High affinity mouse-human chimeric Fab against hepatitis B surface antigen

Aim: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. Methods: We cloned the VH and VL genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. Results: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. Conclusion: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses

Executive Functions and Prefrontal Cortex: A Matter of Persistence?

Executive function is thought to originates from the dynamics of frontal cortical networks. We examined the dynamic properties of the blood oxygen level dependent time-series measured with functional MRI (fMRI) within the prefrontal cortex (PFC) to test the hypothesis that temporally persistent neural activity underlies performance in three tasks of executive function. A numerical estimate of signal persistence, the Hurst exponent, postulated to represent the coherent firing of cortical networks, was determined and correlated with task performance. Increasing persistence in the lateral PFC was shown to correlate with improved performance during an n-back task. Conversely, we observed a correlation between persistence and increasing commission error – indicating a failure to inhibit a prepotent response – during a Go/No-Go task. We propose that persistence within the PFC reflects dynamic network formation and these findings underline the importance of frequency analysis of fMRI time-series in the study of executive functions

On inconsistency of estimators of parameters of non-homogeneous Poisson process models for software reliability

Non-homogeneous Poisson Process (NHPP) models form a significant subclass of the many software reliability models proposed in the literature. We prove an important limitation of NHPP models for which the expected number of failures in infinite testing is finite. Specifically, the parameters of those models cannot be estimated consistently as the testing time approaches infinity. We also discuss certain parameter-based asymptotic properties of the maximum likelihood estimators of the model parameters and some logical implications of NHPP model assumptions.

Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen

Background: Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. Methods: A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. Results: Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. Conclusion: Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization

Generation and characterization of a high-affinity chimaeric antibody against hepatitis B surface antigen

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V<SUB>H</SUB> (heavy-chain variable region) and V<SUB>L</SUB> (light-chain variable region) genes of this mouse antibody, and fused them with C<SUB>H</SUB>1 (heavy-chain constant domain 1) of human IgG1 and C<SUB>L</SUB> (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C<SUB>H</SUB>2 and C<SUB>H</SUB>3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate (≈5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody

Characterization and molecular modelling of a highly stable anti hepatitis B surface antigen scFv

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precurser revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation