Influence of respiratory syncytial virus strain differences on pathogenesis and immunity

Molecular epidemiology studies have provided convincing evidence of antigenic and sequence variability among respiratory syncytial virus (RSV) isolates. Circulating viruses have been classified into two antigenic groups (A and B) that correlate with well-delineated genetic groups. Most sequence and antigenic differences (both inter- and intra-groups) accumulate in two hypervariable segments of the G-protein gene. Sequences of the G gene have been used for phylogenetic analyses. These studies have shown a worldwide distribution of RSV strains with both local and global replacement of dominant viruses with time. Although data are still limited, there is evidence that strain variation may contribute to differences in pathogenicity. In addition, there is some but limited evidence that RSV variation may be, at least partially, immune (antibody) driven. However, there is the paradox in RSV that, in contrast to other viruses (e.g., influenza viruses) the epitopes recognized by the most effective RSV-neutralizing antibodies are highly conserved. In contrast, antibodies that recognize strain-specific epitopes are poorly neutralizing. It is likely that this apparent contradiction is due to the lack of a comprehensive knowledge of the duration and specificities of the human antibody response against RSV antigens. Since there are some data supporting a group- (or clade-) specific antibody response after a primary infection in humans, it may be wise to consider the incorporation of strains representative of groups A and B (or their antigens) in future RSV vaccine development.Work in the Madrid laboratory is currently funded by grants GR09/0039 from Instituto de Salud Carlos III and SAF2009-11632 and SAF2012-31217 from Plan Nacional de I+D+i. Work in the Atlanta laboratory is supported by the following grants: NIH 1R01AI087798 and NIH 1U19AI095227.S

Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development

Extraordinary progress in the structure and immunobiology of the human respiratory syncytial virus glycoproteins has been accomplished during the last few years. Determination of the fusion (F) glycoprotein structure folded in either the prefusion or the postfusion conformation was an inspiring breakthrough not only to understand the structural changes associated with the membrane fusion process but additionally to appreciate the antigenic intricacies of the F protein. Furthermore, these developments have opened new avenues for structure-based designs of promising hRSV vaccine candidates. Finally, recent advances in our knowledge of the attachment (G) glycoprotein and its interaction with cell-surface receptors have revitalized interest in this molecule as a vaccine, as well as its role in hRSV immunobiology.Work in the Madrid lab is currently funded by grant SAF2015-67033-R from Plan Nacional de I+D+I. J.S.M is supported in part by award P20GM113132 from the National Institute of General Medical Sciences of the National Institutes of Health.S

Proteína F del VRSH en conformación pre-fusión estabilizada y anticuerpos neutralizantes específicos frente a la misma.

