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

Pre-clinical development of BCG.HIVA(CAT), an antibiotic-free selection strain, for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVACAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVACAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVACAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVACAT vaccine strain. The BCG.HIVACAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVACAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8+ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVACAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth

Full blood count values as a predictor of poor outcome of pneumonia among HIV-infected patients

Background To evaluate the predictive value of analytical markers of full blood count that can be assessed in the emergency department for HIV infected patients, with community-acquired pneumonia (CAP). Methods Prospective 3-year study including all HIV-infected patients that went to our emergency department with respiratory clinical infection, more than 24-h earlier they were diagnosed with CAP and required admission. We assessed the different values of the first blood count performed on the patient as follows; total white blood cells (WBC), neutrophils, lymphocytes (LYM), basophils, eosinophils (EOS), red blood cells (RBC), hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, red blood cell distribution width (RDW), platelets (PLT), mean platelet volume, and platelet distribution width (PDW). The primary outcome measure was 30-day mortality and the secondary, admission to an intensive care unit (ICU). The predictive power of the variables was determined by statistical calculation. Results One hundred sixty HIV-infected patients with pneumonia were identified. The mean age was 42 (11) years, 99 (62%) were male, 79 (49%) had ART. The main route of HIV transmission was through parenteral administration of drugs. Streptococcus pneumonia was the most frequently identified etiologic agent of CAP The univariate analysis showed that the values of PLT (p < 0.009), EOS (p < 0.033), RDW (p < 0.033) and PDW (p < 0.09) were predictor of mortality, but after the logistic regression analysis, no variable was shown as an independent predictor of mortality. On the other hand, higher RDW (OR = 1.2, 95% CI 1.1-1.4, p = 0.013) and a lower number of LYM (OR 2.2, 95% CI 1.1-2.2; p = 0.035) were revealed as independent predictors of admission to ICU. Conclusion Red blood cell distribution and lymphocytes were the most useful predictors of disease severity identifying HIV infected patients with CAP who required ICU admission. Electronic supplementary material The online version of this article (10.1186/s12879-018-3090-0) contains supplementary material, which is available to authorized users

Host-dependent editing of SARS-CoV-2 in COVID-19 patients

SARS-CoV-2; Mutacions; QuasiespècieSARS-CoV-2; Mutaciones; CuasiespecieSARS-CoV-2; Mutations; QuasispeciesA common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.This study was supported by the Direcció General de Recerca i Innovació en Salut (DGRIS) of the Catalan Health Ministry, Generalitat de Catalunya, through the Vall d’Hebron Institut de Recerca (VHIR); the European Regional Development Fund (ERDF) “A way to achieve Europe” by the Spanish Network for Research in Infectious Diseases [grant number REIPI RD16/0016/0003]; Instituto de Salud Carlos III [grant number FIS PI19/00301]; and Centro para el Desarrollo Tecnologico Industrial (CDTI) from the Ministry of Economic Affairs and Digital Transformation [grant number IDI-20200297]

Host-dependent editing of SARS-CoV-2 in COVID-19 patients

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants

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]

Advances and challenges in recombinant Mycobacterium bovis

Introduction: Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome, tuberculosis, and malaria are responsible for most human deaths produced by infectious diseases worldwide. Vaccination against HIV requires generation of memory T cells and neutralizing antibodies, mucosal immunity, and stimulation of an innate immune responses. In this context, the use of Mycobacterium bovis bacillus Calmette–Guérin (BCG) as a live vaccine vehicle is a promising approach for T-cell induction. Areas covered: In this review, we provide a comprehensive summary of the literature regarding immunogenicity studies in animal models performed since 2005. Furthermore, we provide expert commentary and 5-year view on how the development of potential recombinant BCG-based HIV vaccines involves careful selection of the HIV antigen, expression vectors, promoters, BCG strain, preclinical animal models, influence of preexisting immunity, and safety issues, for the rational design of recombinant BCG:HIV vaccines to prevent HIV transmission in the general population. Expert commentary: The three critical issues to be considered when developing a rBCG:HIV vaccine are codon optimization, antigen localization, and plasmid stability in vivo. The use of integrative expression vectors are likely to improve the mycobacterial vaccine stability and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth