Neutralizing chimeric heavy-chain antibody targeting the L-HN domain of Clostridium botulinum neurotoxin type F.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2 µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5 mg/kg F20 at 1 h, 2 h, 3 h, and 4 h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.

A potential broad-spectrum neutralizing antibody against Betacoronavirus.

Three pandemics caused by human Betacoronavirus had broken out in the past two decades. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was one of the novel epidemic strains which caused the third pandemic, coronavirus disease 2019 (COVID-19), a global public health crisis. So far, more than millions of people have been infected. Considering the public health and economic impact of Betacoronavirus pandemic, drugs with broad-spectrum activity against these coronaviruses are urgently needed. In this study, two monoclonal antibodies targeting SARS-CoV-2 spike protein receptor-binding domain (RBD) with good neutralizing activity were used to construct a novel immunoglobulin-like bispecific antibody BI31. The neutralizing effect of BI31 against the pseudovirus and the authentic virus is better than that of its parent antibodies alone and in combination. What surprised us most was that the newly constructed bispecific antibody also had the neutralizing activity against SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) that the parent antibodies did not have. These suggested that the BI31 can not only be developed as a therapeutic drug against COVID-19 but it could also become a broad-spectrum therapeutic antibody against Betacoronavirus.

Correction: Sun et al. Ergosterol Isolated from Antrodia camphorata Suppresses LPS-Induced Neuroinflammatory Responses in Microglia Cells and ICR Mice. Molecules 2023, 28, 2406.

The original version of this article unfortunately contains an error in Figures 4B,C,H-J and 5C,D [...].

Ergosterol Isolated from Antrodia camphorata Suppresses LPS-Induced Neuroinflammatory Responses in Microglia Cells and ICR Mice.

Inflammation caused by microglial activation is important in neurodegenerative diseases. In this research, we tried to identify safe and effective anti-neuroinflammatory agents by screening a natural compounds library and found that Ergosterol can inhibit the nuclear factor kappa-light-chain enhancer of the activated B cells (NF-κB) pathway induced by lipopolysaccharide (LPS) in microglia cells. Ergosterol has been reported to be an effective anti-inflammatory agent. Nevertheless, the potential regulatory role of Ergosterol in neuroinflammatory responses has not been fully investigated. We further investigated the mechanism of Ergosterol that regulates LPS-induced microglial activation and neuroinflammatory reactions both in vitro and in vivo. The results showed that Ergosterol can significantly decrease the pro-inflammatory cytokines induced by LPS in BV2 and HMC3 microglial cells, possibly by inhibiting the NF-κB, protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, we treated Institute of Cancer Research (ICR) mice with a safe concentration of Ergosterol following LPS injection. Ergosterol treatment significantly decreased microglial activation-associated ionized calcium-binding adapter molecule-1 (IBA-1), NF-κB phosphorylation, and pro-inflammatory cytokine levels. Moreover, Ergosterol pretreatment clearly reduced LPS-induced neuron damage by restoring the expression of synaptic proteins. Our data may provide insight into possible therapeutic strategies for neuroinflammatory disorders.

Generation and characterization of humanized synergistic neutralizing antibodies against SARS-CoV-2.

The emerging coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causative agent of coronavirus disease 2019 (COVID-19), which has become a severe threat to global public health and local economies. In this study, several single-chain antibody fragments that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein were identified and used to construct human-mouse chimeric antibodies and humanized antibodies. These antibodies exhibited strong binding to RBD and neutralization activity towards a SARS-CoV-2 pseudovirus. Moreover, these antibodies recognize different RBD epitopes and exhibit synergistic neutralizing activity. These provide candidate to combination use or bispecific antibody to potential clinical therapy for COVID-19.

Beyond inhibition against the PD-1/PD-L1 pathway: development of PD-L1 inhibitors targeting internalization and degradation of PD-L1.

