scholarly journals An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomohiro Kotaki ◽  
Takeshi Kurosu ◽  
Ariadna Grinyo-Escuer ◽  
Edgar Davidson ◽  
Siti Churrotin ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Variable Region ◽  
Cross Reactivity ◽  
Affinity Maturation ◽  
Interferon Α ◽  
Fusion Loop

AbstractDengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 μg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

scholarly journals An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency

2020 ◽  
Author(s):  
Tomohiro Kotaki ◽  
Takeshi Kurosu ◽  
Ariadna Grinyo ◽  
Edgar Davidson ◽  
Siti Churrotin ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Variable Region ◽  
Cross Reactivity ◽  
Affinity Maturation ◽  
Interferon Α ◽  
Fusion Loop

AbstractDengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization ability (NT50 < 0.1 µg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly improved the survival rate of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 molecules that had lost their in vitro ADE activity showed significantly enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits several promising features for therapeutic application including a low NT50 value, potential for pan-flavivirus infection treatment, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

scholarly journals Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Takeshi Kurosu ◽  
Keiko Hanabara ◽  
Azusa Asai ◽  
Sabar Pambudi ◽  
Supranee Phanthanawiboon ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Evaluation Process ◽  
Denv Infection ◽  
Interferon Α ◽  
Double Knockout ◽  
Virus Envelope

AbstractIn a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/β–γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

scholarly journals A new class of errant DNA polymerases provides candidates for somatic hypermutation

2001 ◽  
Vol 356 (1405) ◽  
pp. 47-51 ◽  
Author(s):  
Brigette Tippin ◽  
Myron F. Goodman
Keyword(s):  
Somatic Hypermutation ◽  
Dna Polymerases ◽  
Variable Region ◽  
Affinity Maturation ◽  
Immunoglobulin Genes ◽  
New Class ◽  
Eukaryotic Dna ◽  
Factor Governing

The mechanism of somatic hypermutation of the immunoglobulin genes remains a mystery after nearly 30 years of intensive research in the field. While many clues to the process have been discovered in terms of the genetic elements required in the immunoglobulin genes, the key enzymatic players that mediate the introduction of mutations into the variable region are unknown. The recent wave of newly discovered eukaryotic DNA polymerases have given a fresh supply of potential candidates and a renewed vigour in the search for the elusive mutator factor governing affinity maturation. In this paper, we discuss the relevant genetic and biochemical evidence known to date regarding both somatic hypermutation and the new DNA polymerases and address how the two fields can be brought together to identify the strongest candidates for further study. In particular we discuss evidence for the in vitro biochemical misincorporation properties of human Rad30B/Pol ι and how it compares to the in vivo somatic hypermutation spectra.

scholarly journals A Recombinant Zika Virus Envelope Protein with Mutations in the Conserved Fusion Loop Leads to Reduced Antibody Cross-Reactivity upon Vaccination

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 603
Author(s):  
Beatrice Sarah Berneck ◽  
Alexandra Rockstroh ◽  
Jasmin Fertey ◽  
Thomas Grunwald ◽  
Sebastian Ulbert
Keyword(s):  
Zika Virus ◽  
Insect Cell ◽  
Neutralizing Antibodies ◽  
Structural Similarity ◽  
Cross Reactivity ◽  
Virus Envelope ◽  
E Protein ◽  
Neutralizing Capacity ◽  
Fusion Loop

Zika virus (ZIKV) is a zoonotic, human pathogenic, and mosquito-borne flavivirus. Its distribution is rapidly growing worldwide. Several attempts to develop vaccines for ZIKV are currently ongoing. Central to most vaccination approaches against flavivirus infections is the envelope (E) protein, which is the major target of neutralizing antibodies. Insect-cell derived, recombinantly expressed variants of E from the flaviviruses West Nile and Dengue virus have entered clinical trials in humans. Also for ZIKV, these antigens are promising vaccine candidates. Due to the structural similarity of flaviviruses, cross-reactive antibodies are induced by flavivirus antigens and have been linked to the phenomenon of antibody-dependent enhancement of infection (ADE). Especially the highly conserved fusion loop domain (FL) in the E protein is a target of such cross-reactive antibodies. In areas where different flaviviruses co-circulate and heterologous infections cannot be ruled out, this is of concern. To exclude the possibility that recombinant E proteins of ZIKV might induce ADE in infections with related flaviviruses, we performed an immunization study with an insect-cell derived E protein containing four mutations in and near the FL. Our data show that this mutant antigen elicits antibodies with equal neutralizing capacity as the wildtype equivalent. However, it induces much less serological cross-reactivity and does not cause ADE in vitro. These results indicate that mutated variants of the E protein might lead to ZIKV and other flavivirus vaccines with increased safety profiles.

