The prophylactic and empirical use of antifungal agents in general, and azoles and echinocandins in particular, has contributed to the emergence of strains with elevated MICs (microbiological resistance), whose clinical impact and relevance are increasingly concerning. Moreover, therapeutic failure or clinical resistance is frequently observed even in the absence of microbiological resistance.
One of the factors that may explain clinical resistance in the absence of microbiological resistance is inadequate drug exposure at the site of infection, together with the pathogen’s adaptive response to host defences or to the administered antifungal therapy.
This research line, active since 2010, aims to advance the understanding of clinical resistance in Aspergillus fumigatus infection through the study of antifungal dose–response relationships by evaluating PK/PD parameters and their role in treatment efficacy. In addition, it investigates the role of biofilms as adaptive strategies that enhance fungal resistance to antifungal agents and to host immune cells.

Exposure: Pharmacokinetics of Antifungal Agents
There is now substantial evidence that the administration of an effective antifungal agent at the appropriate dose is essential to achieve adequate drug concentrations at the site of infection. Through the development of this research line (active since 2009), our group has gained extensive expertise in the development and application of chromatographic methods (HPLC/UPLC–UV/fluorescence) for antifungal quantification, which are useful for therapeutic drug monitoring (TDM).

Techniques that allow monitoring of blood drug concentrations constitute essential tools in patient management, enabling dose optimization by determining whether drug exposure is inadequate—potentially contributing to therapeutic failure and the emergence of resistance—or, conversely, excessive, thereby increasing the risk of significant toxicity.

In addition, the application of disciplines such as pharmacogenetics to antifungal therapy is promoting personalized treatment approaches and improving the management of severe invasive fungal infections (IFIs).
The use of alternative animal models, with ethical approval and under regulatory frameworks, together with methodological advances, has facilitated the preliminary study of dose–response relationships for both established antifungal agents and newly developed compounds.

Response: Monitoring Treatment Efficacy
An aspect of major relevance in the management of severe invasive fungal infections (IFIs) is the evaluation of the efficacy of the administered therapy. This research line further explores the characterization of circulating fungal-specific biomarkers that may help distinguish infection from fungal colonization and assess the effectiveness of the established treatment.
Within this framework, our efforts have focused on the study and characterization of compounds related to fungal secondary metabolism, such as gliotoxin and its derivative bis(methylthio)gliotoxin, detected in tissue and blood samples from patients with aspergillosis and in animal models, as well as other metabolites associated with fungal virulence (e.g., fumagillin and pseurotin A).

More recently, this research line has expanded to investigate adaptive strategies such as the formation of complex biofilm structures—often multispecies in nature and involving intricate interaction mechanisms—which may explain fungal resistance to antifungal agents and host immune cells, thereby contributing to therapeutic failure (clinical resistance).

Classical viral vaccines rely on the induction of neutralizing antibodies. In the case of infection by the human immunodeficiency virus (HIV), the viral spike has evolved to evade recognition by these antibodies. Despite these obstacles, certain monoclonal antibodies capable of neutralizing the majority of primary HIV-1 isolates have been successfully isolated and have demonstrated efficacy both in controlling viremia and in providing protection against infection in animal models. These are known as broadly neutralizing antibodies (bNAbs).

In order to identify the factors involved in the induction of these antibodies and to develop preventive strategies based on bNAb induction, we are pursuing the following research lines:

1. Determination of factors associated with the induction of effective humoral responses in different scenarios: recent infection, chronic infection, co-infection with hepatitis C virus, reinfection, and pediatric infection, among others.
2. Study of the effect of feminizing hormone therapy on the immune system of transgender women.
3. Development of HIV-1 vaccine prototypes based on viral spike proteins incorporated into Virus-Like Particles (VLPs) from two sources:
   a) Selected from a library of randomly mutated spikes to enhance the accessibility of epitopes recognized by bNAbs.
   b) Derived from viruses present in individuals with broad neutralizing responses in recent infection.
4. Isolation and characterization of new bNAbs against HIV-1 from individual B cells of individuals with an efficient neutralizing response. These antibodies could be used in both preventive and therapeutic strategies.
5. Isolation of new monoclonal antibodies against other human pathogenic viruses, adapting the technology developed for HIV-1 antibody isolation, in collaboration with researchers from the National Center for Microbiology.
6. Use of gene therapy vectors (Recombinant Adeno-Associated Viruses; rAAVs) to incorporate bNAbs and apply them in prophylactic and therapeutic strategies.