La presente invención proporciona una proteína de fusión (proteína F) del virus respiratorio sincitial humano (VRSH) en conformación pre-fusión estabilizada, útil para identificar o diseñar anticuerpos y otras moléculas que se unan a ella para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus de la familia Paramyxoviridae, preferiblemente del género Pneumovirus, más preferiblemente por el VRSH. La presente invención se refiere también a un método de obtención de esta proteína así como a anticuerpos y a aptámeros frente a la misma, los cuales son útiles para el diagnóstico, tratamiento y/o prevención de las infecciones mencionadas.REIVINDICACIONES: 1. Proteína F del VRSH en conformación pre-fusión que presenta al menos un 80% de homología con la SEQ ID NO: 1, que comprende: a. al menos un puente disulfuro intermonomérico en la región que comprende los aminoácidos 450 a 550 de su secuencia aminoacídica, y b. al menos dos aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 106 a 109 de su secuencia aminoacídica y al menos cuatro aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 131 a 136 de su secuencia aminoacídica, sustituidos por asparagina o por glutamina. 2. Proteína según la reivindicación 1 que comprende dos puentes disulfuro intermonoméricos formados mediante la sustitución de los aminoácidos Leu481, Asp489, Ser509 y Asp510 por cisteínas. 3. Proteína según cualquiera de las reivindicaciones 1 ó 2, que comprende los aminoácidos básicos Arg106, Arg108, Arg109, Lys131, Lys132, Arg133, Lys134, Arg135 y Arg136 sustituidos por asparagina. 4. Proteína según cualquiera de las reivindicaciones 1 a 3 que además comprende una cola de histidinas en el extremo C-terminal. 5. Proteína según la reivindicación 4 donde la cola de histidinas se ha formado mediante la adición de la SEQ ID NO: 2 en el extremo C-terminal de la proteína. 6. Proteína según cualquiera de las reivindicaciones 1 a 5 que consiste en la secuencia aminoacídica SEQ ID NO: 3. 7. Secuencia nucleotídica aislada que codifica para la proteína según cualquiera de las reivindicaciones 1 a 6. 8. Anticuerpo frente a la proteína según cualquiera de las reivindicaciones 1 a 6. 9. Aptámero frente a la proteína según cualquiera de las reivindicaciones 1 a 6. 10. Método de obtención de la proteína según cualquiera de las reivindicaciones 1 a 6 que comprende: a. sustituir, en una secuencia aminoacídica de la proteína F del VRSH que presenta al menos un 80% de homología con la SEQ ID NO: 1, al menos dos aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 106 a 109 y al menos cuatro aminoácidos básicos del punto de corte de furina que comprende los aminoácidos 131 a 136, por asparagina o por glutamina, b. introducir al menos un puente disulfuro intermonomérico en la región que comprende los aminoácidos 450 a 550 de la secuencia aminoacídica de la proteína F del VRSH del paso (a) , c. expresar la proteína F del VRSH modificada en los pasos (a) y (b) en un sistema de expresión, y d. purificar la proteína expresada en el paso (c) . 11. Método según la reivindicación 10, donde los aminoácidos básicos del paso (a) son Arg106, Arg108, Arg109, Lys131, Lys132, Arg133, Lys134, Arg135 y Arg136 y se sustituyen por asparagina. 12. Método según cualquiera de las reivindicaciones 10 u 11, donde en el paso (b) se introducen dos puentes disulfuro intermonoméricos mediante la sustitución de los aminoácidos Leu481, Asp489, Ser509 y Asp510 de la secuencia aminoacídica de la proteína F del VRSH del paso (a) por cisteínas. 13. Método según cualquiera de las reivindicaciones 10 a 12, que además comprende añadir una cola de histidinas en el extremo C-terminal de la secuencia aminoacídica de la proteína F del VRSH del paso (b) . 14. Método según la reivindicación 13, donde la cola de histidinas se añade mediante la adición de la SEQ ID NO: 2 en el extremo C-terminal de la secuencia aminoacídica de la proteína F del VRSH del paso (b) . 15. Método según cualquiera de las reivindicaciones 10 a 14 donde el sistema de expresión del paso (c) es el virus vaccinia. 16. Uso de la proteína según cualquiera de las reivindicaciones 1 a 6 para la identificación o diseño de compuestos o composiciones para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus pertenecientes al género Pneumovirus. 17. Uso de la proteína según la reivindicación 16 donde el virus es el VRSH. 18. Uso de la proteína según cualquiera de las reivindicaciones 1 a 6 para la elaboración de un medicamento. 19. Uso de la proteína según la reivindicación 18 para la elaboración de un medicamento para el diagnóstico, prevención y/o tratamiento de infecciones producidas por virus pertenecientes al género Pneumovirus. 20. Uso de la proteína según la reivindicación 19 donde el virus es el VRSH. 21. Uso de la proteína según cualquiera de las reivindicaciones 18 a 20 donde el medicamento es una vacuna. 22. Uso del anticuerpo según la reivindicación 8 o del aptámero según la reivindicación 9 para la detección de la proteína F de virus pertenecientes al género Pneumovirus. 23. Uso del anticuerpo o del aptámero según la reivindicación 22 donde el virus es el VRSH. 24. Uso del anticuerpo según la reivindicación 8 o del aptámero según la reivindicación 9 para la elaboración de un medicamento. 25. Uso del anticuerpo o del aptámero según la reivindicación 24 para la elaboración de un medicamento para el tratamiento y/o prevención de infecciones producidas por virus pertenecientes al género Pneumovirus. 26. Uso del anticuerpo o del aptámero según la reivindicación 25 donde el virus es el VRSH.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idiomaInstituto de Salud Carlos IIISolicitud de Patent

