High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires

Abstract

The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies.

Introduction

Listeria monocytogenes is a Gram-positive, food-borne bacterial pathogen which is the causative agent of listeriosis (Farber and Peterkin, 1991). Although this bacterium is ubiquitously found in the environment, individuals with healthy immune systems are able to combat infection. However, the very young, the very old and pregnant women are high risk populations particularly sensitive to L. monocytogenes; infections can result in a mortality rate between 25% and 30%, and spontaneous abortions in pregnant women (Drevets and Bronze, 2008).

Regular, thorough screening for the presence of food-borne bacterial pathogens is critical to safeguard food sources which are to be widely distributed within a population. The importance of such requirements has been highlighted in the recent L. monocytogenes outbreaks which occurred in Canada (Warriner and Namvar, 2009) and the US (Laksanalamai et al., 2012). Although outbreaks are relatively rare, distribution of L. monocytogenes tainted food products has devastating health and economic consequences (Hoffmann et al., 2012).

The current methodologies used to screen for the presence of Listeria spp. and in particular, L. monocytogenes, require specialized equipment and lengthy protocols (Jadhav et al., 2012). Development of a rapid, sensitive and easy-to-use detection platform not reliant on expensive technologies, that could give a visual yes or no result, would be of great value to those tasked with routine screening procedures.

Many biosensor technology platforms rely on appropriate bio-recognition elements for both efficiency and selectivity. Antibodies have been used as bio-recognition reagents since the 1960s (Yalow and Berson, 1960), and have recently been successfully used to detect pathogenic bacteria with great sensitivity (Zhao et al., 2004).

In the Camelidae family, a subset of circulating gamma-immunoglobulin is heavy-chain only (HCAb) (Vincke and Muyldermans, 2012). The variable heavy chain domain of a heavy chain antibody (VHH) retains antigen binding ability and exhibits high chemical, proteolytic and thermal stability (Dumoulin et al., 2002, Goldman et al., 2006, Harmsen et al., 2006, van der Linden et al., 1999), making it an ideal bio-recognition reagent platform for biosensor production.

Here, we report the isolation and characterization of VHHs from a previously constructed naive llama-alpaca-camel (LAC) phage library (Kumaran et al., 2012) and a phagemid library constructed using immunoglobulin RNA templates derived from B cells isolated from an immunized llama which bind the Listeria invasin, Internalin B (InlB). InlB is a member of the Internalin family of proteins, first discovered in L. monocytogenes. Internalins are characterized by the presence of a leucine-rich repeat (LRR) domain, and their function in pathogenesis (Bierne et al., 2007). Internalin A (InlA) and InlB have been characterized, revealing their role in early L. monocytogenes infection. InlA initiates infection by binding to E-cadherin present on intestinal epithelial cells (Mengaud et al., 1996), followed by InlB binding to c-Met (Shen et al., 2000) to induce bacterial uptake by non-phagocytic cells (Li et al., 2005, Mostowy et al., 2011, Veiga and Cossart, 2005, Pentecost et al., 2010). Once the bacterium has established infection of the host, InlB facilitates the entry of L. monocytogenes across the blood–brain barrier (Greiffenberg et al., 1998), the materno-fetal barrier (Disson et al., 2008, Lecuit et al., 2004) and into the liver (Dramsi et al., 1995), producing the trademark signs of listeriosis. The tandem function of InlA and InlB is observed at the genomic level, as they are on the same operon (Gaillard et al., 1996), under the control of strong transcription factors (Lingnau et al., 1995, McGann et al., 2007, McGann et al., 2008). inlB is exclusive to the L. monocytogenes genome (Glaser et al., 2001), whereas inlA has been discovered in non-pathogenic Listeria (Volokhov et al., 2007). Most importantly for antibody isolation (Jonquières et al., 1999), InlA and InlB are structurally related, with InlA containing 15 LRRs (Schubert et al., 2002) compared to 7 in InlB (Freiberg et al., 2004).

Although conventional antibodies which recognize InlB have been reported (Braun et al., 1999, Karamonová et al., 2003, Tully et al., 2006, Tully et al., 2008, Hearty et al., 2006), to the best of our knowledge, this is the first report of the isolation of VHHs shown to bind to InlB. In addition, we demonstrate that antibody repertoires from passively immunized animals are good sources of specific binders to environmental pathogens, demonstrating that active immunization of animals may not be necessary in such cases. This idea supports the recent report by Sabir et al. who have also proposed the same utilization for naive camelid repertoires (Sabir et al., 2014). The antibodies isolated in this study are reagents which can potentially be used in the development of rapid, highly sensitive, specific and cost-effective L. monocytogenes detection technologies. Furthermore, we demonstrate that so-called naive, i.e., naturally immunized, antibody repertoires can be robust sources of binders against environmental, bacterial pathogens.