Peer review of: Serotype specific epitopes identified by neutralizing antibodies underpin immunogenic (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

The manuscript by Wang and colleagues is a natural continuation of a companion manuscript 
describing the Echovirus 30 (E30) interaction with its attachment and uncoating receptors: CD55 
and RnFc. The study describes the interactions of E30 with two potent neutralizing antibodies that 
block capsid binding to the two receptors. Both antibodies attach to the capsid in the canyon 
region, partially overlapping with receptors binding footprints – an area with high variability across 
different echoviruses. 
While the results are less exciting than the those presented in the first manuscript, the study 
brings to light important details on the molecular mechanism of antibody neutralization of 
enteroviruses. Such information is essential for rational designing vaccines with large applicability 
spectrum. 
The manuscript is well constructed, the figures are clear and very informative. However, I want to 
raise several points that will need to be corrected before publication. 

Major point: 
What it makes this manuscript particularly valuable for picornavirologists is the comprehensive 
analysis done with all the neutralizing antibodies against enteroviruses. My suggestion would be to 
develop that part and maybe introduce in the main text a new figure that would illustrate the ideas 
presented in lines 301-315. 

Minor points: 
Line 158: Reverse transcription polymerase chain reaction before using the abbreviation RT-PCR 
would be nice. 
Line 293-295: "These previously reported observations together with our current structural 
analysis suggest antibodies targeting epitopes "inside the canyon" are capable of conferring cross-
protection against EVs". I could be wrong, but I find this to be a strong statement considering 
4B10 (binding inside the canyon) didn't provide cross-protection against other EV-B strains in the 
cell-based viral neutralization assays (Line 131-132). Previous reports and the findings from this 
paper support the hypothesis they have made in the line 323-326 . 
Line 335: 1,500xg should be spaced to 1,500 x g (same issue in lines 336 and 337) 
Line 336: the gradient range before the continuous sucrose density is preferred. For example, 10-
40% 
Line 338: Phosphate-buffered saline before PBS abbreviation 
Line 341: droped should be corrected to dropped 
Line 342: subject should be subjected 
Line 344: Mabs should be MAbs. That's what it was used throughout the paper. It must be 
consistent. 
Line 352: How did the authors obtained these complexes? I assume mixing E30-F particles with 
the Fabs for 30 mins/1 h at 4 °C. I think they should mention it somewhere here. 
Line 356: correct close to dose 
Line 370: Please indicate the version of the Relion. 
Line 411: plaque-forming unit before the PFU abbreviation 
Line 433: Please use Particle stability thermal release assay instead of thermal stability assay. This 
indicates the thermal stability assay we know (using SYTO9, SYPRO Orange/Red). Otherwise there 
are some other microscale fluorescent thermal stability assays with other dyes. 
Line 444: subject should be corrected to subjected 

Please add spaces between numbers and units throughout the methods. Especially between the 
number and °C – multiple cases in the methods section. 

Fig1: Please increase the vertical spacing between the figures? They seem too clustered. 

Fig2c: bottom left: CD55, 1A1, ICAM5 labels are too close to each other-almost overlapped. 
Fig3c and d: please correct "pentomer" to pentamer 
Supp fig 1 c: One on the left is SYTO9 and on the right is SYPRO-Red. It should be mentioned in 
the figure legend. The authors failed to mention about SYPRO-Red in the methods. 
Line 844: kD should be kDa and introduce a space between numbers and units. 

Reviewer #2 (Remarks to the Author): 

This study immunized the mice and elicited two highly potent serotype-specific neutralizing Abs, 
referred to as 6C5 and 4B10, both of which could neutralize E30 infection efficiently by blocking 
viral binding to its two types of receptors. The SPR assays showed the competitive binding results, 
both 6C5 and 4B10 efficiently prevented E30 attachment to the cell surface and could displace the 
viral particles that had already attached to the cell surface in a dose-dependent manner. Using the 
Cryo-EM micrographs to see the E30 in complex with its NAbs 6C5 and 4B10 and define the key 
amino residues for the antigen-antibody interaction. 
This study is very systematic and well define the specificity of 6C5 and 4B10 to E30. 

The results have showed the main effect of mAbs neutralizing activity depended by blocking 
receptor biding and not destabilization of the viral structure. 
In Figure 3, there are no RNA released after the E30 incubated with mAbs. It still requires a 
positive/ negative control to show the E30 released the RNA. For example, you could show the E30 
released the RNA in 1-3 °C or any condition can make the E30 release the RNA. We would like to 
see the differecne and make sure the assay is weel done. 

It is very interesting to heard that the Abs destabilized the virions at low temperature. Would it be 
possible to show the result of intact antibodies and Fab fragments of 6C5 and 4B10 destabilized 
E30 virions by 1-3 °C. This observation is very different with EV71. In general, the EV71 were 
uncoated by long incubation time at 37 °C or bound with mAbs for a short incubation time. 
Please show Yyour result or explain what the differences in between, thanks. 

The supplement figures 1.C were not labeled clearly for E30 complex with mAb, please make the 
label clearly and describe more in detail because we did not know the real means in the figures.