Vectores virales basados en poxvirus como agentes oncolíticos

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-01-2017Esta tesis tiene embargado el acceso al texto completo hasta el 27-07-2019El cáncer supone actualmente uno de los principales problemas de salud a nivel mundial. A pesar de la efectividad de las terapias anti-tumorales empleadas habitualmente, la cifra anual de muertes asociadas a cáncer continúa siendo devastadora. A este respecto, actualmente se han obtenido tratamientos con resultados muy prometedores, encontrándose entre ellos los poxvirus y la oncolisis viral. En el presente trabajo se han desarrollado varios vectores virales candidatos basados en la cepa WR del virus vaccinia como agentes oncolíticos, en los que se ha llevado a cabo la deleción dirigida de genes para poder conseguir un virus seguro en tejidos sanos sin que las deleciones afecten al perfil replicativo en un contexto tumoral, de esta forma sacando provecho de las condiciones particulares del microambiente tumoral dadas por las marcas distintivas del cáncer. Entre los genes delecionados para conseguir dicho objetivo, se encuentran genes virales relacionados con el metabolismo de nucleótidos (A48R y J2R), factores mitogénicos (C11R), genes que intervienen en el cambio metabólico celular (C16L) o el bloqueo de la respuesta anti-viral celular (B18R y F1L). Tras la generación, caracterización y evaluación de los virus candidatos propuestos, se ha demostrado que el virus WR-Δ3-Luc, con 4 genes delecionados –A48R, B18R, C11R y J2R–, se presenta como el virus que mejor se ajusta para su uso en viroterapia oncolítica. El virus WR-Δ3-Luc ha demostrado en ensayos in vitro un comportamiento similar al virus salvaje WR, respecto a su capacidad de replicación en células primarias y tumorales, a la traducción de proteínas virales y a la inhibición de la apoptosis. En ensayos in vivo, entre los virus analizados, el virus WR-Δ3-Luc ha mostrado la mayor reducción de la virulencia; asimismo el virus mutante de deleción muestra títulos más bajos que el virus WR wt en ovarios, exudados peritoneales y cerebro en el ensayo de biodistribución tras una administración sistémica. Cuando se han analizado las poblaciones celulares del sistema inmune en el sitio de infección, el virus WR-Δ3-Luc muestra un perfil de migración similar al que presenta el virus WR wt, destacando por encima de éste último en el incremento de los niveles de migración de neutrófilos. A la hora de estudiar el comportamiento en tumores, los ensayos de trasplante de células de melanoma murino B16F10 muestran una capacidad superior del virus WR-Δ3-Luc en el control del desarrollo tumoral (por encima de la observada en con el virus WR wt); mostrando a su vez que el virus mutante de deleción WR-Δ3-Luc presenta los mismos niveles de replicación en tumor que los observados en el virus WR wt, y niveles superiores en el reclutamiento en tumores de neutrófilos y linfocitos B. Por último, se ha detectado en bazos de animales con tumores tratados con el virus WR-Δ3-Luc, linfocitos T específicos para los antígenos asociados a tumor gp100 y TRP-2, lo que demuestra la capacidad del virus candidato de romper la inmuno-tolerancia e inducir respuesta adaptativa específica frente a antígenos expresados en el tumor. Así, estos estudios ponen de manifiesto las virtudes del virus WR-Δ3-Luc en su uso potencial como vector oncolítico.Cancer is one of the major health problems plaguing our society today. The death toll attributed to cancer each year continues to be devastatingly high, despite the advent of a large number of effective anti-tumour therapies in the recent years. Some of these therapies have shown considerable promise, including those based on poxvirus and viral oncolysis. This work describes the development of viral vector candidates based on the WR strain of vaccinia virus as potential oncolytic agents. This is achieved through the targeted deletion of specific genes that, while rendering the virus safe and secure in somatic tissues, also helps the virus exploit the characteristics of a tumour micro-environment provided by the hallmarks of cancer by preserving its replication competence within the tumour. Among the genes thus deleted are those associated with nucleotide metabolism (A48R y J2R), mitogenic factors (C11R), those that intervene in the cellular metabolic programming (C16L) or block the cellular anti-viral response (B18R y F1L). Following the successful generation, characterization and evaluation of the proposed viral vectors, the WR-Δ3-Luc virus with four deleted genes (A48R, B18R, C11R and J2R) emerged as the candidate of choice for use in viral oncolytic therapy. In vitro, this virus showed similar replication capacity in primary and tumour cells, translation of viral proteins and inhibition of apoptosis to that shown by the wildtype WR virus. Among the viruses studied in vivo, the WR-Δ3-Luc virus demonstrated the greatest reduction of virulence; likewise, this deletion mutant gave lower viral titres than the wildtype WR in ovaries, peritoneal washes and cerebral tissue in a bio-distribution assay following a systemic administration. When the cellular population of the immune system at the site of infection was analysed, the WR-Δ3-Luc virus, while showing a recruitment profile similar to that of the WR, outperformed the latter in terms of the increase in the neutrophil migration levels. In studies using the B16F10 murine melanoma cells transplant, designed to understand the viral behaviour in tumours, the WR-Δ3-Luc virus demonstrated a superior capacity for controlling tumour growth (compared to that observed with the WR wt virus); while at the same time showing the same replication capacity in tumours as that of the WR wt, and higher levels of neutrophil and lymphocyte B recruitment. Finally, the presence of T lymphocytes specific to the tumour associated antigens gp100 and TRP-2 detected in the spleen of animals with tumours treated with the WR-Δ3-Luc virus highlights the ability of this viral candidate to break the immune-tolerance and to induce a tumour antigen-specific adaptive response. Therefore, these studies demonstrate the significant potential of the WR-Δ3-Luc virus as an oncolytic vector for use in therapy against cancer

MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells

Mammalian cells release different types of vesicles, collectively termed extracellular vesicles (EVs). EVs contain cellular microRNAs (miRNAs) with an apparent potential to deliver their miRNA cargo to recipient cells to affect the stability of individual mRNAs and the cells’ transcriptome. The extent to which miRNAs are exported via the EV route and whether they contribute to cell-cell communication are controversial. To address these issues, we defined multiple properties of EVs and analyzed their capacity to deliver packaged miRNAs into target cells to exert biological functions. We applied well-defined approaches to produce and characterize purified EVs with or without specific viral miRNAs. We found that only a small fraction of EVs carried miRNAs. EVs readily bound to different target cell types, but EVs did not fuse detectably with cellular membranes to deliver their cargo. We engineered EVs to be fusogenic and document their capacity to deliver functional messenger RNAs. Engineered fusogenic EVs, however, did not detectably alter the functionality of cells exposed to miRNA-carrying EVs. These results suggest that EV-borne miRNAs do not act as effectors of cell-to-cell communication. Author summary: The majority of metazoan cells release vesicles of different types and origins, such as exosomes and microvesicles, now collectively termed extracellular vesicles (EVs). EVs have gained much attention because they contain microRNAs (miRNAs) and thus could regulate their specific mRNA targets in recipient or acceptor cells that take up EVs. Using a novel fusion assay with superior sensitivity and specificity, we revisited this claim but found no convincing evidence for an efficient functional uptake of EVs in many different cell lines and primary human blood cells. Even EVs engineered to fuse and deliver their miRNA cargo to recipient cells had no measurable effect on target mRNAs in very carefully controlled, quantitative experiments. Our negative results clearly indicate that EVs do not act as vehicles for miRNA-based cell-to-cell communication

Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection

Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.Peer reviewe

Influence of MUC5B gene on antisynthetase syndrome

ABSTRACT: MUC5B rs35705950 (G/T) is strongly associated with idiopathic pulmonary fibrosis (IPF) and also contributes to the risk of interstitial lung disease (ILD) in rheumatoid arthritis (RA-ILD) and chronic hypersensitivity pneumonitis (CHP). Due to this, we evaluated the implication of MUC5B rs35705950 in antisynthetase syndrome (ASSD), a pathology characterised by a high ILD incidence. 160 patients with ASSD (142 with ILD associated with ASSD [ASSD-ILD+]), 232 with ILD unrelated to ASSD (comprising 161 IPF, 27 RA-ILD and 44 CHP) and 534 healthy controls were genotyped. MUC5B rs35705950 frequency did not significantly differ between ASSD-ILD+ patients and healthy controls nor when ASSD patients were stratified according to the presence/absence of anti Jo-1 antibodies or ILD. No significant differences in MUC5B rs35705950 were also observed in ASSD-ILD+ patients with a usual interstitial pneumonia (UIP) pattern when compared to those with a non-UIP pattern. However, a statistically significant decrease of MUC5B rs35705950 GT, TT and T frequencies in ASSD-ILD+ patients compared to patients with ILD unrelated to ASSD was observed. In summary, our study does not support a role of MUC5B rs35705950 in ASSD. It also indicates that there are genetic differences between ILD associated with and that unrelated to ASSD.We are indebted to the patients and healthy controls for their essential collaboration to this study. We also thank the National DNA Bank Repository (Salamanca) for supplying part of the control samples. This study was partially supported by grants from the Foundation for Research in Rheumatology (FOREUM). RL-M is a recipient of a Miguel Servet type I programme fellowship from the ‘Instituto de Salud Carlos III’ (ISCIII), co-funded by the European Social Fund (ESF, ‘Investing in your future’) (grant CP16/00033). SR-M is supported by funds of the RETICS Program (RD16/0012/0009), co-funded by the European Regional Development Fund (ERDF). VP-C is supported by a pre-doctoral grant from IDIVAL (PREVAL 18/01). VM is supported by funds of a Miguel Servet type I programme (grant CP16/00033) (ISCIII, co-funded by ESF). LL-G is supported by funds of PI18/00042 (ISCIII, co-funded by ERDF). OG is Staff Personnel of Xunta de Galicia (Servizo Galego de Saude, SERGAS) through a research-staff stabilization contract (ISCIII/SERGAS). OG,is member of RETICS Programme, RD16/0012/0014 (RIER: Red de Investigación en Inflamación y Enfermedades Reumáticas) via Instituto de Salud Carlos III (ISCIII) and FEDER. The work of OG (PI17/00409), was funded by Instituto de Salud Carlos III and FEDER. OG is a beneficiary of a project funded by Research Executive Agency of the European Union in the framework of MSCA-RISE Action of the H2020 Programme (Project number 734899). OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade e Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10

