11 research outputs found
Towards updatable, multivalent Covid vaccines: A platform process to produce trimeric spike protein of SARS-COV-2 variants expressed in HEK293 stable cell clones
Within 2.5 years since SARS-COV-2 emergence, COVID-19 pandemic has caused more than 6.3 million registered deaths and 530 million registered cases. The quick development of safe and effective vaccines was very important to mitigate the sanitary crisis. However, the continuous emergence of virus variants with increasing transmissibility continues to cause periodic outbreaks worldwide. The original vaccines keep protecting from severe disease and death caused by variants, but not from transmission and mild disease. Thus, new and improved vaccines are necessary, and updatable, multivalent pan-variant vaccines might be one way to control SARS-COV-2.
The spike (S) protein of SARS-COV-2, a highly glycosylated and very large protein (1380 amino acids), is a key target for diagnostics, therapeutics (e.g. monoclonal antibodies) and vaccines. Very early in the pandemic, Wrapp et al. (https://doi.org/10.1126/science.abb2507) produced the recombinant spike protein as a trimer stabilized in the prefusion conformation and determined its structure by cryoelectron microscopy, providing evidence that it binds to the ACE-2 receptor. The stabilized spike trimer forms the basis of most approved COVID vaccines, across vaccine platforms.
In the present work, we studied the expression and purification of the trimeric prefusion-stabilized spike protein in HEK293 cell lines and developed a platform process applicable to SARS-COV-2 variants (current - and potentially future - ones). Parental HEK293 cells (NRC, Canada or Thermofisher, USA) were transfected by lipofection using Lipofectamine 3000 (Thermofisher Scientific, USA) with a plasmid containing the gene encoding the spike protein. At first, for the ancestral strain (Wuhan), we used a plasmid intended for transient expression kindly provided by VRC/NIAID/NIH (USA). However, for the variants, we ordered synthetic genes (Genscript, USA) that were subcloned in a plasmid intended for stable expression. After genetic modification, stably transfected cells were maintained in the presence of G418 sulfate selection agent. After 3-4 weeks, stable cell pools were obtained and submitted to single cell deposit (FACS Aria, BD Biosciences) in order to obtain clonally-derived cell lines. Documented research cell banks of selected clones were cryopreserved. Batch and fed-batch cultivations were investigated in shake flasks and bioreactors, using the chemically defined HEK TF culture medium and HEK FS feed solution (both Sartorius Xell, Germany). For protein purification, different chromatographic techniques were investigated using Akta Purifier and Akta Pilot systems. Detection of the spike protein secreted in cell culture supernatant was performed by immunoblot, whereas UV280 (Nanodrop, Thermofisher, USA) was used for protein quantitation in purified samples.
After first expressing the spike protein in February 2020 by transient transfection, we developed a stable cell pool by co-transfecting the same transient expression plasmid and an empty stable expression plasmid. This stable cell pool allowed the generation of Wuhan protein that was used to develop serological tests and a hyperimmune equine serum (Cunha et al., doi: 10.1016/j.isci.2021.103315; Alvim et al., in press) and was donated so far to over 90 laboratories in Brazil for basic or applied research.
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A numerical study of the development of bulk scale-free structures upon growth of self-affine aggregates
During the last decade, self-affine geometrical properties of many growing
aggregates, originated in a wide variety of processes, have been well
characterized. However, little progress has been achieved in the search of a
unified description of the underlying dynamics. Extensive numerical evidence
has been given showing that the bulk of aggregates formed upon ballistic
aggregation and random deposition with surface relaxation processes can be
broken down into a set of infinite scale invariant structures called "trees".
These two types of aggregates have been selected because it has been
established that they belong to different universality classes: those of
Kardar-Parisi-Zhang and Edward-Wilkinson, respectively. Exponents describing
the spatial and temporal scale invariance of the trees can be related to the
classical exponents describing the self-affine nature of the growing interface.
Furthermore, those exponents allows us to distinguish either the compact or
non-compact nature of the growing trees. Therefore, the measurement of the
statistic of the process of growing trees may become a useful experimental
technique for the evaluation of the self-affine properties of some aggregates.Comment: 19 pages, 5 figures, accepted for publication in Phys.Rev.
Conformational stability of SARS-CoV-2 glycoprotein spike variants
Summary: The severe acute respiratory syndrome spread worldwide, causing a pandemic. SARS-CoV-2 mutations have arisen in the spike, a glycoprotein at the viral envelope and an antigenic candidate for vaccines against COVID-19. Here, we present comparative data of the glycosylated full-length ancestral and D614G spike together with three other transmissible strains classified by the World Health Organization as variants of concern: beta, gamma, and delta. By showing that D614G has less hydrophobic surface exposure and trimer persistence, we place D614G with features that support a model of temporary fitness advantage for virus spillover. Furthermore, during the SARS-CoV-2 adaptation, the spike accumulates alterations leading to less structural stability for some variants. The decreased trimer stability of the ancestral and gamma and the presence of D614G uncoupled conformations mean higher ACE-2 affinities compared to the beta and delta strains. Mapping the energetics and flexibility of variants is necessary to improve vaccine development
Intradermal Immunization of SARS-CoV-2 Original Strain Trimeric Spike Protein Associated to CpG and AddaS03 Adjuvants, but Not MPL, Provide Strong Humoral and Cellular Response in Mice
Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19
Cancer incidence in Italian contaminated sites
INTRODUCTION: The incidence of cancer among residents in sites contaminated by
pollutants with a possible health impact is not adequately studied. In Italy,
SENTIERI Project (Epidemiological study of residents in National Priority
Contaminated Sites, NPCSs) was implemented to study major health outcomes for
residents in 44 NPCSs.
METHODS: The Italian Association of Cancer Registries (AIRTUM) records cancer
incidence in 23 NPCSs. For each NPCSs, the incidence of all malignant cancers
combined and 35 cancer sites (coded according to ICD-10), was analysed
(1996-2005). The observed cases were compared to the expected based on age
(5-year period,18 classes), gender, calendar period (1996-2000; 2001-2005),
geographical area (North-Centre and Centre-South) and cancer sites specific
rates. Standardized Incidence Ratios (SIR) with 90% Confidence Intervals were
computed.
RESULTS: In both genders an excess was observed for overall cancer incidence (9%
in men and 7% in women) as well as for specific cancer sites (colon and rectum,
liver, gallblad-der, pancreas, lung, skin melanoma, bladder and Non Hodgkin
lymphoma). Deficits were observed for gastric cancer in both genders, chronic
lymphoid leukemia (men), malignant thyroid neoplasms, corpus uteri and connective
and soft-tissue tumours and sarcomas (women).
DISCUSSION: This report is, to our knowledge, the first one on cancer risk of
residents in NPCSs. The study, although not aiming to estimate the cancer burden
attributable to the environment as compared to occupation or life-style, supports
the credibility of an etiologic role of environmental exposures in contaminated
sites. Ongoing analyses focus on the interpretation of risk factors for excesses
of specific cancer types overall and in specific NPCSs in relation to the
presence of carcinogenic pollutants
Cancer incidence in Italian contaminated sites
The incidence of cancer among residents in sites contaminated by pollutants with a possible health impact is not adequately studied. In Italy, SENTIERI Project (Epidemiological study of residents in National Priority Contaminated Sites, NPCSs) was implemented to study major health outcomes for residents in 44 NPCSs. Methods: The Italian Association of Cancer Registries (AIRTUM) records cancer incidence in 23 NPCSs. For each NPCSs, the incidence of all malignant cancers combined and 35 cancer sites (coded according to ICD-10), was analysed (1996-2005). The observed cases were compared to the expected based on age (5-year period, 18 classes), gender, calendar period (1996-2000; 2001-2005), geographical area (North-Centre and Centre-South) and