Resistance mechanisms of Aspergillus fumigatus to antifungals

Aspergillus fumigatus is a universally distributed opportunistic fungal pathogen with a significant global incidence and extremely high mortality. The widespread and extensive use of azole antifungals has led to the emergence of A. fumigatus azole resistant, resulting in serious consequences for patients infected with these isolates, who are left with limited therapeutic options.       
Initially, the emergence of resistant strains was very sporadic and showed point mutations in key areas of the Cyp51A enzyme (G54, G138, F219, M220, G448S) in strains isolated from patients undergoing long-term treatment with azoles. This clinical pathway is due to the selective pressure that azoles exert on A. fumigatus within the patient. However, since 2014, resistance has grown significantly, and almost all azole-resistant A. fumigatus strains have a combined mechanism of modifications in the promoter and the coding portion of cyp51A (TR34/L98H or TR46/Y121F/T289A). Both resistance mechanisms are frequently detected in strains from patients who have never been exposed to antifungal therapy. In these cases, the involvement of an environmental route is raised, in which the unintentional exposure of A. fumigatus to DMIs (imidazole and triazole) in the field would be favoring the resistance emergence.

Origin and Evolution of A. fumigatus Resistance to Antifungals

Nowadays, the isolation of A. fumigatus strains resistant to antifungals is an increasing global emergence. The continuous exposure of A. fumigatus to environmental fungicides, used for crop protection against other fungal species that cause agricultural damage, is believed to be selecting multi drug resistant strains. The main azole resistance mechanisms in A. fumigatus are strains with modifications of the azole target (cyp51A gene), mainly the TR34/L98H, followed by TR46/Y121F/A289T. Both types of mechanisms are responsible for panazole resistance and cross resistance to DMIs used for crop protection (imidazoles and triazoles). More recently, resistance to several fungicide classes such as, Bencimidazoles (MBC), Estrobilurinas (QoIs), sucinato deshidrogenase inhibitors (SDHIs) and  Dicarboximides, has also been acknowledged.

Genomic characterization (NGS) of strains from both clinical and environmental sources allows linking genomic differences with the acquisition of resistance to different fungicides. Adding data on susceptibility to non-azole antifungals provides a more precise picture of the phylogenetic relationships among strains, as distinct subclades are formed in which strains multi-resistant to non-azole antifungals grouped with azole-resistant strains with TRs resistance mechanisms. This formation of specific clades with strains that differ in geographic origin and year of isolation suggests the existence of a common link, an evolutionary origin according to which the strains have developed under similar circumstances that converge in a series of multi-resistance mechanisms to fungicides from different families. The resistance of A. fumigatus to non-azole fungicides, that are exclusively used in the environment, confirms that the strains with TRs resistance mechanisms are selected and developed in the environment where they are exposed to the selective pressure of multiple fungicides.

Tolerance and Persistence to Azole Antifungals in Aspergillus fumigatus

Tolerance and persistence are two phenomena by which pathogenic organisms can survive the microbicidal action of antimicrobials that should kill them over an extended period. In our laboratory, we investigate the ability of certain A. fumigatus isolates to exhibit tolerance and persistence to azoles, which are the first-line antifungal treatment for aspergillosis infections.

We are developing methodologies to detect and study tolerance and persistence, both in the laboratory and in clinical diagnosis. Using these methods, we are exploring the underlying molecular and genomic mechanisms that enable these phenomena. In addition, we are investigating the potential relevance of tolerance and persistence in the efficacy of antifungal treatment.

Differential Modulation of Persulfidation in the Fungus and Host as a Novel Antifungal Strategy

Persulfidation is a post-translational modification in which an activated sulfur group (S₂-), through the action of an enzyme, performs a specific nucleophilic attack on thiol (-SH) groups of cysteine residues in target proteins, forming a persulfide group (-SSH). This modification has been shown to modulate the intrinsic activity of proteins, playing a crucial role in various cellular mechanisms and physiological functions.

In our previous research, we demonstrated that correct levels of persulfidation are important both for A. fumigatus virulence and for orchestrating an adequate immune response in the host. Based on this, our research explores the hypothesis that differential modulation of persulfidation could constitute a novel antifungal treatment strategy.

We are investigating the ability of compounds to inhibit fungal enzymes responsible for persulfidation, aiming to reduce persulfidation levels and thereby decrease A. fumigatus virulence. Additionally, we are studying the use of sulfur donors as a potential means to enhance persulfidation in pulmonary host cells, with the goal of strengthening the immune response.

Evolution of Cross-Resistance to the New Antifungals Olorofim and Manogepix

Azole resistance is already present worldwide. Studies have shown that the most common resistance mechanisms—tandem repeats in the promoter of the gene encoding the azole target—have developed in agricultural settings due to the indiscriminate use of pesticides from the same family as clinical azoles.