Role of MUC1 rs4072037 polymorphism and serum KL-6 levels in patients with antisynthetase syndrome

Mucin 1/Krebs von den Lungen-6 (KL-6) is proposed as a serum biomarker of several interstitial lung diseases (ILDs), including connective tissue disorders associated with ILD. However, it has not been studied in a large cohort of Caucasian antisynthetase syndrome (ASSD) patients. Consequently, we assessed the role of MUC1 rs4072037 and serum KL-6 levels as a potential biomarker of ASSD susceptibility and for the differential diagnosis between patients with ILD associated with ASSD (ASSD-ILD?+) and idiopathic pulmonary fibrosis (IPF). 168 ASSD patients (149 ASSD-ILD?+), 174 IPF patients and 523 healthy controls were genotyped for MUC1 rs4072037 T?>?C. Serum KL-6 levels were determined in a subgroup of individuals. A significant increase of MUC1 rs4072037 CC genotype and C allele frequencies was observed in ASSD patients compared to healthy controls. Likewise, MUC1 rs4072037 TC and CC genotypes and C allele frequencies were significantly different between ASSD-ILD+ and IPF patients. Additionally, serum KL-6 levels were significantly higher in ASSD patients compared to healthy controls. Nevertheless, no differences in serum KL-6 levels were found between ASSD-ILD+ and IPF patients. Our results suggest that the presence of MUC1 rs4072037 C allele increases the risk of ASSD and it could be a useful genetic biomarker for the differential diagnosis between ASSD-ILD+ and IPF patients

High Quality Long-Term CD4+ and CD8+ Effector Memory Populations Stimulated by DNA-LACK/MVA-LACK Regimen in Leishmania major BALB/c Model of Infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4+ and CD8+ T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4+ and CD8+ effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4+ and CD8+ T cells. Anti-vector responses were largely CD8+-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4+ and CD8+ T cells with an effector phenotype, could be relevant in protection against leishmaniasis

HLA association with the susceptibility to anti-synthetase syndrome.

Objective: To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). Methods: We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. Results: A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P = 1.56E–09, odds ratio–OR [95% confidence interval–CI] = 2.54 [1.84–3.50] and 21.4% versus 5.5%, P = 18.95E–18, OR [95% CI] = 4.73 [3.18–7.05]; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P = 0.0003, OR [95% CI] = 0.48 [0.31–0.72]). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P = 0.001, OR [95% CI] = 2.54 [1.39–4.81]). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Conclusions: Our results support the association of the HLA complex with the susceptibility to ASSD.This study was partially supported by grants from the Foundation for Research in Rheumatology (FOREUM); SR-M is supported by funds of the RETICS Program [grant number RD16/0012/0009] from the `Instituto de Salud Carlos III´ (ISCIII), co-funded by the European Regional Development Fund (ERDF); BA-M is a recipient of a ‘López Albo’ Post-Residency Programme funded by Servicio Cántabro de Salud; VP-C is supported by a pre-doctoral grant from IDIVAL [grant number PREVAL 18/01]; LL-G is supported by funds of ISCIII, co-funded by ERDF [grant number PI18/00042]; OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade e Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10; EAR is partially supported by Versus Arthritis [grant number 20719] and by Scleroderma and Raynaud's UK [grant number BR11]; RL-M is a recipient of a Miguel Servet type I programme fellowship from the ISCIII, co-funded by the European Social Fund (ESF, ‘Investing in your future’) [grant number CP16/00033]

