Karen Delgadillo-Gutiérrez’s research while affiliated with National Polytechnic Institute and other places

Mycobacteria, including nontuberculous mycobacteria (NTM) and Mycobacterium tuberculosis complex (MTB), are global pathogens of major concern due to their intrinsic drug resistance and their capacity to cause a wide range of severe infections. The treatment of mycobacterial infections is particularly challenging because of the multidrug resistance. Efflux pumps are involved in drug resistance by actively expelling antibiotics. A promising strategy to decrease drug resistance is the inhibition of efflux pump activity by efflux pump inhibitors. In this chapter, we will review the current knowledge on efflux pumps and their impact on clinical drug resistance, as well as the potential of efflux pump inhibitors to mitigate resistance. The search for novel compounds as efflux pump inhibitors or the inclusion of existing inhibitors in the current drug therapy for mycobacterial infections has become a major goal in the treatment of these diseases.

Tuberculosis is a disease caused by Mycobacterium tuberculosis, representing the second leading cause of death by an infectious agent worldwide. The available vaccine against this disease has insufficient coverage and variable efficacy, accounting for a high number of cases worldwide. In fact, an estimated third of the world’s population has a latent infection. Therefore, developing new vaccines is crucial to preventing it. In this study, the highly antigenic PE_PGRS49 and PE_PGRS56 proteins were analyzed. These proteins were used for predicting T- and B-cell epitopes and for human leukocyte antigen (HLA) protein binding efficiency. Epitopes GGAGGNGSLSS, FAGAGGQGGLGG, GIGGGTQSATGLG (PE_PGRS49), and GTGWNGGKGDTG (PE_PGRS56) were selected based on their best physicochemical, antigenic, non-allergenic, and non-toxic properties and coupled to HLA I and HLA II structures for in silico assays. A construct with an adjuvant (RS09) plus each epitope joined by GPGPG linkers was designed, and the stability of the HLA-coupled construct was further evaluated by molecular dynamics simulations. Although experimental and in vivo studies are still necessary to ensure its protective effect against the disease, this study shows that the vaccine construct is dynamically stable and potentially effective against tuberculosis.

Influenza is a relevant problem for public and animal health, with a significant economic impact. In recent years, outbreaks of avian influenza virus have resulted in devastating losses in the poultry industry worldwide, and although its transmission to humans is very rare, there is always a potential risk for an even more severe outbreak. Currently, vaccination is considered the most effective tool for the control and prevention of influenza infections in both humans and animals. The maintenance of animal welfare and the successful implementation of animal health programs depend on the timely administration of vaccines, which must comply with quality specifications indicated by health authorities; for example, the capability to ensure a minimum antibody titer. The production of viral antigens used in these tests can pose a biosafety risk, and some viral strains can be difficult to grow. Therefore, new biotechnological alternatives are required to overcome these disadvantages. In this study, we produced pseudotypes carrying H5 and H7 hemagglutinins from lowly and highly pathogenic avian influenza viruses. These pseudotypes were used in neutralization assays to detect neutralizing antibodies in avian sera, which were confirmed positive by inhibition of the hemagglutination test. Our results showed that the pseudotype neutralization assay is a viable alternative for the detection of neutralizing antibodies, by demonstrating subtype specificity and requiring reduced biosafety requirements. Therefore, it represents a versatile platform that can facilitate technology transfer protocols between laboratories, and an immediate application in serological tools for quality control of veterinary vaccines against avian influenza.

New technologies in vaccinology are capable of achieving fast development, as well as large-scale production of effective and safe vaccines. Reverse vaccinology is an in silico methodology, which studies different characteristics of infectious agents, in order to identify antigens that are good vaccine candidates, without the need of traditional culture. This strategy is based on bioinformatics tools, that in a simple, safety and inexpensive way, reduces time and effort significantly in the new vaccine design, against traditional vaccinology. In recent years, the rapid spread of infections by emerging pathogens requires prompt development of new vaccines. Bioinformatic strategies joined with the latest next-generation vaccines allow the selection of vaccine candidates in a short time, which is relevant in the development of new vaccines against pathogens with pandemic potential.

Retroviral pseudotypes are broadly used as safe instruments to mimic the structure and surface of highly pathogenic viruses. They have been employed for the discovery of new drugs, as diagnostic tools in vaccine studies, and part of serological assays. Because of their widespread use in research and their potential as tools for quality control, it is important to know their shelf life, stability, and best storage conditions. In this study, we produced pseudotypes carrying the lacZ reporter gene and the hemagglutinin (HA) of avian influenza virus subtypes H5 and H7 to investigate their stability under various storage conditions. We produced pseudotypes with titers of approximately 10⁶ RLU/mL, which decreased to 10⁵–10⁶ RLU/mL after short-term storage at 4 °C (up to 4 weeks). Stability was maintained after long-term storage at −20 °C (up to 12 months), even under storage variations such as freeze-thaw cycles. We conclude that, although the titers decreased by 1 log10 under the different storage conditions, the remaining titers can be readily applicable in many techniques, such as neutralization assays. These findings show that large quantities of retroviral pseudotypes can be safely stored for short- or long-term use, allowing standardization and reduced variation in assays involving retroviral pseudotypes.