Virus like particles expressed in insect cells and mammalian cells as a plataform for the development of a Zika vaccine

The Zika virus (ZIKV) is an emergent mosquito-borne virus of the flaviviridae family that has caused severe challenges to global health since 2015. It causes Guillain-Barré syndrome and congenital malformations. Although case numbers have decreased, it is important to develop a vaccine for outbreaks. One alternative is the use of virus like particles (VLP) as vaccines. ZIKV is enveloped and is composed of three main structural proteins: enveloped (E), pre-membrane (M), and capsid. The main target of neutralizing antibodies is the E glycoprotein, which is glycosylated in some strains. The N-glycosylation profile is determined by the producer host cell. ZIKV has both insect and human hosts, and the N-glycosylation profile of the E protein produced by each host is expected to be different. It can be expected that glycosylation pattern has an impact on immune response against the E protein, but its effect on the immunogenicity against VLP of ZIKV has not been determined. For this reason, the present work seeks in the first instance, to design and produce VLP of ZIKV (ZIK VLP) in insect and human cells. To produce ZIK VLP, a chimeric gene was designed containing the M and E ZIKV genes fused to the transmembrane (TM) domain of Japanese encephalitis virus (JEV), ss- M-E (minus) ZIKV, TM JEV. After that, a recombinant baculovirus that contains the chimeric sequence was generated for VLP expression in insect cells. Production kinetics were followed, and the best conditions for VLP production were determined. For expression in human cells, the chimera was introduced into lentiviral vectors and was produced in HEK-293T/17 cells and used for the stable transfection of HEK-293 cells producing ZIKV VLP. High producing clones were selected by flow citometry. ZIK VLP were purified and characterized. In this work, strategies were developed for the efficient production of PPV in both systems, which can be used for further research. Ongoing studies are focused on determining the glycosylation profile of VLP expressed in both systems and on investigating the impact of glycosylation pattern of ZIK VLP immunogenicity in an animal model

Rotavirus VP6 nanotubes show an antigen form-dependent adjuvant activity: Zika virus envelope protein monomer vs Zika virus-like particles

The structural protein of rotavirus (RV) VP6 can self-assemble into tubular polymeric structures under specific conditions of pH and ionic strength when expressed in recombinant systems. Previous studies have shown that RV VP6 nanotubes (VP6NT) have an adjuvant effect on the immunogenicity of norovirus virus-like particles (VLPs) in mice (Blazevic et al., 2011; Malm et al., 2016). The present study focused on the determination of adjuvant activity of VP6NT on the immunogenicity of monomers of the viral envelope (E) protein or of Zika virus (ZikV) VLPs. ZikV infection can cause congenital malformations in fetuses and the Guillain-Barré syndrome in adults, as the most severe consequences. To date, there is no treatment or vaccine available against ZikV. Several vaccine candidates against this virus have been reported and E protein has been selected as the primary antigenic determinant. Please click Download on the upper right corner to see the full abstract

Defining the multiplicity and type of infection for the production of Zaire Ebola virus-like particles in the insect cell baculovirus expression system

Ebola virus hemorrhagic fever affects thousands of people worldwide with high mortality rates. The Ebola virus has a short incubation time between 2-21 days and death usually occurs within 4-10 days1. Ebola virus disease is characterized by a sudden onset of fever, weakness, headache, diarrhea and vomiting, internal and external bleeding2. In the Filovirus family, Zaire Ebola virus (ZEBOV) is the most aggressive and virulent species, its fatality rates have been reported to be up to 90%3. Even when important advances in vaccine development have occurred, the need of safe and effective vaccines persists4. An alternative is the production of virus-like particles, which are formed by the recombinant virus structural proteins that self-assemble into highly immunogenic structures5. The ZEBOV contains three main structural proteins: the glycoprotein (GP), the viral matrix protein 40 (VP40) and the nucleoprotein (NP). GP induces humoral and cellular responses by itself but when VP40 is co-expressed, the immune response increases in a mouse model6. NP determines the structure of the resulting VLP. To our knowledge, there is no information about the production conditions that result in coexpression and assembly of ZEBOV recombinant proteins. In this work, a multifactorial experimental design was used to evaluate 32 different conditions for the production of the ZEBOV structural proteins utilizing the insect cell-baculovirus expression system technology (BEST). Multiplicity (MOI = 0.1 or 5 ufp/cell) and consecutive times of infection (0 or 6 hours after the first infection) were the principal factors, and the production of each recombinant protein and assembly of VLP were the evaluated responses. We observed that multiplicity of infection had an impact over expression of the recombinant proteins, higher multiplicities increased yield and VLP assembly. In contrast, later times of infection reduced the production of each protein. The initial presence of VP40 resulted in a higher concentration of NP. The conditions where the simultaneous expression of the three structural proteins and where VLP were detected were identified. The highest MOIs for bacVP40 and bacGP were needed. bacNP should be added during the initial infection with an MOI of 0.1, or at 6 hpi at MOI of 5. The obtained ZEBOV-VLPs were similar to native virus. The obtained VLP are a candidate vaccine under evaluation. Research performed thanks to the financial support of PAPIIT-UNAM IT200418 and CONACyT 247101. References: 1. Shuaib F, Gunala R, Musa EO, Mahoney FJ, et al., 2014. Ebola virus disease outbreak-Nigeria, July–September 2014. Morb. Mortal. Wkly. Rep. 63 (39),867–872. 2. Qiu X, Audet J, Wong G. Fernando L, et al., 2013. Sustained protection virus infection following treatment of infected nonhuman primates with ZMAb. Sci. Rep. 3, 3. Richardson JS, Wong G, Pillet S, Schindle S, et al., 2011. Evaluation of different strategies for post-exposure treatment of Ebola virus infection in rodents. J.Bioterror. Biodef. S1, 007 4. Ige, Ohimain E, 2016. Recent advances in the development of vaccines for Ebola virus disease. Virus Research 211: 174-185. 5. Palomares LA, Ramírez OT, 2009. Challenges for the production of virus-like particles in insect cells: The case of rotavirus-like particles. Biochem. Eng. J. 45: 158-167. 6. Wahl-Jensen, V. et al (2005). Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages. Journal of Virology, 79(4), 2413-241

