ABSTRACT
Exquisite specificity is the hallmark of antigen-antibody recognition. However, breakdown in the specific recognition potential culminating in the binding to multiple antigens by a single antibody has been observed, even after the maturation of the humoral response. While such a broad specificity may be expected to assist the host to counter the antigenic variations associated with an immune-evading pathogen, escape from immune surveillance by subtle epitopic mutations in pathogens like HIV and influenza virus has been clearly established. In the light of this dichotomy, the issues of degeneracy/specificity in the humoral response against such epitopes were analysed using three HIV-neutralizing epitopes and their variants as a model system. Cross-reactivity was observed in the polyclonal response against two of the epitopes. Multi-reactive mAb KEL10 was isolated against one of the epitopes, ELDKWA from this response. It is evident that even after the affinity maturation, antibodies showing binding to multiple variants of an immunizing peptide epitope existed. Binding kinetics and in silico structural analyses indicated that conserved interactions across epitopes and limited conformational flexibility in the paratope may account for the observed multi-reactivity. Though the affinity maturation process is expected to incorporate an extent of specificity to the paratope, there appear to be still some B-cell clones producing antibodies with subtle flexibility in their binding site, as demonstrated in case of KEL10. Generation of such antibodies against effective immunogens could be a possible approach for countering the antibody neutralization escape by various immune-evading pathogens.
Subject(s)
Antigen-Antibody Reactions/immunology , Binding Sites, Antibody/immunology , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/immunology , HIV-1/chemistry , HIV-1/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Cross Reactions/immunology , Female , HIV Antibodies/chemistry , HIV Envelope Protein gp41/immunology , Immune Sera/immunology , Kinetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/immunology , Pliability , Protein Conformation , TemperatureABSTRACT
The 3' poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.
Subject(s)
Nucleotides , Vaccines , RNA, Messenger/genetics , Workflow , Electrophoresis, Capillary/methodsABSTRACT
The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.