Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles

VPH; Pichia pastoris; PurificaciónVPH; Pichia pastoris; PurificacióHPV; Pichia pastoris; PurificationCurrently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 681137. In addition, we acknowledge support by Instituto de Salud Carlos III (RETIC-RIS RD12/0017, FIS PI14/00494, and FIS PI20/00217), Direcció General de Recerca i Innovació en Salut (DGRIS), Catalan Health Ministry Generalitat de Catalunya, and Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number IDI-20200297

The frequency of defective genomes in Omicron differs from that of the Alpha, Beta and Delta variants

Evolution; Genetics; Molecular biologyEvolució; Genètica; Biologia molecularEvolución; Genética; Biología molecularThe SARS-CoV-2 Omicron variant emerged showing higher transmissibility and possibly higher resistance to current COVID-19 vaccines than other variants dominating the global pandemic. In March 2020 we performed a study in clinical samples, where we found that a portion of genomes in the SARS-CoV-2 viral population accumulated deletions immediately before the S1/S2 cleavage site (furin-like cleavage site, PRRAR/S) of the spike gene, generating a frameshift and appearance of a premature stop codon. The main aim of this study was to determine the frequency of defective deletions in prevalent variants from the first to sixth pandemic waves in our setting and discuss whether the differences observed might support epidemiological proposals. The complete SARS-CoV-2 spike gene was deeply studied by next-generation sequencing using the MiSeq platform. More than 90 million reads were obtained from respiratory swab specimens of 78 COVID-19 patients with mild infection caused by the predominant variants circulating in the Barcelona city area during the six pandemic waves: B.1.5, B.1.1, B.1.177, Alpha, Beta, Delta, and Omicron. The frequency of defective genomes found in variants dominating the first and second waves was similar to that seen in Omicron, but differed from the frequencies seen in the Alpha, Beta and Delta variants. The changing pattern of mutations seen in the various SARS-CoV-2 variants driving the pandemic waves over time can affect viral transmission and immune escape. Here we discuss the putative biological effects of defective deletions naturally occurring before the S1/S2 cleavage site during adaption of the virus to human infection.This study was partially supported by Pla Estratègic de Recerca i Innovació en Salut (PERIS) – Direcció General de Recerca i Innovació en Salut (DGRIS), Catalan Health Ministry, Generalitat de Catalunya; the Spanish Network for the Research in Infectious Diseases (REIPI RD16/0016/0003) from the European Regional Development Fund (ERDF); Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business, grant number IDI-20200297; Grant PI19/00301 from Instituto de Salud Carlos III cofinanced by the European Regional Development Fund (ERDF), and Gilead’s biomedical research project GLD21/00006. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID’s EpiCov Database on which this research is based

A year living with SARS-CoV-2: an epidemiological overview of viral lineage circulation by whole-genome sequencing in Barcelona city (Catalonia, Spain)

Catalonia; SARS-CoV-2; Molecular epidemiologyCataluña; SARS-CoV-2; Epidemiología molecularCatalunya; SARS-CoV-2; Epidemiologia molecularHerein, we describe the genetic diversity of circulating SARS-CoV-2 viruses by whole-genome sequencing (WGS) in Barcelona city (Catalonia, Spain) throughout the first four pandemic waves. From weeks 11/2020–24/2021, SARS-CoV-2-positive respiratory samples were randomly selected per clinical setting (80% from primary care or 20% from the hospital), age group, and week. WGS was performed following the ARTICv3 protocol on MiSeq or NextSeq2000 Illumina platforms. Nearly complete consensus sequences were used for genetic characterization based on GISAID and PANGOLIN nomenclatures. From 2475 samples, 2166 (87%) were fully sequenced (78% from primary care and 22% from hospital settings). Multiple genetic lineages were co-circulating, but four were predominant at different periods. While B.1.5 (50.68%) and B.1.1 (32.88%) were the major lineages during the first pandemic wave, B.1.177 (66.85%) and B.1.1.7 (83.80%) were predominant during the second, third, and fourth waves, respectively. Almost all (96.4%) were carrying D614G mutation in the S protein, with additional mutations that define lineages or variants. But some mutations of concern, such as E484K from B.1.351 and P.1 lineages are currently under monitoring, together with those observed in the receptor-binding domain or N-terminal domain, such as L452R and T478K from B.1.617.2 lineage. The fact that a predominant lineage was observed in each pandemic wave suggests advantageous properties over other contemporary co-circulating variants. This genetic variability should be monitored, especially when a massive vaccination campaign is ongoing because the potential selection and emergence of novel antigenic SARS-CoV-2 strains related to immunological escapement events.This work was supported by Spanish Network for the Research in Infectious Diseases: [Grant Number REIPI RD16/0016/0003]; Centro para el Desarrollo Tecnológico Industrial (CDTI) from the Spanish Ministry of Economy and Business [Grant Number IDI-20200297]; Direcció General de Recerca i Innovació en Salut (DGRIS); European Development Regional Fund (ERDF) “A way to achieve Europe” by Spanish Network for Research in Infectious Diseases [REIPI RD16/0016/0003]