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Sviluppo ed umanizzazione di un anticorpo in formato scFv contro β-1,3 glucano
Get PDFINTRODUCTION
Fungal diseases caused by opportunistic pathogenic species of fungi, are increasingly impacting on health of populations, particularly in patients under immunosuppressive chemotherapy and hospitalized. Classic therapies are often unable to control these infections and may be associated with severe side effects and the emergence of resistant fungal strains.
There is increasing interest in the development of new biological tools, such as monoclonal antibodies, to fight these opportunistic fungi. These molecules may be used to replace or integrate the classical antifungal therapy.
Our work aimed to develop and humanize a single chain Fv (scFv) derived from the murine antibody 2G8, which specifically recognizes β1,3-glucan, a major cell wall polysaccharide crucial for growth and survival of the most common pathogenic species of fungi, such as Candida Albicans, Aspergillus fumigatus and Criptococcus neoformans.
METHODS:
The murine framework (FRW 1, 2, 3, 4) regions of 2G8 were humanized using bioinformatics tools by choosing the human variable domains with high sequence homology.
The humanized scFv 2G8 was obtained by CDR-graft method of the murine CDRs into humanized framework regions.
scFv 2G8 was expressed in E. Coli as periplasmic-secreted protein, and purified through 6 His-tag, using immobilized metal chelate affinity chromatography (IMAC) with Ni-Sepharose column.
Biological activity of the molecule was tested by ELISA and in vitro growth inhibition analysis.
RESULTS:
Recombinant protein was purified from insoluble fraction in denatured conditions.
The purified scFv showed a specific binding activity for β1,3-glucans as determined by ELISA test.
scFv 2G8 at the concentration of 100 μg/ml showed a growth inhibition of 33% on Candida Albicans cells with germ tubes.
CONCLUSIONS:
Future studies will concern: site-directed mutagenesis, new in silico modeling studies, new in vitro studies and VL-linker-VH orientation
Development of new biological drugs for the treatment of fungal infections
Get PDFThe microbiological world and especially the fungal panorama is currently facing hard times with the rapid
development and spread of resistance and the birth of new species intrinsically and sometimes multidrug-
resistant. Noted the poor drug arsenal and the lack of effectiveness, an innovative approach had to be found.
The application of biological drugs in this field has always been scarce and limited to a few attempts that
almost always ended up with failures. In this study, we started from a candidate rich in activity potential but
poor in safety and clinical future, the murine monoclonal antibody 2G8. 2G8 demonstrated to be able to bind
selectively β-1,3-glucans, vital components of the cell wall of pathogenic fungi, and to be efficient in
controlling fungal infections. Nevertheless, the murine nature was its major bottleneck, therefore I started my
Ph.D. project with the aim to develop, produce and characterize a humanized antibody starting from 2G8. Two
different antibody formats were evaluated: the single-chain fragment variable (scFv) and the full-length. These
choices reflected two different ways of administration, hence diverse infection severity and urgency level. The
full-length format was designed for systemic applications while the scFv, with its briefer half-life, for topical
use. The canonical CDR-grafting permitted the birth of a humanized single-chain fragment variable (hscFv).
The orientation as VL-linker-VH and the presence of two ubiquitin monomers at the N-terminus and the His-
tag at the C-terminus granted higher quantity, stability, and solubility. With many efforts, we managed to have
a clean product (~40 mg from 1L of bacterial culture) with two steps of purification, a negative passage in Q
Sepharose and a positive passage in Ni2+ Sepharose. hscFv is still able to bind β-1,3-glucans both as coated
antigens and on C. auris and C. albicans cells and to affect the growth of C. auris when combined with
caspofungin and amphotericin B. A black mark is represented by its tendency to aggregate forming disulphide
bonds and, in a minor way, aspecific interactions. Studies on the best formulation led to the substitution of the
reducing agent and to relatively long stability and retention of activity when stored at 4°, -20° and -80° C.
In the meanwhile, with a novel humanisation approach combining different humanization methods, the
humanised full-length monoclonal antibody H5K1 was generated. It was soon compared to the murine 2G8
from which it took immediate distance in terms of performances showing better IC50, AUC and Kd. Among
pathogenic fungi, Candida spp. are the causes of many nosocomial infections and even though C. albicans is
still the prevalent specie, over the past few years a mycological shift toward the non-albicans Candida (NAC)
species has been recorded. Apropos Candida auris is one of the three leading causes of morbidity and mortality
worldwide and is often associated with intrinsic multidrug-resistance and Candida glabrata is associated with
higher mortality compared to other NACs. Both these species were considered as a fungal model to evaluate
hmAb H5K1 effectiveness. H5K1 can bind selectively β-1,3-glucans on C. auris, C. albicans and C. glabrata
cells and on Aspergillus fumigatus and Fusarium solani conidia. In particular, C. auris, C. albicans and C.
glabrata had almost 100% of positive cells with a major binding propensity for budded cells and hyphae.
Surprisingly H5K1 demonstrated activity alone in C. auris growth and adhesion inhibition and cell wall
perturbation and in C. glabrata biofilm matrix alteration. However, the best activity was revealed in
combination with amphotericin B and echinocandins on both C. auris and C. glabrata (of the latter two strains
had low susceptibility for echinocandins and one was a biofilm hyper producer). H5K1 establishes additivity
with echinocandins and synergy with amphotericin B potentiating its fungicidal activity as well. This happens
also at low concentrations of both hmAb H5K1 and of the antifungal drugs being an advantage considering
the side effects of the available antifungal drugs used for prolonged periods and at high doses. In addition to
its intrinsic and combinatorial efficacy, hmAb H5K1 keeps being a full-length antibody then able to be
recognized by macrophages. As a matter of fact, after C. auris opsonization, macrophage efficiency increased,
and the phagocytosis was enhanced.
In conclusion, hscFv and hmAb H5K1, both born from the murine hmAb 2G8, demonstrated comparable and
sometimes better performances than their parental. They are effective in vitro especially in combination with
amphotericin B and echinocandins showing to be able to modulate not just the fungal growth and adhesion but
also the biofilm formation. The two different antibody formats are destined for different applications but in
view of this fact, the full-length H5K1, suitable for systemic use, can also enhance the activity of phagocytic
cells of the immune system.
These data showed suggest that hscFv and hmAb H5K1 could be new drug candidates for the treatment of
fungal infections and especially, candidiasis. Although the encouraging results, hscFv must be further explored
while, noted the promising preclinical outcomes of hmAb H5K1, we are optimistic and hopeful about its rapid
moving to clinical phases with the name of Dia-T51
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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