Ernesto T. A. Marques

Computed Tomographic Findings in Microcephaly Associated with Zika Virus

This report describes the characteristics of infants who were exposed to the Zika virus in utero and were born with abnormalities of the central nervous system, as seen on computed tomography.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Teratogenic effects of the Zika virus and the role of the placenta

The mechanism by which the Zika virus can cause fetal microcephaly is not known. Reports indicate that Zika is able to evade the normal immunoprotective responses of the placenta. Microcephaly has genetic causes, some associated with maternal exposures including radiation, tobacco smoke, alcohol, and viruses. Two hypotheses regarding the role of the placenta are possible: one is that the placenta directly conveys the Zika virus to the early embryo or fetus. Alternatively, the placenta itself might be mounting a response to the exposure; this response might be contributing to or causing the brain defect. This distinction is crucial to the diagnosis of fetuses at risk and the design of therapeutic strategies to prevent Zika-induced teratogenesis.

From re-emergence to hyperendemicity: the natural history of the dengue epidemic in Brazil.

Background Dengue virus (DENV) was reintroduced into Brazil in 1986 and by 1995 it had spread throughout the country. In 2007 the number of dengue hemorrhagic fever (DHF) cases more than doubled and a shift in the age distribution was reported. While previously the majority of DHF cases occurred among adults, in 2007 53% of cases occurred in children under 15 years old. The reasons for this shift have not been determined. Methods and Findings Age stratified cross-sectional seroepidemiologic survey conducted in Recife, Brazil in 2006. Serostatus was determined by ELISA based detection of Dengue IgG. We estimated time-constant and time-varying forces of infection of DENV between 1986 and 2006. We used discrete-time simulation to estimate the accumulation of monotypic and multitypic immunity over time in a population previously completely susceptible to DENV. We projected the age distribution of population immunity to dengue assuming similar hazards of infection in future years. The overall prevalence of DENV IgG was 0.80 (n = 1427). The time-constant force of infection for the period was estimated to be 0.052 (95% CI 0.041, 0.063), corresponding to 5.2% of susceptible individuals becoming infected each year by each serotype. Simulations show that as time since re-emergence of dengue goes by, multitypic immunity accumulates in adults while an increasing proportion of susceptible individuals and those with monotypic immunity are among young age groups. The median age of those monotypically immune can be expected to shift from 24 years, 10 years after introduction, to 13 years, 50 years after introduction. Of those monotypically immune, the proportion under 15 years old shifts from 27% to 58%. These results are consistent with the dengue notification records from the same region since 1995. Interpretation Assuming that persons who have been monotypically exposed are at highest risk for severe dengue, the shift towards younger patient ages observed in Brazil can be partially explained by the accumulation of multitypic immunity against DENV-1, 2, and 3 in older age groups, 22 years after the re-introduction of these viruses. Serotype specific seroepidemiologic studies are necessary to accurately estimate the serotype specific forces of infection.

Seroprevalence and risk factors for dengue infection in socio-economically distinct areas of Recife, Brazil.

Brazil currently accounts for the majority of dengue cases reported in the Americas, with co-circulation of DENV 1-3. Striking variation in the epidemiological pattern of infection within cities has been observed. Therefore, investigation of dengue transmission in small areas is important to formulate control strategies. A population-based household survey was performed in three diverse socio-economic and environmental areas of Recife, a large urban center of Brazil, between 2005 and 2006. Dengue serostatus and individual- and household-level risk factors for infection were collected in residents aged between 5 and 64 years. A total of 2833 individuals were examined, and their residences were geo-referenced. Anti-dengue IgG antibodies were measured using commercial ELISA. The dengue seroprevalence and the force of infection were estimated in each area. Individual and household variables associated with seropositivity were assessed by multilevel models for each area. A spatial analysis was conducted to identify risk gradients of dengue seropositivity using Generalized Additive Models (GAM). The dengue seroprevalence was 91.1%, 87.4% 74.3%, respectively, in the deprived, intermediate and high socio-economic areas, inversely related to their socio-economic status. In the deprived area, 59% of children had already been exposed to dengue virus by the age of 5 years and the estimated force of infection was three times higher than that in the privileged area. The risk of infection increased with age in the three areas. Not commuting away from the area was a risk factor for seropositivity in the deprived area (OR=2.26; 95% CI: 1.18-4.30). Number of persons per room was a risk factor for seropositivity in the intermediate (OR=3.00; 95% CI: 3.21-7.37) and privileged areas (OR=1.81; 95% CI: 1.07-3.04). Living in a house, as opposed to an apartment, was a risk factor for seropositivity in the privileged area (OR=3.62; 95% CI: 2.43-5.41). The main difference between the privileged and other areas could be attributed to the much larger proportion of apartment dwellers. Intensive vector control, surveillance and community education should be considered in deprived urban areas where a high proportion of children are infected by an early age.

