Biology subjects

de Silva, A. M.

Publications and source records attributed to de Silva, A. M..

8 recordsLinked to original sources

bioRxiv2024

Multivalent administration of dengue E dimers on liposomes elicits type-specific neutralizing responses without immune interference

The four serotypes of dengue virus (DENV1-4) are a major health concern putting 50% of the global population at risk of infection. Crucially, DENV vaccines must be tetravalent to provide protection against all four serotypes because immunity to only one serotype can enhance infections caused by heterologous serotypes. Uneven replication of live-attenuated viruses in tetravalent vaccines can lead to disease enhancement instead of protection. Subunit vaccines are a promising alternative as the vaccine components are not dependent on viral replication and antigen doses can be controlled to achieve a balanced response. Here, we show that a tetravalent subunit vaccine of dengue envelope (E) proteins computationally stabilized to form native-like dimers elicits type-specific neutralizing antibodies in mice against all four serotypes. The immune response was enhanced by displaying the E dimers on liposomes embedded with adjuvant, and no interference was detected between the four components.

microbiology↗

bioRxiv2024

Stabilized dengue virus 2 envelope subunit vaccine redirects the neutralizing antibody response to all E-domains

The four-dengue virus (DENV) serotypes cause several hundred million infections annually. Several live-attenuated tetravalent dengue vaccines (LAVs) are at different stages of clinical testing and regulatory approval. A major hurdle faced by the two leading LAVs is uneven replication of vaccine serotypes stimulating a dominant response to one serotype at the expense of the other three, leading to the potential for vaccine antibody (Ab) enhanced more severe infections by wild type DENV serotypes that fail to replicate in the vaccine. Protein subunit vaccines are a promising alternative since antigen dosing can be precisely controlled. However, DENV envelope (E) protein subunit vaccines have not performed well to date, possibly due to differences between the monomeric structure of soluble E and the E homodimer of the viral surface. Previously, we have combined structure-guided computational and experimental approaches to design and produce DENV2 E antigens that are stable homodimers at 37 and stimulate higher levels of neutralizing Abs (NAbs) than the WT E antigen in mice. The goal of this study was to evaluate if DENV2 E homodimers stimulate NAbs that target different epitopes on E protein compared to the WT E monomer. Using DENV4/2 chimeric viruses and Ab depletion methods, we mapped the WT E-elicited NAbs to simple epitopes on domain III of E. In contrast, the stable E homodimer stimulated a more complex response towards all three surface-exposed domains of the E protein. Our findings highlight the impact of DENV2 E oligomeric state on the quality and specificity of DENV NAbs, and the promise of DENV E homodimers as subunit vaccines. ImportanceThe ideal dengue virus (DENV) vaccine should elicit a balanced and highly protective immune response against all 4 DENV serotypes. Current tetravalent live-attenuated DENV vaccines have faced challenges due to uneven replication of vaccine virus strains stimulating a strong immune response to one serotype and weak responses to the other three. Protein subunit vaccines provide novel opportunities to stimulate a balanced response because dosing can be precisely controlled and independent of vaccine virus replication. Here, we compare immune responses elicited by a new DENV serotype 2 protein vaccine designed to match the structure of proteins on the viral surface. We find that proteins designed to match the viral surface stimulate better immune responses targeting multiple sites on the viral surface compared to previous protein vaccines. Our results justify further testing and development of these second-generation DENV protein subunit vaccines.

microbiology↗

bioRxiv2024

CD4+ T Cell Depletion Before Primary Dengue and/or Secondary Zika Infection Reveals Mechanistic Correlates of Antibody Functionality in Rhesus Macaques.

