scholarly journals In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

2019 ◽  
Vol 18 (1) ◽  
pp. 266-273 ◽  
Author(s):  
Jonathan Hurtado ◽  
Dhiraj Acharya ◽  
Huafang Lai ◽  
Haiyan Sun ◽  
Somanath Kallolimath ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nicotiana Benthamiana ◽  
Chikungunya Virus ◽  
Wild Type ◽  
In Vivo Efficacy

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

2018 ◽  
Author(s):  
Vally Kommineni ◽  
Matthew Markert ◽  
Zhongjie Ren ◽  
Sreenath Palle ◽  
Berenice Carrillo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Cost Effective ◽  
Fold Increase ◽  
Expression Platform ◽  
Dependent Cell ◽  
Anti Cd20

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e. effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

scholarly journals In Vivo Packaging of Brome Mosaic Virus RNA3, but Not RNAs 1 and 2, Is Dependent on a cis-Acting 3′ tRNA-Like Structure

2006 ◽  
Vol 81 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Padmanaban Annamalai ◽  
A. L. N. Rao
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Untranslated Region ◽  
Northern Blot Analysis ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Cis Acting

ABSTRACT The four encapsidated RNAs of brome mosaic virus (BMV; B1, B2, B3, and B4) contain a highly conserved 3′ 200-nucleotide (nt) region encompassing the tRNA-like structure (TLS) which is required for packaging in vitro (Y. G. Choi, T. W. Dreher, and A. L. N. Rao, Proc. Natl. Acad. Sci. USA 99:655-660, 2002). To validate these observations in vivo, we performed packaging assays using Agrobacterium-mediated transient expression of RNAs and coat protein (CP) (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005). Coexpression of TLS-less constructs of B1 or B2 or B3 and CP mRNAs in Nicotiana benthamiana leaves resulted in packaging of TLS-less B1 and B2 but not B3, suggesting that packaging of B3 requires the TLS in cis. This conjecture was confirmed by the efficient packaging of a B3 chimera in which the viral TLS was replaced with a cellular tRNATyr. When N. benthamiana leaves were infiltrated with a mixture of transformants containing wild-type B1 (wtB1) plus wtB2 plus a TLS-less B3 (wtB1+wtB2+TLS-lessB3), the 3′ end of progeny B3 was restored by heterologous recombination with that of either B1 or B2. This intrinsic cis-requirement of TLS in promoting B3 packaging was further confirmed when a mixture containing agrotransformants of TLS-less B1+B2+B3 was supplemented with either wtB4 or a 3′ 200-nt or 3′ 336-nt untranslated region (UTR) of B3. Northern blot analysis followed by sequencing of B3 progeny revealed that replication of TLS-less B3, but not TLS-less B1 or B2, was fully restored due to recombination with TLS from transiently expressed wtB4 or the B3 3′ UTR. Collectively, these observations suggested that the requirement of a cis-acting TLS is distinct for B3 compared with B1 or B2.

scholarly journals Structural basis of Chikungunya virus inhibition by monoclonal antibodies

2020 ◽  
Vol 117 (44) ◽  
pp. 27637-27645
Author(s):  
Qun Fei Zhou ◽  
Julie M. Fox ◽  
James T. Earnest ◽  
Thiam-Seng Ng ◽  
Arthur S. Kim ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Chikungunya Virus ◽  
Neutralizing Antibody ◽  
Structural Basis ◽  
Chikv Infection ◽  
Receptor Binding Site ◽  
Viral Pathogen ◽  
Virus Attachment

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

scholarly journals In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

2019 ◽  
Vol 20 (1) ◽  
pp. 194 ◽  
Author(s):  
Vally Kommineni ◽  
Matthew Markert ◽  
Zhongjie Ren ◽  
Sreenath Palle ◽  
Berenice Carrillo ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Cost Effective ◽  
Fold Increase ◽  
Expression Platform ◽  
Dependent Cell ◽  
Anti Cd20

