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Antigenic profiling of the porcine reproductive and respiratory syndrome virus

Merijn Vanhee (UGent)
(2011)
Author
Promoter
(UGent)
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Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and boars, and is involved in the porcine respiratory disease complex, causing huge economical losses to the swine industry. Despite the development and broad implementation of vaccines, the virus could not be eradicated, and the high variability and fast evolution of the virus compromises the efficacy of the current vaccines in the field. Since PRRSV engages a complex interaction with the host’s immune system, and exploits several strategies to evade protective immunity, the development of new and better PRRSV vaccines is highly relying on a good understanding of the virus-specific immune response. The general goal of this work was to create new insights into one particular hallmark of PRRSV-specific immunity, namely the weak virus-neutralizing antibody response upon infection or vaccination in pigs, in sharp contrast with the robust non-neutralizing antibody response. Chapter 1 summarizes the current literature on general aspects of the virus and the disease, PRRSV-specific immunity, and PRRSV vaccines. Furthermore, the current knowledge on the PRRSV-specific neutralizing antibody response is extensively discussed. In Chapter 2, the particular problem underlying the work in this thesis is described, and a number of aims are stated. The next two chapters consist of research results addressing these aims. In Chapter 3, it was investigated to which extent virus-neutralizing antibodies can be induced by immunization of pigs with inactivated PRRSV. This study addresses the question whether the poor development of virus-neutralizing antibodies is dependent on structural properties of the virus, or rather on replication-dependent mechanisms. Experimental vaccines were produced, based on formerly optimized virus inactivation procedures and by using a number of different adjuvants. Vaccination of PRRSV-negative piglets systematically led to a strong virus-specific antibody response, but the virus-neutralizing antibody response was very weak or absent, indicating that the poor immunogenicity of neutralizing antibody targets in PRRSV is to a large extent inherent to the structural properties of the virus. Still, vaccination with a high dose of inactivated PRRSV in combination with one particular adjuvant induced virus-neutralizing antibodies, and provided a certain level of protection against viremia upon infection. These results formed the basis for further vaccine research at the Laboratory of Virology. Chapter 4 deals with the identification of non-neutralizing and neutralizing antibody targets in the European prototype PRRSV strain Lelystad virus (LV). In a first study (§ 4.1), the antibody response against linear epitopes in the GP4 protein was investigated. It was found that antibodies are to a large extent produced against one particular region of this protein that was earlier described as neutralizing antibody target. Since this region is highly variable, the antibody response against GP4 was also investigated for other virus strain. The conclusion of this study was that the variable region in GP4 of EU-type PRRSV induces neutralizing antibodies upon infection in pigs, but only against strictly homologous virus. In a second study (§ 4.2), the antibody response against the entire set of envelope proteins (GP2, E, GP3, GP4, GP5 and M) upon infection with LV was investigated. Twenty-one antigenic regions were identified that are capable of inducing antibodies in pigs. Peptide-purified antibodies against four antigenic regions turned out to reduce in vitro virus replication. In addition to the known neutralizing epitope in GP4, two neutralizing antibody targets were identified in GP2, and one in GP3. No neutralizing epitopes were found in E, GP5 and M. Since the neutralizing epitope in GP3 was one of the more immunogenic regions, the antibody response against this epitope was investigated more extensively in different virus strains. Taken together, it can be concluded from Chapter 4 that four linear neutralizing antibody targets are present in the LV envelope proteins: two weakly immunogenic epitopes in GP2, one more immunogenic epitope in GP3, and one strongly immunogenic but highly variable epitope in GP4. Chapter 5 provides a general discussion on the research data generated in this thesis. In § 5.1, it is discussed what can be learned from the vaccination studies in Chapter 3. § 5.2 summarizes the results from Chapter 4, and discusses them in a broader context. First, a synoptic visual overview is given of the non-neutralizing and neutralizing antibody targets that were identified in the viral envelope proteins. Next, some hypotheses are presented on how neutralizing antibodies against different parts of the viral proteins may interfere with viral replication. Subsequently, the current knowledge on PRRSV antigenicity is discussed in light of virus evolution, and some thoughts are made on how the high mutation rate and large variability of the virus may be involved in immune evasion. To conclude, it is critically evaluated to which extent the data generated in this thesis can contribute to the development of new and better PRRSV vaccines.