Dengue (DENV) virus and Zika virus (ZIKV) are two flaviviruses of major public health concern. One drawback designing effective vaccines is our limited understanding of the mechanisms ruling protection or harm among DENV serotypes, or between DENV and ZIKV. Here, we depleted rhesus macaques of CD4+ T cells in vivo before primary DENV infection and/or secondary ZIKV challenge to recreate a sub-optimal priming of the humoral immune response. Our results support that CD4+ T cells are needed to induce a quantitative and type-specific effective humoral immune response against primary DENV, but also against secondary ZIKV in DENV-experimented subjects. Our results also indicate a limited contribution of the DENV-Memory B cells to anti-ZIKV response. Furthermore, our results suggest that a suboptimal B cell priming during a primary DENV infection does differentially impact different antibody (Abs) properties. While binding or neutralization of ZIKV or DENV during a subsequent exposure to ZIKV is not affected by the lack of CD4+ T - B cells interaction during a primary DENV infection, that interaction is critical to guarantee the Abs specificity. Also, we found that depleting CD4+ T cells before DENV primary infection but not before ZIKV challenge significantly increases Abs cross-reactivity against DENV-EDIII domain and DENV-NS1 protein but not against ZIKV-EDIII domain or NS1 protein. Furthermore, there was more cross-reactivity among the DENV-NS1 proteins than against DENV-EDIII domains, suggesting that during a primary DENV infection CD4+ T cells have a different weight in the responses against EDIII domain and NS1 protein. The proper Abs binding and neutralization with increased cross-reactivity profile was associated with limited frequency of circulating peripheral T helper cells (pTfh) with T helper 1 phenotype (CD4+/CXCR5+/CXCR3+) and expressing markers related to B cell activation (CXCR5+/CXCR3+/PD-1+/ICOS+) in the group depleted of CD4+ T cells only before primary DENV infection. However, memory B cells - but not Antibody Secreting Cells (ASC) activation 7 days after the infection - positively correlate with those two populations of pTfh. Finally, when Abs cross-reactivity values were incorporated in a Principal Component Analysis (PCA), the DENV-CD4+ T depleted group separates from the other two groups with similar Abs binding and neutralization profiles. Our result strongly suggests that during a heterologous sequential DENV/ZIKV infections Abs binding, and neutralization, may be regulated by different factors than their specificity. Before, the induction of cross-neutralizing Abs has been described in the context of secondary DENV infection. Here, for the first time, we are reproducing the experimental conditions leading to the generation of such Abs population in vivo. In summary, we show that suboptimal immune priming during a primary flavivirus infection has functional consequences during a secondary heterologous infection. Finally, we shown that CD8+ T cells are essential to guarantee an optimal Abs neutralization activity. These results have huge implications understanding the immune response to DENV vaccines (and maybe ZIKV), including why an optimal vaccine or natural-induced neutralizing response not necessarily protects or enhances pathogenesis during a subsequent natural heterologous exposure.

immunology↗

bioRxiv2023

A semi-quantitative, rapid, point of care SARS-CoV-2 serologic assay predicts neutralizing antibody levels

The ongoing COVID-19 pandemic has caused millions of deaths and the continued emergence of new variants suggests continued circulation in the human population. In the current time of vaccine availability and new therapeutic development, including antibody-based therapies, many questions about long-term immunity and protection remain uncertain. Identification of protective antibodies in individuals is often done using highly specialized and challenging assays such as functional neutralizing assays, which are not available in the clinical setting. Therefore, there is a great need for the development of rapid, clinically available assays that correlate with neutralizing antibody assays to identify individuals who may benefit from additional vaccination or specific COVID-19 therapies. In this report, we apply a novel semi-quantitative method to an established lateral flow assay (sqLFA) and analyze its ability to detect the presence functional neutralizing antibodies from the serum of COVID-19 recovered individuals. We found that the sqLFA has a strong positive correlation with neutralizing antibody levels. At lower assay cutoffs, the sqLFA is a highly sensitive assay to identify the presence of a range of neutralizing antibody levels. At higher cutoffs, it can detect higher levels of neutralizing antibody with high specificity. This sqLFA can be used both as a screening tool to identify individuals with any level of neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or as a more specific tool to identify those with high neutralizing antibody levels who may not benefit from antibody-based therapies or further vaccination.

immunology↗

bioRxiv2022

Structure and Neutralization Mechanism of a Human Antibody Targeting a Complex Epitope on Zika Virus

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

microbiology↗

bioRxiv2021

SARS-CoV-2 mRNA Vaccine Induces Robust Specific and Cross-reactive IgG and Unequal Strain-specific Neutralizing Antibodies in Naïve and Previously Infected Recipients

