[Development of a second generation prophylactic vaccine against human papillomavirus]. / Desarrollo de una vacuna profiláctica de segunda generación contra el papilomavirus humano.

Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection.

A high-throughput screening for inhibitors of riboflavin synthase identifies novel antimicrobial compounds to treat brucellosis.

Brucella spp. are pathogenic intracellular Gram-negative bacteria adapted to life within cells of several mammals, including humans. These bacteria are the causative agent of brucellosis, one of the zoonotic infections with the highest incidence in the world and for which a human vaccine is still unavailable. Current therapeutic treatments against brucellosis are based on the combination of two or more antibiotics for prolonged periods, which may lead to antibiotic resistance in the population. Riboflavin (vitamin B2) is biosynthesized by microorganisms and plants but mammals, including humans, must obtain it from dietary sources. Owing to the absence of the riboflavin biosynthetic enzymes in animals, this pathway is nowadays regarded as a rich resource of targets for the development of new antimicrobial agents. In this work, we describe a high-throughput screening approach to identify inhibitors of the enzymatic activity of riboflavin synthase, the last enzyme in this pathway. We also provide evidence for their subsequent validation as potential drug candidates in an in vitro brucellosis infection model. From an initial set of 44 000 highly diverse low molecular weight compounds with drug-like properties, we were able to identify ten molecules with 50% inhibitory concentrations in the low micromolar range. Further Brucella culture and intramacrophagic replication experiments showed that the most effective bactericidal compounds share a 2-Phenylamidazo[2,1-b][1,3]benzothiazole chemical scaffold. Altogether, these findings set up the basis for the subsequent lead optimization process and represent a promising advancement in the pursuit of novel and effective antimicrobial compounds against brucellosis.

Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domain of the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-binding activity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2 interaction has a strong electrostatic component, which correlates with the high proportion of acidic residues found in the antibody combining site. Further circular dichroism experiments in the presence of phosphate show that, in addition to electrostatic screening of key potential interactions, ionic strength affects the conformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlates with the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface. Noticeably, this finding is well correlated with the strong entropic component of the interaction. Site directed mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix of E2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociation constant of the E2-ED15 interaction is 10(-7) M and the association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower than those generally reported in the most extensively described "nonflexible" antibody-protein interactions, indicating the presence of a slow conformational rearrangement on the antigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition is discussed.

A viral DNA-binding domain elicits anti-DNA antibodies of different specificities.

We have previously demonstrated that immunization of normal mice with a defined protein:DNA complex comprising the C-terminal DNA-binding domain of the human papillomavirus E2 protein and its cognate site 35 oligonucleotide, results in high antibody titers against both E2 and its target DNA sequence. Here we show that repeated immunization with the isolated form of the E2 domain also elicits anti-DNA antibodies, but in this case, no preferential binding for a given sequence was observed, indicating that these antibodies have broad specificity for DNA. Taken together our results indicate that this viral protein can induce two classes of anti-DNA antibody responses: one directed against endogenous DNA and other in which anti-site 35-specific antibodies are produced. In both cases, the character of the resulting anti-DNA response seems to be directed by the DNA molecule that the protein binds in vivo. Evaluation of the fine specificity of the antibodies induced by the free and bound states of this single foreign DNA-binding protein would contribute to the understanding of the processes involved in the acquisition of particular DNA specificities by anti-DNA antibodies.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.

Desarrollo de una vacuna profiláctica de segunda generación contra el papilomavirus humano / Development of a second generation prophylactic vaccine against human papillomavirus

Los papilomavirus humanos (HPV) son el agente etiológico del cáncer cervical (CC), la segunda causa de muerte por cáncer en mujeres. Se estima que medio millón de nuevos cánceres se diagnostica cada año, ocurriendo la mayoría de ellos en países en vías de desarrollo debido a la ausencia o ineficiencia de los programas masivos de detección temprana. Recientemente se han introducido en el mercado dos vacunas profilácticas contra las principales cepas oncogénicas de HPV, la cepa 16 y 18, responsables por el 80% de todos los CC. Estas vacunas se obtienen en forma recombinante y han demostrado ser extremadamente seguras y eficaces. Sin embargo, su impacto inmediato en la incidencia de la infección por HPV en países en vías de desarrollo será mínimo, debido principalmente al alto costo de las mismas. Existe la necesidad de contar con vacunas de segunda generación, de bajo costo y de aplicación masiva que permitan disminuir sensiblemente el número de CC en la población. Con este objetivo hemos desarrollado una plataforma de expresión recombinante que permite obtener partículas tipo virus (VLPs) con las cuales es posible formular vacunas efectivas y accesibles contra la infección por HPV.

Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection.