Challenge infection model for MERS-CoV based on naturally infected camels

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 from a pneumonic patient who subsequently died, in the Kingdom of Saudi Arabia (KSA) [1]. Since its emergence, the virus has infected more than 2450 individuals in 27 countries [2]. Outbreaks occurred mainly in the Arabian Peninsula in large crowded hospitals with one large outbreak in the Republic of Korea [3]. To date, bats were suggested, but have not been confirmed, as the virus natural reservoir with some suggestive experimental data [4], whilst dromedary camels are the only confirmed intermediate animal host. 54.9% of human primary cases have reported contact with camels [5] and the index patient in the Korean outbreak traveled back from the Gulf countries where MERS-CoV is endemic and circulating in dromedary camels [6]. Dromedaries in Africa and Arabia have a high rate of seroprevalence ranging from 74 to 100% [7‐11]. More importantly, these animals seem to have been infected with MERS-CoV by as early as 1983 according to serological data on archived dromedary sera. Samples from several countries in Africa and Arabia, collected in different years between 1983 and 2010, were seropositive for MERS-CoV with a range of seroprevalence between 29 and 97% [8, 12‐16]. Moreover, a recent study looking at dromedaries associated with confirmed human cases in Saudi Arabia found that 70% of these camels were seropositive and that viral RNA could be detected in 12% of these camels [6, 17].

Adult camels and older calves are more likely to be seropositive as compared to younger calves; also viral RNA is more likely to be detected in younger, seronegative, calves [6, 10, 17, 18]; however, this tendency was not significantly different in other studies [9]. Calves initially possess maternal anti-MERS-CoV antibodies that wane by five to six months of age [19] leaving them susceptible to infection. However, MERS-CoV reinfection into seropositive camels has been reported [20], indicating that pre-existing immunity does not prevent new MERS viral infection in camels although the viral load may be reduced. Currently, there is no approved antiviral therapy or vaccine against MERS-CoV in humans or camels. Therefore, vaccines against camel MERS-CoV infections are being developed with the aim of reducing viral transmission and introduction into humans [21]. Three vaccine candidates have been evaluated in dromedaries so far; a DNA based vaccine [22], a poxviral vectored vaccines [23], and an adenoviral vectored vaccine (data is expected to be available soon from our team). The two published vaccines elicited antibody immune responses in camels and one was partially protective in reducing the viral load in camels upon experimental challenge [23]; however, the experimental challenge might not represent the natural infection in camels. To guide MERS vaccine development in camels, many questions still need to be addressed in experimental settings such as what is the best target population for vaccination (younger calves versus older calves or adult camels)? What is the infectious viral dose that needs to be assessed in vaccine efficacy studies? Here, in an attempt to investigate these questions, we first explore the camel population and density in KSA as well as conducting a cross sectional seroprevalence study in young dromedary to explore the younger camel population as a target for vaccine development. Second, we aimed to utilise MERS-CoV naturally infected camels as a model of challenge for vaccine efficacy studies. Unlike lab experimental challenge, the natural challenge model would mimic the natural setting of MERS-CoV infection in camels. Therefore, this paper reports a cross sectional seroprevalence study in young camel and examines the utility of natural infection in camels as a potential challenge model for MERS vaccine assessment. It also includes information on the dynamics of MERS-CoV natural infection, incubation period, and shedding time.

Dromedary camels are the only confirmed animal host for MERS-CoV and the source of human infections. Here, we report the latest available census on the dromedary population in KSA. We speculate, based on our local knowledge and experience, that the reasons behind camel high population in Eastern, Riyadh, Makkah, and Qassim provinces is the following: Eastern province is largely an empty desert where camel keepers would have more space and natural resources to move their camels; Makkah province would be a large spot and market for animal sacrifice during the sacred Hajj pilgrimage; Riyadh is the most populated province, including the capital city of Riyadh, that has camel markets, camel festivals, and many camel abattoirs and butcher shops; Qassim province is geographically central and it has the biggest camel market in KSA and most probably worldwide.

In this study, we also confirm that seroprevalence rate of MERS-CoV in young dromedaries in two regions of KSA, in 2017, is 90% (Qassim and Jouf had a rate of 94.5 and 77%, respectively), which is in agreement with several previous reports on dromedaries in general (young and adult) in Africa and Arabia [7‐11]. Qassim region was selected because it has a large camel market, which is surrounded by camel farms whereas Jouf was selected based on previous data that this region had lower MERS seroprevalence rate [6, 17]. These data have important implications on MERS vaccine development for camels. Although a significant population of camels has pre-existing antibodies to MERS-CoV, these antibodies do not seem to be protective and re-infections in camels have now been documented [20, 28]. Therefore, MERS-CoV vaccines in camels might be designed to boost the natural immune responses in dromedary camels that are usually seropositive. Additionally, naive camels (which are considered a minor population of camels) might require robust vaccination platforms or regimens in order to achieve high and protective titres of antibodies. However, cell-mediated immunity should also be evaluated in naturally infected camels to elucidate reasons behind the re-infection of seropositive camels as well as to guide vaccine development. It is also important to note that with a serpositivity rate of 90%, naïve camels are a small population of camels in Saudi Arabia and it is extremely difficult to source these camels for vaccine studies. Therefore, vaccine studies could be first aimed at seropositive animals to prove that MERS vaccines can, indeed, be efficacious in the camels with pre-existing immunity.