SAMENVATTING Het porcien reproductief en respiratoir syndroom virus (PRRSV) veroorzaakt reproductiestoornissen bij zeugen en beren, en is betrokken bij het porcien respiratoir ziektecomplex, wat leidt tot enorme economische verliezen in de varkenshouderij. Ondanks de ontwikkeling en brede toepassing van vaccins kon het virus niet worden uitgeroeid, en de grote variabiliteit en snelle evolutie van het virus staan de werkzaamheid van de huidige vaccins in het veld in de weg. Aangezien PRRSV een complexe interactie aangaat met het immuunsysteem van de gastheer en meerdere strategieën gebruikt om de beschermende immuniteit te ontwijken, is de ontwikkeling van nieuwe en betere PRRSV vaccins sterk afhankelijk van een goed inzicht in de virusspecifieke immuunrespons. De algemene doelstelling van deze thesis was het creëren van nieuwe inzichten in één specifiek kenmerk van de PRRSV-specifieke immuniteit, namelijk de zwakke virusneutraliserende antistofrespons na infectie of vaccinatie in varkens, in scherp contrast met de robuuste niet-neutraliserende antistofrespons. Hoofdstuk 1 geeft een samenvatting van de huidige literatuur over algemene aspecten van het virus en de ziekte, de PRRSV-specifieke immuniteit, en PRRSV vaccins. Verder wordt de huidige kennis over de PRRSV-specifieke neutraliserende antistofrespons uitgebreid besproken. In Hoofdstuk 2 wordt het specifiek probleem dat aan de basis ligt van deze thesis beschreven, en worden een aantal doelstellingen vooropgesteld. De volgende twee hoofdstukken omvatten onderzoeksresultaten die aan deze doelstellingen tegemoetkomen. In Hoofdstuk 3 werd onderzocht in welke mate virusneutraliserende antistoffen kunnen worden geïnduceerd door immunisatie van varkens met geïnactiveerd PRRSV. Deze studie komt tegemoet aan de vraag of de zwakke ontwikkeling van virusneutraliserende antistoffen afhankelijk is van structurele eigenschappen van het virus, of eerder van replicatie-afhankelijke mechanismen. Experimentele vaccins werden aangemaakt op basis van eerder geoptimaliseerde virusinactivatiemethodes, en gebruik makend van een aantal verschillende adjuvantia. Vaccinatie van PRRSV-negatieve biggen leidde telkens tot een sterke virusspecifieke antistofrespons, maar de virusneutraliserende antistofrespons was daarentegen erg zwak of afwezig, wat aangeeft dat de zwakke immunogeniciteit van neutraliserende epitopen in PRRSV grotendeels afhankelijk is van structurele eigenschappen van het virus. Toch konden door vaccinatie met een hoge dosis geïnactiveerd PRRSV in combinatie met één specifiek adjuvans virusneutraliserende antistoffen worden geïnduceerd, en er kon een zekere graad van bescherming worden geboden tegen viremie na infectie. Deze resultaten hebben de basis gevormd voor verder vaccinonderzoek aan het Laboratorium voor Virologie. Hoofdstuk 4 handelt over de identificatie van doelwitten voor niet-neutraliserende en neutraliserende antistoffen in de Europese prototype PRRSV stam Lelystad virus (LV). In een eerste studie (§ 4.1) werd de antistofrespons tegen lineaire epitopen in het GP4 eiwit onderzocht. Daaruit bleek dat antistoffen voornamelijk worden gevormd tegen één bepaalde regio in dit eiwit, die eerder werd beschreven als doelwit voor neutraliserende antistoffen. Aangezien deze regio sterk variabel is, werd