Multiplex Assay for Simultaneous Detection of Antibodies against Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein and Glycoproteins in Ruminants

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread tick-borne zoonotic virus that causes Crimean-Congo hemorrhagic fever (CCHF). CCHF is asymptomatic in infected animals but can develop into severe illness in humans, with high case-fatality rates. Due to complex environmental and socio-economic factors, the distribution of CCHFV vectors is changing, leading to disease occurrence in previously unaffected countries. Neither an effective treatment nor a vaccine has been developed against CCHFV; thus, surveillance programs are essential to limit and control the spread of the virus. Furthermore, the WHO highlighted the need of assays that can cover a range of CCHFV antigenic targets, DIVA (differentiating infected from vaccinated animals) assays, or assays for future vaccine evaluation. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies in ruminants to three recombinantly produced CCHFV proteins: the nucleocapsid (N) protein and two glycoproteins, GN ectodomain (GNe), and GP38. This triplex assay was used to assess the antibody response in naturally infected animals. Out of the 29 positive field sera to the N protein, 40% showed antibodies against GNe or GP38, with 11 out of these 12 samples being positive to both glycoproteins. To determine the diagnostic specificity of the test, a total of 147 sera from Spanish farms free of CCHFV were included in the study. This multiplex assay could be useful to detect antibodies to different proteins of CCHFV as vaccine target candidates and to study the immune response to CCHFV in infected animals and for surveillance programs to prevent the further spread of the virus. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever, which is one of the most important tick-borne viral diseases of humans and has recently been found in previously unaffected countries such as Spain. The disease is asymptomatic in infected animals but can develop into severe illness in humans. As neither an effective treatment nor a vaccine has been developed against CCHFV, surveillance programs are essential to limit and control the spread of the virus. In this study, a multiplex assay detecting antibodies against different CCHFV antigens in a single sample and independent of the ruminant species has been developed. This assay could be very useful in surveillance studies, to control the spread of CCHFV and prevent future outbreaks, and to better understand the immune response induced by CCHFV.

It is a matter of fact that CCHFV serology is currently under rapid evolution with several kits developed (in particular by IDVet) trying both to define the right line for positivity (ruminants over the world have not the same serological background) and an easy use for various animal species. The principal interest of the present manuscript is clearly to allow a parallel analysis of the serological response to 3 main viral antigens (both N and Gs). However, the present form is clearly incomplete and perfectible. I hope that the following remarks will allow to improve the interest for this work.
Antigens production and coating • General remark : CCHFV displays a large diversity and this may influence the serological response. Is it possible to clarify why the Bagdad strain has been used for the N and the Ibadan strain for the Gs.
• Lines 173-4 : the Gn and GP38 are purified through a step at a very acidic pH 2.6 before neutralisation at pH 10. Assuming that the virus-cell envelope-membrane fusion preceding the RNP entry into the cytoplasm necessitates a conformational change of G under acidic pH, is it possible that such pH treatment would interfere with the G antigens conformation and therefore on their recognition by serum antibodies. • Figure 2B : is it possible to discuss the two bands observed for the GP38-GST. Only the GST alone is discussed. • Figure 3 : while fig 1 and 2 repeatedly suggest the GP38 is expressed in lower amounts than Gn, the WB with the pool of positive sera clearly shows the inverse. Despite the fact that the following steps are probably equilibrating the response (50µg of Gn, 25µg for N and G38), this is an important information which has to be linked to the previous remark about possible conformational modifications provoked by the purification treatment. Is Gn as correctly folded than GP38 and N ? Is this interfering with the further serological results ? This observation is outlined by authors on lines 404-06. Their explanations proposed in lines 375-84 (less Gn immunogenicity or different glycosylation) are possible but may be not the only ones. • Lines 192-3 and later in the text : "The three target proteins were covalently coupled to three different regions 192 (regions #15, #20, #25) of carboxylated magnetic microspheres (Luminex)". This sentence is not clear to me (as a reader) even if a reference is given. Different beads of different colours for each antigens? Different region of each bead!!!!! Clarify please. • Figure 4 : the panels and writings are very small. Triplex assay, Figure 5 • The ID Screen CCHF Double Antigen Multi-species (IDVet) is used as the reference technique to decide on "CCHFV positivity". This may be discussed since it has a global tendency to give a high back-ground. • Technically, I would suggest to put the positive samples in a different colour in order to distinguish them more easily. Also, if it is possible to join the 3 panels with very slight vertical lines (or another way) to join the identical samples, this would facilitate the understanding of the explanations given in Results and also in Discussion. • Lines 330-2 : is it possible to discuss a possible effect of the species. Is the background the same for cow, sheep and goat ? BTW, it would be also interesting to design then on figure 5 to illustrate this possible species effect. • It may be interesting in the discussion to develop a bit more about the IDVet "negative" ELISA samples that are positive for the 2 Gs but not with N. In which animal species ? From which country (link with current transmission to human ?). Why a response against G and not against N ?
Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
Microbiology Spectrum -Spectrum-02600-22 "Multiplex assay for simultaneous detection of antibodies against Crimean-Congo hemorrhagic fever virus nucleocapsid protein and glycoproteins in ruminants" This manuscript Spectrum-02600-22 by Hoste et al. describes the development of a multiplex serological assay allowing to simultaneously detect antibodies against different antigens of CCHFV in the ruminant serum. The first part details (1) the antigen production, in E.coli for the Nucleoprotein, in SF9 insect cells for two Glycoprotein segments matching Gn and Gc; (2) the coating of the recombinant antigens on beads for multiplex analysis. In the second part, the triplex assay is evaluated on a total of 176 ruminant field serum samples including 29 «positive» according to the IDVet CCHF Double Antigen Multi-species kit and 147 negative field serum samples.
It is a matter of fact that CCHFV serology is currently under rapid evolution with several kits developed (in particular by IDVet) trying both to define the right line for positivity (ruminants over the world have not the same serological background) and an easy use for various animal species. The principal interest of the present manuscript is clearly to allow a parallel analysis of the serological response to 3 main viral antigens (both N and Gs). However, the present form is clearly incomplete and perfectible. I hope that the following remarks will allow to improve the interest for this work.
Antigens production and coating • General remark : CCHFV displays a large diversity and this may influence the serological response. Is it possible to clarify why the Bagdad strain has been used for the N and the Ibadan strain for the Gs.
We agree that CCHFV strains are genetically highly diverse, and this surely can influence the serological response. However, the N protein sequences are not very variable and the N protein sequences from Baghdad strain and Nigeria/IbAr10200/1970 are 95% identical (blast alignment on the sequences below) and the Baghdad strain used in this paper was already available in the lab. For the Gs, we have used IbAr as it was the model for CCHFV and the most studied strain for which we could determine the ectodomain of the G N e and the GP38. The homology between the G N e and GP38 of IbAr and Baghdad strain is 92% identical. The main difference in the identity of the M segment is mostly due to the murine-like domain.
• Lines 173-4 : the Gn and GP38 are purified through a step at a very acidic pH 2.6 before neutralisation at pH 10. Assuming that the virus-cell envelope-membrane fusion preceding the RNP entry into the cytoplasm necessitates a conformational change of G under acidic pH, is it possible that such pH treatment would interfere with the G antigens conformation and therefore on their recognition by serum antibodies.
We agree that such pH treatment as described by the reviewer could interfere with the conformation of the glycoproteins recombinantly produced in this article. However, the G N e and GP38 are purified through a very acidic pH 2.6 before being neutralized by addition of 3 M Tris-HCl, pH 10 until the pH reached 7, but the pH was not brought under basic conditions. The correction was made to L170, p8. We apologize if the sentence in the M&M was not clear • Figure 2B : is it possible to discuss the two bands observed for the GP38-GST. Only the GST alone is discussed. This point has been corrected and is discussed L372-375, p16. The two bands observed for the GP38-GST on Figure 2B around the 50 kDa line probably correspond to a glycosylated and a nonglycosylated form of GP38-GST or a partial degradation of GP38 during the purification process.
• Figure 3 : while fig 1 and 2 repeatedly suggest the GP38 is expressed in lower amounts than Gn, the WB with the pool of positive sera clearly shows the inverse. Despite the fact that the following steps are probably equilibrating the response (50µg of Gn, 25µg for N and G38), this is an important information which has to be linked to the previous remark about possible conformational modifications provoked by the purification treatment. Is Gn as correctly folded than GP38 and N ? Is this interfering with the further serological results ? This observation is outlined by authors on lines 404-06. Their explanations proposed in lines 375-84 (less Gn immunogenicity or different glycosylation) are possible but may be not the only ones.
On the SDS-PAGE and on the WB (Figure 1 and 2B), we can see that GP38 is expressed in lower amounts than G N e (fainter band compared to G N e). For the WB with the pool of positive sera (Figure 3), the same amount of protein was loaded per well (0.5 μg of each protein), to avoid accounting for the expression yield. On this WB, strong bands for CCHFV N protein and GP38 can be seen, meaning that they are better recognized by the antibodies present in the CCHFV pool sera than G N e which shows a fainter band. We agree that this result could be due to a lower immunogenicity of G N e, different glycosylation or incorrect folding of G N e compared to the other two proteins tested. This lower response of G N e in the WB, but also when setting up the Luminex assay, explains why we had to increase the concentration of G N e when labelling the Luminex beads, to increase the signal obtained with G N e in the Luminex assay. This led to signals obtained with G N e close to the ones obtained with GP38 in the Luminex assay. Other factors such as animal age, phase of the infection, time between exposure or different localities, and different decay rates of the antibody responses could also explain the lower antibody response to G N e compared to GP38 and the N protein. This was added L387-390, p17.
• Lines 192-3 and later in the text : "The three target proteins were covalently coupled to three different regions 192 (regions #15, #20, #25) of carboxylated magnetic microspheres (Luminex)". This sentence is not clear to me (as a reader) even if a reference is given. Different beads of different colours for each antigens? Different region of each bead!!!!! Clarify please.
The corrections were made L188-192, p8 to clarify this part. The microspheres include two internal dyes where a first excitation wavelength allows the identification of each microsphere from 100 unique microsphere sets. Each antigen (N protein, G N e and GP38) was coupled to a specific set of beads (#15, #20, #25, also called bead regions) which can be recognized by the equipment. Then, a second excitation wavelength allows the observation of the fluorescent reporter molecule (in our case Streptavidin R-phycoerythrin), allowing the detection of the analyte captured on the surface of the microsphere.
• Figure 4 : the panels and writings are very small. The corrections were made. Triplex assay, Figure 5 • The ID Screen CCHF Double Antigen Multi-species (IDVet) is used as the reference technique to decide on "CCHFV positivity". This may be discussed since it has a global tendency to give a high back-ground.
According to the manufacturer, the ID Screen CCHF Double Antigen Multi-species has a high specificity (100%) and sensitivity (98.9 %) across multiple species.
If the reviewer means that the assay has a tendency to give a high noise-to-signal ratio, we have not observed a high noise-to-signal ratio in all the negative sera we have been testing with the ID Screen CCHF Double Antigen Multi-species. In a study performed by one of our collaborators with over 300 negative samples tested in this assay, the signal obtained was: from 0,036 to 0,059 = 297 sera (90,27%); from 0,060 to 0,1 = 22 sera (6,68%); and from 0,1 to 0,4 = 10 sera (3,29%), with no false-positive.
If the reviewer means that the assay has a tendency to give a high number of false-positives, from our side, we have tried to confirm the results obtained with the ID Screen CCHF Double Antigen Multi-species with other techniques, mainly by IFA using the animal-adapted EUROIMMUN IFA kit and in some cases, the animal-adapted Vector-Best ELISA. The IFA assay confirmed about half of the positive samples identified with the ID Screen CCHF Double Antigen Multi-species. This could indeed indicate a lack of specificity of the CCHF Double Antigen Multi-species, but also may be due to a lack of sensitivity of the IFA assay. Some positive sera (ID Screen +) giving either a positive or negative result in the IFA (IFA + or IFA -) were also tested in the Vector-Best ELISA, which confirmed the ID Screen + as true positives. In our opinion, the IFA is less sensitive than either ELISA. However, as there is no "gold standard" technique to rely on as a reference, we are always working with some level of uncertainty.
• Technically, I would suggest to put the positive samples in a different colour in order to distinguish them more easily. Also, if it is possible to join the 3 panels with very slight vertical lines (or another way) to join the identical samples, this would facilitate the understanding of the explanations given in Results and also in Discussion.
We agree and have changed the figure accordingly. All the positive samples are now regrouped on the same figure ( Figure 5A) and the negative samples are regrouped in second figure ( Figure 5B). For better clarity, the median fluorescence intensity was converted to a median fluorescence intensity-to-cut-off ratio. The title of Figure 5 has been changed accordingly: Figure 5. Screening of the positive and negative field sera for antibodies against CCHFV GP38, CCHFV G N e and CCHFV N protein in the CCHFV triplex assay. A: median fluorescence intensity-to-cut-off ratio for the positive field samples. B: median fluorescence intensity-to-cut-off ratio for the negative field samples. The dashed line corresponds to the cut-off which was calculated as the mean obtained for the negative field samples plus two standard deviations. The signal was measured as MFI of at least 50 events of each bead region. MFI: median fluorescence intensity.
• Lines 330-2 : is it possible to discuss a possible effect of the species. Is the background the same for cow, sheep and goat ? BTW, it would be also interesting to design then on figure 5 to illustrate this possible species effect.
We agree and have made the changes to Figure 5A to show the different species of the positive field samples. The background of the animals is not known as the positive samples are samples that have been collected in the field in Macedonian farms. The following sentence was added L397-401, p17 to discuss the possible effect of the species: "The immune response and more specifically the antibody response against CCHFV in farm animals needs to be further investigated. It is not yet known whether different farm animal species develop specific anti-CCHFV N protein, anti-CCHFV GP38 and anti-CCHFV G N e antibodies, the time points postinfection at which these can be detected and the persistence of these antibodies".
• It may be interesting in the discussion to develop a bit more about the IDVet "negative" ELISA samples that are positive for the 2 Gs but not with N. In which animal species ? From which country (link with current transmission to human ?). Why a response against G and not against N ?
We agree with this comment and the following sentences have been added in the discussion at L414-423, p18 linked to the previous comment by the reviewer. "One cattle and two sheep originating from Spanish farm free of diseases were positive in the triplex assay for both GP38 and G N e and negative in both the reference assay and in the triplex assay for the N protein. These samples could be false positive results, however, CCHFV has been reported as circulating in Spain and endemic human cases have already been reported (Negredo et al., 2019). As mentioned previously, the immune response of animals to CCHFV is not known, nor the persistence of antibodies to CCHFV antigens. However, a study from Emmerich et al. suggests that during the acute phase of CCHFV infection, antibodies are first raised against CCHFV antigens such as the envelope glycoprotein, before being raised against CCHFV N protein (Emmerich et al., 2018). These hypotheses should be assessed for animals as well."