Biology subjects

Pinto, E.

Publications and source records attributed to Pinto, E..

7 recordsLinked to original sources

bioRxiv2022

Immunogenicity of COVID-eVax Is Moderately Impacted by Temperature and Molecular Isoforms

DNA integrity is a key issue in gene therapy and genetic vaccine approaches based on plasmid DNA. In contrast to messenger RNA that requires a controlled cold chain for efficacy, DNA molecules are considered to be more stable. In this study, we challenged this concept by characterizing the immunological response induced by a plasmid DNA vaccine delivered using electroporation. As a model, we used COVID-eVax, which is a plasmid DNA vaccine that targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Increased nicked DNA was produced by using either an accelerated stability protocol or a lyophilization protocol. Surprisingly, the immune response induced in vivo was only minimally affected by the percentage of open circular DNA. This result suggests that plasmid DNA vaccines, such as COVID-eVax that has completed a phase I clinical trial, retain their efficacy upon storage at higher temperatures and this feature may facilitate their use in low-/middle-income countries.

immunology↗

bioRxiv2022

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 600 million cases and over 6 million deaths worldwide. Vaccination has been the main strategy used to contain the spread of the virus, and to avoid hospitalizations and deaths. Currently, there are two mRNA-based and one adenovirus vectored vaccines approved and available for use in the U.S. population. The versatility, low cost and rapid-to-manufacture attributes of DNA vaccines are important advantages over other platforms. However, DNA vaccination must meet higher efficiency levels for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been successfully used in the veterinary field. Here we evaluated the safety, immunogenicity and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate for delivered by intramuscular injection followed by electroporation (Vet-ePorator) in ferrets. The results demonstrated that the linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects, and was able to elicit neutralizing antibodies and T cell responses using a low dose of the linear DNA construct in prime-boost regimen, and significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

immunology↗

bioRxiv2022

A linear DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain elicits protective immunity in domestic cats

Since its first detection in China in late 2019, SARS-CoV-2, the etiologic agent of COVID-19 pandemic, has infected a wide range of animal species, especially mammals, all over the world. Indeed, as reported by the American Veterinary Medical Association, besides human-to-human transmission, human-to-animal transmission has been observed in some wild animals and pets, especially in cats. With animal models as an invaluable tool in the study of infectious diseases combined with the fact that the intermediate animal source of SARS-CoV-2 is still unknown, researchers have demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a randomized Phase I/II clinical study conducted in domestic cats to assess safety and immunogenicity of a linear DNA ("linDNA") vaccine encoding the RBD domain of SARS-CoV-2. No significant adverse events occurred and both RBD-specific binding/neutralizing antibodies and T cells were detected. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.

immunology↗

bioRxiv2022

Linear DNA amplicons as a novel cancer vaccine strategy

DNA-based vaccines represent a simple, safe and promising strategy for harnessing the immune system to fight infectious diseases as well as various forms of cancer and thus are considered an important tool in the cancer immunotherapy toolbox. Nonetheless, the manufacture of plasmid DNA vaccines has several drawbacks, including long lead times and the need to remove impurities from bacterial cultures. Here we report the development of polymerase chain reaction (PCR)-produced amplicon expression vectors as DNA vaccines and their in vivo application to elicit antigen-specific immune responses in animal cancer models. Amplicons encoding tumor-associated-antigens, such as telomerase reverse transcriptase or neoantigens expressed by murine tumor cell lines were able to elicit antigen-specific immune responses and proved to significantly impact tumor growth when administered in combination with immune-checkpoint inhibitors (ICIs). These results strongly support the further exploration of the use of PCR-based amplicons as an innovative immunotherapeutic approach to cancer treatment.

immunology↗

bioRxiv2021

COVID-eVax, an electroporated plasmid DNA vaccine candidate encoding the SARS-CoV-2 Receptor Binding Domain, elicits protective immune responses in animal models of COVID-19

The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already been approved by European and American authorities for preventing COVID-19 but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle, a technology previously utilized for cancer vaccines. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 Spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax - a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein RBD - induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function and significantly lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started in Italy.

immunology↗

bioRxiv2020

L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

O_SCPLOWLC_SCPLOW-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous O_SCPLOWLC_SCPLOW-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of O_SCPLOWLC_SCPLOW-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess O_SCPLOWLC_SCPLOW-DOPA, both the betaxanthin and hygroaurin adducts of O_SCPLOWLC_SCPLOW-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other O_SCPLOWLC_SCPLOW-DOPA dioxygenases. The rate constants for the conversion of O_SCPLOWLC_SCPLOW-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

biochemistry↗

bioRxiv2020

Comprehensive database of secondary metabolites from cyanobacteria

Cyanobacteria form harmful mass blooms in freshwater and marine environments around the world. A range of secondary metabolites has been identified from cultures of cyanobacteria and biomass collected from cyanobacterial bloom events. A comprehensive database is necessary to correctly identify cyanobacterial metabolites and advance research on their abundance, persistence and toxicity in natural environments. We consolidated open access databases and manually curated missing information from the literature published between 1970 and March 2020. The result is the database CyanoMetDB, which includes more than 2000 entries based on more than 750 literature references. This effort has more than doubled the total number of entries with complete literature metadata and structural composition (SMILES codes) compared to publicly available databases to this date. Over the past decade, more than one hundred additional secondary metabolites have been identified yearly. We organized all entries into structural classes and conducted substructure searches of the provided SMILES codes. This approach demonstrated, for example, that 65% of the compounds carry at least one peptide bond, 57% are cyclic compounds, and 30% carry at least one halogen atom. Structural searches by SMILES code can be further specified to identify structural motifs that are relevant for analytical approaches, research on biosynthetic pathways, bioactivity-guided analysis, or to facilitate predictive science and modeling efforts on cyanobacterial metabolites. This database facilitates rapid identification of cyanobacterial metabolites from toxic blooms, research on the biosynthesis of cyanobacterial natural products, and the identification of novel natural products from cyanobacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/038703v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@3504f7org.highwire.dtl.DTLVardef@d92680org.highwire.dtl.DTLVardef@6ea103org.highwire.dtl.DTLVardef@10cd983_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