New nanoparticles for vaccination against Listeriosis and Tuberculosis

RESUMEN: The emergence of non-tubercolus mycobacteria is becoming and increasing problem for healthcare system around the world and tuberculosis is still one of the most significant infectious diseases specially in developing countries. Until this day there is a lack of an effective vaccine against these pathogens, the only available vaccine BCG is unable to confer long lasting and complete protection. Nanoparticles have emerged as a new platform for the development of new therapeutic strategies due to their reduced toxicity, capacity to target selected cells and to be conjugated with a wide range of biomolecules. We propose a new strategy for vaccination based in the use of gold nanoparticles (GNP) carrying the bacterial peptides 1-15 of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH1-22) of M. tuberculosis, and the peptide GAPDH1-15 of Listeria monocytogenes. This peptide has a high homology between different species of mycobacteria as well as L. monocytogenes which makes it a good candidate for the development of multivalent vaccines. In this work we demonstrate the capacity of these vaccines to induce an effective immune response and confer protection in a murine model against infection by dermal mycobacteria M. marinum and M. chelonae.ABSTRACT: La aparición de micobacterias no tubercolusas se está convirtiendo en un problema cada vez mayor para el sistema de salud en todo el mundo y la tuberculosis sigue siendo una de las enfermedades infecciosas más importantes, especialmente en los países en desarrollo. Hasta el día de hoy falta una vacuna eficaz contra estos patógenos, la única vacuna disponible BCG es incapaz de conferir una protección duradera y completa. Las nanopartículas han surgido como una nueva plataforma para el desarrollo de nuevas estrategias terapéuticas debido a su reducida toxicidad, capacidad para dirigirse a células seleccionadas y conjugarse con una amplia gama de biomoléculas. Proponemos una nueva estrategia de vacunación basada en el uso de nanopartículas de oro (GNP) portadoras de los péptidos bacterianos 1-15 de la gliceraldehído-3-fosfato deshidrogenasa (GAPDH1-22) de M. tuberculosis y el péptido GAPDH1-15 de Listeria monocytogenes. Este péptido tiene una alta homología entre diferentes especies de micobacterias así como con L. monocytogenes lo que lo convierte en un buen candidato para el desarrollo de vacunas multivalentes. En este trabajo demostramos la capacidad de estas vacunas para inducir una respuesta inmune eficaz y conferir protección en un modelo murino frente a la infección por micobacterias dérmicas M. marinum y M. chelonae

Epitopes for Multivalent Vaccines Against Listeria, Mycobacterium and Streptococcus spp: A Novel Role for Glyceraldehyde-3-Phosphate Dehydrogenase

The glycolytic enzyme and bacterial virulence factor of Listeria monocytogenes, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Lmo2459), ADP-ribosylated the small GTPase, Rab5a, and blocked phagosome maturation. This inhibitory activity localized within the NAD binding domain of GAPDH at the N-terminal 1?22 peptides, also conferred listeriosis protection when used in dendritic cell-based vaccines. In this study, we explore GAPDH of Listeria, Mycobacterium, and Streptococcus spp. taxonomic groups to search for epitopes that confer broad protection against pathogenic strains of these bacteria. GAPDH multivalent epitopes are selected if they induce inhibitory actions and wide-ranging immune responses. Proteomic isolation of GAPDH from dendritic cells infected with Listeria, Mycobacterium, or Streptococcus confirmed similar enzymatic, Rab5a inhibitory and immune stimulation abilities. We identified by bioinformatics and functional analyses GAPDH N-terminal 1?22 peptides from Listeria, Mycobacterium, and Streptococcus that shared 95% sequence homology, enzymatic activity, and B and T cell immune domains. Sera obtained from patients or mice infected with hypervirulent pathogenic Listeria, Mycobacterium, or Streptococcus presented high levels of anti-GAPDH 1?22 antibodies and Th2 cytokines. Monocyte derived dendritic cells from healthy donors loaded with GAPDH 1?22 peptides from Listeria, Mycobacterium, or Streptococcus showed activation patterns that correspond to cross-immunity abilities. In summary, GAPDH 1?22 peptides appeared as putative candidates to include in multivalent dendritic based vaccine platforms for Listeria, Mycobacterium, or Streptococcus

First computational design using lambda-superstrings and in vivo validation of SARS-CoV-2 vaccine

