Multiplex Serology for Sensitive and Specific Flavivirus IgG Detection: Addition of Envelope Protein Domain III to NS1 Increases Sensitivity for Tick-Borne Encephalitis Virus IgG Detection

Round 1

Reviewer 1 Report

In the paper “Multiplex serology for sensitive and specific flavivirus IgG detection: addition of envelope protein domain III to NS1 increases sensitivity for Tick-borne encephalitis virus IgG detection”, Valle et al show that evaluating a combination of antibody responses to EDIII and NS1 could eventually help to improve the interpretation of serological monitoring for anti-TBEV IgG responses.

This is an important concept as TBE diagnostics and seroprevalence monitoring is complicated by both 1) substantial cross-reactivity between antibody responses to various flaviviruses, and 2) that TBE vaccination elicits IgG responses to the E protein (and to a small extent NS1), which make it difficult to distinguish between responses to vaccination versus infection. Therefore, the development of “better” assays is certainly still needed.

Overall, I felt that this was a nice study and, in principle, worthy of publication. I felt that some aspects of the “story” could, perhaps, be changed a bit to improve clarity and to better support the conclusions of the study. Some comments:

I would suggest to replace “a-specific” (page 2, line15) with “non-specific”

I had to read through the text a couple of time to understand the actual assay (of course the descriptions of how the recombinant proteins were generated is important to include, but this is more tangential). Relatedly, It wasn’t 100% clear to me what constituted a positive microarray result (a combination of positivity to NS1 and EDIII?). Perhaps the authors could state this a bit more clearly or Perhaps a diagram depicting the array and workflow would be helpful here?

Although the sample from the “Dutch forestry study” are described in another reference, it would be nice to also include them in Table 1 or in a second or supplementary table so the reader can better understand the cohort without having to look up another paper.

Was anyone from the “TBEV vaccinated group” also vaccinated for YF or JE? This could be important to include.

Would it be possible to show the EDIII titer data also as heatmaps as for the NS1 data? This would make the logical comparisons between the two easier. It would also be nice to include the patient number from each cohort on the axis so then you could compare the same patient in terms of both EDIII and NS1 results. Furthermore, it would be nice to include the heatmaps, as well as the summary violin plots alongside so the reader can look at the overall pattern (heatmaps) as well as the corresponding statistical comparisons in titers (violin plots).

Single stars are presented in violin plots (*), but their meaning isn’t indicated in the caption (probably p<0.5, but it should be included anyway).

For this line: “’Dutch forestry workers’ study who did not report TBEV vaccination” (page 11, line 395) – From this phrasing it is unclear whether information on vaccination wasn’t collected or whether these workers reported not being vaccinated.

A model/algorithm to interpret results of the microarray would be great to include as a figure at the end and would also be an easy to reference for others.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors describe an improved test to detect and differentiate flavivirus IgG antibodies against different flaviviruses including rEIII domain to rNS1. The authors conclude that the addition of rEIII domain will increase sensitivity of the test.

There is one major point and some are minor points to mention:

Major issue: As it is known that the NS1 IgG appear late during acute infection it would be interesting to state when the sera from acute patients were taken. Were the 3 late samples of the three consecutive serum pairs among the positive ones? Might the low sensitivity of the rNS1 test due to too early taken samples? Please comment.

Minor issues:

Line 38: more detailled to state that the reported infections are the ones with clinically overt infections.

Line 44: Ixodes ricinus is the main vector in Europe, but there are some others like Dermacentor reticulatus.

Line 44: TBEV-EU is mainl biphasic. TBEV-FE is often monophasic.

Line 233: Which strain of TBEV-EU was used for PRNT. Please add as ou also described for WNV.

Author Response

"Please see the attachment."

Author Response File: Author Response.pdf