004.12.001DOI Listing
March 2005

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

J Biol Chem 2003 Nov 2;278(47):46357-68. Epub 2003 Sep 2.

Departamento de Biologia Celular, Universidade de Brasilia, Brasilia DF 70910-900, Brazil.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M307624200DOI Listing
November 2003
-->