Characterization of Therapeutic Monoclonal Antibodies Reveals Differences Between In Vitro and In Vivo Time-Course Studies

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

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Monoclonal antibodies targeting the influenza virus N6 neuraminidase

10.1101/2021.02.15.431355 ◽

2021 ◽

Author(s):

Shirin Strohmeier ◽

Fatima Amanat ◽

Juan Manuel Carreño ◽

Florian Krammer

Keyword(s):

Monoclonal Antibodies ◽

Influenza A ◽

Neutralization Assay ◽

Escape Mutant ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Lateral Ridge

AbstractInfluenza A viruses are a diverse species that include 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 NA subtypes are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding and inhibition of the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 and H5N6 mouse challenge models when the antibodies were given prophylactically.ImportanceThe N6 NA has recently gained prominence due to the emergence of highly pathogenic H5N6 viruses. Currently, there is limited characterization of the antigenicity of avian N6 neuraminidase. Our data is an important first step towards a better understanding of the N6 NA antigenicity.

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Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

10.1101/2021.02.02.428995 ◽

2021 ◽

Author(s):

Tal Noy-Porat ◽

Adva Mechaly ◽

Yinon Levy ◽

Efi Makdasi ◽

Ron Alcalay ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Phage Display Library ◽

Host Cells ◽

Terminal Domain ◽

Isolation And Characterization ◽

Recent Emergence ◽

Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

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999 In Vitro and In Vivo Characterization of Neutralizing Monoclonal Antibodies Against Clostridium difficile Toxins a and B

Gastroenterology ◽

10.1016/s0016-5085(13)60657-5 ◽

2013 ◽

Vol 144 (5) ◽

pp. S-185

Author(s):

Dominiek Staelens ◽

Marlies Van de Wouwer ◽

Els Brouwers ◽

Silvia Caluwaerts ◽

Borden Lacy ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clostridium Difficile ◽

In Vivo Characterization

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Immunolocalization and characterization of the low molecular weight antigen (4–5 kDa) ofToxoplasma gondiithat elicits an early IgM response upon primary infection

Parasitology ◽

10.1017/s0031182000068220 ◽

1994 ◽

Vol 108 (2) ◽

pp. 139-145 ◽

Cited By ~ 26

Author(s):

S. Tomavo ◽

G. Couvreur ◽

M. A. Leriche ◽

A. Sadak ◽

A. Achbarou ◽

...

Keyword(s):

Electron Microscopy ◽

Molecular Weight ◽

Monoclonal Antibodies ◽

Primary Infection ◽

Low Molecular Weight ◽

Immuno Electron Microscopy ◽

Surface Localization

SUMMARYA striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the lowMrantigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability betweenToxoplasma gondiitachyzoites grownin vivoandin vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

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Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation.

Journal of Experimental Medicine ◽

10.1084/jem.178.4.1283 ◽

1993 ◽

Vol 178 (4) ◽

pp. 1283-1292 ◽

Cited By ~ 163

Author(s):

Y Matsuzaki ◽

J Gyotoku ◽

M Ogawa ◽

S Nishikawa ◽

Y Katsura ◽

...

Keyword(s):

T Cells ◽

Time Course ◽

Cell Types ◽

Double Positive ◽

Differentiation Potential ◽

Fetal Thymus ◽

Extensive Growth

We found that c-kit-positive, lineage marker-negative, Thy-1lo cells are present in both bone marrow and thymus ("BM c-kit" and "thymus c-kit" cells). Although the two cell types are phenotypically similar, only BM c-kit cells showed the potential to form colonies in vitro as well as in vivo. However, both of them revealed extensive growth and differentiation potential to T cells after direct transfer into an irradiated adult thymus, or a deoxyguanosine-treated fetal thymus. Time course analysis showed that thymus c-kit cells differentiated into CD4CD8 double-positive cells approximately 4 d earlier than BM c-kit cells did. In addition, anti-c-kit antibody blocked T cell generation of BM c-kit cells but not of thymus c-kit cells. Intravenous injection of thymus c-kit resulted in the generation of not only T cells, but B as well as NK1.1+ cells. These data provide evidence that thymus c-kit cells represent common lymphoid progenitors with the differentiation potential to T, B, and possibly NK cells. The c-kit-mediated signaling appears to be essential in the transition from BM c-kit to thymus c-kit cells.

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