cancer sites specific rates. Standardized Incidence Ratios (SIR) with 90% Confidence Intervals were computed. Results: In both genders an excess was observed for overall cancer incidence (9% in men and 7% in women) as well as for specific cancer sites (colon and rectum, liver, gallbladder, pancreas, lung, skin melanoma, bladder and Non Hodgkin lymphoma). Deficits were observed for gastric cancer in both genders, chronic lymphoid leukemia (men), malignant thyroid neoplasms, corpus uteri and connective and soft-tissue tumours and sarcomas (women). Discussion: This report is, to our knowledge, the first one on cancer risk of residents in NPCSs. The study, although not aiming to estimate the cancer burden attributable to the environment as compared to occupation or life-style, supports the credibility of an etiologic role of environmental exposures in contaminated sites. Ongoing analyses focus on the interpretation of risk factors for excesses of specific cancer types overall and in specific NPCSs in relation to the presence of carcinogenic pollutants
Musculoskeletal manifestations of childhood cancer and differential diagnosis with juvenile idiopathic arthritis (ONCOREUM): a multicentre, cross-sectional study
Summary
Background
Presenting symptoms of childhood cancers might mimic those of rheumatic diseases. However, the evidence available to guide differential diagnosis remains scarce. Preventing wrong or delayed diagnosis is therefore important to avoid incorrect administration of glucocorticoid or immunosuppressive therapy and worsening of prognosis. As such, we aimed to assess the prevalence and characteristics of presenting musculoskeletal manifestations in patients at cancer onset and to identify the factors that differentiate childhood malignancies with arthropathy from juvenile idiopathic arthritis.
Methods
We did a multicentre, cross-sectional study at 25 paediatric haemato-oncology centres and 22 paediatric rheumatology centres in Italy. We prospectively recruited patients who were younger than 16 years that were newly diagnosed with cancer or juvenile idiopathic arthritis. We excluded patients with glucocorticoid pre-treatment (>1 mg/kg per day of oral prednisone or equivalent for â„2 consecutive weeks). We collected data for patients with a new diagnosis of cancer or juvenile idiopathic arthritis using an electronic case report form on a web-based platform powered by the Cineca Interuniversity Consortium. The primary outcome was to describe the frequency and characteristics of musculoskeletal manifestations at cancer onset; and the secondary outcome was to identify factors that could discriminate malignancies presenting with arthropathy, with or without other musculoskeletal symptoms, from juvenile idiopathic arthritis using multivariable logistic regression analysis.
Findings
Between May 1, 2015, and May 31, 2018, 1957 patients were eligible, of which 1277 (65%) had cancer and 680 (35%) had juvenile idiopathic arthritis. Musculoskeletal symptoms occurred in 324 (25% [95% CI 23·0â27·8]) of 1277 patients with cancer, of whom 207 had arthropathy. Patients with malignant bone tumours had the highest frequency of musculoskeletal symptoms (53 [80%] of 66), followed by patients with Langerhans histiocytosis (16 [47%] of 34), leukaemia (189 [32%] of 582), soft-tissue sarcomas (16 [24%] of 68), and neuroblastoma (21 [19%] of 109). In the 324 patients with cancer and musculoskeletal symptoms, the most common complaints were joint pain (199 [61%]), followed by limb bone pain (112 [35%]). Joint involvement had a prevalent monoarticular pattern (100 [48%] of 207) and oligoarticular pattern (86 [42%] had 2â4 joints involved and 20 [10%] had >4 joints involved), with the most frequently involved joints being the hip (88 [43%] of 207) and knee (81 [39%]). On multivariable analysis, limb bone pain was the independent variable most strongly associated with cancer (odds ratio [OR] 87·80 [95% CI 18·89â408·12]), followed by weight loss (59·88 [6·34â565·53]), thrombocytopenia (12·67 [2·40â66·92]), monoarticular involvement (11·30 [4·09â31·19]), hip involvement (3·30 [1·13â9·61]), and male sex (2·40 [1·03â5·58]). Factors independently associated with juvenile idiopathic arthritis were morning stiffness (OR 0·04 [95% CI 0·01â0·20]), joint swelling (0·03 [0·01â0·09]), and involvement of the small hand joints (0·02 [0â1·05]).
