Immunogenicity and Blocking Efficacy of Norovirus GII.4 Recombinant P Protein Vaccine

Round 1

Reviewer 1 Report

 

Dear Editor-in-Chief,

I have now reviewed the manuscript entitled: Immunogenicity and blocking efficacy of Norovirus GII.4 recombinant P-protein vaccine” by Yu Zhendi et al. (Manuscript vaccines-2367374).  The manuscript authors present the in vitro expression and purification of the recombinant P-protein, its in vitro binding to ELISA-plate well coated human blood group antigens. The produced recombinant P-protein was further used as antigen for immunizing rabbits to obtain in vitro human NoV- and murine norovirus neutralizing serum antibodies. Neutralizing antibody activity was studied using HBGA coated ELISA plate wells and soluble virus or P-protein binding assay.

 

Questions:

In Materials and methods several issues are missing.

2.3 Determining the receptor binding …. Line 77: After washing, secondary antibodies HRP conjugated goat anti-mouse IgG and HRP-conjugated goat anti-mouse IgM were added ….

Question 1A): What was the reason for both HRP-conjugated goat anti-mouse IgG and anti-IgM used ? Question 1B) Were the monoclonal antibodies a mixture of mouse IgG and mouse IgM? Question 1C) How did the authors know if the reactivity was dependent of the IgM or the IgG-reactivity?

 

2.4. Production and determination of neutralizing antibodies to NoVs recombinant P protein.

NoV recombinant P protein was made into a water-in-oil immune emulsion (Freunds complete adjuvant).

Question2A): Which concentration of P-protein was mixed with the Freunds adjuvant?

Question 2B). Which concentration of Freunds ajuvant was used?

Question 2C). How large volumes of the P-protein + Freunds adjuvant was injected in each rabbit?.

Question 2D). What was the reason for performing three 1 weekly distant booster immunizations. Perhaps the titers of serum would have been good already after two boosters?

 

2.5. Detecting blocking efficacy of neutralizing antibody.

Question 3):Line 95: The authors claim that they coated P protein on ELISA plates. How much P-protein was added to each plate well, and in which volume and dilution-buffer was the P-antigen dissolved in during coating?

 

 

RESULTS

Question 4A): In Figure 1 the authors present the content of P-protein in their cell-lysates and purified eluates. In Figure 1B, eluted 1, 2 and 3 show the presence of P-protein. What was the protein concentration obtained in the eluate fractions 1, 2 and 3?

 

Question 4B): Figure 1: It looks like the protein fractions were stained in some way? Or was the SDS-PAGE-protein content visible without protein staining?

 

Question 5A: Line 171: The authors discuss the use of Normal serum. The authors need to explain which kind of Normal serum they were using, and from where did they get hold of Normal serum?

Question 5B): In which way were the used rabbit serum not normal serum?

 

Question 6:  Line 175-177. The authors discuss here neutralizing antibody titers in a unclear form. They present the load of antibodies in a treatment group (2,50 Log?) and in a control group (4,79 Log?)? What do they mean with Log? Should it be Log₂ or Log₁₀ or some other kind of Log?

 

Question 7A: Figure 3. Line 180-181. The Figure 3A and B is described as showing neutralizing antibody titers from cell culture experiments in vitro. However, the data are quite odd since low serum dilutions (1/2 to 1/16) still do not even neutralize more than maximally 48% of the virus detected?  Can the authors explain why it seem possible to inhibit virus-binding with only <50% even at ½-serum dilution?

Question 7B: How many repetitions of the inhibition assay were performed with each of the serum samples?

 

Question 8): Figure 5.  The Figure 5B is described as showing neutralizing antibody titers from cell culture experiments in vitro. However, the data are quite odd since low serum dilutions (1/2 to 1/16) still do not even closely neutralize more than maximally 50% of the virus detected. Non-clear dose-response in serum titer is shown in the figureB (also the p-value indicate weak inhibition significance activity). The data may rather suggest that there is some level of serum-antibodies that inhibit the detectability of infections virus protein-receptor protein instead of true virus-neutralization? Can the authors better explain why a <50% neutralization titer is considered as neutralizing?

 

Question 9).   Conclusions, Line 251-251. The authors claim that the P-protein immunization resulted in a good immune response in rabbits. However, the binding absorbance reactivity in a low serum-dilution show only weak reactivity in their ELISA, and there is no investigation of the T-cell immunity or memory response against the P-protein? Also, there is no discussion about the level of neutralizing antibodies that correlate with protection from infection or disease?

Question 9): The authors should explain what they mean with a “good” immune response?

 

Quality of english language text acceptable.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript “Immunogenicity and Blocking Efficacy of Norovirus GⅡ.4 Re- 2 combinant P Protein Vaccine” is a good study. The authors have identified that the recombinant P protein expressed in E. coli can induce antibodies to block 17 HuNoV and MNV. The recombinant P protein of NoVs GII.4 has the value of vaccine development. The manuscript can be improved by rectifying the following points.

1. The abstract needs a one-liner objective and description of important findings.

2. Provide the animal approval number in section 2.1

3. The appropriate words must be italicized (for example, “in vitro” in lines 211 and 244)

4. The discussion part is not convincing nor reflects the study's importance properly. The authors can enrich the discussion with the following points.

(a) Advantages over previous work

(b) Foreseeable applications (like used to treat/prevent other diseases) of the research work with proper references

(c) Shortcomings, and

(d) Prospects

Minor editing of English language required

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The revised manuscript vaccines-2367374 by Yu Z. et al.  have significantly clarified the methods, results and discussion section. My recommendation is that the manuscript can now be recommended to be published.

English language with sufficient quality.