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation

We used optimized DNA expression vectors to compare two gene delivery methodologies in rhesus macaques, namely direct DNA injection and in vivo adaptive constant-current electroporation via the intramuscular route. The use of in vivo electroporation increased levels of gene expression and immune responses. We used an optimized HIV gag expression plasmid to show the development of new cellular immune responses in SIV-infected animals controlling viremia. Furthermore, after vaccination with SIV expression plasmids the recall responses to the SIV antigens were very high, indicating that DNA is a strong boost in the presence of antiretroviral treatment in SIV-infected animals. There was substantial animal-to-animal variability in DNA expression, revealed by plasma measurements of IL-15 produced by co-injected IL-15 DNA. IL-15 expression levels correlated with peak immune responses. Electroporation led to an expansion of antigen-specific CD4+ and CD8+ T cells of both central and effector memory phenotype. These results indicate that improved gene delivery and expression by electroporation dramatically increases immunogenicity of DNA vaccines. Electroporation is thus an important method to improve the effectiveness of DNA vaccination.

Positive IgM for Zika virus in the cerebrospinal fluid of 30 neonates with microcephaly in Brazil.

Artigo liberado em acesso aberto como parte do acordo para tornar publico todos os dados produzidos sobre o virus zika - Compartilhamento de dados em emergencias de saude publica - http://www.wellcome.ac.uk/News/Media-office/Press-releases/2016/WTP060169.htm

HIV-1 p55Gag Encoded in the Lysosome-associated Membrane Protein-1 as a DNA Plasmid Vaccine Chimera Is Highly Expressed, Traffics to the Major Histocompatibility Class II Compartment, and Elicits Enhanced Immune Responses

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.

Alternative complement pathway deregulation is correlated with dengue severity.

Background The complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity. Methods and Results Patients with DHF had lower levels of complement factor 3 (C3; p = 0.002) and increased levels of C3a, C4a and C5a (p<0.0001) when compared to those with the less severe form, DF. There were no significant differences between DF and DHF patients in the levels of C1q, immunocomplexes (CIC-CIq) and CRP. However, small but statistically significant differences were detected in the levels of MBL. In contrast, the levels of two regulatory proteins of the alternative pathway varied widely between DF and DHF patients: DHF patients had higher levels of factor D (p = 0.01), which cleaves factor B to yield the active (C3bBb) C3 convertase, and lower levels of factor H (p = 0.03), which inactivates the (C3bBb) C3 convertase, than did DF patients. When we considered the levels of factors D and H together as an indicator of (C3bBb) C3 convertase regulation, we found that the plasma levels of these regulatory proteins in DHF patients favored the formation of the (C3bBb) C3 convertase, whereas its formation was inhibited in DF patients (p<0.0001). Conclusion The data suggest that an imbalance in the levels of regulatory factors D and H is associated with an abnormal regulation of complement activity in DHF patients.

Dengue 2 PreM-E/LAMP chimera targeted to the MHC class II compartment elicits long-lasting neutralizing antibodies.

A dengue 2 plasmid DNA vaccine (pD2) expressing the pre-membrane and envelope proteins (preM-E) was modified by replacing the dengue transmembrane and cytoplasmic sequences with those of the mouse lysosome-associated membrane protein (pD2/LAMP). Immunofluorescence and confocal microscopy of human 293, NIH 3T3, and macrophage IC21 cell lines transfected with pD2/LAMP showed that the preM-E/LAMP protein chimera was present in vesicles containing endogenous LAMP and major histocompatability complex class II (MHC II), in contrast to the non-vesicular localization of native preM-E protein lacking the LAMP targeting sequence. Mice immunized with pD2 showed an antigen-specific immunoglobulin response but the neutralizing antibodies titers (plaque reduction neutralization test, PRNT(50)) elicited by the native protein were minimal. In contrast, vaccination with pD2/LAMP resulted in PRNT(50) of 270, 320 and 160 at approximately 1, 3 and 8 months after two immunizations with 50 microg DNA, and approached 100% neutralization at 1:20 dilution. Additional immunization with pD2/LAMP, after 8 months, increased the neutralizing antibody titers to >640. Comparable neutralizing antibody responses were induced by two vector backbones, pVR1012 and pVax-1, at 5 and 50 microg of DNA. The neutralizing responses to the pD2/LAMP chimera were greatly superior to those elicited by pD2 in all conditions. These results underscore the importance of MHC class II presentation of DNA-encoded dengue-virus envelope protein for production of neutralizing antibodies.