Baculovirus display of single chain antibody (scFv) using a novel signal peptide

Kuntida Kitidee,Saw-See Hong,Sawitree Nangola,Pierre Boulanger,Chatchai Tayapiwatana,Gaëlle Gonzalez

doi:10.1186/1472-6750-10-80

Abstract

BackgroundCells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.ResultsTwo versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.ConclusionExpression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.

Highlights

Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable antibodies directed towards viral structural or regulatory proteins, or viruscoded enzymes
Expression and characterization of scFvG2/p17 and scFvE2/p17 molecules in recombinant BV-infected Sf9 cells The monoclonal antibody secreted by the MH-SVM33C9 hybridoma cell line reacts with the highly conserved and accessible epitope 121DTGHSSQVSQNY132 corresponding to the C-terminus of HIV-1 MAp17 [22,23,24]
The N-terminal octadecapeptide sequence in scFvE2/p17 read 1MEASLAAQAAQIQLVQSG18, and 1MGLAAQAAQIQLVQSGPE18 in scFvG2/p17. Both subclones were modified at the C-terminus by the addition of a HAtag, the Influenza A virus hemagglutinin epitope YPYDVPDYA, and when inserted into the baculoviral genome, generated two recombinant BVs, BV-E2/p17 and BV-G2/p17, respectively

Summary

Introduction

Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or viruscoded enzymes. Among all the antiretroviral molecules, antibodies occupy a special position as they can inhibit HIV-1 replication by interfering with multiple steps of virus-cell interaction. The design of virus-resistant cells via intracellular expression of specific single chain fragment variable (scFv) antibodies directed to the virus has been successfully used to block HIV-1 replication in vitro [2,3,4]. The viral proteins which have been targeted by these intrabodies include structural proteins, such as the envelope glycoprotein gp120 [5] or the matrix protein MAp17 [6], the viral enzyme reverse transcriptase [7], and the auxiliary proteins Tat [8,9] and Vif [10,11]

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