Development Of A Novel CLIA-based Diagnostic Kit For Sensitive Detection Of VZV-gE Specific IgG,IgA,and IgM,and Production Of VZV-gE Nanobodies

The varicella-zoster virus(VZV),also known as human herpesvirus-3(HHV-3),belongs to the α-herpesviridae subfamily,together with herpes simplex viruses(HSVs).VZV is responsible for varicella disease or chickenpox in children,adolescents,and young adults.The latent viral resurgence years later commonly occurs in adults or the elderly and is responsible for zoster,also known as shingles.Varicella is less prevalent in adults but common in pregnant and immunocompromised adults.These people constitute people at risk,and they are susceptible to severe VZV-infection cases.Although considered among the mild symptom diseases,VZV-related diseases are highly morbid,with severe complications.The most life-threatening complications are mainly associated with long-term viral neurovirulence.In varicella infection cases,these complications mainly include mental development deficits,meningoencephalitis,and postinfectious encephalopathy.In herpes zoster infection cases,these complications mainly include vasculitis,zoster sine herpete(ZSH),and postherpetic neuralgia or PHN(difficult to treat).A lack of early VZV diagnostic mainly results in treatment delays,which usually leads to fatalities,especially in temperate regions,where the VZV infection is highly prevalent,particularly in newborns,elders,organ transplant recipients,and immunocompromised people.While vaccination has resulted in a considerable decrease in VZV incidence,immunobiological diagnostics are requested for vaccine efficacy assessment and routine epidemiological surveillance.However,the current diagnostic tests are limited in terms of diagnostic abilities.Interestingly,the most commercially diagnostic tests have low sensitivity and specificity.Moreover,commercially high specific diagnostic tests are not widely available worldwide,and some of them are difficult to handle,time-consuming,and labor-intensive.Thus,there is a considerable need for routine,simple,rapid,reliable,and high-sensitive diagnostic tests for the specific determination of VZV infection in clinical laboratories.Besides,the current therapeutic aspect of VZV infection is based and limited on symptomatic treatments,which means that the virus is not neutralized and can still trigger disease-associated discomforts.Moreover,individuals,including pregnant and breastfeeding women and immunodeficient people,are not eligible for VZV treatment,which exposes them to disease complications in VZV infection.By focusing on nanobodies as an alternative way in immunotherapy,it is possible to gain an advantage for completely neutralizing the virus in the deeper tissues such as neuronal cells,where it stays in the latent phase.In this study,I first developed a novel diagnostic kit for the sensitive detection of VZV glycoprotein E specific IgG,IgA,and IgM based on the chemiluminescent immuneanalysis(CLIA)approach.Secondly,I produced nanobodies from alpaca with high affinities to VZV glycoprotein E for a potent immunotherapeutic VZV-infection treatment.Overall,our developed CLIA-based diagnostic kits showed diagnostic sensitivities of 95.24%,95.24%,and 97.62%,and specificities of 100%,98.01%,and 98.90%,for detection of VZV-gE-specific IgG,IgA,and IgM,respectively.The best performance was obtained when the VZV-gE-specific IgG,IgA,and IgM detections were combined,resulting in sensitivity,specificity,and an overall agreement of 97.62%,100%,and 99.80%,respectively.Interestingly,the combined measurement with our developed diagnostic kits revealed a high potency to detect very low immunoglobulin titers from patient samples,which are hardly diagnosed positive by the current diagnostic tests,including ELISA.Furthermore,from a random population containing individuals with variable VZV-specific immunoglobulin titers(from very low to higher),the CLIA approach can detect VZV-gE-specific IgG,IgA,and IgM with sensitivities of 74.19%,93.55%,and 69.35%and specificities of 96.03%,98.01%,and 99.78%,respectively.Interestingly,when IgG,IgA,and IgM detection were combined,the diagnostic sensitivity,specificity,and overall agreement were 98.39%,100%,and 99.81%,respectively.Thus,the CLIA-based VZV-gE-specific IgG and IgM detection approach provide additional values in terms of sensitivity and specificity for routine VZV diagnosis and VZV-infection assessment of unvaccinated individuals.Moreover,the nanobodies produced from alpaca demonstrated the formation of therapeutic immune complexes with VZV-gE.Thus,I suggest that these nanobodies,especially NbG14 and NbG8,could be of interest in immunotherapy to mitigate the VZV infection,hamper viral reactivationassociated zoster,and related complications.