Role of MUC1 rs4072037 polymorphism and serum KL-6 levels in patients with antisynthetase syndrome

Mucin 1/Krebs von den Lungen-6 (KL-6) is proposed as a serum biomarker of several interstitial lung diseases (ILDs), including connective tissue disorders associated with ILD. However, it has not been studied in a large cohort of Caucasian antisynthetase syndrome (ASSD) patients. Consequently, we assessed the role of MUC1 rs4072037 and serum KL-6 levels as a potential biomarker of ASSD susceptibility and for the differential diagnosis between patients with ILD associated with ASSD (ASSD-ILD +) and idiopathic pulmonary fibrosis (IPF). 168 ASSD patients (149 ASSD-ILD +), 174 IPF patients and 523 healthy controls were genotyped for MUC1 rs4072037 T > C. Serum KL-6 levels were determined in a subgroup of individuals. A significant increase of MUC1 rs4072037 CC genotype and C allele frequencies was observed in ASSD patients compared to healthy controls. Likewise, MUC1 rs4072037 TC and CC genotypes and C allele frequencies were significantly different between ASSD-ILD+ and IPF patients. Additionally, serum KL-6 levels were significantly higher in ASSD patients compared to healthy controls. Nevertheless, no differences in serum KL-6 levels were found between ASSD-ILD+ and IPF patients. Our results suggest that the presence of MUC1 rs4072037 C allele increases the risk of ASSD and it could be a useful genetic biomarker for the differential diagnosis between ASSD-ILD+ and IPF patients

HLA association with the susceptibility to anti-synthetase syndrome

Objective: To investigate the human leukocyte antigen (HLA) association with anti-synthetase syndrome (ASSD). Methods: We conducted the largest immunogenetic HLA-DRB1 and HLA-B study to date in a homogeneous cohort of 168 Caucasian patients with ASSD and 486 ethnically matched healthy controls by sequencing-based-typing. Results: A statistically significant increase of HLA-DRB1*03:01 and HLA-B*08:01 alleles in patients with ASSD compared to healthy controls was disclosed (26.2% versus 12.2%, P=1.56E-09, odds ratio-OR [95% confidence interval-CI]=2.54 [1.84-3.50] and 21.4% versus 5.5%, P=18.95E-18, OR [95% CI]=4.73 [3.18-7.05]; respectively). Additionally, HLA-DRB1*07:01 allele was significantly decreased in patients with ASSD compared to controls (9.2% versus 17.5%, P=0.0003, OR [95% CI]=0.48 [0.31-0.72]). Moreover, a statistically significant increase of HLA-DRB1*03:01 allele in anti-Jo-1 positive compared to anti-Jo-1 negative patients with ASSD was observed (31.8% versus 15.5%, P=0.001, OR [95% CI]=2.54 [1.39-4.81]). Similar findings were observed when HLA carrier frequencies were assessed. The HLA-DRB1*03:01 association with anti-Jo-1 was unrelated to smoking history. No HLA differences in patients with ASSD stratified according to the presence/absence of the most representative non-anti-Jo-1 anti-synthetase autoantibodies (anti-PL-12 and anti-PL-7), arthritis, myositis or interstitial lung disease were observed. Conclusions: Our results support the association of the HLA complex with the susceptibility to ASSD

Development of a Safe and Effective Vaccinia Virus Oncolytic Vector WR-Δ4 with a Set of Gene Deletions on Several Viral Pathways

Despite the effectiveness of classic treatments and available diagnostic tools, cancer continues to be a leading world health problem, with devastating cancer-related death rates. Advances in oncolytic virotherapy have shown promise as potentially effective treatment options in the fight against cancer. The poxviruses have many features that make them an attractive platform for the development of oncolytic vectors, with some candidates currently in clinical trials. Here, we report the design and generation of a new oncolytic vector based on the vaccinia virus Western Reserve (WR) strain. We show that the WR-Δ4 virus, with the combined deletion of four specific viral genes that act on metabolic, proliferation, and signaling pathways (A48R, B18R, C11R, and J2R), has effective anti-tumor capabilities in vivo. In WR-Δ4-infected mice, we observed strong viral attenuation, reduced virus dissemination, and efficient tumor cell growth control in the B16F10 syngeneic melanoma model, with enhanced neutrophil migration and activation of tumor antigen-specific immune responses. This approach provides an alternative strategy toward ongoing efforts to develop an optimal oncolytic poxvirus vector. Keywords: oncolytic vectors, vaccinia virus, virotherapy, immune response