Development of protein microarrays for the investigation of host-pathogen interactions

Abstract

Varicella-Zoster virus (VZV) is a human herpesvirus (causing chickenpox and shingles upon reactivation) whose diagnosis is particularly important in pregnancy and immunocompromised patients. Current sérodiagnostic assays are either based on crude whole cell lysates or glycoprotein preparations lacking high specificity and sensitivity. The work presented here investigates the humoral immune response to VZV infection in more detail, to better understand the mechanisms of the adaptive immune response to lead to improved diagnostic tests and vaccine development.

In a proof-of-concept study, two protein microarray platforms representing the full VZV proteome were developed. The first one is a nucleic acid programmable protein array (NAPPA), in which a full VZV ORFeome clone collection is printed and then transcribed/translated in situ into a protein microarray. The second microarray platform is a modified sandwich protein array (MoSA) in which proteins expressed in vitro were subsequently printed in an array format. They overcome limitations, such as time and cost-­intensive protein expression and purification, of traditional approaches.

These arrays were used for the screening of characterised and uncharacterised patients’ sera, in order to identify immunogenic proteins involved in viral pathogenesis. A detailed analysis from individuals with either no, a previous or an acute VZV infection was performed on NAPPA arrays. This analysis identified a set of new potential antigenic VZV structural and non-structural proteins (such as glycoproteins 5, 14, 31, 37, 68 and ORFs 28, 62 and 63), indicating that the immune response to VZV might actually be considerably broader than previously known.

Complementing this approach, MoSA arrays were used to analyse 2 defined groups of sera, i.e. patients either with shingles and post-herpetic neuralgia or fully recovered after reactivation. This analysis identified a subset of new antigens that may be useful for monitoring the progression of VZV infections towards complications and therefore allows for appropriate timely treatments. Furthermore, the effect of the newly identified antigenic protein, ubiquitin protease ORF 22, was studied showing a proviral effect on VZV replication.

Together, these findings demonstrate that both NAPPA and MoSA are versatile research tools, characterised by benefits over classical methods, to speed up the development of novel diagnostic tests. These findings could lead to the identification of proteins involved in viral pathogenesis, which may improve therapeutic intervention.