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The identification of specific biomarkers for Zika infection and its clinical complications is fundamental to mitigate the infection spread, which has been associated with a broad range of neurological sequelae. We present the characterization of antibody responses in serum samples from individuals infected with Zika, presenting non-severe (classical) and severe (neurological disease) phenotypes, with high-density peptide arrays comprising the Zika NS1 and NS2B proteins. The data pinpoints one strongly IgG-targeted NS2B epitope in non-severe infections, which is absent in Zika patients, where infection progressed to the severe phenotype. This differential IgG profile between the studied groups was confirmed by multivariate data analysis. Molecular dynamics simulations and circular dichroism have shown that the peptide in solution presents itself in a sub-optimal conformation for antibody recognition, which led us to computationally engineer an artificial protein able to stabilize the NS2B epitope structure. The engineered protein was used to interrogate paired samples from mothers and their babies presenting Zika-associated microcephaly and confirmed the absence of NS2B IgG response in those samples. These findings suggest that the assessment of antibody responses to the herein identified NS2B epitope is a strong candidate biomarker for the diagnosis and prognosis of Zika-associated neurological disease.

In this manuscript we describe the engineering of a biomarker for the diagnosis and prognosis of Zika-associated neurological disease. Although the causal association between congenital Zika virus (ZIKV) infection and neurological manifestations has been well documented in the recent years, biomarkers for proper diagnostic and disease outcome still remain to be defined. Combining high-density peptide array and multivariate analysis, we have identified an ZIKV epitope that is associated to a lack of IgG antibody response in patients with severe neurological symptoms. An engineered chimera was developed to discriminate between mild and severe clinical forms of the disease.

The emergence of a new human coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has imposed great pressure on the health system worldwide. The presence of glycoproteins on the viral envelope opens a wide range of possibilities for the application of lectins to address some urgent problems involved in this pandemic. In this work, we discuss the potential contributions of lectins from nonmammalian sources in the development of several fields associated with viral infections, most notably COVID-19. We review the literature on the use of nonmammalian lectins as a therapeutic approach against members of the Coronaviridae family, including recent advances in strategies of protein engineering to improve their efficacy. The applications of lectins as adjuvants for antiviral vaccines are also discussed. Finally, we present some emerging strategies employing lectins for the development of biosensors, microarrays, immunoassays and tools for purification of viruses from whole blood. Altogether, the data compiled in this review highlight the importance of structural studies aiming to improve our knowledge about the basis of glycan recognition by lectins and its repercussions in several fields, providing potential solutions for complex aspects that are emerging from different health challenges.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2–0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.

Antibodies against the HIV-1 2F5 epitope are known as one of the most powerful and broadly protective anti-HIV antibodies. Therefore, vaccine strategies that include the 2F5 epitope in their formulation require a robust method to detect specific anti-2F5 antibody production by B cells. Towards this goal, we have biotinylated a previously reported computer-designed protein carrying the HIV-1 2F5 epitope aiming the further development of a platform to detect human B-cells expressing anti-2F5 antibodies through flow cytometry. Biophysical and immunological properties of our devised protein were characterized by computer simulation and experimental methods. Biotinylation did not affect folding and improved protein stability and solubility. The biotinylated protein exhibited similar binding affinity trends compared to its unbiotinylated counterpart and was recognized by anti-HIV-1 2F5 antibodies expressed on the surface of patient-derived peripheral blood mononuclear cells. Moreover, we present a high affinity marker for the identification of epitope-specific B cells that can be used to measure the efficacy of vaccine strategies based on the HIV-1 envelope protein.

Research is an important component of an effective response to the increasing frequency of widespread infectious disease outbreaks. In turn, the ability to do such studies relies on willingness of partners in different regions to collaborate and the capacity to mount a rapid research response. The EU-funded ZIKAlliance Consortium has initiated a multicountry epidemiological, clinical, and laboratory research agenda to determine the incidence, risk factors, and outcomes of Zika virus infection in pregnant women and their children. We reviewed the timeline of patient cohort initiation in relation to the Zika virus epidemic and mapped key events regarding funding, regulatory approvals, and site preparation during this timeline. We then assessed barriers and delays that the international research team experienced through a systematic telephone interview. We have identified three major bottlenecks in the implementation of a swift response: the absence of a timeline for the funding process, delays in regulatory and ethical approval, and the challenging logistics of laboratory support, including diagnostics. These bottlenecks illustrate the clear and urgent need for implementing a strong and permanent global emerging infectious diseases research capacity that has structured funding, enables long-term partnerships, and develops basic clinical and laboratorial research and a response infrastructure that is ready to deploy.