Currently, two new clinical antifungals with novel molecular mechanisms of action have been introduced: olorofim and manogepix. However, analogous compounds with the same mechanism of action, ipflufenoquin and aminopyrifen, have also been developed for use as pesticides. This situation puts us at risk of repeating the same mistake made with azoles.

In this international collaborative project, we study the evolution of resistance and cross-resistance to these clinical and environmental antifungals. Our goal is to design strategies to minimize the emergence of resistance in the environment and develop early detection methods for antifungal resistance.

Mechanisms of pathogenic fungal host adaptation: Morphogenesis in Cryptococcus neoformans

One of the main mechanisms by which fungi are able to cause disease in humans is their ability to evade the immune response and adapt to the environmental conditions found in the host. In this regard, one of the yeasts that has the greatest ability to adapt to the host is Cryptococcus neoformans. This fungus is found in the environment, and is acquired by inhalation, although the most typical picture is meningitis in immunocompromised patients, mainly HIV+. The main phenotypic characteristic is the presence of a polysaccharide capsule surrounding the cell, which is considered a virulence factor. In addition, C. neoformans is able to increase cell size significantly forming “titan” cells, which can reach a diameter of more than 70 microns. In the laboratory, we are interested in the role of these titan cells in the virulence of C. neoformans. Recently, we have described in vitro media in which C. neoformans forms pseudo-titan cells, which has allowed us to identify new factors and pathways involved in this process.

Mechanisms of action of antifungals

In parallel, we have a line whose main objective is to characterize the mechanisms of action of antifungals. Specifically, we have focused our work on the effect of Amphotericin B (AmB). For decades it has been thought that this antifungal causes cell death after binding to ergosterol and pore formation. Our results indicate that this antifungal also induces strong oxidative stress in the cell, which occurs before cell integrity is lost. Furthermore, we have shown that oxidative stress is necessary for the fungicidal action of AmB. These results open the door to design new strategies to improve its efficiency in patients.

New therapeutic strategies

Work with AmB has led to research aimed at improving antifungal therapies. In particular, we have used the strategy of “off-patent” drug repositioning to search for new activities. Using this approach, we have identified several drugs that increase the effectiveness of AmB against major pathogenic yeasts, such as the antibiotic erythromycin. This approach has allowed us to identify drugs with antifungal activity against emerging pathogens, such as Candida auris.

A) Effect of vaccination on the prevalence and distribution of Human Papillomavirus (HPV) genotypes. HPV vaccination was introduced in Spain in 2007-2008 for the prevention of cervical cancer and other cancers associated with these viral infections. The use of HPV vaccination is expected to lead to a decrease in vaccine genotypes in the population. However, it may also lead to an increase in other non-vaccine genotypes, similar to the change in vaccine serotypes observed in pneumococcal infections. This requires continuous surveillance of genotype frequency and data to monitor the efficacy of the HPV vaccination program.

B) Study of the distribution and dynamics of HPV infections in risk groups. There are some particularly vulnerable groups, some of them difficult to access (sex workers, transgender groups, etc.), in which HPV infections deserve special attention. The prevalence of HPV infection is especially high in people living with HIV and/or among men who have sex with men. Knowledge of the distribution and dynamics of infections is especially interesting in these groups, as they may help to improve current algorithms for the prevention of anogenital cancer.

C) Study of infection by HPV genotypes and their relationship with progression to neoplastic processes. The oncogenic capacity of some HPV genotypes and their involvement in the production of anogenital cancer is well known. In addition, there are other oncological processes, such as non-melanoma skin cancer, in which HPV could be implicated. Thus, members of the gamma-24 HPV species have recently been associated with skin cancer. It is to be hoped that the appearance of new genotypes and the performance of more extensive studies may lead to the identification of new associations between HPV and neoplastic processes.

D) Study of co-infections by different HPV genotypes. The presence of co-infections of different HPV genotypes is a very frequent finding, both in skin samples and in different mucous membranes. The great genetic diversity of HPV limits the ability of classical molecular methods to perform a comprehensive detection and study of the genotypes present. However, the use of massive sequencing makes it possible to eliminate some of these biases and to obtain more detailed information on the existing HPV populations, as well as to analyze interactions between the different genotypes.

E) Description of new HPV genotypes/variants. Currently at the International HPV Reference Center (Karolinska Institute, Sweden) more than 220 HPV genotypes are described, distributed in 5 different genera. However, improved molecular detection techniques, as well as the use of massive sequencing, are allowing this number to increase rapidly. The study of new genotypes and variants is essential for the validation and quality control of available diagnostic methods. Similarly, their characterization and the study of possible associations of HPV with pathologies other than those already known is a field of great interest for research.