567 MDK1319/MDK-701: A potent fully efficacious peptidyl agonist of IL-7Rαγc, designed with no reference to cytokine or receptor structure and unrelated to IL-7, fused to an Fc-domain for PK enhancement

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A601-A601
Author(s):  
William Dower ◽  
Alice Bakker ◽  
Steven Cwirla ◽  
Prarthana Joshi ◽  
Praechompoo Pongtornpipat ◽  
...  
Keyword(s):  
T Cell ◽  
Neutralizing Antibodies ◽  
Human Lymphocytes ◽  
Receptor Activation ◽  
Biological Properties ◽  
Cross Reactivity ◽  
Lymphocyte Subpopulation ◽  
Minimal Risk

BackgroundIL-7 receptor activation is essential for the proper development and homeostasis of T-cell subpopulations, and maintenance of the TCR clonal repertoire. Emerging evidence indicates potential clinical utility of IL-7 for immunotherapy of lymphopenia, oncology, and other indications. Here we report the discovery of MDK1319, a small novel peptidyl agonist of IL-7R. This peptide is structurally unrelated to IL-7, with a MW less than 5000D. To improve in vivo properties, we fused MDK1319 to an IgG-Fc-domain to construct MDK-701, which exhibits biological properties similar to those of IL-7 in vitro, and when administered to non-human primates.MethodsPeptides were selected from peptide libraries by screens designed to identify molecules binding simultaneously to the Rα and γc subunits of the human IL-7 receptor. Synthetic peptides, and peptides fused to IgG Fc-domains were evaluated for efficacy, potency, and quality of signaling in IL-7-responsive cell lines and human lymphocytes. PK/PD properties in non-human primates were also determined.ResultsMDK1319 and MDK-701 activate the major IL-7R signaling pathways, JAK-STAT (pSTAT5), and PI3K (pAKT), and induce proliferation in human PBMCs, exhibiting lymphocyte subpopulation selectivity, kinetics, efficacy, and potency similar to those of IL-7. Agonism is attributable to direct activation of IL-7R, as shown by dependence on the presence of the IL-7Rα subunit for response in test cells, and insensitivity to IL-7 neutralizing antibodies. MDK1319 and MDK-701 do not activate nor inhibit any other (off target) Rγc family receptors at concentrations 100-fold greater than required for maximal IL-7R activation. MDK-701 administered to cynomologous macaques (single dose, IV at 1 mg/kg) exhibits a circulating terminal half life of ~32 hr; and induces peripheral lymphocyte profiles similar to IL-7 treatment, including initial reduction (tissue migration), followed by sustained elevation of peripheral lymphocytes remaining above baseline for 29 days, with no observed adverse effects.ConclusionsIn addition to the utility of Fc-fusion MDK-701 for monotherapy, the small peptidyl nature of the active peptide MDK1319, fusable to recombinant protein partners, offers opportunities for incorporation into bispecific molecules, linking IL-7 activity to a variety of useful functions. These include synergistic cytokine activities, checkpoint blockade, and tissue targeting. Cells engineered to secrete MDK1319 display autocrine stimulation potentially useful in T-cell therapeutics. The structural novelty of MDK1319 substantially decreases risk of cross reactivity of any anti-drug immune response with endogenous IL-7, and may provide a safer alternative to modified forms of IL-7 reported to produce significant anti-IL-7 immunogenicity.Ethics ApprovalAnimal studies were performed by Envol Biomedical or Charles Rivers Laboratories, as approved by the Institution Ethics Boards with the following study and approval numbers:Envol Biomedical 7037-20 MDK2002; 7037-20: PK/PD Cynomolgus monkeysCharles Rivers Laboratories 20200121001K; US19001: PK Mouse The use of human PBMC in this study was authorized under Minimal Risk Research Related Activities at Stanford Blood Center (SQL 79075)

scholarly journals Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4021 ◽  
Author(s):  
Subenya Injampa ◽  
Nataya Muenngern ◽  
Chonlatip Pipattanaboon ◽  
Surachet Benjathummarak ◽  
Khwanchit Boonha ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Dengue Virus ◽  
Mammalian Cells ◽  
Human Monoclonal Antibody ◽  
Site Directed Mutagenesis ◽  
Neutralizing Activity ◽  
Dengue Disease ◽  
Denv Serotypes ◽  
Fusion Loop

