Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response

Abstract

To reduce the embryonic pathogenicity of Newcastle disease virus (NDV), escape mutants of the La Sota strain were produced with selected monoclonal antibodies. Immunoselection resulted in the elimination of an epitope by single amino acid substitution (F and HN molecule) or in a conformational change (HN molecule). The embryonic pathogenicity of these escape mutants was reduced and their dose was optimised for in ovo vaccination. Because antibody responses and protection of in ovo vaccinated chicks were similar to controls vaccinated at hatch with the La Sota strain, immunoselection appears a valuable technique to produce attenuated NDV strains, which are candidate in ovo vaccines.

Introduction

Newcastle disease (ND) is a highly contagious viral disease of poultry and other bird species caused by specified viruses of the avian paramyxovirus type I (APMV-I) serotype of the genus Avulavirus belonging to the family Paramyxoviridae [1]. The virus has a wide host range and over 250 species of birds have been reported as susceptible to infection [2]. ND virus (NDV) isolates are classified into forms or pathotypes of disease based on clinical signs in chickens [3].

In the poultry industry, vaccination against ND with live vaccines is common practice and even obligatory in many countries. The objective is to establish a mild infection in the flock, preferably in each bird at the time of application [4]. By the in ovo vaccination technology, this could be realised in a safe, efficacious and convenient way. This is illustrated by the fact that more than 80% of the U.S. broiler industry had converted to the in ovo vaccination process in 1999 to control Marek's disease [5]. Studies within the last few years have shown, however, that only few live vaccines that are routinely administered to hatched chickens may also be injected into embryonated eggs during the late stages of embryonation without a lethal effect [6]. NDV strains of low virulence such as the Bl strain [7], [8] strain and NDV clone-30 [9], that are routinely administered to hatched chicks cannot be employed for in ovo vaccination in their current form due to their embryonic lethality. To attenuate NDV strains, different approaches have been applied. Ahmad and Sharma described a Hitchner Bl derived NDV strain for in ovo vaccination, mutated by the chemical agent ethyl methanesulfonate [7]. By reverse genetics Mebatsion et al. [9], [10] introduced a mutation in the conserved editing site of the P gene mRNA of NDV clone-30 which reduces its V protein expression and embryonic pathogenicity [9]. After serial passages, restoration of the pathogenicity by a compensating second-site mutation was, however, observed [10].

Monoclonal antibodies (MAb) have been successfully used for selecting antigenic variants (also known as escape mutants) of NDV [11], [12], [13], [14]. Escape mutants with reduced pathogenicity of dengue-2 virus [15], chicken anaemia virus [16] and NDV [13] could be selected using neutralising MAb. In this work, we examined the possibility of deliberately further attenuating the NDV La Sota vaccine strain by immunoselection with neutralising MAb, making it a suitable vaccine for in ovo vaccination. The F or HN-glycoproteins, whose interaction determines infectivity of the virus [17], [18], [19], [20], were targeted using the same neutralising MAb that allowed selecting less pathogenic variants from the very virulent Italian strain of NDV [13]. The production of strains with mutated F and HN-glycoproteins, so-called double escape mutants, was envisaged to further reduce virulence and reduce risks of reversal. For the obtained escape mutants, the relation between the introduced mutations and their biological activity was investigated and they were used to examine whether protective immune responses against NDV could be induced by vaccination of 18-day-old embryos.