Identification of a Zika NS2B epitope as a biomarker for severe clinical phenotypes.

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.

The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the spike protein.

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. Importantly, we detected the community transmission of different P.1 lineages bearing NTD indels ∆69-70 (which can impact several SARS-CoV-2 diagnostic protocols), ∆144 and ins214ANRN, and a new VOI N.10 derived from the B.1.1.33 lineage carrying three NTD deletions (∆141-144, ∆211, and ∆256-258). These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil generates new viral lineages that might be more resistant to antibody neutralization than parental variants of concern.

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

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.

Immune evasion of SARS-CoV-2 variants of concern is driven by low affinity to neutralizing antibodies.

SARS-CoV-2 VOC immune evasion is mainly due to lower cross-reactivity from previously elicited class I/II neutralizing antibodies, while increased affinity to hACE2 plays a minor role. The affinity between antibodies and VOCs is impacted by remodeling of the electrostatic surface potential of the Spike RBDs. The P.3 variant is a putative VOC.

SARS-CoV-2 VOCs immune evasion from previously elicited neutralizing antibodies is mainly driven by lower cross-reactivity due to spike RBD electrostatic surface changes.

O rápido espalhamento das novas variantes do novo coronavírus SARS-CoV-2, como exemplificado pela alta prevalência da variante P.1 ainda nos dois primeiros meses após seu surgimento, junto a relatos de reinfecção causada por estas variantes, levanta para a ciência a questão de quais seriam os mecanismos por trás deste cenário. É de especial interesse para estas pesquisas o estudo das mutações no genoma das variantes ­ em particular, no gene da glicoproteína Spike (proteína S), que promove a entrada nas células humanas a partir da interação com a Enzima Conversora de Angiotensina 2 (hACE2), molécula que atua como receptor do vírus. A capacidade de se ligar à hACE2 pode tornar-se maior ou menor de acordo com as alterações na estrutura da proteína S, que variam entre linhagens do SARS-CoV-2 e cada uma de suas variantes. Essas alterações na estrutura da proteína S podem também resultar em uma menor capacidade de anticorpos gerados em resposta a uma infecção ­ ou, possivelmente, mesmo após a vacinação ­ de se ligarem à proteína S e neutralizar a capacidade do vírus de causar infecção. A presente publicação ­ agora também disponível em sua versão revisada por pares ­ investiga estes dois possíveis efeitos das mutações da proteína S detectadas em variantes como a P.1, variantes da linhagem B.1.351 e a B.1.1.7: a de uma interação "mais forte" com o receptor hACE2 ou a de uma interação "mais fraca" com os anticorpos anti-proteína-S. No link do ChemRxiv ainda em estágio pre-print, antes da revisão por pesquisadores independentes, o artigo descreve uma série de experimentos feitos com modelagem computacional das moléculas envolvidas (hACE2, anticorpos e as diversas "versões" da proteína S, referentes a cada uma das variantes estudadas). Com base na simulação computacional da interação entre as moléculas, foi possível verificar que a alteração da estrutura da proteína S não teve efeitos marcantes na interação com o receptor hACE2. Por outro lado, ao simular a interação dos anticorpos gerados em resposta a linhagens iniciais do SARS-CoV-2 com a proteína S das novas variantes, foi possível ver que há uma diminuição na ligação entre as moléculas, um achado que aponta para um potencial de "escape" da resposta imune. Segundo essa hipótese, as novas variantes seriam mais eficazes em fugir da neutralização proporcionada por anticorpos, e este seria um mecanismo mais relevante para explicar seu rápido espalhamento pela população. É importante ressaltar que, baseados em algumas medidas de afinidade entre variações da proteína S e o receptor hACE2, outros grupos de pesquisa previamente sugeriram que o aumento da transmissibilidade estaria relacionado com uma maior afinidade entre a proteína viral e o receptor humano, em contraste com os resultados do presente estudo. Contudo, estes estudos não exploraram a interação das diferentes proteínas S com os anticorpos neutralizantes. O artigo aponta ainda a nova variante denominada P.3 como uma potencial Variante de Preocupação (VOC), tendo em vista que a maioria dos anticorpos analisados no estudo não foram capazes de se ligar eficientemente à proteína S desta linhagem nas simulações realizadas.

The influence of biotinylation on the ability of a computer designed protein to detect B-cells producing anti-HIV-1 2F5 antibodies.

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.

Impact of preexisting dengue immunity on Zika virus emergence in a dengue endemic region.

The clinical outcomes associated with Zika virus (ZIKV) in the Americas have been well documented, but other aspects of the pandemic, such as attack rates and risk factors, are poorly understood. We prospectively followed a cohort of 1453 urban residents in Salvador, Brazil, and, using an assay that measured immunoglobulin G3 (IgG3) responses against ZIKV NS1 antigen, we estimated that 73% of individuals were infected during the 2015 outbreak. Attack rates were spatially heterogeneous, varying by a factor of 3 within a community spanning 0.17 square kilometers. Preexisting high antibody titers to dengue virus were associated with reduced risk of ZIKV infection and symptoms. The landscape of ZIKV immunity that now exists may affect the risk for future transmission.

Rational Zika vaccine design via the modulation of antigen membrane anchors in chimpanzee adenoviral vectors.

Zika virus (ZIKV) emerged on a global scale and no licensed vaccine ensures long-lasting anti-ZIKV immunity. Here we report the design and comparative evaluation of four replication-deficient chimpanzee adenoviral (ChAdOx1) ZIKV vaccine candidates comprising the addition or deletion of precursor membrane (prM) and envelope, with or without its transmembrane domain (TM). A single, non-adjuvanted vaccination of ChAdOx1 ZIKV vaccines elicits suitable levels of protective responses in mice challenged with ZIKV. ChAdOx1 prME ∆TM encoding prM and envelope without TM provides 100% protection, as well as long-lasting anti-envelope immune responses and no evidence of in vitro antibody-dependent enhancement to dengue virus. Deletion of prM and addition of TM reduces protective efficacy and yields lower anti-envelope responses. Our finding that immunity against ZIKV can be enhanced by modulating antigen membrane anchoring highlights important parameters in the design of viral vectored ZIKV vaccines to support further clinical assessments.

Mapping Putative B-Cell Zika Virus NS1 Epitopes Provides Molecular Basis for Anti-NS1 Antibody Discrimination between Zika and Dengue Viruses.

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.