Analysis of genetic variability and mapping of point mutations in influenza virus by the RNase A mismatch cleavage method

We have applied the RNase A mismatch cleavage method to analyze genetic variability in RNA viruses by using influenza virus as a model system. Uniformly labeled RNA probes synthesized from a cloned hemagglutinin gene of a given viral strain were hybridized to RNA isolated from other strains of characterized or uncharacterized genetic composition. The RNA.RNA heteroduplexes containing a variable number of base mismatches were digested with RNase A, and the resistant products were analyzed by denaturing polyacrylamide gel electrophoresis. We show that many of these single base mismatches are cleaved by RNase A, generating unique and characteristic patterns of resistant RNA fragments specific for each of the different viral strains. Comparative analysis of the cleavage patterns allows a qualitative estimation of the genetic relatedness and evolution of field strains. We also show that cleavage by RNase A at single base mismatches can readily detect and localize point mutations present in monoclonal antibody-resistant variants. This method should have wide applications in the study of RNA viruses, not only for epidemiological analysis but also in some diagnostic problems, such as characterization of phenotypic mutants.This work was supported by National Institutes of Health Grant CA33021 awarded by the Nationa l Cancer Institute to M.P. and by grants from the Comision Asesora de Investigacion Cientificay Tecnica (Grant 608/438) and Fondo de Investigaciones Sanitarias to J.O. and J.A.M.C.L.-G. was a recipient of a NATO short-term post doctoral fellow-ship while on leave from the Centro Nacional de Microbiologia, Majadahonda, Madri

Exploring the antigenic relatedness of influenza virus haemagglutinins with strain-specific polyclonal antibodies

Alternative methods to the standard haemagglutination inhibition (HI) and neutralization tests to probe the antigenic properties of the influenza virus haemagglutinin (HA) were developed in this study. Vaccinia virus recombinants expressing reference HAs were used to immunize rabbits from which polyclonal antibodies were obtained. These antibodies were subtype specific but showed limited intra-subtype strain specificity in ELISA. The discriminatory capacity of these antibodies was, however, markedly increased after adsorption to cells infected with heterologous influenza viruses, revealing antigenic differences that were otherwise undistinguishable by standard HI and neutralization tests. Furthermore, the unadsorbed antibodies could be used to select escape mutants of the reference strain, which after sequencing unveiled amino acid changes responsible of the noted antigenic differences. These procedures therefore provide alternative methods for the antigenic characterization of influenza HA and might be useful in studies of HA antigenic evolution.This work was supported by grants (JAM) GR09/0039, (IC) GR09/0040 and (JAM) SAF2012-31217.S

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation

UNLABELLED: Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15(-/-)) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE: At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.This work was supported by grant PI11/00590 from Fondo de Investigación Sanitaria to I.M.S

Social, economic, and health impact of the respiratory syncytial virus: a systematic search

