Using the Gut Antigen HA86 as a Biomarker for Hyalomma Tick Bites, the Principal Vectors of the Crimean-Congo Hemorrhagic Fever Virus

Research Article

J Dis Markers. 2023; 8(2): 1058.

Using the Gut Antigen HA86 as a Biomarker for Hyalomma Tick Bites, the Principal Vectors of the Crimean-Congo Hemorrhagic Fever Virus

Moufid Mhamadi1,2#*; Aminata Badji3#; Moundhir Mhamadi4; Alioune Gaye3; Idrissa Dieng3; El Hadji Ndiaye3; Mignane Ndiaye1; Babacar Faye2; Ousmane Faye1; Amadou Alpha Sall1; Gary Kobinger5; Hugues Fausther-Bovendo5; Mawlouth Diallo3; Oumar Faye1

1Department of Virology, Institut Pasteur de Dakar, Dakar 12900, Senegal

2Department of Parasitology, Université Cheikh Anta Diop de Dakar, Dakar 10700, Senegal

3Department of Medical Zoology, Institut Pasteur de Dakar, Dakar 12900, Senegal

4Institut Pasteur de Dakar, DIATROPIX, Dakar 12900, Senegal

5University of Texas Medical Branch, Galveston, TX 77555-0132, USA

*Corresponding author: Moufid Mhamadi Department of Virology, Institut Pasteur de Dakar, Dakar 12900, Senegal. Email: moufid.mhamadi@pasteur.sn

#These authors have equally contributed to this article.

Received: October 13, 2023 Accepted: November 22, 2023 Published: November 29, 2023

Abstract

Hyalomma ticks are the principal vectors of the Crimean-Congo hemorrhagic fever virus. Antibody responses and the viral genome, are usually used for virus surveys. These markers cannot assess the role of ticks in the burden of CCHFV. It has been documented that tick bites induce a host immunological response, and numerous antigens have been described. To note, the gut protein Bm86 of Boophilus micropulus induces such an immune response, and several orthologues have been identified. Furthermore, a cross-immunological response against orthologues was reported. Based on these findings, we hypothesized that the Bm86 of Hyalomma ticks could serve as a biomarker of exposure to Hyalomma tick bites.

For this purpose, 2432 ticks, 435 human and 480 animal sera, collected in Agnam (Northern Senegal) between February 2021 and April 2022 for arbovirus and viral hemorrhagic fever surveys were used.

We found that the development of HA86 IgG in livestock sheep is influenced by exposure to Hyalomma ticks. Additionally, the anti-HA86 antibodies did not persist for more than 2 months, and no cross-HA86 response wasn’t detected between Hyalomma and Rhipicephalus species. These results suggest that HA86, could be used as a biomarker for Hyalomma tick bites.

Keywords: Ticks; Hyalomma; Biomarker; HA86; Antibody

Context

Crimean-Congo Hemorrhagic Fever (CCHF) is the most prevalent tick-borne arbovirus in the world. Hard ticks (Ixodidae) of the Hyalomma genus are the main vectors of the disease [9]. It has been established that the Crimean-Congo Hemorrhagic Fever Virus (CCHFV) can be transmitted to the host by a competent tick carrying the CCHFV during a blood meal on a vertebrate host [9]. In humans, the infection could also occur after contact with infected tissues or fluids, such as during the slaughter of infected livestock or when caring for infected patients [17]. Several components are inoculated into the host during this blood meal, leading to a chain of reactions at the host level, which cause the production of markers of infection or exposure. Virological markers, such as the presence of viral genome or antibodies against the virus, are typically used to diagnose infection or exposure to CCHFV. Studies on the kinetics of this virus show that viral RNA can be detected in the blood by RT-PCR for up to 18 days after the onset of the first symptoms. IgM and IgG antibodies remain detectable for at least 4 to 6 months and 5 years, respectively [4]. These virological markers allow determination of the burden of CCHFV on the population's health. However, these markers cannot directly determine the impact of vector populations on the epidemiology of CCHFV. It is worth noting that few studies have documented exposure to tick bites, even though the immunological response to tick bites was described nearly a century ago [16].

For the record, many proteins in the gut of ticks have been shown to be immunogenic for vertebrate hosts [5-7,11]. It has been established that vertebrate hosts develop an immune response against these antigens after multiple infestations [12]. To date, the gut protein Bm86 from Boophilus micropulus ticks is the most well-documented. Its immunogenic role has been utilized to develop livestock vaccines against B. micropulus tick infestations [12]. Studies have shown that immunity induced by Bm86 protected cattle against other species of the same genus, such as B. appendiculus, B. annulatus, and B. decoloratus [5,15]. Similarly, a cross-immune response against ticks of the Hyalomma genus was documented with the Hd-86 ortholog of Bm-86 identified in Hyalomma scupense (syn. H. detritum) [8]. The high percentage of amino acid conservation (between 87 and 91%) between this Bm-86 ortholog of ticks could explain this cross-immune response [2,3]. Thus, we hypothesize that this cross-antibody response could be used as a biomarker for tick bite exposure. In this work, we propose to develop a serological test for the detection of antibody responses against a Bm-86 antigen of Hyalomma ticks, the main vectors of CCHFV.

Methodology

Hyalomma Bm86-HIS Orthologue

As no commercial Bm86 like protein of Hyalomma ticks was available, we aim to develop it. For this purpose, the Hyalomma Bm86 like protein, codons 1-646 of the Hyalomma anatolicum BM86-like gut protein (AAL36024.1) were synthesized and a Histidine tag (HIS) was added downstream. Then, HEK293T cells were cultured in 24-well plates and transfected using polyethyleneimine (PEI, Polysciences, France) with a 1:3 ratio of PEI to Bm86-HIS plasmid according to the manufacturer's instructions. Supernatants were collected 3 days after transfection and clarified by centrifugation at 8000g. The Bm86-HIS protein was then purified using nickel agarose resin (Ni-NTA) (Qiagen, Toronto, Canada) according to the manufacturer's instructions. The purity of the Bm86-HIS protein obtained was analyzed by SDS-PAGE. The generated antigen was labeled HA86.

Samples Used for the Study

The samples used from our study were collected in the One Health site established in the Agnam area as previously described [14]. The Agnam area is an arid zone located in the region of Matam (15°06′ 18″ N and 13°38′ 30″ W) in the northeast of Senegal. CCHFV circulates in this area on human-animal and Hyalomma impeltatum constitutes the dominant tick species in Agnam. Other species of this genus have been listed: H. marginatum rufipes and H. truncatum [14]. For the development of the test and for the evaluation of populations exposition to Hyalomma tick bites, we selected human and animal sera as well as ticks collected between February 2021 to April 2022 in Agnam areas. A total of 413 human samples, 480 samples from the 32-sentinel sheep, and 2432 ticks were selected for this study.

Detection of Anti-HA86 Antibodies

For the detection of Immunoglobulin G (IgG) antibodies against Hyalomma tick antigen 86 (HA86), an indirect ELISA was used. Briefly, 96-well plates were coated with 500 ng/mL of the HA86 protein diluted in Phosphate Buffer Saline 1x (PBS). These plates were then sealed and incubated overnight at 4 °C. Then plates were washed 3 times by adding 300μL of washing buffer (PBS 1x + Tween 20 at 0.05%) and the antigenic sites were blocked using a blocking buffer (PBS 1x + Tween 20 at 0.05% + 5% skimmed milk). After 1 hour of incubation at 37°C, plates were washed 3 times then we added the 1/100 diluted sera and we incubated them again at 37°C for 1 hour. After washing, a species-specific antibody (rabbit anti-sheep IgG (Biorad) or a goat anti-human IgG (KPL)) was conjugated with horseradish peroxidase then the plates were incubated at 37°C for 1 hour. The plates were then washed and 3, 3 ′, 5, 5′-tétraméthylbenzidine (TMB SIGMA Aldrich, USA) was added. The reaction was stopped with sulfuric acid after 5 minutes. Optical Densities were read with the 450/620 filters and the cut-off was determined using the finite mixture model.

Data Analysis

The data generated were entered in Excel and the statistical analyzes were performed on the R software using the R studio interface. Using the finite mixture model on Optical Densities (OD) as previously described [13], the cut-off threshold was determined. Subsequently, the difference in antibody levels and HA86 IgG carriage between 2 groups was analyzed by logistic regression. Linear regression was used in order to determine the influence of number of ticks on antibody levels. The correlation of HA86 specific IgG between 2 independent groups was calculated with a Pearson test. The development of HA86 immunity during the time of exposition to ticks bites was represented by the Kaplan Maier curve and compared by Log-rank (Mantel-Cox) test. The effect of time exposition to ticks bites on the development of HA86 immunity was tested by Khi-square test. The significance level was set at 0.05% (p<0.05).

Results

Ticks Distribution

Out of the 2432 ticks selected, seven species were identified between February 2021 and April 2022: H. impeltatum (78.86%), H. marginatum rufipes (0.20%), H. truncatum (1.02%), R. guilhoni (12.33%), R. mushamae (3.45%), R. sulcatus (2.17%), and R. evertsi evertsi (1.93%). H. impeltatum was collected every month except for September and October 2021. Among the species of the Rhipicephalus genus, Rhipicephalus guilhoni was identified throughout the entire 14-month survey. The other species were rarely collected (Figure 1).