Tumor cells hijack the programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway to suppress the immune response through overexpressing PD-L1 to interact with PD-1 of T cells. With in-depth ongoing research, tumor-intrinsic PD-L1 is found to play important roles in tumor progression without interaction with PD-1 expressed on T cells, which provides an additional important target and therapeutic approach for development of PD-L1 inhibitors. Existing monoclonal antibody (mAb) drugs against the PD-1/PD-L1 pathway generally behave by conformationally blocking the interactions of PD-1 with PD-L1 on the cell surface. Beyond general inhibition of the protein-protein interaction (PPI), inhibitors targeting PD-L1 currently focus on the functional inhibition of the interaction between PD-1/PD-L1 and degradation of tumor-intrinsic PD-L1. This perspective will clarify the evolution of PD-L1 inhibitors and provide insights into the current development of PD-L1 inhibitors, especially targeting internalization and degradation of PD-L1.

Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B.

Background: Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Methods: Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. Results: The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. Conclusion: The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Neutralizing Chimeric Anti-F1 Monoclonal Antibody against Yersinia pestis Infection.

Drug-resistant Yersinia pestis (Y. pestis) poses a threat to the use of antibiotics to treat Y. pestis infections. Passive immunization with neutralizing monoclonal antibodies (mAbs) is one approach to the treatment of infectious diseases. In this study, a murine single-chain fragment variable (scFv) phage antibody library targeting the F1 antigen was constructed and screened. Therapeutic intravenous injection of 400 µg chimeric mAb S1 through tail veins provided complete protection against Y. pestis (strain: 91001) challenge in a pneumonic plague mouse model. Timely antibody treatment eliminated the bacteria and reduced lung inflammation. Our data suggest that chimeric mAb S1 is a candidate treatment for Y. pestis infection that warrants further study.

Characterization of neutralizing chimeric heavy-chain antibodies against tetanus toxin.

Tetanus toxin (TeNT) is one of the most toxic proteins. Neutralizing antibodies against TeNT are effective in prevention and treatment. In this study, 14 anti-tetanus nanobodies were obtained from a phage display nanobody library by immunizing a camel with the C-terminal receptor-binding domain of TeNT (TeNT-Hc) as the antigen. After fusion with the human Fc fragment, 11 chimeric heavy-chain antibodies demonstrated nanomolar binding toward TeNT-Hc. The results of toxin neutralization experiments showed that T83-7, T83-8, and T83-13 completely protected mice against 20 × the median lethal dose (LD50) at a low concentration. The neutralizing potency of T83-7, T83-8, and T83-13 against TeNT is 0.4 IU/mg, 0.4 IU/mg and 0.2 IU/mg, respectively. In the prophylactic setting, we found that 5 mg/kg of T83-13 provided the mice with full protection from tetanus, even when they were injected 14 days before exposure to 20 × LD50 TeNT. T83-7 and T83-8 were less effective, being fully protective only when challenged 7 or 10 days before exposure, respectively. In the therapeutic setting, 12 h after exposure to TeNT, 1 ~ 5 mg/kg of T83-7, and T83-8 could provide complete protection for mice against 5 × LD50 TeNT, while 1 mg/kg T83-13 could provide complete protection 24 h after exposure to 5 × LD50 TeNT. Our results suggested that these antibodies represent prophylactic and therapeutic activities against TeNT in a mouse model. The T83-7, T83-8, and T83-13 could form the basis for the subsequent development of drugs to treat TeNT toxicity.

A human bispecific antibody neutralizes botulinum neurotoxin serotype A.

Botulinum neurotoxin (BoNT) shows high lethality and toxicity, marking it as an important biological threat. The only effective post-exposure therapy is botulinum antitoxin; however, such products have great potential for improvement. To prevent or treat BoNT, monoclonal antibodies (mAbs) are promising agents. Herein, we aimed to construct a bispecific antibody (termed LUZ-A1-A3) based on the anti-BoNT/A human monoclonal antibodies (HMAb) A1 and A3. LUZ-A1-A3 binds to the Hc and L-HN domains of BoNT/A, displaying potent neutralization activity against BoNT/A (124 × higher than that of HMAb A1 or HMAb A3 alone and 15 × higher than that of the A1 + A3 combination). LUZ-A1-A3 provided effective protection against BoNT/A in an in vivo mouse model. Mice were protected from infection with 500 × LD50 of BoNT/A by LUZ-A1-A3 from up to 7 days before intraperitoneal administration of BoNT/A. We also demonstrated the effective therapeutic capacity of LUZ-A1-A3 against BoNT/A in a mouse model. LUZ-A1-A3 (5 µg/mouse) neutralized 20 × LD50 of BoNT/A at 3 h after intraperitoneal BoNT/A administration and complete neutralized 20 × LD50 of BoNT/A at 0.5 h after intraperitoneal BoNT/A administration. Thus, LUZ-A1-A3 is a promising agent for the pre-exposure prophylaxis and post-exposure treatment of BoNT/A.

Humanization and characterization of a murine monoclonal neutralizing antibody against human adenovirus 7.

Human adenovirus type 7 (HAdV7) is commonly associated with febrile acute respiratory disease (ARD) outbreaks. We have reported that 10G12, a mouse monoclonal antibody (mAb) specifically recognizing and neutralizing HAdV7, is a promising candidate for humanization. In this study, we engineered the six variants of 10G12 with increased degree of humanization and investigated their biological activity. The humanized monoclonal antibody (mAb) 10G12-M2 was shown to retain the parental antibody's high binding affinity, specificity and potent efficacy of viral suppression. The mAb 10G12-M2 recognized a conformational neutralization epitope of the hexon protein. Complex structure-based molecular docking simulation showed that the hexon protein formed several interactions with 10G12-M2, including hydrogen bonds and salt bridges interaction. Physicochemical properties analysis of 10G12-M2 demonstrated that it is stable and desirable lead candidate. In general, 10G12-M2 had excellent biological activity after humanization combined with the potential for use in prophylactic or therapeutic applications against HAdV7.

A humanized anti-human adenovirus 55 monoclonal antibody with good neutralization ability.

Background: Human adenovirus type 55 (HAdV55) has a re-emerged as pathogen causing an acute respiratory disease presenting as a severe lower respiratory illness that can cause death. To date, there is no HAdV55 vaccine or treatment available for general use. Methods: Herein, a monoclonal antibody specific for HAdV55, mAb 9-8, was isolated from an scFv-phage display library derived from mice immunized with the purified inactived-HAdV55 virions. By using ELISA and a virus micro-neutralization assay, we evaluated the binding and neutralizing activity of mAb 9-8 following humanization. Western blotting analysis and antigen-antibody molecular docking analysis were used to identify the antigenic epitopes that the humanized monoclonal antibody 9-8-h2 recognized. After that, their thermal stability was determined. Results: MAb 9-8 showed potent neutralization activity against HAdV55. After humanization, the humanized neutralizing monoclonal antibody (9-8-h2) was identified to neutralize HAdV55 infection with an IC50 of 0.6050 nM. The mAb 9-8-h2 recognized HAdV55 and HAdV7 virus particles, but not HAdV4 particles. Although mAb 9-8-h2 could recognize HAdV7, it could not neutralize HAdV7. Furthermore, mAb 9-8-h2 recognized a conformational neutralization epitope of the fiber protein and the crucial amino acid residues (Arg 288, Asp 157, and Asn 200) were identified. MAb 9-8-h2 also showed favorable general physicochemical properties, including good thermostability and pH stability. Conclusions: Overall, mAb 9-8-h2 might be a promising molecule for the prevention and treatment of HAdV55.

A human monoclonal antibody to neutralize all four serotypes of dengue virus derived from patients at the convalescent phase of infection.

Dengue virus (DENV) is a prevalent mosquito-transmitted human pathogen, causing about 100 million cases of acute dengue fever and 21,000 deaths annually worldwide. Therapeutic neutralizing antibodies against dengue virus might be effective to treat severe dengue fever. Here, we showed that human monoclonal antibody (HMAb) 9C7 bound to all four intact serotypes of DENV but not to the recombinant envelope protein, suggesting HMAb 9C7 recognized a conformational epitope of the envelope protein. Taken together our results suggested that HMAb 9C7 neutralized all four serotypes of DENV in vitro and, for DENV-1, indicated activity at the pre- and post-attachment steps in the viral life cycle. HMAb 9C7 potently protected suckling mice from lethal challenge with all four serotypes of DENV. FcγRII-mediated uptake of immune complexes and antibody-dependent enhancement at low doses of the antibody were abolished by two Leu-to-Ala (9C7-LALA) mutations or deletion of nine amino acids (9C7-9del) in HMAb 9C7 Fc. Therefore, HMAb 9C7 represented a promising prophylactic and therapeutic agent against all four serotypes of DENV.