BACKGROUND: Bronchiolitis caused by the respiratory syncytial virus (RSV) and its related complications are common in infants born prematurely, with severe congenital heart disease, or bronchopulmonary dysplasia, as well as in immunosuppressed infants. There is a rich literature on the different aspects of RSV infection with a focus, for the most part, on specific risk populations. However, there is a need for a systematic global analysis of the impact of RSV infection in terms of use of resources and health impact on both children and adults. With this aim, we performed a systematic search of scientific evidence on the social, economic, and health impact of RSV infection. METHODS: A systematic search of the following databases was performed: MEDLINE, EMBASE, Spanish Medical Index, MEDES-MEDicina in Spanish, Cochrane Plus Library, and Google without time limits. We selected 421 abstracts based on the 6,598 articles identified. From these abstracts, 4 RSV experts selected the most relevant articles. They selected 65 articles. After reading the full articles, 23 of their references were also selected. Finally, one more article found through a literature information alert system was included. RESULTS: The information collected was summarized and organized into the following topics: 1. Impact on health (infections and respiratory complications, mid- to long-term lung function decline, recurrent wheezing, asthma, other complications such as otitis and rhino-conjunctivitis, and mortality; 2. Impact on resources (visits to primary care and specialists offices, emergency room visits, hospital admissions, ICU admissions, diagnostic tests, and treatments); 3. Impact on costs (direct and indirect costs); 4. Impact on quality of life; and 5. Strategies to reduce the impact (interventions on social and hygienic factors and prophylactic treatments). CONCLUSIONS: We concluded that 1. The health impact of RSV infection is relevant and goes beyond the acute episode phase; 2. The health impact of RSV infection on children is much better documented than the impact on adults; 3. Further research is needed on mid- and long-term impact of RSV infection on the adult population, especially those at high-risk; 4. There is a need for interventions aimed at reducing the impact of RSV infection by targeting health education, information, and prophylaxis in high-risk populations.The project was funded by an unrestricted grant from AbbVie.S

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.This work was supported in part by grants GR09/0023 (A. N.), GR09/0039 (J. A. M.) and GR09/0040 (I. C.) from Instituto de Salud Carlos III under a special research programme on pandemic flu. Additionally, the Biología Viral Unit is supported currently by grant SAF2012-31217 from Plan Nacional I+D+i.S

Marine Synechococcus sp. Strain WH7803 Shows Specific Adaptative Responses to Assimilate Nanomolar Concentrations of Nitrate

Marine Synechococcus, together with Prochlorococcus, contribute to a significant proportion of the primary production on Earth. The spatial distribution of these two groups of marine picocyanobacteria depends on different factors such as nutrient availability and temperature. Some Synechococcus ecotypes thrive in mesotrophic and moderately oligotrophic waters, where they exploit both oxidized and reduced forms of nitrogen. Here, we present a comprehensive study, which includes transcriptomic and proteomic analyses of the response of Synechococcus sp. strain WH7803 to nanomolar concentrations of nitrate, compared to micromolar ammonium or nitrogen starvation. We found that Synechococcus has a specific response to a nanomolar nitrate concentration that differs from the response shown under nitrogen starvation or the presence of standard concentrations of either ammonium or nitrate. This fact suggests that the particular response to the uptake of nanomolar concentrations of nitrate could be an evolutionary advantage for marine Synechococcus against Prochlorococcus in the natural environment. IMPORTANCE Marine Synechococcus are a very abundant group of photosynthetic organisms on our planet. Previous studies have shown blooms of these organisms when nanomolar concentrations of nitrate become available. We have assessed the effect of nanomolar nitrate concentrations by studying the transcriptome and proteome of Synechococcus sp. WH7803, together with some physiological parameters. We found evidence that Synechococcus sp. strain WH7803 does sense and react to nanomolar concentrations of nitrate, suggesting the occurrence of specific adaptive mechanisms to allow their utilization. Thus, very low concentrations of nitrate in the ocean seem to be a significant nitrogen source for marine picocyanobacteria

Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced lung cancer

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n = 3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (pcombined  = 5.66 × 10-5 ; ORcombined  = 2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (pcombined  = 1.02 × 10-4 ; ORcombined  = 2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10DmRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p = 0.01 and p < 0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p = 0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.This work was supported by the Spanish Society of Medical Oncology; Fundación SEOM and Fundación Salud 2000; and Government of Navarra.S