73 Parole chiave Cavalli, Risposta immunologica, Vaccino, Virus della West Nile. Riassunto Il virus della West Nile (WNV) è un virus a RNA del genere Flavivirus, famiglia Flaviviridae che, trasmesso da zanzare, può causare morbilità e mortalità significative in uccelli, umani e cavalli. Questi ultimi sono ospiti a fondo cieco. Ad oggi non esiste una cura per la malattia e le misure preventive sono fondamentali; per i cavalli, occorre ridurre al minimo l'esposizione ai vettori o vaccinare. In Europa sono in uso tre vaccini per cavalli che, quando sono stati utilizzati, hanno impedito la manifestazione dei segni clinici della WND. Per valutare la risposta immunologica alla vaccinazione sono stati scelti quaranta cavalli sierologicamente negativi al WNV e divisi in due gruppi da venti unità. Un gruppo è stato vaccinato (richiamo dopo 28 giorni) con un intero ceppo virale inattivato e il secondo gruppo con uno vivo ricombinante per il canarypox virus che esprime i geni codificanti per le proteine virali preM / E del virus della West Nile. Per 360 giorni IgM, IgG e anticorpi neutralizzanti sono stati Risposta immunologica indotta da due vaccini per il virus della West Nile: inattivato e ricombinante Keywords West Nile virus, Vaccine, Horses, Immunological response. Summary West Nile virus (WNV) is an RNA virus belonging to Flavivirus genus, family Flaviviridae. Transmitted by mosquitoes, the virus may cause significant morbidity and mortality in birds, humans and horses. Humans and horses are dead‑end hosts. As no specific treatment for the disease is currently available, preventive measures are critical. In horses the prevention can be achieved by minimizing the exposure to the vectors or through vaccination. In Europe three products have been registered for horses. When used, they are capable of preventing WND clinical signs. To evaluate the immunological response following vaccination, 40 WNV serologically negative horses were selected and divided in two groups of 20 animals. One group was vaccinated (booster after 28 days) with a whole inactivated viral strain and the second group with a live recombinant canarypox virus expressing the genes coding for the WNV preM/E viral proteins. IgM, IgG and neutralizing antibodies were monitored by class specific ELISAs and serum neutralization assay for 360 days. In both groups, IgM antibodies were first detected 7 days post vaccination lasting up to 42 dpv and 52 dpv in the animals vaccinated with the inactivated and the recombinant product respectively. A similar (P < 0.05%) number of horses [30%; 95% Confidence interval (CI):14.59%‑52.18%] showed IgM antibodies after vaccination with the recombinant vaccine (Rec‑WNV) compared to the group (32%; 95% CI: 15.39‑54.28%) vaccinated with the inactivated vaccine (K‑WNV). Both vaccines induced a detectable IgG antibody response as early as 7 days following the vaccinations till the end of the trial. Both vaccines induced the rise in neutralizing antibodies even though neutralizing antibody responses induced by the live canarypox virus‑vectored vaccine were higher (mean titres 1:298 vs 1:18.9) and lasted longer than did those induced by the killed‑virus vaccines. In fact, one year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus‑based vaccine but not the group vaccinated with the killed product. The use of vaccines is a valuable tool to prevent WNV disease in horses and the availability of different products facilitate the control of the disease in endemic areas. Veterinaria Italiana 2019, 55 (1), 73‑79. doi: 10.12834/VetIt.1820.9611.1 Accepted: 24.10.2018 | Available on line: 31.03.2019 1Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy. 2Istituto Zooprofilattico Sperimentale della Sicilia ‘A. Mirri’, via Gino Marinuzzi 3, 90129 Palermo, Italy. *Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332446, e‑mail: f.monaco@izs.it. Federica Monaco1*, Giuseppa Purpari2, Annapia Di Gennaro1, Francesco Mira2, Patrizia Di Marco2, Annalisa Guercio2 and Giovanni Savini1 Immunological response in horses following west Nile virus vaccination with inactivated or recombinant vaccines 74 Veterinaria Italiana 2019, 55 (1), 73‑79. doi: 10.12834/VetIt.1820.9611.1 West Nile virus vaccines in horses Monaco et al. previously Duvaxyn® WNV, Pfizer, US) (EMEA 2008), the recombinant canarypox virus vCP2017 strain, that expresses the WNV prM/prE genes (Recombitek equine WNV vaccine, Merial) (EMEA 2011) and the inactivated chimaeric flavivirus strain of Yellow fever virus presenting the genes for the structural proteins E and prM of WNV (Equilis® West Nile, Intervet International BV, Netherlands) (EMEA 2013). These products are capable of protecting horses against possible development of clinical manifestations of the infection which can lead to a severe and long lasting neurological impairment of the animal. Aim of this study was to look into the dynamic of the serological response in horses vaccinated with two different products widely used in EU member countries to protect horse population, the inactivated vaccine (Equip® WNV) and the recombinant canarypox virus expressing the WNV prM/prE genes (Recombitek equine WNV). Materials and methods Forty healthy horses, serologically negative to WNV, were randomly selected and divided into two groups of twenty animals. One group was vaccinated with the inactivate WNV strain, the other with the recombinant canarypox expressing the WNV prM/prE genes. Both groups received a booster shot 28 days after the first dose. Following the vaccination, all horses were regularly bled up to 1 year to monitor the immune response. To this purpose, blood samples were collected by jugular venipuncture in dry tubes at the indicated time points (Figure 1). Sera were prepared and stored at ‑20  °C until analysis. A commercial ELISA kit (ID Screen West Nile Competition Multi‑species ‑ ID‑Vet, Montpellier, France) was used to detect IgG immunoglobulins while the West Nile Virus IgM Introduction West Nile virus (WNV) is a member of the Flavivirus genus family Flaviviridae. Included in the Japanese encephalitis group, it is one of the most widespread arbovirus in the world. In Europe, WNV has been reporting since the mid 1960s (Filipe et  al. 1969, Joubert et  al. 1970) but the virus circulation has increased dramatically in the last decades (Calistri et al. 2010, Savini et al. 2018). In Italy, WNV epidemics caused by genetically divergent isolates have been recorded since 2008 (Savini et al. 2008, Monaco et al. 2010, Savini et  al. 2012) and most of the territory is nowadays endemic. The infection in nature in a mosquito‑bird cycle involving Culex species of ornitophilic mosquitoes as main vectors and an extensive variety of birds as reservoir hosts (Komar et  al. 2001, Mancini et  al. 2017). Mammals including humans and equidae are susceptible to the infection and can show clinical symptoms ranging from a flu‑like syndrome to a fatal meningoencephalitis (Komar 2000, Debiasi and Tyler 2006). Because of the low grade of viraemia and the lack of viral shedding, the virus cannot be transferred further and an infected horse acts as a dead‑end host (Bunning et  al. 2002). Nevertheless, the development of the severe clinical symptoms might raise devastating emotional effects and significant financial burden to owners. There is no specific anti‑viral treatment for the disease and prevention can be achieved by minimizing the exposure to the vector or, in equines, through vaccination (Amanna and Slifka 2014). To date, three vaccines have obtained the marketing authorization in European Union (EU) member countries: the inactivated vaccine, produced from the VM‑2 strain (Equip® WNV, Zoetis, Belgium monitorati con ELISA specifici per classe e test di sieroneutralizzazione. In entrambi i gruppi, gli anticorpi IgM sono stati rilevati per la prima volta dopo 7 giorni di vaccinazione con una durata fino a 42 dpv e 52 dpv negli animali vaccinati rispettivamente con il prodotto inattivato e il prodotto ricombinante. Dopo la vaccinazione, sia il gruppo sottoposto al ricombinante (Rec‑WNV) sia quello trattato con l’inattivato (K‑WNV) ha mostrato anticorpi IgM in un numero simile (P < 0,05%) di cavalli con risultati rispettivamente pari a 30%; 95% intervallo di confidenza (CI): 14,59%‑52,18% e a 32%; IC 95%: 15,39‑54,28%. Entrambi i vaccini hanno indotto una risposta anticorpale IgG rilevabile già 7 giorni dopo le vaccinazioni fino alla fine dello studio. Entrambi i vaccini hanno indotto l'aumento degli anticorpi neutralizzanti, anche se le risposte al vaccino vivo ricombinante sono state più elevate (titoli medi 1: 298 vs 1: 18,9) e sono durate più a lungo di quelle indotte dal vaccino con il virus inattivato. A un anno dalla vaccinazione infatti, non erano rilevabili nei cavalli di quest’ultimo gruppo si riscontravano in quelli del primo gruppo. I vaccini sono uno strumento prezioso per prevenire la malattia del WNV nei cavalli e la disponibilità di diversi prodotti facilita il controllo della malattia nelle aree endemiche. 75Veterinaria Italiana 2019, 55 (1), 73‑79. doi: 10.12834/VetIt.1820.9611.1 Monaco et al. West Nile virus vaccines in horses after vaccination with the recombinant vaccine (Rec‑WNV ) compared to the group (32%; 95% CI: 15.39‑54.28%) vaccinated with the inactivated vaccine (K‑WNV ). In both groups, IgM antibodies were first detected after 7 days. However, in the group vaccinated with the inactivated product, the IgM antibodies were detected up to 42 dpv (in 1 animal) while in the group immunized with the recombinant product IgM antibodies were detected up to 52 dpv (in 5 animals) (Figure 1). In the K‑WNV group, although the IDEXX ELISA was capable of detecting a higher percentage of positive animals (32%; 95% CI:  15.39‑54.28%) than the ID‑VET kit (21%; 95% CI: 8.66‑43.66%), this difference was not statistically significant (P > 0.05%) (Figure 1). Both vaccines induced a detectable IgG response as early as 7 days following the vaccinations whatever the vaccine used. In the K‑WNV group, all animals seroconverted on day 18 pv while in the Rec‑WNV group, all the animals become IgG positive following the booster injection (on day 35 pv). IgG antibodies were detected in the animals of both groups until the end of the trial (one year after vaccination). In the group vaccinated with the modified canarypox strain, neutralizing antibodies were first detected on day 18 pv in 1 horse. From day 42 pv till the end of the trial, neutralizing titers were detected in all vaccinated animals. The peak was observed on day 56 pv (mean antibody titer  =  1:298) (Figure  2). In contrast, not all the animals vaccinated with the killed WNV strain developed a neutralizing response following vaccination. In fact, neutralizing titers were first detected on day 21 pv and 3 weeks later (day 42  pv) all except one animal seroconverted. After peaking on day 42 (mean antibody titer  =  1:18.9), the neutralizing titers decreased and, at the end of the trial, only 4 animals showed detectable titers (1:5) (Figure 2). None of the animals in the trial developed Antibody ELISA kit (IDEXX Laboratories, Inc., Maine, US) to evaluate the IgM response in both groups. IgM response in the group of animals receiving the recombinant product was also assessed by the ID Screen West Nile IgM Capture (IDvet, Grabels, France). The tests were performed according to the manufacturer instructions. To confirm the presence of WNV antibodies, to define their titers and to exclude any cross reaction with other co‑circulating related flaviviruses such as Usutu (Savini et al. 2011), serum samples were also examined by serum neutralization (SN) assay in microtitre format (Di Gennaro et  al. 2014). For each proportion, 95% confidence intervals (CI) were calculated through the Bayesian approach using the Beta (s+1, n‑s+1) distribution where s is the total number of positives and n is the total number of tested animals. Any WNV circulation in the area where the animals were kept was investigated according to the procedure defined in the WNV National Surveillance Plan (Italian Ministry of Health, 20161). Results No adverse effects were observed in the animals following vaccination. IgM antibodies were detected in both groups of animals following vaccination (Figure  1). A similar (P  <  0.05%) proportion of horses [30%; 95% Confidence interval (CI):  14.59%‑52.18%] developed IgM antibodies 1 Italian Ministry of Health. 2016. Piano Nazionale integrato di sorveglianza e risposta al virus della West Nile. Circolare 10/08/2016, n. 23689. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 50 100 150 200 250 300 350 0 7 11 14 18 21 28 Days post vaccination M ea n n eu tr al iz in g t it re P er ce n ta g e o f h o rs es w it h n eu tr al iz in g a n ti b o d ie s 35 42 49 56 90 360 % positive horses K-WNV % positive horses Rec-WNV Mean titre K-WNV Mean tire Rec-WNV Figure 2. Mean titres and percentage of animals with detectable neutralizing antibodies in two groups of horses following vaccination against the West Nile virus (WNV) with an inactivated (K-WNV) or a recombinant canaripox WNV vaccine (Rec-WNV). 0 7 11 14 18 21 28 35 42 52 56 K-WNV (1) 0 0.1 0.32 0.16 0.11 0.11 0.11 0 0 0 0 K-WNV (2) 0 0.1 0.21 0.21 0.16 0.11 0.11 0.11 0.05 0 0 Rec-WNV 0 0.05 0 0.25 0.3 0.25 0.1 0.2 0.25 0.25 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 % p o si ti ve h o rs es Days post vaccination Figure 1. Detection of IgM antibodies in 2 groups of horses (n = 20) vaccinated either with an inactivated West Nile virus vaccine (K-WNV) or with a recombinant canarypox WNV vaccine (Rec-WNV). K-WNV (1) and Rec-WNV = IgM response detected by ‘West Nile Virus IgM Antibody’ Test (IDEXX Laboratories, Inc., Maine, US). K-WNV (2) = IgM response detected by ID Screen West Nile IgM Capture (IDvet, Grabels, France). 76 Veterinaria Italiana 2019, 55 (1), 73‑79. doi: 10.12834/VetIt.1820.9611.1 West Nile virus vaccines in horses Monaco et al. This study confirmed, once more, how difficult is to predict the IgM kinetic following vaccination suggesting that, in endemic areas or within a WNV surveillance plans, it is not possible to differentiate infected horses from recently vaccinated horses based on the presence/absence of IgM antibodies. As a consequence, positive IgM results should be carefully interpreted by verifying the vaccination history of the horses especially when vaccination against WNV is commonly practiced. When dealing with WNV IgM antibodies, particular attention should also be paid to the IgM ELISAs used since variations may exist in the IgM ELISA performances which often demonstrate different diagnostic sensitivity and potential for false positive results (Davidson et  al. 2005, Beck et  al. 2017). In this study, although in the number of positive animals detected by the two ELISAs after the vaccination with the inactivated product was different, this difference was not statistically significant. Both vaccines were capable of evoking an IgG  response in all vaccinated animals even if differences between groups were observed in the timeframe elapsed to stimulate the complete seroconversion. All horses immunized with the inactivated product developed a detectable IgG  response starting from the 18th dpv while 35 days were necessary to achieve the complete seroconversion in the group which received the recombinant vaccine. Such difference may rely on the different nature of the two vaccines. The viral antigens are ‘ready to be used’ in the inactivated vaccines, while with the recombinant product the same antigens need to be expressed by the vaccinated host vaccinated. Differences were also noted in the duration and intensity of the neutralizing antibodies response elicited by the two vaccines. Both vaccines induced the rise in neutralizing antibodies as observed by other authors (Davies et al. 2008, Joò et al. 2017, Seino et al. 2007) even though neutralizing antibody responses induced by the live canarypox virus‑vectored vaccine were higher and lasted longer than did those induced by the killed‑virus vaccines. One year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus‑based vaccine but not in the group vaccinated with the killed product. Long‑term immunity is not a characteristic of inactivated vaccines, and field studies demonstrated the drop of neutralizing titers 5 to 7 months after vaccination (Davidson et  al. 2005). In our K‑WNV group of horses, the presence of neutralizing antibodies was detected only in four animals with titers below the threshold (< 1:10). It is known that the protective immune response to WNV requires both innate and adaptive immunity (De Filette et al. 2012) and there is strong evidence neutralizing antibodies against Usutu virus nor WNV circulation was detected in the area during the study period. Discussion In accordance with the Directive 2003/99/EC on monitoring of zoonoses and zoonotic agents, European Member States collect data in order to define hazards, to evaluate exposures and to assess risks related to zoonoses and zoonotic agents. As a consequence, Member States have implemented surveillance programs and, since 2012, they have been collecting data and reporting WND cases. In Austria, France, Greece, Italy, and United Kingdom, an integrated animal‑human‑vector approach is already in place (Gossner et  al. 2017). Veterinary surveillance usually relies on the combination of passive measures, based on reporting clinical WND cases in horses, and active surveillance, based on detecting seroconversion in sentinel horses (Humblet et  al. 2016). The serological tests most commonly used are ELISAs (for detection of IgG and IgM antibodies) for screening and VNT as confirmatory test. The humoral response following WNV infection includes the production of IgM antibodies, which are detectable 4‑7  days after WNV infection, and of IgG, detectable 5  to  7  days after the infection (Bunning et al. 2002). As the IgM lifespan is considered to be less than three months in horses, the presence of IgM antibodies in this species is regarded as a valuable indicator of recent infections (Castillo‑Olivares and Wood 2004) and, as a consequence, the WNV IgM ELISA the test of choice for diagnosis of recent infection. It is also claimed that vaccination only occasionally elicits an IgM response making it possible to differentiate acutely‑infected from recently vaccinated horses by using an IgM‑based ELISA (EMEA 2008). The possibility to differentiate a recently infected horse from one that has been (recently) vaccinated is particularly useful to early identify viral circulation either in WNV‑free or endemic areas. In this study, WNV recombinant and killed vaccines were examined and even if the IgM response of the inactivated vaccine was lower, shorter and involving fewer animals, both products were capable of stimulating the production of IgM  antibodies in vaccinated horses. These results were in line with what found in similar trials by other authors (Porter et al. 2004, Jonquiere et al. 2011, Joò et  al. 2017). Surprisingly, in few animals vaccinated with the recombinant vaccine, the IgM antibodies persisted for 52 days after vaccination showing a kinetic similar to that observed in naturally infected horses in which IgM antibodies can be detected 3  months after infection (Ostlund et  al. 2000). 77Veterinaria Italiana 2019, 55 (1), 73‑79. doi: 10.12834/VetIt.1820.9611.1 Monaco et al. West Nile virus vaccines in horses reported, the cell‑mediated response is still poorly explored (Nelson et al. 2010). The use of vaccines has been demonstrated as a valuable preventative strategy against WNV disease in horses (Grosenbaugh et al. 2004, Siger et al. 2004, Joò et  al. 2017). The 2018 has been regarded as an exceptional year referring to WNV circulation in EU countries (Haussig et  al. 2018, Riccardo et  al. 2018) with the infection of a high number of horses even in endemic areas (https://westnile.izs.it/j6_wnd/ docBolletMeditPeriodico?annoDocumento=2018). Thus, the availability of different products provides a valuable tool to reduce the impact of the severe clinical symptoms in this species Funding This work was supported by the Italian Ministry of Health (MSRCSI 01, 2012). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the OIE Reference Laboratory for West Nile Fever of the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. that neutralizing antibodies provide long‑term protection from clinical signs of the disease (Pierson and Diamond 2015). Different neutralizing responses however do not necessarily reflect dissimilarities in the protective capacity induced by the two vaccines. Many trials demonstrated that the level of neutralizing antibody titer is not predictive of protective immunity in horses or hamsters (Tesh et  al. 2002, Seino et  al. 2007) since protection may occur in the absence of detectable antibodies. The requirements of significant levels of neutralizing Ab at the time of exposure may not be critical as long as vaccination properly primes the immune system and response is rapid (Minke et al. 2004). The absence or scarce level of humoral response does not preclude the efficacy of the cellular response. In fact, the role of cell mediated immunity in protecting against WNV and other related flaviviruses has been demonstrated in experimental murine studies (Diamond et  al. 2003, Shrestha and Diamond 2004). 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