The Immunobiology group has been working for years on the following lines of research:
1) The mechanisms of haematopoietic cell generation throughout ontogeny and the influence that the first haematopoietic cells exert on the innate and adaptive immune system present in the adults. We have identified and characterised a new population of B lymphocytes called B1-Rel (B220lo), which produce high levels of natural IgG/IgA antibodies. We sought to understand their role in the immune response in animal models of infection, analysing their impact on immune cell populations and on the production of soluble mediators (cytokines and immunoglobulins). In this regard, we have evaluated the generation of embryonic megakaryocytes (and their differentiation niches), their functionality and that of platelets, and their influence on haematopoietic development. For lymphoid populations, we have carried out extensive characterisation by flow cytometry and single cell RNA sequencing (scRNAseq) methodology. To carry out these cellomic studies, we have designed complex panels for use in multiparametric phenotypic analysis, and single cell cytometry and RNAseq omics technologies on purified cell populations.

In parallel, we are interested in understanding local immune responses in respiratory infections at times of particular susceptibility due to the fragility of the immune system (childhood and old age), both in mouse animal models, which allow their manipulation, and in humans. 

2) Mouse models studied during neonatal life, in which we evaluated the effect of antibiotic (AB) treatment and addressed the role of TLR receptors in innate, pseudo-innate and adaptive immune cell populations. In these models, we observed that AB administration was able to modulate B-lymphoid populations, as well as their ability to secrete proinflammatory cytokines in culture and their differentiation into plasma cells, with differentiated immunoglobulin repertoires. Furthermore. These effects were mediated through the Toll-like receptor-2 (TLR2).

3) Mouse models with accelerated senescence (SAMP8) and senescent animals (over 20 months of age) to map lymphoid populations and soluble mediators of the immune response (immunoglobulins and cytokines). In these models, the B lymphoid populations (B1Rel and marginal zone B lymphocytes) are observed to be altered, accompanied by an increase in IgG1 with great restriction of their VDJ repertoires.

4) Role of the B1Rel population in animal models of local or systemic infection. We analysed the response to Streptoccoccus pneumoniae (SPN) locally in the lung and systemically in the spleen, as well as the role of TLR4 in these responses.

5) In humans, we are studying immune responses in children with respiratory syncytial virus (RSV) viral primo-infection. In this case we studied the immune response that occurs locally in the nasal mucosa (by analysis of nasal washings, NW) in a cohort of infected children versus healthy controls, stratified by age. We found that lymphomyeloid cells accumulate in these nasal washings in patients with diverse lymphocyte populations, as well as cytokines and immunoglobulins.

6) Analysis and characterisation of extracellular vesicles produced during respiratory infection both in lung supernatants from models of SPN infection and in LN in the case of children with RSV infection.

7) In parallel, we carry out studies of the genetic rearrangements of immunoglobulins and their use in the generation of chimeric receptors for possible use in immunotherapy.

Research Lines:

1.    Molecular mechanisms associated to the protection of HIV-1 infection in limb-girdle muscular dystrophy dominant D2 (LGMDD2) patients.
2.    Generation of neutralizing antibodies for therapeutic use based on the broad-spectrum neutralizing response against founder viruses.
3.    Characterization of the immune memory against SARS-CoV-2 in a population over 65 years of age.
4.    Screening and characterization of new anti-latency drugs against HIV-1.
5.    Study of viral entry and HIV tropism in viruses of special epidemiological relevance in Spain. 
6.    Genetic mechanisms of protection and control of HIV-1 infection in populations with extreme phenotypes.

Clinical studies:

1.    Phase 1 clinical trial to evaluate the safety and immunogenicity of HIV-1 envelope-based 763SIP8/MPLA-5 vaccine as a preventive vaccine in healthy uninfected adults. 
2.    ENE-COVID-Senior: Prospective observational study in a cohort of elderly nursing home residents to establish their immune status after receiving a complete vaccination regimen.

Implementation of new technologies:

1.    Identification of HIV-1 integration sites by deep sequencing.
2.    Single cell transcriptomics with simultaneous TCR/BCR sequencing.
3.    Epidemiological intelligence for prediction of SARS-CoV-2 variants likely to emerge in different vaccination settings.
 