CRISPR/Cas9 as a tool to correct point mutations in a recombinant monoclonal antibody gene in the genome of CHO cells

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Recent Changes in Breast Cancer Incidence in Spain, 1980–2004

BACKGROUND: Since the 1980s, Spain experienced two decades of sharply increasing breast cancer incidence. Declines in breast cancer incidence have recently been reported in many developed countries. We examined whether a similar downturn might have taken place in Spain in recent years. METHODS: Cases of invasive female breast cancer were drawn from all population-based Spanish cancer registries that had at least 10 years of uninterrupted registration over the period 1980-2004. Overall and age-specific changes in incidence rates were evaluated using change-point Poisson models, which allow for accurate detection and estimation of trend changes. All statistical tests were two-sided. RESULTS: A total of 80,453 incident cases of invasive breast cancer were identified. Overall age- and registry-adjusted incidence rates rose by 2.9% (95% confidence interval [CI] = 2.7% to 3.1%) annually during the 1980s and 1990s; there was a statistically significant change in this trend in 2001 (95% CI = 1998 to 2004; P value for the existence of a change point <.001), after which incidence declined annually by 3.0% (95% CI = 1.8% to 4.1%). This trend differed by age group: There was a steady increase in incidence for women younger than 45 years, an abrupt downturn in 2001 for women aged 45-64 years, and a gradual leveling off in 1995 for women aged 65 years or older. Separate analyses for registries that had at least 15 years of uninterrupted registration detected a statistically significant interruption of the previous upward trend in breast cancer incidence in provinces that had aggressive breast cancer screening programs and high screening participation rates, including Navarra (change point = 1991, P < .001), Granada (change point = 2002, P = .003), Bizkaia (change point = 1998, P < .001), Gipuzkoa (change point = 1998, P = .001), and Araba (change point = 1997, P = .002). CONCLUSIONS: The recent downturn in breast cancer incidence among Spanish women older than 45 years is best explained by a period effect linked to screening saturation.Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP) (AC07-005 to M.P., PM07-004 to R.P-B.) and Carlos III Institute of Health (ISCIII-CIBERESP collaborative agreement “Acción Transversal del Cancer”).S

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved understanding of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, interrogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain transcriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/

Common variants in Alzheimer’s disease and risk stratification by polygenic risk scores

Funder: Funder: Fundación bancaria ‘La Caixa’ Number: LCF/PR/PR16/51110003 Funder: Grifols SA Number: LCF/PR/PR16/51110003 Funder: European Union/EFPIA Innovative Medicines Initiative Joint Number: 115975 Funder: JPco-fuND FP-829-029 Number: 733051061Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease

New insights into the genetic etiology of Alzheimer's disease and related dementias

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies

Background Genome-wide association studies (GWAS) in Parkinson's disease have increased the scope of biological knowledge about the disease over the past decade. We aimed to use the largest aggregate of GWAS data to identify novel risk loci and gain further insight into the causes of Parkinson's disease. Methods We did a meta-analysis of 17 datasets from Parkinson's disease GWAS available from European ancestry samples to nominate novel loci for disease risk. These datasets incorporated all available data. We then used these data to estimate heritable risk and develop predictive models of this heritability. We also used large gene expression and methylation resources to examine possible functional consequences as well as tissue, cell type, and biological pathway enrichments for the identified risk factors. Additionally, we examined shared genetic risk between Parkinson's disease and other phenotypes of interest via genetic correlations followed by Mendelian randomisation. Findings Between Oct 1, 2017, and Aug 9, 2018, we analysed 7·8 million single nucleotide polymorphisms in 37 688 cases, 18 618 UK Biobank proxy-cases (ie, individuals who do not have Parkinson's disease but have a first degree relative that does), and 1·4 million controls. We identified 90 independent genome-wide significant risk signals across 78 genomic regions, including 38 novel independent risk signals in 37 loci. These 90 variants explained 16–36% of the heritable risk of Parkinson's disease depending on prevalence. Integrating methylation and expression data within a Mendelian randomisation framework identified putatively associated genes at 70 risk signals underlying GWAS loci for follow-up functional studies. Tissue-specific expression enrichment analyses suggested Parkinson's disease loci were heavily brain-enriched, with specific neuronal cell types being implicated from single cell data. We found significant genetic correlations with brain volumes (false discovery rate-adjusted p=0·0035 for intracranial volume, p=0·024 for putamen volume), smoking status (p=0·024), and educational attainment (p=0·038). Mendelian randomisation between cognitive performance and Parkinson's disease risk showed a robust association (p=8·00 × 10−7). Interpretation These data provide the most comprehensive survey of genetic risk within Parkinson's disease to date, to the best of our knowledge, by revealing many additional Parkinson's disease risk loci, providing a biological context for these risk factors, and showing that a considerable genetic component of this disease remains unidentified. These associations derived from European ancestry datasets will need to be followed-up with more diverse data. Funding The National Institute on Aging at the National Institutes of Health (USA), The Michael J Fox Foundation, and The Parkinson's Foundation (see appendix for full list of funding sources)