Development of an experimental inactivated PRRSV vaccine that induces virus-neutralizing antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce reproductive disorders and is involved in the porcine respiratory disease complex, causing tremendous economic losses to the swine industry. Inactivated PRRSV vaccines are preferred over attenuated vaccines because of their safety and flexibility towards emerging virus strains, but the efficacy of current inactivated PRRSV vaccines is questionable. In this study, experimental inactivated PRRSV vaccines were developed, based on two formerly optimized inactivation procedures: UV irradiation and treatment with binary ethylenimine (BEI). In a first experiment, it was shown that vaccination with UV- or BEI-inactivated virus in combination with Incomplete Freund's Adjuvant induced virus-specific antibodies and strongly primed the virus-neutralizing (VN) antibody response. Subsequently, the influence of adjuvants on the immunogenicity of neutralizing epitopes on the inactivated virus was investigated. It was shown that vaccination with BEI-inactivated virus in combination with a commercial oil-in-water adjuvant induced high titers (3.4 log(2)) of VN antibodies in 6/6 pigs, instead of only priming the neutralizing antibody response. After challenge, neutralizing antibody titers in these vaccinated animals rose to a mean value of 5.5 log(2), and the duration of the viremia was reduced to an average of 1 week. This study shows that, by the use of an optimized inactivation procedure and a suitable adjuvant, inactivated PRRSV vaccines can be developed that induce VN antibodies and offer partial protection upon challenge

Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is divided into a European and North American genotype. East European PRRSV isolates have been found to be of the European genotype, but form different subtypes. In the present study, PRRSV was isolated from a Belarusian farm with reproductive and respiratory failure and designated "Lena". Analyses revealed that Lena is a new East European subtype 3 PRRSV isolate. The main purpose of this investigation was to study the pathogenesis and antigenic characteristics of PRRSV (Lena). Results: Obvious clinical and virological differences were observed between the animals inoculated with a recent European subtype 1 PRRSV isolate (Belgium A) and animals inoculated with PRRSV (Lena). Three out of six pigs inoculated with PRRSV (Belgium A) had anorexia and low fever at 3, 4 and 5 days post-inoculation (dpi). High fever, anorexia and depression were prominent signs in most pigs inoculated with PRRSV (Lena) between 2 and 28 dpi. Four pigs out of ten died during the experiment. Arcanobacterium pyogenes was isolated from lungs of one animal that died, and Streptococcus suis was isolated from lungs of one animal that was euthanized. The difference in viral titres in sera from PRRSV (Belgium A) and PRRSV (Lena)-infected pigs was statistically significant (p < 0.05) at 7, 10, 14 and 21 dpi. The highest viral titres in sera ranged from 10(4.8) to 10(6.1) TCID50/ml for PRRSV (Lena) whereas they ranged from 10(3.1) to 10(4.8) TCID50/ml for PRRSV (Belgium A). The replication of PRRSV (Lena) was further studied in depth. Viral titres ranged from 10(2.5) TCID50/100 mg to 10(5.6) TCID50/100 mg in nasal secretions between 3 and 14 dpi and from 10(2.8) TCID50/100 mg to 10(4.6) TCID50/100 mg in tonsillar scrapings between 3 and 21 dpi. High viral titres were detected in lungs (10(2.3)-10(7.7) TCID50/g tissue), tonsils (10(2.0)-10(6.2) TCID50/g tissue) and inguinal lymph nodes (10(2.2)-10(6.6) TCID50/g tissue) until 35, 28 and 35 dpi, respectively. To examine the antigenic heterogeneity between the East European subtype 3 isolate Lena, the European subtype 1 strain Lelystad and the North American strain US5, sets of monospecific polyclonal antisera were tested in immunoperoxidase monolayer assays (IPMAs) with homologous and heterologous viral antigens. Heterologous antibody titres were significantly lower than homologous titres (p = 0.01-0.03) for antisera against PRRSV (Lena) at all sampling time points. For antisera against PRRSV (Lelystad) and PRRSV (US5), heterologous antibody titres were significantly lower than homologous titres at 14 and 21 dpi (p = 0.01-0.03) and at 10 and 14 dpi (p = 0.04), respectively. Conclusions: Lena is a highly pathogenic East European subtype 3 PRRSV, which differs from European subtype 1 Lelystad and North American US5 strains at both the genetic and antigenic level

Persistent domestic circulation of African swine fever virus in Tanzania, 2015–2017

Background African swine fever (ASF) is a highly fatal viral hemorrhagic disease of domestic pigs that threatens livelihoods and food security. In Africa, ASF virus (ASFV) circulates in sylvatic (transmission between warthogs and soft argasid ticks) and domestic (transmission between domestic pigs) cycles, with outbreaks resulting from ASFV spill-over from sylvatic cycle. A number of outbreaks were reported in different parts of Tanzania between 2015 and 2017. The present study investigated ASFV transmission patterns through viral DNA sequencing and phylogenetic analysis. A total of 3120 tissue samples were collected from 2396 domestic pigs during outbreaks at different locations in Tanzania between 2015 and 2017. Partial sequencing of theB646L(p72) gene was conducted for diagnostic confirmation and molecular characterization of ASFV. Phylogenetic analysis to study the relatedness of current ASFV with those that caused previous outbreaks in Tanzania and representatives of all known 24 ASFV was performed using the Maximum Composite Likelihood model with 1000 bootstrap replications in MEGA 6.0. Results ASFV was confirmed to cause disease in sampled domestic pigs. ASFV genotypes II, IX, and X were detected from reported outbreaks in 2015-2017. The current ASFV isolates were similar to those recently documented in the previous studies in Tanzania. The similarities of these isolates suggests for continuous circulation of ASFV with virus maintenance within the domestic pigs. Conclusions Genetic analysis confirmed the circulation of ASFV genotypes II, IX, and X by partialB646L(p72) gene sequencing. The similarities of current isolates to previously isolated Tanzanian isolates and pattern of disease spread suggest for continuous circulation of ASF with virus' maintenance in the domestic pigs. Although certain viral genotypes seem to be geographically restricted into certain zones within Tanzania, genotype II seems to expand its geographical range northwards with the likelihood of spreading to other states of the East African Community. The spread of ASFV is due to breach of quarantine and transportation of infected pigs via major highways. Appropriate control measures including zoosanitary measures and quarantine enforcement are recommended to prevent ASF domestic circulation in Tanzania

RNA-Sequence analysis of primary alveolar macrophages after in vitro infection with porcine reproductive and respiratory syndrome virus strains of differing virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects porcine alveolar macrophages (PAMs), resulting in porcine reproductive and respiratory syndrome (PRRS) in pigs. Most of the transcriptomic studies on PAMs infected with PRRSV conducted thus far have made use of microarray technology. Here, we investigated the transcriptome of PAMs in vitro at 12 h post-infection with two European PRRSV strains characterized by low (Lelystad, LV) and high (Lena) virulence through RNA-Seq. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with the two strains. Gene ontology analysis confirmed that infection of PAMs with both the Lena and LV strains affected signaling pathways directly linked to the innate immune response, including interferon regulatory factors (IRF), RIG1-like receptors, TLRs and PKR pathways. The results confirmed that interferon signaling is crucial for transcriptional regulation during PAM infection. IFN-beta 1 and IFN-alpha omega, but not IFN-alpha, were up-regulated following infection with either the LV or Lena strain. The down-regulation of canonical pathways, such as the interplay between the innate and adaptive immune responses, cell death and TLR3/TLR7 signaling, was observed for both strains, but Lena triggered a stronger down-regulation than LV. This analysis contributes to a better understanding of the interactions between PRRSV and PAMs and outlines the differences in the responses of PAMs to strains with different levels of virulence, which may lead to the development of new PRRSV control strategies

Antigenic profiling of the porcine reproductive and respiratory syndrome virus

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