German Centre for Infection Research European Union's Horizon research and innovation program (ZIKAlliance) EVAg We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is required for adequate estimates of Zika virus-associated disease and determination of patient care. Charite, German Ctr Infect Res, Berlin, Germany Hosp Univ Prof Edgard Santos, LAPI, Salvador, BA, Brazil Fdn Prosangue Hemoctr Sao Paulo, Sao Paulo, Brazil Inst Oswaldo Cruz, Rio De Janeiro, Brazil Fed Univ Para, Belem, Para, Brazil Univ Brasilia, Brasilia, DF, Brazil Univ Sao Paulo, Sao Paulo, Brazil Fundacao Oswaldo Cruz, Pernambuco, Brazil Univ Fed Bahia, Salvador, BA, Brazil Diagnost Amer DASA, Sao Paulo, Brazil Hosp Israelita Albert Einstein, Sao Paulo, Brazil Fac Med Sao Jose do Rio Preto, Sao Jose De Rio Preto, Brazil Fdn Med Trop Dr Heitor Vieira Dourado, Manaus, Amazonas, Brazil Fundacao Oswaldo Cruz, Salvador, BA, Brazil Univ Fed Sao Paulo, Sao Paulo, Brazil Aix Marseille Univ, Marseille, France Assistance Publ Hop Marseille, Marseille, France Univ Bonn, Med Ctr, Bonn, Germany Robert Koch Inst, Berlin, Germany Univ Fed Sao Paulo, Sao Paulo, Brazil ZIKAlliance: 734548 EVAg: 653316 Web of Science

B-cell epitope sequences from Zika virus (ZIKV) NS1 protein have been identified using epitope prediction tools. Mapping these sequences onto the NS1 surface reveals two major conformational epitopes and a single linear one. Despite an overall average sequence identity of ca. 55% between the NS1 from ZIKV and the four dengue virus (DENV) serotypes, epitope sequences were found to be highly conserved. Nevertheless, nonconserved epitope-flanking residues are responsible for a dramatically divergent electrostatic surface potential on the epitope regions of ZIKV and DENV2 serotypes. These findings suggest that strategies for differential diagnostics on the basis of short linear NS1 sequences are likely to fail due to immunological cross-reactions. Overall, results provide the molecular basis of differential discrimination between Zika and DENVs by NS1 monoclonal antibodies.

Class II transactivator (CIITA) induces transcription of major histocompatibility complex (MHC) II genes and can potentially be used to improve genetic immunotherapies by converting non-immune cells into cells capable of presenting antigens to CD4+ T cells. However, CIITA expression is tightly controlled and it remains unclear whether distinct non-immune cells differ in this transactivator regulation. Here we describe the development of gene delivery systems capable of promoting the efficient CIITA expression in non-immune cell lines and in primary human cells of an ex vivo skin explant model. Different human cell types undergoing CIITA overexpression presented high-level de novo expression of MHC II, validating the delivery systems as suitable tools for the CIITA evaluation as a molecular adjuvant for gene therapies.

Suggest new approaches focus on engineer antigenic structures capable of exposing a stable conformation of these epitopes and thus inducing B-cell secretion of HIV-1 pnAbs. We postulated that conformationally stable structures are better immunogens and can elicit the production of better antibodies and results show that MD simulations can help engineer vaccine antigens capable of enhancing protective antibody responses.

Epitope exposure strategies often fail due to high conformational instability, resulting in loss of native conformation and fast degradation. Protein scaffolding, where a structural element of an antigen protein is transplanted into a scaffold acceptor protein, has been employed as an alternative solution. However, while this approach aims to preserve the epitope structure, it frequently results in unstable chimeric scaffolds. To overcome this issue, we employed a novel computational approach to rationally engineer conformational antigens into a highly stable scaffold protein. This strategy is showcased to display conformational HIV-1 gp41-based epitopes in their native structure. From HIV-1 antigen sequence databases, we have identified short sequences with the most probable antibody-recognizable regions. These sequences were inserted into or replaced regions of the original Top7 protein with analogous secondary structure assignment. Molecular dynamics simulations were used to characterize the protein stability and structural dynamic of the chimeric proteins, leading to a selection of promising candidates whose immunogenic epitopes are suitably exposed for antibody recognition. These computer-designed recombinant proteins were produced in bacteria using codon optimized DNA sequences and their diagnostic performance was assessed by liquid microarray against a human cohort of 47 sera samples. Our results show that the Top7 protein is a suitable scaffold to provide the required structural stability to predetermined target shapes and sequences, allowing the potential use of the chimeric proteins as antigens for specific antibody recognition and/or stimulation of immune response.