Research ArticleSelection and characterization of highly specific recombinant antibodies against West Nile Virus E protein
Introduction
West Nile virus (WNV) is a member of the Flavivirus genus, one of the four genera belonging to the Flaviviridae family (Simmonds et al., 2017). The Flavivirus genus includes more than 70 arthropod-borne viruses characterized by a positive-stranded RNA genome of 11 Kb and an enveloped icosahedral capsid (Gould and Solomon, 2008; Heinz and Stiasny, 2012). Together with Zika virus (ZIKV), Dengue virus (DENV), Tick-borne encephalitic virus (TBEV), Yellow fever virus (YFV) and Japanese encephalitis virus (JEV), WNV represents one of the most important and wide spread human pathogenic flavivirus (Chaskopoulou et al., 2016; Gould and Solomon, 2008; Reisen, 2013).
According to the Centers for Disease Control and Prevention (CDC), about 80 % of WNV infected people do not develop specific symptoms, while around 20 % develop fever associated with headache, body aches, joint pains, vomiting, diarrhea or rash. In 1% of cases, WNV infection leads to a severe illness affecting the central nervous system (CNS), such as encephalitis and meningitis (Bai et al., 2019; Lustig et al., 2018). WNV is widely distributed in almost all the continents and Italy is one of the European countries that are endemic for WNV (Marini et al., 2018; Zannoli and Sambri, 2019). 2018 has witnessed a high increase of WNV, with 1.491 human cases reported in EU countries. Among them Italy reported 569 cases, Greece 309, Croatia 53 and Slovenia 3. In addition, 171 deaths due to WNV infection have been reported, mostly by Greece (45) and Italy (42). During the transmission season, 279 outbreaks among equids have been reported, 145 by Italy, 15 by Greece and 1 by Slovenia (data from European Centre for Disease Prevention and Control, last update 15th of November 2018).
To date, despite the development of four licensed veterinary WNV vaccines, there are still no approved vaccines or antivirals available for humans (Kaiser and Barrett, 2019a). In addition, the short-time viremia that follows infection makes an early diagnosis by virus isolation or molecular assays extremely difficult. For this reason, the detection of specific antibodies against the virus, based on enzyme-linked immunosorbent assays (ELISAs), is the most wide used diagnostic tool (Sambri et al., 2013). In particular, as most of the immune response elicited after infection is directed against the viral surface, commercially available ELISA tests mainly rely on the detection of anti-envelope antibodies by using inactivated virus or purified envelope (E) protein.
The E protein contains 500 amino residues and presents a final molecular weight of 53−60 kDa depending on the glycosylation status. It comprises three regions (DI, DII and DIII) collectively defined ectodomains (sE) and two transmembrane domains that are connected to the sE by a stem anchor. A N-linked glycosylation motif in the DI domain, between amino acid (aa) positions 154–156, is present in most WNV strains that have been isolated during significant outbreaks of human disease (Nybakken et al., 2006). However, the antigenic similarity between envelope protein of several flaviviruses leads to the production of cross-reactive antibodies (Crill and Chang, 2004; Mansfield et al., 2011) affecting the specificity of results.
Improvement of serodiagnosis for flaviviruses infections can be achieved by developing adequate antigens and controls. The use of E protein mutants represents a potential approach to reduce flaviviruses cross-reactivity, as demonstrated in previous studies (Rockstroh et al., 2019, 2015). An alternative approach is the employment of the DIII domain of the E protein, which exhibits the highest sequence variability among flaviviruses and, as demonstrated by several works, is able to improve diagnostic specificity (Beasley et al., 2004; Chávez et al., 2010; Holbrook et al., 2004; Ludolfs et al., 2007; Piyasena et al., 2017; Rebollo et al., 2018).
In our recent work (Rizzo et al., 2019) we described a fast and easy protocol to produce the DIII domain and the E protein of TBEV, with a preserved antigenicity with murine and human serum samples. In the present work, we applied the same protocol for WNV E and DIII production, confirming their antigenic properties with human sera from infected patients. Later the E protein was used to select a panel of highly specific anti-WNV antibodies from a human naïve phage-display library (Sblattero and Bradbury, 2000). Seven specific antibodies were identified and characterized for their properties by defining the binding site on the target, affinity and impact on WNV neutralization.
Section snippets
Cloning, expression and purification of WNV sE, DI/DII, DIII and DI/DII peptides in bacteria
The nucleotide sequence of the ectodomain of the Envelope protein (sE) of WNV (strain NY99, NCBI reference genome: NC_009942.1, aa 1–400) was codon-optimized for E. coli and obtained as synthetic sequence in the pMAT vector (Invitrogen) between the BssHII and NheI restriction sites. The sequences of DI/DII domains (aa 1 – 297), DIII domain (aa 298 – 400) and six DI/DII peptides (aa 1–51, aa 1–133, aa 1–195, aa 52–133, aa 134–195, aa 196–297) were obtained by PCR using the pMAT-sE plasmid as a
WNV sE and DIII antigens production and testing
The viral E protein is the main target of the immune response following flavivirus infection and it is also the main antigen used in immunodiagnostic tests (Lustig et al., 2018). In order to obtain specific antibodies to the E protein, we initially applied our previously developed protocol (Rizzo et al., 2019) for the production of recombinant antigens. We focus on the ectodomain (sE) comprising three sub-domains (DI, DII and DIII) (Supplementary Fig. 1 A) and to the DIII domain alone as it
Discussion
WNV is one of the most important members of the flavivirus genus, in terms of disease impact and geographical distribution (Bai et al., 2019), and to date no vaccines or therapeutic treatments have been licensed for humans (Kaiser and Barrett, 2019b). A vaccine formulation based on recombinant canarypox virus expressing the prM/E genes of WNV is licensed for veterinary use in horses (Minke et al., 2004), highlighting the importance of the E protein for the induction of a protective immune
Funding
This work was supported by the Beneficientia Stiftung, Vaduz, Lichtenstein, and by the FLAVIPOC and SEVARE projects from the Regione FVG of Italy.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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