Coronavirus disease 2019 (COVID-19) is the greatest threat to global health at the present time, and considerable public and private effort is being devoted to fighting this recently emerged disease. Despite the undoubted advances in the development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uncertainty remains about their future efficacy and the duration of the immunity induced. It is therefore prudent to continue designing and testing vaccines against this pathogen. In this article we computationally designed two candidate vaccines, one monopeptide and one multipeptide, using a technique involving optimizing lambda-superstrings, which was introduced and developed by our research group. We tested the monopeptide vaccine, thus establishing a proof of concept for the validity of the technique. We synthesized a peptide of 22 amino acids in length, corresponding to one of the candidate vaccines, and prepared a dendritic cell (DC) vaccine vector loaded with the 22 amino acids SARS-CoV-2 peptide (positions 50-71) contained in the NTD domain (DC-CoVPSA) of the Spike protein. Next, we tested the immunogenicity, the type of immune response elicited, and the cytokine profile induced by the vaccine, using a non-related bacterial peptide as negative control. Our results indicated that the CoVPSA peptide of the Spike protein elicits noticeable immunogenicity in vivo using a DC vaccine vector and remarkable cellular and humoral immune responses. This DC vaccine vector loaded with the NTD peptide of the Spike protein elicited a predominant Th1-Th17 cytokine profile, indicative of an effective anti-viral response. Finally, we performed a proof of concept experiment in humans that included the following groups: asymptomatic non-active COVID-19 patients, vaccinated volunteers, and control donors that tested negative for SARS-CoV-2. The positive control was the current receptor binding domain epitope of COVID-19 RNA-vaccines. We successfully developed a vaccine candidate technique involving optimizing lambda-superstrings and provided proof of concept in human subjects. We conclude that it is a valid method to decipher the best epitopes of the Spike protein of SARS-CoV-2 to prepare peptide-based vaccines for different vector platforms, including DC vaccines.Luis Martínez and Iker Malaina were supported by the Basque Government, grants IT974-16 and KK-2018/00090 and by the UPV/EHU and Basque Center of Applied Mathematics, grants US18/21 and US21/27. Carmen Alvarez-Dominguez was funded by the Instituto de Salud Carlos III, grants DTS18-00022 and PI19-01580, co-funded in part with European FEDER funds “A new way of making Europe”, the Instituto de Investigación Marqués de Valdecilla, grant INNVAL20/01, and the COST European action ENOVA CA-16231. David Salcines-Cuevas was supported by a predoctoral contract for the BioHealth research program of the Cantabria government. Hector Teran-Navarro salary was supported by the Instituto de Investigación Marqués de Valdecilla, grant INNVAL19/26. Andrea Zeoli was an Erasmus student from the University of Milan “La Statale” (Milan, Italy) performing a stay at IDIVAL.Peer reviewe

Caracterización de la expresión génica en hojas verdes y albinas de plántulas obtenidas mediante cultivo in vitro de anteras de cebada

La producción de doblehaploides (DH) permite obtener plantas completamente homocigotas en una sola generación acelerando la obtención de nuevas ariedades en programas de mejora. En el caso de la cebada (y otros cereales), la producción de DH mediante androgénesis de la microspora tiene como problema asociado la generación de plantas albinas que sobreviven poco tiempo en cultivo in vitro. En este trabajo se pretende estudiar los mecanismos moleculares asociados al albinismo en plantas DH de cebada generada mediante androgénesis. Mediante PCR semicuantitativa se ha medido la expresión de 56 genes, seleccionados en trabajos previos, en dos líneas DH, DH6148 que se caracteriza por un alto porcentaje de embriogénesis y bajo porcentaje de albinismo y DH6183 con bajo porcentaje de embriogénesis y alto porcentaje de albinismo. Los resultados del estudio de la expresión genética han mostrado una disminución de genes asociados a la fotosíntesis en hojas albinas frente a hojas verdes obtenidas mediante cultivo in vitro de la línea DH6183. Por el contrario también se ha observado que genes implicados en la expresión génica en cloroplastos se encuentran sobreexpresados en plantas albinas. Las hojas albinas además parecen presentar una síntesis de almidón activa, esto junto a la deficiencia en mecanismos fotosintéticos indica que los plástidos en plantas albinas presentan características más similares a amiloplastos que a cloroplastos. Las diferencias de expresión no se limitan a las plantas albinas ya que también se han observado respuestas ligadas al estrés debido a las condiciones de cultivo así como diferencias asociadas al genotipo. Estas últimas son de especial relevancia ya que indican la necesidad de tener en cuenta los fenotipos utilizados en el estudio de los mecanismos asociados al albinismo