The Laboratory of Medical Entomology (LME) develops an intense reference and research activity, focused on the field of disease vectors of interest in Public Health. The LME has an insectary where biological cycles of insect vectors are currently maintained, allowing the performance, among others, of vector competence and xenodiagnostic studies. The LME supports the national health system by offering techniques available in the portfolio of services for the taxonomic identification of arthropods of health interest. In addition, it performs entomological surveillance of outbreaks, supporting Surveillance Plans. In particular, the LME plays a leading role in the Entomological Surveillance Plan for Leishmaniasis in the Community of Madrid. On the other hand, the LME offers scientific advice to the CCAES (Centro de Coordinación de Alertas y Emergencias Sanitarias, Ministerio de Sanidad, Consumo y Bienestar Social), and participates in the elaboration of reports and rapid risk assessments.

The main research lines of the Laboratory of Medical Entomology are:

1. Maintenance of insect vector colonies: phlebotomine sand flies (Phlebotomus perniciosus, Phlebotomus papatasi and Phlebotomus argentipes, vectors of Leishmania infantum, Leishmania major and Leishmania donovani, respectively), Culex and Aedes mosquitoes (vectors of various arboviruses) and Rhodnius prolixus (vector of Trypanosoma cruzi). 
2. Biology of disease vectors of public health interest: biology, vector competence, experimental infections. The CNM has a BSL3 safety laboratory to carry out vector competence studies with culicidae and phlebotomine sand flies.
3. Entomological sampling, infectivity of potential reservoirs of leishmaniasis.
4. Insecticides and repellents: evaluation of their efficacy. 
5. Characterization of saliva proteins of hematophagous Diptera: genomics, proteomics, biochemistry and gene editing. Study of salivary proteins as markers of bite exposure, virulence factors and/or vaccines.
6. Xenodiagnosis of leishmaniasis.
7. Molecular biology and taxonomy of phlebotomine sand flies. Molecular detection of Leishmania infantum in phlebotomine sand flies and characterization of Leishmania spp. Molecular identification of blood ingested by vectors.

Research

The Molecular Virology group focuses its research on the study of HIV-1 genetic variation and viral evolution using both in vitro and ex vivo approaches, structured around the following research lines:

- Non-progressor patients. These patients maintain control of the disease in the absence of antiretroviral therapy and have therefore been proposed as a model of functional cure. Our objective is to study the contribution of viral factors to disease control through biological characterization and analysis of viral evolution in individuals with undetectable viral loads (elite controllers, EC), compared with individuals showing other patterns of viral control.

- Viral envelope. This viral protein is key in determining viral fitness. Therefore, its functionality significantly affects infection progression. In collaboration with Dr. Blanco and Dr. Valenzuela, we study which specific events (CD4 binding, fusogenicity, etc.) are associated with envelope functionality. To this end, we have analyzed envelopes from individuals with different patterns of disease progression. Some of these have been contributed to the AIDS Research Network envelope biobank for broader use.

- Dual infection. Infection with more than one viral variant (either through co-infection or superinfection) may have consequences for infection pathogenesis. Within our group, different aspects of DI have been analyzed, including its detection in non-progressor patients, its prevalence and incidence in Spain, and its influence on the neutralizing antibody response.

- Molecular Epidemiology. The group has analyzed viral evolution throughout the epidemic in Spain and in other countries (the Netherlands, Italy, Germany, Uruguay, Panama, Brazil, etc.).

- Role of amino acid residues in reverse transcriptase. We study the role of specific amino acid residues in HIV-1 reverse transcriptase in enzymatic function and replication capacity using an infectious molecular clone previously obtained by the group.

- “In vitro” variability. Serial passage studies have been used to detect the mechanisms responsible for the gain or loss of viral fitness.

- Antiviral studies. We have analyzed the selection of resistance mutations in vitro against different antivirals, as well as the effect of these mutations on viral fitness, and the activity of new antivirals such as ATR inhibitors.

Virological Diagnosis and Reference in HIV and HTLV Infections

The research group provides diagnostic and reference activities through the service portfolio of the National Center for Microbiology to the entire Spanish National Health System.

These services include:

• Diagnosis and reference of HIV infection (types 1 and 2) through detection of specific antibodies and detection of proviral DNA by PCR.

• Diagnosis and reference of HTLV-I/II infection through detection of specific antibodies and detection of proviral DNA by PCR. Quantification of HTLV-1 proviral load by real-time PCR.

European Union Reference Laboratory (EURL) in the field of in vitro diagnostic medical devices for microbiological diagnosis (IVD) of HIV and HTLV (Regulation 2023/2713 of December 5th, 2023). Our role is to confirm the reliability and effectiveness of devices for detecting these pathogens and to ensure their specific performance requirements through laboratory testing before they can be marketed within the European Union.

