Design and Optimization of a Monkeypox virus Specific Serological Assay

Round 1

Reviewer 1 Report

In this manuscript, Taha at al present a peptide-ELISA test capable of detecting antibodies against MPXV and discriminate them from other OPXV antibodies. The rationale of test can be of interest for detection of bona-fide MPXV seropositive individuals in endemic areas. The results constitute a valuable contribution to Orthopoxvirus diagnostics since the authors were able to identify three peptides that can discriminate between MPXV and OPXV antibodies according to the results presented. One of the strengths of the paper is the large number of serum samples tested and the fact that the samples come from very diverse backgrounds.

The article is relevant for the field, the results are well presented and overall, it is well structured. In addition, cited references are mostly recent publications and cover adequately the state of the art. Although manuscript is very clear, some explanations about the reason for subject selection appear too it complicated. The experimental design is correct. Manuscript results are reproducible and many details are given in the methods section. All the data is well presented, including figures and tables.

Few points are described below which must be clarified.

It is not clear why the authors divided the responses in strong and weak in Figure 1, instead of just showing them all together. Also, how the authors decided the threshold COV values for each kind of response.

In line 354 it is stated that say the optimum peptide concentration for the assay was determined. I think the article should include a figure that shows how that concentration was fixed and how sensitivity and specificity of the assay change when the peptide concentration per well is increased.

Line 387-390, the sentence starting “Of the total 63 subjects positive...” is confusing and should be rewritten.

I am curious to know why the authors only included two subjects that had been recently vaccinated of the n=26 subjects of the vaccine study group.

Based on the results I think it is crucial to prove the validity of the ELISA by testing it with sera from subjects previously exposed to other OPXV, to confirm the assay is specific. Therefore, sera from recently vaccinated subjects should also be included, to test the specificity in situations where when antibody levels might be higher.

Author Response

1. During the initial investigation and optimization, the authors were trying to determine if this assay would work beyond proof of principle and the weak versus strong responses were based on what would have possibly fallen within the equivocal range in other assays. Each sample was run with and without peptide coating the well to introduce individual controls as well as the traditional plate controls. The uncoated well (in duplicate) was averaged and the plate background plus the standard deviation was subtracted from the sample background to define the cutoff per sample.
2. We can include this figure in the supplementary material.
3. We have done this.
4. This work was done prior to the 2022 mpox outbreak when vaccination was uncommon outside of childhood vaccination. This work was also done with individuals recently vaccinated with Acambis 2000. Further work needs to be done to evaluate this assay with those vaccinated with the newer Jynneos vaccine as well as recently vaccinated and samples from the most recent outbreak. We recognize the value of additional testing to address recent vaccination and the rollout of Jynneos vaccination for mpox (originally noted in lines 414-421)
5. This assay was performed using previous variola virus (smallpox) survivors, sera from the 2003 mpox outbreak, sera from an outbreak in Kole, as well as sera from childhood vaccinees and recently vaccinated with Acambis 2000. The authors agree that given the most recent 2022 mpox outbreak and the rollout of the Jyenneos vaccine that additional work should be done and will be pursued in the future. We recognize the value of additional testing to address recent vaccination and the rollout of Jynneos vaccination for mpox (originally noted in lines 414-421).
6.  

Reviewer 2 Report

An important and useful article, the results of which are well verified. Text editorial changes required:

Author Response

1. We have corrected line 51 and anywhere else that refers to the 2022 Mpox Outbreak. The virus itself is still referred to as Monkeypox virus so we have left that as is. The outbreak and disease are now Mpox, and the virus is monkeypox virus. We have made corrections to ensure uniformity in correct nomenclature throughout the paper.
2. This change has been made.
3. We have made this correction as well.

Reviewer 3 Report

This work is comprehensive and of great significance. The article is well written and the authors provide a new serological assay approach for Mpox virus.

Please confirm:

line167: please confirme time for TMB incubation, 1min?

Author Response

We have clarified that the user should follow the manufacturer’s instructions.

Reviewer 4 Report

Taha and colleagues provided a nice manuscript describing a peptide-based Elisa assay for serology of MPXV specific antibody detection.

comments:

According to the ICTV the virus species name is still monkeypox virus. WHO changed the name of the disease induced by MPXV from monkeypox to Mpox. Therefore, the authors should rephrase the manuscript accordingly.

Results are presented and discussed in a convincing manner. 

As the 2022 pandemic was induced and maintained by human to human transmission it is reasonable to generate an assay of human serology. However, analysis of  animal sera in endemic situtations in Africa would clearly strengthen surveillance strategies. As sera from animals were used intitally to screen for reactive peptides, effort to implement a system that analyses indepent from host species would be highly appreciated.  (keyword nowadays: One Health). 

Author Response

1. The outbreak and disease are now Mpox, and the virus is monkeypox virus. We have made corrections to ensure uniformity in correct nomenclature throughout the paper.
2. We agree with the reviewers' comment. The utility of this assay for screening animal sera will be evaluated in the future.