Vaccination of alpacas against Rift Valley fever virus: Safety, immunogenicity and pathogenicity of MP-12 vaccine
Introduction
Rift Valley fever (RVF) is a disease of high impact for both livestock and humans within Africa and Arabia. The disease is caused by the Rift Valley fever virus (RVFV), an Arbovirus (arthropod-borne) of the family Bunyaviridae, genus Phlebovirus. Its tripartite ambisense RNA-genome encompasses a large (L)-, medium (M)- and a small (S) segment. The L segment is encoding the RNA-dependent RNA polymerase. The M segment encodes for the glycoproteins Gn and Gc, two major targets for neutralization, and for a nonstructural protein NSm, that is responsible for apoptosis inhibition. Finally the S segment is encoding the nucleoprotein NP and another nonstructural protein, NSs, known to determine virulence through inhibition of host immune response [1].
RVFV is presumably transmitted by more than 30 different mosquito species [2]. The virus is endemic throughout many sub-Saharan African countries and recurrently causes substantial outbreaks [3]. An introduction of RVFV to naïve regions such as Europe or the United States would be of massive concern. Humans are usually infected by contact to viremic animals and occasionally by mosquito bites. Although most human infections proceed as mild flu-like illnesses, severe progression of meningoencephalitis, retinitis or hemorrhagic fever syndromes can also be observed [4], [5]. Clinical signs in livestock mainly depend on species and age of infected animals. Most severe symptoms are seen in small ruminants, in which so called ‘abortion storms’ with up to 100% mortality rates in fetuses and neonates are characteristic [6]. The particular role of camelids in the epidemiology of RVFV was discussed controversially during the past decades. A RVFV infection of dromedary camels was first described 1962 in Kenya, where animals were conspicuous because of abortions [7]. Thereupon no severe clinical manifestations were observed but several studies found camels to carry RVFV-specific antibodies [8], [9], [10]. However, the significant role of camelids in transmission and spread of the virus were already demonstrated by their involvement during the introduction of RVFV to Egypt [11], [12]. During an epidemic of RVF in Mauritania, severe clinical manifestations, such as ocular discharge, foot lesions, hemorrhages, neurological disorders as well as fatal hyperacute progressions were observed for the first time. An association between infected camels and subsequent human infections highlights the previously underestimated role of camelids in terms of RVFV epidemiology [13]. Therefore safe and efficacious vaccines for camelids are urgently needed. However, to date only few data about application of RVFV vaccines for camelids are available [14].
The best protection against fatal RVFV infections is usually facilitated by life-attenuated vaccines, such as Clone 13 or MP-12 [15]. MP-12 was generated by serial passage of the ZH548 strain in the presence of the chemical mutagen 5-fluorouracil [16]. It is one of the best characterized RVFV-vaccines and it is currently conditionally licensed for veterinary applications in the United States. The efficacy of the vaccine was proven in numerous studies in sheep, cattle and macaques [17], [18], [19], [20], [21] and recently a phase 2 clinical trial in humans was performed [22]. However, teratogenic effects and abortions in sheep were observed when administered during the first trimester of gestation [23]. Additionally studies of Wilson et.al suggested the necessity of performing independent safety testings of veterinary vaccines on target species, as remaining replication of MP-12 in the liver was verified [24].
In this study we analyze the safety, immunogenicity and pathogenicity of the MP-12 in alpacas as model-organism for dromedary camels, as important RVFV target species. For this purpose three male alpacas were immunized and the immune response was analyzed. Moreover virus shedding and replication in tissues of the animals after MP-12 challenge were examined.
Section snippets
Virus and cell culture
The MP-12 strain of RVFV was kindly provided by Richard Elliot, University of Glasgow, Centre for virus research, United Kingdom. MP-12 was propagated in Vero 76 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany).
The virus titer was determined using a 50% Tissue Culture Infective Dose (TCID50) assay, calculated as described by Spearman and Kaerber. Briefly, 100 μl of 10-fold serial diluted MP-12 were added to 90% confluent monolayers of Vero 76 cells.
Clinical assessment
Following the application of the MP-12, no clinical signs were observed in any of the animals. Similarly, the determination of blood leucocytes gave no meaningful deviations. Liver enzymes in serum were predominantly stable, with one exception of a short-term elevation of alanine aminotransferase in animal #A3 (see supplemental Fig. S1). No remarkable fluctuations were observed for the bile acids and γ-GT.
Serology
Significant serological responses were detectable in all assays for animal #A2 and #A3,
Discussion
In this study, host responses of alpacas after MP-12 immunization were analyzed in terms of safety, immunogenicity and potential pathogenicity.
MP-12 was highly immunogenic in alpacas, as significant seroconversion was recognized. In general, antibodies were detectable from 7 dpi in various assays. At that time both #A2 and #A3 elicited solid levels of neutralizing antibody titers above 1:40, which is defined as minimal acceptable titer for RVF at-risk personnel [33]. Neutralizing titers of #A2
Financial support
This work was supported by the Innovative Medicines Initiative (IMI); Grant no. 115760 – [ZAPI] - 324 DoW V1. This study was furthermore supported by EU grant FP7-613996 Vmerge and is catalogued by the VMERGE Steering Committee as Vmerge 019 (http://www.vmerge.eu<http://www.vmergedata.com/>).
The contents of this publication are the sole responsibility of the authors and don't necessarily reflect the views of the European Commission.
Conflict of interest
None.
Acknowledgment
The authors wish to thank Silvia Schuparis and Birke Böttcher for their excellent technical assistance. Additionally the authors would like to thank Christine Fast and Claudia Schulz for their expertise and support within the experiments.
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