Background Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. Methods In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. Results The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate. Discussion Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.

scholarly journals In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom

2021 ◽  
Author(s):  
Line Ledsgaard ◽  
Andreas H. Laustsen ◽  
Urska Pus ◽  
Jack Wade ◽  
Pedro Villar ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Snake Venom ◽  
Human Monoclonal Antibody ◽  
Specific Treatment ◽  
Affinity Maturation ◽  
Igg Antibody ◽  
Phage Display Technology ◽  
Parental Antibody

AbstractThe monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against α-cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified α-cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.One Sentence SummaryA recombinant human monoclonal immunoglobulin G antibody, discovered and optimized using in vitro methods, was demonstrated to neutralize the lethal effect of whole venom from the monocled cobra in mice via abrogation of α-neurotoxin-mediated neurotoxicity.

scholarly journals Blocking the GITR-GITRL pathway to overcome resistance to therapy in sarcomatoid malignant pleural mesothelioma

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Meilin Chan ◽  
Licun Wu ◽  
Zhihong Yun ◽  
Trevor D. McKee ◽  
Michael Cabanero ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Malignant Pleural Mesothelioma ◽  
Neutralizing Antibodies ◽  
Pleural Mesothelioma ◽  
Spheroid Formation ◽  
Resistance To Therapy ◽  
Sarcomatoid Mesothelioma

AbstractMalignant pleural mesothelioma (MPM) is an aggressive neoplasm originating from the pleura. Non-epithelioid (biphasic and sarcomatoid) MPM are particularly resistant to therapy. We investigated the role of the GITR-GITRL pathway in mediating the resistance to therapy. We found that GITR and GITRL expressions were higher in the sarcomatoid cell line (CRL5946) than in non-sarcomatoid cell lines (CRL5915 and CRL5820), and that cisplatin and Cs-137 irradiation increased GITR and GITRL expressions on tumor cells. Transcriptome analysis demonstrated that the GITR-GITRL pathway was promoting tumor growth and inhibiting cell apoptosis. Furthermore, GITR+ and GITRL+ cells demonstrated increased spheroid formation in vitro and in vivo. Using patient derived xenografts (PDXs), we demonstrated that anti-GITR neutralizing antibodies attenuated tumor growth in sarcomatoid PDX mice. Tumor immunostaining demonstrated higher levels of GITR and GITRL expressions in non-epithelioid compared to epithelioid tumors. Among 73 patients uniformly treated with accelerated radiation therapy followed by surgery, the intensity of GITR expression after radiation negatively correlated with survival in non-epithelioid MPM patients. In conclusion, the GITR-GITRL pathway is an important mechanism of autocrine proliferation in sarcomatoid mesothelioma, associated with tumor stemness and resistance to therapy. Blocking the GITR-GITRL pathway could be a new therapeutic target for non-epithelioid mesothelioma.

scholarly journals Previous Humoral Immunity to the Endemic Seasonal Alphacoronaviruses NL63 and 229E Is Associated with Worse Clinical Outcome in COVID-19 and Suggests Original Antigenic Sin

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 298
Author(s):  
Daniele Focosi ◽  
Angelo Genoni ◽  
Ersilia Lucenteforte ◽  
Silvia Tillati ◽  
Antonio Tamborini ◽  
...  
Keyword(s):  
Humoral Immunity ◽  
Cross Reactivity ◽  
Clinical Severity ◽  
World Health ◽  
Laboratory Findings ◽  
Cellular And Humoral Immunity ◽  
Rate Ratios ◽  
Original Antigenic Sin

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization’s (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

scholarly journals Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanomaterials ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1597
Author(s):  
Thuong Thi Ho ◽  
Van Thi Pham ◽  
Tra Thi Nguyen ◽  
Vy Thai Trinh ◽  
Tram Vi ◽  
...  
Keyword(s):  
High Pressure ◽  
Neutralizing Antibodies ◽  
H5n1 Virus ◽  
Vaccine Development ◽  
Particle Characterization ◽  
Innovative Strategy ◽  
Specific Igg ◽  
Positively Charged

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use.

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