Biology subjects

Alvarez, D. E.

Publications and source records attributed to Alvarez, D. E..

6 recordsLinked to original sources

bioRxiv2024

Improvement of the potency of a N1-methylpseudouridine-modified self-amplifying RNA through mutations in the RNA-dependent-RNA-polymerase

RNA vaccines are sensed as non-self molecules by the innate immune system, and balancing control of the immune activation and vaccine safety and efficacy has remained a challenge, especially for self-amplifying RNAs (saRNAs). Incorporation of modified nucleotides has been widely used to temper immune activation of RNA vaccines. However, it was previously reported that incorporation of modified nucleotides to saRNAs impeded antigen expression. Here, we used a reporter replicon of the attenuated TC-83 strain of Venezuelan equine encephalitis virus (VEEV) to investigate the impact of modified nucleotide incorporation on the replication capacity of the saRNA in transfected cells. {psi} and {psi}-modified molecules showed a profound defect in RNA synthesis compared to unmodified saRNA. Interestingly, the levels of RNA synthesis of m5C-modified RNAs were similar to unmodified molecules, positioning m5C as a promising candidate for saRNA modification. To overcome the impact of {psi} or m1{psi}-modified nucleotide incorporation in RNA synthesis, we explored two alternative approaches: engineering the UTR sequences and tuning polymerase fidelity. Our results uncover a previously unappreciated link between polymerase fidelity and saRNA amplification. Overall, we provide new insights for the design of saRNAs with high levels of heterologous protein expression and potential vaccine applications.

molecular biology↗

bioRxiv2024

New highly selective antivirals for Chikungunya Virus identified from the screening of a drug-like compound library

Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.

microbiology↗

bioRxiv2023

The chikungunya virus E1 glycoprotein fusion loop and hinge alter glycoprotein dynamics leading to cell and host specific changes in infectivity.

Alphaviruses infect both mammals and insects, yet the distinct mechanisms that alphaviruses use to infect different hosts are not well defined. In this study, we characterize CHIKV E1 variants in the fusion loop (E1-M88L) and hinge region (E1-N20Y) in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. Through cell culture assays, we found that CHIKV E1-N20Y enhanced infectivity in mosquito cells while the CHIKV E1-M88L variant enhanced virus binding and infectivity in both BHK-21 and C6/36 cells, and led to changes in the virus cholesterol-dependence in BHK-21 cells. Given these in vitro results and that residue E1-M88L is in a defined Mxra8 interacting domain, we hypothesized that this residue may be important for receptor usage. However, while the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, it was attenuated in vitro in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type dependent manner to alter entry. Finally, using molecular dynamics to understand how potential changes in the E1 glycoprotein may impact the CHIKV glycoprotein E1-E2 complex, we found that E1-M88L and other E1 domain II variants lead to changes in both E1 and E2 dynamics. Taken together, these studies show that key residues in the CHIKV E1 fusion loop and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry. ImportanceArthropod-borne viruses (arboviruses) are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus class II glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, to address these gaps in our knowledge, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo. Here, we show that changes in the CHIKV E1 fusion loop and hinge contribute to host-specific entry and E1-E2 dynamics, furthering our knowledge of how alphaviruses infect mammals and insects.

microbiology↗

bioRxiv2023

Dengue virus preferentially uses human and mosquito non-optimal codons

Codon optimality refers to the effect codon composition has on messenger RNA (mRNA) stability and translation level and implies that synonymous codons are not silent from a regulatory point of view. Here, we investigated the adaptation of virus genomes to the host optimality code using mosquito-borne dengue virus (DENV) as a model. We defined which codons are associated with unstable and stable (non-optimal and optimal codons, respectively) mRNAs in mosquito cells and showed that DENV preferentially uses non-optimal codons and avoids codons that are defined as optimal in either human or mosquito cells. Human genes enriched in the codons preferentially and frequently used by DENV are up-regulated during infection, and so is the tRNA decoding the non-optimal and DENV preferentially used codon for arginine. We found that synonymous mutations towards DENVs preferred non-optimal codons (e.g., AGA) increase fitness of DENV during serial passaging in human or mosquito cells. Finally, our analyses revealed that hundreds of viruses preferentially use non-optimal codons, with those infecting a single host displaying an even stronger bias, suggesting that synonymous codon choice is a key aspect of host-pathogen interaction.

molecular biology↗

bioRxiv2023

A Gamma-adapted recombinant subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from SARS-CoV-2 infection.

The COVID-19 pandemic continues with the emergence of successive new variants of concern (VOC). One strategy to prevent breakthrough infections is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD recombinant vaccine candidate derived from the Gamma SARS-CoV-2 variant adjuvanted with alum. Gamma RBD-derived antigen elicited better neutralizing antibody and T cell responses than formulation containing ancestral RBD antigen. The Gamma-adapted subunit vaccine elicited a long-lasting antibody response with cross-neutralizing activity against different VOC including the Omicron variant. Additionally, Gamma variant RBD-adapted vaccine elicited robust T cells responses with induction of Th1 and CD8+ T cell responses in spleen and lung. Vaccine-induced immunity protected K18-hACE2 mice from intranasal challenge with SARS-CoV-2 increasing survival, reducing body weight loss and viral burden in the lungs and brain. Importantly, the subunit vaccine demonstrated a potent effect as heterologous booster of different vaccine platforms including the non-replicating adenovirus vaccine ChAdOx1-S, the mRNA vaccine BNT162b2 and the inactivated SARS-CoV-2 vaccine BBIBP-CorV, increasing cross-reactive antibody responses. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate to combat SARS-CoV-2 variants including Omicron.

immunology↗

bioRxiv2021

A novel bacterial protease inhibitor adjuvant in RBD-based COVID-19 vaccine formulations increases neutralizing antibodies, specific germinal center B cells and confers protection against SARS-CoV-2 infection.

In this work we evaluated recombinant receptor binding domain (RBD) based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus Alum, AddaS03, AddaVax or the combination of Alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and Alum as adjuvants have a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus Alum, AddaVax or AddaS03. Antibodies induced with the formulation containing U-Omp19 not only increased their neutralization capacity against the wild-type virus but also cross neutralized alpha, lambda and gamma variants with similar potency. Also, addition of U-Omp19 to vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+Alum formulation induced RBD-specific Th1 and CD8+ T cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal SARS-CoV-2 challenge of K18-hACE2 mice.

immunology↗