Arbovirus y Enfermedades víricas importadas

​El laboratorio de Arbovirus y Enfermedades Víricas Importadas (AEVI) del CNM desarrolla su trabajo dentro de la línea de investigación “Virus emergentes transmitidos por vector y/o reservorio, de importancia en salud pública”, que se sustenta en las áreas de epidemiología molecular, desarrollo metodológico, detección en vectores y reservorios y, otros aspectos relacionados con estas zoonosis con una aplicación clara hacia la investigación, prevención, preparación, control y respuesta a las amenazas o brotes causados por  estos virus.
Arbovirus como Dengue, Chikungunya o Zika son virus endémicos en todo el cordón tropical/sub-tropical del planeta en continua expansión a latitudes más lejanas debido al calentamiento global y a la dispersión y colonización de nuevos hábitats llevada a cabo por sus vectores artrópodos y son transportados a otras zonas del planeta a través de pacientes virémicos por lo que si, como ocurre en España, se cuenta con vectores transmisores establecidos, se puede propiciar el establecimiento de circulación autóctona. Además de estos virus exóticos, en España circulan endémicamente los arbovirus Toscana, West Nile y el virus de la Fiebre hemorrágica de Crimea-Congo, entre otros.
La OMS elaboró un listado en 2019 con las 10 amenazas para la Salud Global que consideraron más importantes, entre las que se  encuentran los virus Dengue, Ébola y Zika. El principal temor es que la falta de preparación cause una epidemia. Además, algunos de estos virus como Dengue y Chikungunya son considerados también “Enfermedades Tropicales Desatendidas” que ponen en peligro la salud de muchas personas en países empobrecidos sin que se estudien con los recursos necesarios.

La importancia para la salud pública de estos patógenos, y la necesidad de prepararse frente a ellos, se refleja también en la lista de actividades prioritarias de investigación y desarrollo de la OMS que actualmente incluye, entre otros, el Ébola, el virus de la Fiebre Hemorrágica de Crimea Congo, el virus de Zika y la enfermedad por el virus de Nipah,  debido a la amenaza que suponen para la Salud Pública por su potencial epidémico o porque no hay medidas de control suficientes. Además del peligro real que representan en zonas endémicas, algunos de los virus mencionados (Ébola, Zika, West Nile, Crimea-Congo y Dengue) han supuesto, y continúan siendo, una amenaza para nuestro país habiendo producido casos esporádicos o brotes localizados de infección autóctona. El riesgo para España de las enfermedades transmitidas por vectores está aumentando de manera muy clara como se ha podido observar en los últimos años, y la previsión es que siga aumentando.
Todos estos virus son virus zoonóticos emergentes y nuestro grupo de investigación lleva décadas trabajando en diferentes aspectos en relación con estos patógenos. El riesgo de emergencia y expansión de estos virus se basa de sus ciclos complejos de transmisión, por lo que nuestros estudios se basan tanto en el ser humano, como en los reservorios y los vectores que los transmiten. De esta base, parten transversalmente las líneas de actuación que van enfocadas a estudios de epidemiología molecular, desarrollo metodológico, detección de virus en vectores y hospedadores, caracterización de los mismos y estudios de competencia vectorial, con una aplicación dirigida hacia la investigación, prevención y respuesta a las amenazas o brotes causados por estos virus. El trabajo que desarrollamos se articula en torno a 3 objetivos principales:

Objetivo 1. Búsqueda y caracterización de virus emergentes en vectores y/o reservorios. Se lleva a cabo mediante herramientas moleculares incluyendo las nuevas estrategias de NGS, de virus emergentes en vectores y reservorios. De los virus detectados se realiza una caracterización molecular y serológica, llevando a cabo estudios de epidemiología molecular y de relaciones genéticas y antigénicas con virus relacionados.

Objetivo 2. Desarrollo metodológico para detección, identificación y caracterización de virus emergentes. Los métodos desarrollados pueden transferirse al SNS, explotarse comercialmente y/o utilizarse en la Cartera de Servicios del CNM. Estos desarrollos moleculares, y/o serológicos, refuerzan al CNM en su papel como Laboratorio Nacional de Referencia de Zoonosis, con un aporte de herramientas útiles y necesarias para la detección y caracterización de estos agentes. De igual forma, el desarrollo de herramientas tipo flujo lateral, las denominadas “Point Of Care” es una de las necesidades que pretendemos dar solución.

Objetivo 3. Eco-epidemiología de viriasis emergentes.  Debido a los complejos ciclos biológicos de los arbovirus, el estudio de las especies de vectores implicados en nuestro país, así como el origen y evolución de los agentes circulantes, es crucial a la hora de entenderlos y responder a las amenazas que generan. Para ello estudiamos la presencia de estos virus tanto en muestras humanas como de vectores y posibles hospedadores, lo que nos permite, con un enfoque de “Una Salud”, entender los mecanismos que controlan su circulación y que puedan estar implicados en su patogenicidad.

Babesiosis

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Bacterial Genetics

Our group has been studying for more than 30 years the mechanisms of antibiotic resistance in Streptococcus pneumoniae (Spn). Our objectives are to understand the molecular basis of antimicrobial action, to search for new targets of action and new compounds. Seconeolitsine (SCN) is one of these new compounds targeting topoisomerase I (Topo I). As for the search for new targets, our research has focused in recent years on the factors that organize the topology of the chromosome, allowing optimal compaction (about 1000-fold) to harmonize its replication, chromosome segregation and gene expression. This compaction is mediated both by the level of DNA supercoiling (Sc) and by association with nucleoid-binding proteins (NAPs). The level of Sc depends mainly on the enzymatic activities of their DNA topoisomerases, reaching a homeostatic equilibrium by the opposite activities of the topoisomerases that relax DNA (Topo I and Topo IV), and of gyrase, which introduces negative Sc. Our group has characterized the three Spn topoisomerases and two NAPs: HU and SatR. In addition, the availability of antimicrobials that inhibit each of the Spn topoisomerases has allowed us to analyze their transcriptome under conditions of local or global change of the Sc level and to define gene domains of coordinated transcription and similar functions. Fluoroquinolones, which inhibit Topo IV and gyrase, produce local changes in Sc that induce alterations in 6% of the transcriptome, altering metabolic pathways that originate an increase in reactive oxygen species (ROS) that contribute to lethality, in accordance with the general mechanism of bactericidal antibiotics. On the other hand, the induction of global changes in Sc by novobiocin (NOV, gyrase inhibitor), or by SCN (Topo I inhibitor), has allowed us to define topological domains. Global changes in Sc include the regulation of topoisomerase genes: its decrease activates the transcription of gyrase genes (gyrA, gyrB) and inhibits those of Topo IV (parEC) and Topo I (topA); the increase in Sc regulates the expression of topA. Decreased Sc affects 37% of the genome, with >68% of genes clustered in 15 domains. Increased Sc affects 10% of the genome, with 25% of the genes clustered in 12 domains. The AT content in the genome correlates with the domains, being higher in UP domains than in DOWN domains. The genes in the different domains have common functional characteristics, indicating that they have been subjected to topological selective pressure to determine the location of genes involved in metabolism, virulence and competition. 

The current objectives of the group are:
1.    Identification of factors that stabilize chromosome topology: NAPs, ncRNAs, intra-chromosomal interactions.
2.    Regulation of transcription in response to topological stress: in vivo localization of DNA topoisomerases, RNA polymerase and NAPs.
3.    Topo I as a new antimicrobial target and action of SCN. 
4.    Design of antisense RNAs and use of the CRISPR system as new antibacterial agents.

Biología y Variabilidad del VIH

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Enfermedades bacterianas transmitidas por agua y alimentos

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Genomica y Bioinformática

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Hepatitis

1. Diseño de métodos diagnósticos para el estudio de los virus de las hepatitis (VH) A, B, C, D, E: Diseñamos sistemas de PCR para su detección y caracterización.
2. Evaluación de métodos diagnósticos de los VH. Colaboramos con empresas para estudios de sensibilidad y especificidad de equipos diagnósticos.
3.  Estudios de Seroprevalencia de los virus de las hepatitis.
4.  Epidemiología genómica de genomas completos de VHA, VHB, VHC, VHD y VHE en colaboración con el ECDC. Estudios de trazabilidad del VHE.
5. Caracterización molecular de virus de las hepatitis mediante secuenciación masiva:               a) VHB: mutantes de escape HBsAg (prevalencia y efectos en la detección del HBsAg). Estudio de mutaciones en epítopos de estimulación inmune y mutaciones asociadas a evolución clínica desfavorable.
   1. b) VHC: resistencias a los antivirales de acción directa. Análisis molecular de subtipos poco frecuentes.
      c) Estudios filogenéticos del VHD.
       d) Análisis genómico del VHE.
       e) Investigación etiológica de hepatitis no filiadas mediante estudios de metagenómica.

Infección Viral e Inmunidad

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Infecciones relacionadas con la Asistencia Sanitaria

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Infecciones Víricas e Inmunidad en Enfermos Inmunodeprimidos

Nuestras líneas de investigación se centran en distintas áreas del conocimiento relacionadas con hepatitis virales crónicas (Hepatitis B, C y D), VIH, y SARS-CoV-2:

- Inmunopatogenia de las infecciones virales y su relación con eventos clínicos.