N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

scholarly journals Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 88-91 ◽  
Author(s):  
Kipp Weiskopf ◽  
Aaron M. Ring ◽  
Chia Chi M. Ho ◽  
Jens-Peter Volkmer ◽  
Aron M. Levin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Responses ◽  
Cancer Cells ◽  
Macrophage Activation ◽  
Universal Method ◽  
Wild Type ◽  
High Affinity ◽  
Macrophage Phagocytosis

CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This “one-two punch” directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

scholarly journals Bactericidal Activities of BMS-284756, a Novel Des-F(6)-Quinolone, against Staphylococcus aureus Strains with Topoisomerase Mutations

2002 ◽  
Vol 46 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Laura E. Lawrence ◽  
MaryBeth Frosco ◽  
Brenda Ryan ◽  
Susan Chaniewski ◽  
Hyekyung Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
In Vivo Efficacy ◽  
Mutant Strains ◽  
Bactericidal Activities ◽  
Time Kill

ABSTRACT The antistaphylococcal activities of BMS-284756 (T-3811ME), levofloxacin, moxifloxacin, and ciprofloxacin were compared against wild-type and grlA and grlA/gyrA mutant strains of Staphylococcus aureus. BMS-284756 was the most active quinolone tested, with MICs and minimal bactericidal concentrations against S. aureus wild-type strain MT5, grlA mutant MT5224c4, and grlA/gyrA mutant EN8 of 0.03 and 0.06, 0.125 and 0.125, and 4 and 4 μg/ml, respectively. In the time-kill studies, BMS-284756 and levofloxacin exhibited rapid killing against all strains. Ciprofloxacin, however, was not bactericidal for the double mutant, EN8. BMS-284756 and levofloxacin were bactericidal (3 log10 decrease in CFU/ml) against the MT5 and MT5224c4 strains at two and four times the MIC within 2 to 4 h. Against EN8, BMS-284756 was bactericidal within 4 h at two and four times the MIC, and levofloxacin achieved similar results within 4 to 6 h. Both the wild-type strain MT5 and grlA mutant MT5224c4 should be considered susceptible to both BMS-284756 and levofloxacin, and both quinolones are predicted to have clinical efficacy. The in vivo efficacy of BMS-284756, levofloxacin, and moxifloxacin against S. aureus strain ISP794 and its single mutant 2C6(1)-1 directly reflected the in vitro activity: increased MICs correlated with decreased in vivo efficacy. The 50% protective doses of BMS-284756 against wild-type and mutant strains were 2.2 and 1.6 mg/kg of body weight/day, respectively, compared to the levofloxacin values of 16 and 71 mg/kg/day and moxifloxacin values of 4.7 and 61.6 mg/kg/day. BMS-284756 was more potent than levofloxacin and equipotent with moxifloxacin against ISP794 both in vitro and in vivo, while BMS-284756 was more potent than levofloxacin and moxifloxacin against 2C6(1)-1.

In vitro activity of APX2041, a new GWT1-inhibitor and in vivo efficacy of the prodrug APX2104 against Aspergillus fumigatus

2021 ◽  
Author(s):  
Shareef K. Shaheen ◽  
Praveen R. Juvvadi ◽  
John Allen IV ◽  
E. Keats Shwab ◽  
D. Christopher Cole ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Vitro Activity ◽  
Prolonged Survival ◽  
In Vitro Activity ◽  
Wild Type ◽  
In Vivo Efficacy ◽  
Resistant Strains

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a deadly infection for which new antifungal therapies are needed. Here we demonstrate the efficacy of a Gwt1 inhibitor, APX2041, and its prodrug, APX2104, against A. fumigatus . The wild-type, azole-resistant and echinocandin-resistant A. fumigatus strains were equally susceptible to APX2041 in vitro . APX2104 treatment in vivo significantly prolonged survival of neutropenic mice challenged with the wild-type and azole-resistant strains, revealing APX2104 as a potentially promising therapeutic against IA.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Sign in / Sign up

Export Citation Format

Share Document