de antistofrespons tegen GP4 ook onderzocht voor andere virusstammen. Het besluit van deze studie was dat de variabele regio in GP4 van EU-type PRRSV neutraliserende antistoffen induceert na infectie in varkens, maar enkel tegen strikt homoloog virus. In een tweede studie (§ 4.2) werd de antistofrespons tegen de volledige reeks gekende envelop-eiwitten (GP2, E, GP3, GP4, GP5 en M) na LV infectie onderzocht. Eénentwintig antigene regio’s werden geïdentificeerd die antistoffen kunnen induceren bij infectie in varkens. Peptide-opgezuiverde antistoffen tegen vier antigene regio’s bleken in vitro virusreplicatie te reduceren. Naast het gekende neutraliserend epitoop in GP4 werden twee doelwitten voor neutraliserende antistoffen gevonden in GP2, en één in GP3. In E, GP5 en M werden geen neutraliserende epitopen teruggevonden. Daar het neutraliserend epitoop in GP3 één van de meer immunogene regio’s was, werd de antistofrespons tegen dit epitoop verder onderzocht bij verschillende virusstammen. Samenvattend kan uit Hoofdstuk 4 worden besloten dat vier lineaire doelwitten voor neutraliserende antistoffen aanwezig zijn in de LV envelop-eiwitten: twee zwak immunogene epitopen in GP2, één meer immunogeen epitoop in GP3, en één sterk immunogeen maar aanzienlijk variabel epitoop in GP4. Hoofdstuk 5 tenslotte geeft een algemene bespreking van de onderzoeksresultaten in deze thesis. In § 5.1 wordt bediscussieerd wat kan worden geleerd uit de vaccinatie-experimenten in Hoofdstuk 3. § 5.2 vat de resultaten van Hoofdstuk 4 samen, en bespreekt deze in een bredere context. Eerst wordt een beknopt aanschouwelijk overzicht gegeven van de doelwitten voor niet-neutraliserende en neutraliserende antistoffen die werden geïdentificeerd in de virale envelop-eiwitten. Vervolgens worden een aantal hypotheses naar voor geschoven over de manier waarop neutraliserende antistoffen tegen verschillende delen van de virale eiwitten zouden kunnen interfereren met virusreplicatie. Daarna wordt de huidige kennis over PRRSV antigeniciteit bediscussieerd in het licht van virusevolutie, en een aantal bedenkingen worden gemaakt over hoe de hoge mutatiesnelheid en de grote variabiliteit van het virus een rol kunnen spelen bij het ontwijken van het immuunsysteem. Tot slot wordt kritisch geëvalueerd in welke mate de resultaten van deze thesis kunnen bijdragen tot de ontwikkeling van nieuwe en betere PRRSV vaccins.
Keywords
Epitope, Porcine reproductive and respiratory syndrome virus, Antigenic profiling, Antibody, Vaccine

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Citation

Please use this url to cite or link to this publication:

Chicago
Vanhee, Merijn. 2011. “Antigenic Profiling of the Porcine Reproductive and Respiratory Syndrome Virus”. Merelbeke, Belgium: Ghent University. Faculty of Veterinary Medicine.
APA
Vanhee, M. (2011). Antigenic profiling of the porcine reproductive and respiratory syndrome virus. Ghent University. Faculty of Veterinary Medicine, Merelbeke, Belgium.
Vancouver
1.
Vanhee M. Antigenic profiling of the porcine reproductive and respiratory syndrome virus. [Merelbeke, Belgium]: Ghent University. Faculty of Veterinary Medicine; 2011.
MLA
Vanhee, Merijn. “Antigenic Profiling of the Porcine Reproductive and Respiratory Syndrome Virus.” 2011 : n. pag. Print.
