A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines.

In vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r2 = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.

A single-dose intranasal live-attenuated codon deoptimized vaccine provides broad protection against SARS-CoV-2 and its variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) continues its significant health and economic impact globally. Despite the success of spike-protein vaccines in preventing severe disease, long-lasting protection against emerging variants and the prevention of breakthrough infections and transmission remain elusive. We generate an intranasal live-attenuated SARS-CoV-2 vaccine, CDO-7N-1, using codon deoptimization. CDO-7N-1 shows highly attenuated replication and minimal or no lung pathology in vivo over multiple passages. It induces robust mucosal and systemic neutralizing antibody and T-cell subset responses, in mice (female K18-hACE2 and male HFH4-hACE2 mice), hamsters, and macaques triggered by a single immunization. Mice and hamsters vaccinated with CDO-7N-1 are protected from challenge with wild-type (WT) SARS-CoV-2 and other variants of concern. Serum from vaccinated animals neutralizes WT SARS-CoV-2, variants of concern (beta and delta), variants of interest (omicron XBB.1.5) and SARS-CoV-1. Antibody responses are sustained and enhanced by repeated immunization or infection with WT SARS-CoV-2. Immunity against all SARS-CoV-2 proteins by CDO-7N-1 should improve efficacy against future SARS-CoV-2 variants.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen.

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r² values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

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.

Recombinant diabody-based immunocapture enzyme-linked immunosorbent assay for quantification of rabies virus glycoprotein.

The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V(H)) and light chain (V(L)) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human x mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r(2) value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.

Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells.

The Amyloid-beta (Abeta) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Abeta are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Abeta using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Abeta, we identified an scFv that bound oligomeric Abeta specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Abeta aggregation and toxicity, and reduces the toxicity of preformed oligomeric Abeta towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Abeta scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Abeta in AD brains.