This study is also an attempt to support and enable assessment for MERS-CoV transmission as well as drug and vaccine efficacy in camels where there is no high containment lab for large animals in endemic countries (including KSA). It reports the first natural infection challenge model of MERS-CoV in dromedary camels. RT-qPCR for UpE and ORF1a amplicons, which are the accepted method for documenting MERS infections in humans and camels, was used to confirm infections in camels. We found that infected camels with a Ct value ≤25 co-housed as one-third of a herd in a 30 m² can achieve 100% infection in healthy naive camels within 2 days (as measured by the TR-qPCR). Although we utilised camels with low Ct values, we did not quantify the virus or its RNA levels in these camels; and whether higher Ct values (lower viral load) would achieve the same outcome needs to be confirmed. In addition, although we documented the Ct values of viral RNA for 14 days, it is likely that all camels would have cleared the virus within 3 to 5 weeks, MERS-CoV infectious virus was isolated only within one week post experimental infection in dromedaries whereas the viral RNA was detected for 35 days [29]. Thus, the natural course of MERS infection in camels could be around 2 weeks. The natural infection challenge model of MERS-CoV in camels can be utilised for drug and vaccine efficacy studies, in countries where there is a lack of large animal biosafety level 3 labs. We applied strict infection control measures on the study personnel who were all negative for MERS-CoV RNA and antibodies before, during, and at the end of the study.

Alpacas have been suggested as a surrogate model for MERS vaccine evaluation as they facilitate animal-to-animal transmission and were protected from re-infection [30]; however, when the same vaccine was tested in both alpacas and dromedaries there were differences in immunogenicity and efficacy [31]. All alpacas seroconverted and were completely protected whereas only some dromedaries seroconvert and showed partial protection. Dromedaries that were challenged experimentally with 10⁷ TCID50 of a MERS-CoV strain showed nasal discharges and viral RNA detection that peaked for a week then declined in the second week post-challenge; however, the virus was isolated only in the first week post the experimental challenge, in two different studies [23, 29]. Here, our model showed high viral RNA in the first week that decreased in the second week post natural challenge. Although we did not quantify the infectious virus titre in our model, it showed a similar outcome to the experimental model in terms of viral RNA detection. This model would closely mimic the natural context especially with the lack of biosafety containment labs for large animals in endemic countries as well as that the infectious dose for MERS-CoV is not yet defined for the experimental challenge. Nasal discharges were not observed in naturally infected camels in markets in our study, but it was only observed during the natural challenge model in a confined space. This supports previous studies that nasal discharge in dromedaries, infected with MERS-CoV, manifests in experimental settings [23, 29].

Therefore, we propose that this natural challenge model could be used to assess a MERS vaccine efficacy; to perform this, camels receiving a vaccine as well as camels receiving a placebo (control) should be co-housed in one pen in addition to infectious camels that have Ct values below 25. The infectious camels could be half the number of experimental camels, although further work is required to determine the minimal ratio (or number) of infectious camels for such a model. It should also be noted that there might be some difficulties in conducting this model. First, the number of available infectious camels at the challenge time might not be enough; so the timing of such studies must be carefully planned. Second, infectious camels could vary in terms of viral shedding at the time of challenge; so a single infectious camel might be used as a source of infection to have a more defined model, but this has to be carefully assessed before conducting such studies. Following the natural challenge, nasal swab samples collected daily for 14 days post co-housing (natural challenge) should be used to evaluate RNA levels as well as virus titres in vaccinated camels, control camels, and infectious camels. The difference in virus titres post natural challenge between vaccinated and control camels should be then used to calculate the efficacy rate. Virus titre in infectious camels would also be important to determine how long these animals remain infectious. MERS-CoV strains (isolates) in infectious camels could be different, giving the advantage of assessing vaccines against different circulating strains (or isolates). The exact genetic information of these strains can be analysed and reported along with the efficacy data; although we did not study the genetic sequence of the isolates in the current study. There are several vaccine candidates in development for camels as a one health intervention where vaccinating camels is proposed to reduce spillover infections and transmission into humans. Therefore, in the absence of informative research on what is the MERS viral infectious dose in camels, utilising a natural model of challenge would mimic the natural setting and could accelerate vaccine development.

This study reports three main conclusions. First, it reports the population and distribution of dromedary camels in the 13 provinces of Saudi Arabia. Second, it reports that the MERS-CoV seroprevalence rate in young dromedaries in Qassim and Jouf provinces of Saudi Arabia in a given time were 94.5 and 77%, respectively. Third, it details the feasibility of utilising MERS-CoV infected camels as a mean to evaluate vaccine efficacy studies in dromedaries; therefore, it establishes a controlled natural challenge model.