RESULT REPORT

PARRENO V.; SOSA HOLT, CARLA; PUNTEL, MARIANA; LOVE, KERRY

2019-02-04/2020-07-06

1-6

Biológica

Enf.Endemicas-Enf.infecciosas respirat.

IntroductionIn the nineties, it was discovered that camelid possess a functional class of Abs devoid of light chains, referred to as heavy-chain Abs, whose single N-terminal variable domain (VHH or nanobody) is the smallest molecule present in nature capable to bind an antigen. VHH have a molecular weight of only 12?15 kDa rather than the 150 kDa of conventional Abs, and their more compact architecture allows them to access hidden targets inaccessible to conventional Abs.VHH can resist temperatures above 90°C and extreme pH as stomach acidic environment. All these features encourage the use of VHH in oral therapies. VHH consist of only one polypeptide chain, highly hydrosoluble and can be expressed to a high level in several microorganisms which represents an advantage for their application in massive treatment strategies.INTA had developed a set of VHH directed to the hemoagglutinin (HA) of the Human Influenza virus strain pandemic H1N1 A/Argentina/017/2009pdm. All clones were able recognized the virus and the HA -H1 by ELISA. The clone G41 showed neutralizing and hemoagglutination inhibition activity, while clone E13 only showed neutralizing activity (Baztarrica, MSc Thesis, 2017). Both clones were protected in a mouse model with the adapted virus strain, and the PR8 reference strain (Sosa Holt, Puntel, unpublished data).MIT had engineered recombinant Pichia pastoris, a GRAS microorganism, expressing E13 and G41VHH to H1N1 Flu.ObjectiveIn the present report, we evaluated the capability of clone E13 and G41 expressed in Picchia pastoris by MIT vs VHH expressed in E coli by INTA to recognize Flu H1N1 by ELISA, to test their hemoaglutination inhibition and VN activity and protection in a mouse model.MATERIALS & METHODS AND RESULTSVHH production in Pichia pastoris MITThe nucleotide sequences of five nanobodies (VHH) directed to H1N1 Flu were submitted by INTA to MIT team (Table1). Both VHHs were obtained from an immune library derived from a llama immunized with the influenza vaccines used in humans [Baztarriza, MSc Thesis, 2017].manuscrito EN REVISION. Laura E. Crowell, Chaz Goodwine, Sergio A. Rodriguez, Celina Vega, Carla S. Holt, Mariana Puntel, Viviana Parreno, Kerry R. Love, Steven M. Cramer, J. Christopher Love. Development of a platform process for the production and purification of single-domain antibodies.