@phdthesis{1864156,
  abstract     = {Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and boars, and is involved in the porcine respiratory disease complex, causing huge economical losses to the swine industry.  Despite the development and broad implementation of vaccines, the virus could not be eradicated, and the high variability and fast evolution of the virus compromises the efficacy of the current vaccines in the field.  Since PRRSV engages a complex interaction with the host{\textquoteright}s immune system, and exploits several strategies to evade protective immunity, the development of new and better PRRSV vaccines is highly relying on a good understanding of the virus-specific immune response.  The general goal of this work was to create new insights into one particular hallmark of PRRSV-specific immunity, namely the weak virus-neutralizing antibody response upon infection or vaccination in pigs, in sharp contrast with the robust non-neutralizing antibody response.  Chapter 1 summarizes the current literature on general aspects of the virus and the disease, PRRSV-specific immunity, and PRRSV vaccines. Furthermore, the current knowledge on the PRRSV-specific neutralizing antibody response is extensively discussed.  In Chapter 2, the particular problem underlying the work in this thesis is described, and a number of aims are stated.  The next two chapters consist of research results addressing these aims.  In Chapter 3, it was investigated to which extent virus-neutralizing antibodies can be induced by immunization of pigs with inactivated PRRSV.  This study addresses the question whether the poor development of virus-neutralizing antibodies is dependent on structural properties of the virus, or rather on replication-dependent mechanisms.  Experimental vaccines were produced, based on formerly optimized virus inactivation procedures and by using a number of different adjuvants.  Vaccination of PRRSV-negative piglets systematically led to a strong virus-specific antibody response, but the virus-neutralizing antibody response was very weak or absent, indicating that the poor immunogenicity of neutralizing antibody targets in PRRSV is to a large extent inherent to the structural properties of the virus.  Still, vaccination with a high dose of inactivated PRRSV in combination with one particular adjuvant induced virus-neutralizing antibodies, and provided a certain level of protection against viremia upon infection.  These results formed the basis for further vaccine research at the Laboratory of Virology.  Chapter 4 deals with the identification of non-neutralizing and neutralizing antibody targets in the European prototype PRRSV strain Lelystad virus (LV).  In a first study ({\textsection} 4.1), the antibody response against linear epitopes in the GP4 protein was investigated.  It was found that antibodies are to a large extent produced against one particular region of this protein that was earlier described as neutralizing antibody target.  Since this region is highly variable, the antibody response against GP4 was also investigated for other virus strain.  The conclusion of this study was that the variable region in GP4 of EU-type PRRSV induces neutralizing antibodies upon infection in pigs, but only against strictly homologous virus.  In a second study ({\textsection} 4.2), the antibody response against the entire set of envelope proteins (GP2, E, GP3, GP4, GP5 and M) upon infection with LV was investigated.  Twenty-one antigenic regions were identified that are capable of inducing antibodies in pigs.  Peptide-purified antibodies against four antigenic regions turned out to reduce in vitro virus replication.  In addition to the known neutralizing epitope in GP4, two neutralizing antibody targets were identified in GP2, and one in GP3.  No neutralizing epitopes were found in E, GP5 and M.  Since the neutralizing epitope in GP3 was one of the more immunogenic regions, the antibody response against this epitope was investigated more extensively in different virus strains.  Taken together, it can be concluded from Chapter 4 that four linear neutralizing antibody targets are present in the LV envelope proteins: two weakly immunogenic epitopes in GP2, one more immunogenic epitope in GP3, and one strongly immunogenic but highly variable epitope in GP4.  Chapter 5 provides a general discussion on the research data generated in this thesis.  In {\textsection} 5.1, it is discussed what can be learned from the vaccination studies in Chapter 3. {\textsection} 5.2 summarizes the results from Chapter 4, and discusses them in a broader context.  First, a synoptic visual overview is given of the non-neutralizing and neutralizing antibody targets that were identified in the viral envelope proteins.  Next, some hypotheses are presented on how neutralizing antibodies against different parts of the viral proteins may interfere with viral replication.  Subsequently, the current knowledge on PRRSV antigenicity is discussed in light of virus evolution, and some thoughts are made on how the high mutation rate and large variability of the virus may be involved in immune evasion.  To conclude, it is critically evaluated to which extent the data generated in this thesis can contribute to the development of new and better PRRSV vaccines.},
  author       = {Vanhee, Merijn},
  isbn         = {9789058642646},
  keyword      = {Epitope,Porcine reproductive and respiratory syndrome virus,Antigenic profiling,Antibody,Vaccine},
  language     = {eng},
  pages        = {III, 179},
  publisher    = {Ghent University. Faculty of Veterinary Medicine},
  school       = {Ghent University},
  title        = {Antigenic profiling of the porcine reproductive and respiratory syndrome virus},
  year         = {2011},
}