With the advance of SARS-CoV-2 vaccines, the outlook for overcoming the global COVID-19 pandemic has improved. However, understanding of immunity and protection offered by the SARS-CoV-2 vaccines against circulating variants of concern (VOC) is rapidly evolving. We investigated the mRNA vaccine-induced antibody responses against the referent WIV04 (Wuhan) strain, circulating variants, and human endemic coronaviruses in 168 naive and previously infected people at three-time points. Samples were collected prior to vaccination, after the first and after the second doses of one of the two available mRNA-based vaccines. After full vaccination, both naive and previously infected participants developed comparable robust SARS-CoV-2 specific spike IgG levels, modest IgM and IgA binding antibodies, and varying degrees of HCoV cross-reactive antibodies. However, the strength and frequency of neutralizing antibodies produced in naive people were significantly lower than in the previously infected group. We also found that 1/3rd of previously infected people had undetectable neutralizing antibodies after the first vaccine dose; 40% of this group developed neutralizing antibodies after the second dose. In all subjects neutralizing antibodies produced against the B.1.351 and P.1 variants were weaker than those produced against the reference and B.1.1.7 strains. Our findings provide support for future booster vaccinations modified to be active against the circulating variants.

immunology↗

bioRxiv2020

A novel antigenic site spanning domains I and III of the Zika virus envelope glycoprotein is the target of strongly neutralizing human monoclonal antibodies

Zika virus (ZIKV), a mosquito-transmitted flavivirus, caused a large epidemic in Latin America between 2015 and 2017. Effective ZIKV vaccines and treatments are urgently needed to prevent future epidemics and severe disease sequelae. People infected with ZIKV develop strongly neutralizing antibodies linked to viral clearance and durable protective immunity. To understand mechanisms of protective immunity and to support the development of ZIKV vaccines, here we characterize the properties of a strongly neutralizing antibody, B11F, isolated from a recovered ZIKV patient. Our results indicate that B11F targets a complex epitope on the virus that spans domains I and III of the envelope glycoprotein. While previous studies point to quaternary epitopes centered on domain II of ZIKV E glycoprotein as targets of strongly neutralizing and protective human antibodies, we uncover a new site spanning domain I and III as a target of strongly neutralizing human antibodies. ImportancePeople infected with Zika virus develop durable neutralizing antibodies that prevent repeat infections. In the current study, we characterize a ZIKV-neutralizing human monoclonal antibody isolated from a patient after recovery. Our studies establish a novel site on the viral envelope targeted by human neutralizing antibodies. Our results are relevant to understanding how antibodies block infection and for guiding the design and evaluation of candidate vaccines.

immunology↗

bioRxiv2019

Oligomeric state of the ZIKV E protein defines protective immune responses.

The current leading Zika vaccine candidates in clinical testing are based on live or killed virus platforms, which have safety issues, especially in pregnant women. Zika subunit vaccines, however, have shown poor performance in preclinical studies. We hypothesized that Zika Envelope (E) protein subunit vaccines have performed poorly because the antigens tested have been recombinant E monomers that do not display critical quaternary structure epitopes present on Zika E protein homodimers that cover the surface of the virus. To test this hypothesis, we engineered and produced stable recombinant E protein homodimers. Unlike the E monomer, the dimer was recognized by strongly neutralizing monoclonal antibodies isolated from Zika-immune individuals. In a mouse model of vaccination, the dimeric antigen stimulated strongly neutralizing antibodies that targeted epitopes that were similar to epitopes recognized by human antibodies following natural Zika virus infection. In contrast, the monomer antigen stimulated lower levels of neutralizing antibodies directed to simple epitopes on domain III of E protein. In a mouse model of ZIKV challenge, only E dimer antigen stimulated protective antibodies, not the monomer. These results highlight the importance of mimicking the highly structured flavivirus surface when designing subunit vaccines. The flavivirus field has a long history of using E monomers as vaccine antigens with limited success. These results are applicable to developing second generation subunit vaccines against Zika as well as other medically important flaviviruses such as dengue and yellow fever viruses.

microbiology↗