- Impacto en el organismo del control o eliminación de la infección viral.

- Biopsia líquida y ómicas: biomarcadores de enfermedad en infección viral.

- Resistencia a la infección viral y aclaramiento espontáneo.

- Cribado de infección viral y epidemiología molecular de los virus.

- Desarrollo de kits de diagnóstico rápido.

- Respuesta inmune a vacunas.

Infección por CMV en pacientes trasplantados

En los últimos años en nuestro grupo hemos estudiado la cinética de infección por CMV y el desarrollo de la respuesta inmune protectora específica frente a CMV. Como resultados de estos estudios hemos sido capaces de caracterizar la cinética y magnitud de la adquisición de la respuesta inmune frente a CMV y establecer puntos de corte de inmunidad específica que se relacionan con la protección frente a la infección.

El objetivo de esta línea de investigación en los últimos años ha sido definir parámetros relacionados con la respuesta inmune específica frente a CMV y con factores genéticos que puedan estar relacionados con el control de la infección y enfermedad por CMV. El estudio de estas variables de forma conjunta como marcadores de predicción del riesgo de desarrollo de infección/enfermedad y de la evolución de la infección tras el trasplante permitirían establecer un algoritmo para el manejo de los pacientes. Además, permitirían definir niveles mínimos de protección que sirvan de endpoints para el desarrollo de una vacuna. Esta línea de investigación se enmarca dentro del programa de Infecciones en Transplantes de la Red Española de Investigación en Patologías Infecciosas (REIPI), como parte del WP2 denominado Optimización de la prevención de la enfermedad por CMV.

Desarrollo preclínico de vacunas protectoras frente a CMV

La vacuna ideal frente a CMV debería estar compuesta por múltiples antígenos y ser capaz de imitar el efecto producido por la infección natural, y estimular una respuesta inmune específica combinada tanto de células T como de anticuerpos neutralizantes. Sin embargo, a pesar de los esfuerzos realizados y los progresos de los últimos años, los resultados obtenidos en el desarrollo de una vacuna frente a CMV no han mostrado resultados definitivos.

Por tanto, el objetivo de esta línea de investigación es promover aproximaciones innovadoras para el diseño y desarrollo de una vacuna frente a la infección por CMV. Para ello se ha puesto en marcha una aproximación a través de la construcción de un plásmido optimizado para generar una respuesta inmune combinada y amplia específica frente a CMV y la búsqueda de los antígenos candidatos, a través del estudio de la inmunogenicidad in vivo del proteoma completo de CMV.

Desarrollo preclínico de alternativas terapéuticas frente a CMV basadas en la inmunoterapia

La inmunidad mediada por células T constituye una respuesta adaptativa fundamental y representa el mecanismo de defensa más importante frente a la infección viral. Por otra parte, la presencia en suero de anticuerpos neutralizantes se han asociado con tasas más bajas de transmisión del CMV de la madre al feto y en los receptores de trasplante de órgano sólido.

El presente proyecto propone un enfoque novedoso a través de la identificación y  caracterización de la respuesta inmune tanto celular como de  anticuerpos en pacientes que han sido inmunizados de forma natural y están protegidos frente a  la infección por CMV, para abordar el desarrollo preclínico de alternativas terapéuticas basadas en la transferencia adoptiva de células T CD8+ citotóxicas y de anticuerpos monoclonales específicos de CMV con el objetivo de desarrollar una nueva estrategia terapéutica para el tratamiento frente a la infección por este patógeno. ​

Estudio del desarrollo de inmunidad frente a SARS-CoV-2 

Dada la situación producida como consecuencia de la propagación del SARS-CoV-2 urge la realización de estudios serológicos que nos ayuden a determinar el alcance y la duración de la inmunidad adquirida por la población infectada por SARS-CoV-2 que ha superado la enfermedad. En este sentido el estudio de los anticuerpos neutralizantes, que son aquellos que reconocen las proteínas del virus implicadas en el reconocimiento del receptor celular bloqueando su capacidad infectiva, en pacientes recuperados de la infección por SARS-CoV-2 podrían ser clave para explicar el pronóstico de la enfermedad en estos pacientes. Además son necesarios estudios que estudien la cinética de la inmunidad de anticuerpos específicos frente a coronavirus y su relación con la evolución de la enfermedad que nos permitan establecer estrategias de manejo de los pacientes en base a su patrón inmunológico.

También participamos en el desarrollo de un prototipo de vacuna basado en ADN plasmídico que expresa antígenos de SARS-CoV-2 y la caracterización de la respuesta inmune inducida por esta vacuna en un modelo murino de inmunización.

Inmunología Celular

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