Interpretation
Our study provides detailed information about presenting musculoskeletal manifestations of childhood cancers and highlights the clinical and laboratory features that are most helpful in the differential diagnosis with juvenile idiopathic arthritis
Multiple involvement of oxidative stress in Werner syndrome phenotype
Werner syndrome is a genetic disease characterized by early ageing, excess cancer risk, high incidence of type II diabetes mellitus, early atherosclerosis, ocular cataracts, and osteoporosis. The protein encoded by the defective gene, WRN (WRNp) associates with three activities, that is, a RecQ DNA helicase, 3'-5'-exonuclease and ATPase activities. By highlighting the DNA helicase activity, a widespread consensus in WS-associated defect(s) has been established, pointing toward a deficiency in maintaining DNA integrity. However, a possible involvement of redox pathways in WS may be suggested by several lines of evidence that include: (i) the multiple functions and interactions of WRNp with oxidative stress-related activities and factors; (ii) the pleiotropic WS clinical phenotype encompassing a number of oxidative stress-related pathologies; (iii) redox-related toxicity mechanisms of several xenobiotics exerting excess toxicity in WS cells; (iv) recent in vivo and in vitro findings of redox abnormalities in WS patients and in WS cells. The working hypothesis is raised that a deficiency in WRNp, and the pleiotropic clinical phenotype in WS patients may provide the basis to envision an underlying in vivo prooxidant state, which causes oxidative damage to biomolecules, with multiple oxidative stress-related alterations, resulting in multi-faceted clinical consequence
LâattivitĂ dei Centri Antifumo italiani tra problematiche e aree da potenziare: i risultati di unâindagine svolta attraverso un questionario on-line
Introduzione. In Italia sono 295 i Servizi per la cessazione dal
fumo di tabacco (Centri Antifumo - CA) afferenti al Servizio Sanitario
Nazionale (SSN) censiti nel 2011 dallâOsservatorio Fumo,
Alcol e Droga (OssFAD) dellâIstituto Superiore di SanitĂ . La presente
indagine, condotta dallâOssFAD in collaborazione con i CA,
Ăš stata volta a rilevare alcune delle problematiche con le quali il
personale dei CA si confronta per portare avanti la propria attivitĂ
e le iniziative ritenute utili per migliorarla.
Materiali e metodi. Lâindagine Ăš stata condotta dal 7 al 21
maggio 2012, mediante un questionario compilabile on-line
composto da 5 brevi sezioni di domande con un totale di 38
items da completare. Il link al questionario on-line Ăš stato inviato
per e-mail a 322 indirizzi dei CA censiti nel 2011 dallâOssFAD. I
dati raccolti sono stati elaborati statisticamente con il programma
SPSS 20.
Risultati. Allâindagine hanno risposto 146 operatori dei CA
(45,3%). Sebbene ci siano aspetti ormai consolidati dellâattivitĂ
dei CA, sono ancora molte le criticitĂ che gli operatori riscontrano
nella loro attivitĂ . Le principali problematiche che influiscono
in modo fondamentale/rilevante per la buona attivitĂ
del centro sono le âScarse o nulle risorse economicheâ per il
60,7% del personale, âla mancanza di personale dedicatoâ per
il 52,4% del personale; il âriconoscimento/mandato istituzionale
del CAâ per il 40,9% del personale. Tra le azioni ritenute
piĂč efficaci per facilitare lâaccesso ai CA sono risultate la sensibilizzazione
del personale sanitario (91%), in particolare dei medici
di famiglia e lâinserimento delle prestazioni antitabagiche
nei LEA (76,8%).
Conclusioni. Ă auspicabile che lâattivitĂ dei CA riceva una maggiore
attenzione, attraverso la dotazione di strutture, personale
e finanziamenti adeguati a svolgere un importante ruolo nella
tutela e promozione della salute