Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies

A Brucella abortus S19 Glyco-conjugate Vaccine Consisting of Lipopolysaccharide and outer Membrane Protein Protects Mice against Challenge with Brucella abortus

A glyco-conjugate vaccine using the lipopolysaccharide (LPS) and the outer membrane protein (OMP) of Brucella abortus S19 strain was prepared. The vaccine was administered in mice subcutaneously (25 µg LPS per dose). Separate groups of mice were also vaccinated with LPS, OMP or live, attenuated S19 vaccine. Mice were challenged 30 days post vaccination with B. abortus 544 strain. The LPS, OMP and LPS-OMP glyco-conjugate vaccinated mice were protected against the challenge. The percentage of animals protected with the sub-unit vaccines and the glyco-conjugate vaccine were comparable with the live, attenuated vaccine. The glyco-conjugate vaccine was able to induce strong immune response against both the components. The prominent isotypes were IgG1, IgG2a and IgG3. In addition, the glyco-conjugate vaccine was able to induce a cell mediated immune response as indicated by the expression of IFN γ by splenocytes. The study indicated that the glyco-conjugate vaccine may be a useful candidate for prophylactic use

Immunocapture Enzyme-Linked Immunosorbent Assay for Assessment of In Vitro Potency of Recombinant Hepatitis B Vaccines▿

Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELISA) as an unlabeled capture antibody and biotin-labeled detection antibody. The IC-ELISA was standardized and validated using experimental hepatitis B vaccine batches with various HBsAg concentrations per dose and commercial vaccines. The vaccine was treated with an alkaline solubilizer to desorb the HBsAg from Algel-adjuvanted vaccines before testing, and the sensitivity of the test was 5 ng/ml. A good correlation could be observed between the HBsAg estimates derived by both formats, except for the higher HBsAg concentration range, where the IC-ELISA format could estimate closer to the actual values than AxSYM. There was a significant correlation between the estimated relative potencies of the two methods. There was lack of correlation between the in vivo potency and the relative in vitro potency. However, the estimates of IC-ELISA were comparable to the in vivo values when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for deriving in vitro relative potency and antigen concentration in vaccine batches for batch control and release