Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

BACKGROUND: Human papillomaviruses (HPV) are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs) are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. METHODS: The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. RESULTS: ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and solubility in comparison with the parental scFv43. CONCLUSION: The characterization of 5 specific anti-HPV16 E7 scFvs shows features important for their activity in vivo. ScFv43 M2 shows higher thermal stability with respect to the parental scFv43, and scFv51 shows high stability and solubility. These properties make the 2 scFvs the best candidates to be tested for anti-E7 activity in vivo

Expression of Single-Chain Fv Fragments in E. coli Cytoplasm.

International audienceThe most frequently used approach to produce single-chain Fv fragments (scFv) and Fab in Escherichia coli is to express them in the periplasm of the bacteria. We present here an alternative procedure that uses cytoplasmic expression of soluble active scFv. This can be accomplished by using either specially engineered E. coli strains or hyperstable scFvs

Randomized, double-blind, Phase 111, pivotal field trial of the comparative immunogenicity, safety, and tolerability of two yellow fever 17D vaccines (ARILVAXTMand YF-VAX) in healthy infants and children in Peru

We conducted a randomized, double-blind, phase III yellow lever (YF) vaccine trial among 1,107 healthy children in Sullana in northern Peru. The safety and efficacy (by measurement of geometric mean neutralizing antibody titer responses) were determined for two YF vaccines. ARILVAX™ (n = 738) and YF-VAX® (n = 369). Seroconversion rates were higher (94.9%) in ARILVAX™ than in YF-VAX® (90.6%) recipients. The two-sided 95% confidence interval (YF-VAX®-ARILVAX™) was (-12.8% to -2.5%). indicating that the higher seroconversion rate for Arilvax™ was significant. Post-vaccination (30-day) mean log neutralization indices were found to be similar for both products: 1.32 for ARILVAX™ and 1.26 for YF-VAX® (P = 0.1404. by analysis of variance). A similar number of subjects in each group reported at least one adverse event (AE): 441 (59.8%) for ARILVAX™ versus 211 (59.9%) for YF-VAX®. Most (591: 96.7%) of these were of a mild nature and resolved without treatment. There were no treatment-related serious AEs. This is the first randomized, double-blind comparison of two YF vaccines in a pediatric population: both vaccines were shown to be highly immunogenic and well-tolerated