Testing a vaccine candidate against Hepatitis C virus designed by combinatorial optimization

This paper presents a new procedure for vaccine design against highly variable viruses such as Hepatitis C. The procedure uses an optimization algorithm to design vaccines that maximize the coverage of epitopes across different virus variants. Weighted epitopes based on the success ratio of immunological assays are used to prioritize the selection of epitopes for vaccine design. The procedure was successfully applied to design DC vaccines loaded with two HCV peptides, STG and DYP, which were shown to be safe, immunogenic, and able to induce significant levels of anti-viral cytokines, peptide-specific cellular immune responses and IgG antibodies. The procedure could potentially be applied to other highly variable viruses that currently lack effective vaccines

A Comparison Between Recombinant Listeria GAPDH Proteins and GAPDH Encoding mRNA Conjugated to Lipids as Cross-Reactive Vaccines for Listeria, Mycobacterium, and Streptococcus

Cross-reactive vaccines recognize common molecular patterns in pathogens and are able to confer broad spectrum protection against different infections. Antigens common to pathogenic bacteria that induce broad immune responses, such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the genera Listeria, Mycobacterium, or Streptococcus, whose sequences present more than 95% homology at the N-terminal GAPDH(1-22) peptide, are putative candidates for universal vaccines. Here, we explore vaccine formulations based on dendritic cells (DC) loaded with two molecular forms of Listeria monocytogenes GAPDH (LM-GAPDH), such as mRNA carriers or recombinant proteins, and compare them with the same molecular forms of three other antigens used in experimental vaccines, listeriolysin O of Listeria monocytogeness, Ag85A of Mycobacterium marinum, and pneumolysin of Streptococcus pneumoniae. DC loaded with LM-GAPDH recombinant proteins proved to be the safest and most immunogenic vaccine vectors, followed by mRNA encoding LM-GAPDH conjugated to lipid carriers. In addition, macrophages lacked sufficient safety as vaccines for all LM-GAPDH molecular forms. The ability of DC loaded with LM-GAPDH recombinant proteins to induce non-specific DC activation explains their adjuvant potency and their capacity to trigger strong CD4(+) and CD8(+) T cell responses explains their high immunogenicity. Moreover, their capacity to confer protection in vaccinated mice against challenges with L. monocytogenes, M. marinum, or S. pneumoniae validated their efficiency as cross-reactive vaccines. Cross-protection appears to involve the induction of high percentages of GAPDH(1-22) specific CD4(+) and CD8(+) T cells stained for intracellular IFN-gamma, and significant levels of peptide-specific antibodies in vaccinated mice. We concluded that DC vaccines loaded with L. monocytogenes GAPDH recombinant proteins are cross-reactive vaccines that seem to be valuable tools in adult vaccination against Listeria, Mycobacterium, and Streptococcus taxonomic groups

Glyceraldehyde-3-phosphate dehydrogenase common peptides of listeria monocytogenes, mycobacterium marinum and streptococcus pneumoniae as universal vaccines

Universal vaccines can be prepared with antigens common to different pathogens. In this regard, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a common virulence factor among pathogenic bacteria of the genera Listeria, Mycobacterium and Streptococcus. Their N-ter-minal 22 amino acid peptides, GAPDH-L1 (Listeria), GAPDH-M1 (Mycobacterium) and GAPDH-S1 (Streptococcus), share 95–98.55% sequence homology, biochemical and MHC binding abilities and, therefore, are good candidates for universal vaccine designs. Here, we used dendritic cells (DC) as vaccine platforms to test GAPDH epitopes that conferred protection against Listeria monocytogenes, Mycobacterium marinum or Streptococcus pneumoniae in our search of epitopes for universal vaccines. DC loaded with GAPDH-L1, GAPDH-M1 or GAPDH-S1 peptides show high immunogenicity measured by the cellular DTH responses in mice, lacked toxicity and were capable of cross-protec-tion immunity against mice infections with each one of the pathogens. Vaccine efficiency correlated with high titers of anti-GAPDH-L1 antibodies in sera of vaccinated mice, a Th1 cytokine pattern and high frequencies of GAPDH-L1-specific CD4+ and CD8+ T cells and IFN-γ producers in the spleens. We concluded that GAPDH-L1 peptide was the best epitope for universal vaccines in the Listeria, Mycobacterium or Streptococcus taxonomic groups, whose pathogenic strains caused relevant morbidities in adults and especially in the elderly