Evaluation of Immunogenicity and Cross-Protective Efficacy of a CpG-Adjuvanted Trivalent Inactivated Influenza Vaccine in Ferrets

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary: In the submitted manuscript, Qiu and co-authors immunized ferrets with an investigational trivalent inactivated influenza virus vaccine that was adjuvanted with CpG. The authors characterized the vaccine-elicited antibody response via ELISA. Moreover, they also assessed the titer of neutralizing antibody activity against H1N1, H3N2 or an influenza B virus (IBV) in hemagglutination inhibition (HI) assays. Ferrets were then challenged with either an H1N1 virus (A/Victoria/4897/2022), a heterologous H3N2 virus (A/Hong Kong/4801/2014), or an IBV (B/Austria/1359417/2021). Following challenge of ferrets (n=6), nasal washes were collected on Days 1, 3 and 5. Furthermore, on Day 6 post-challenge ferrets were euthanized for collection of blood and lung tissue. During the acute phase of influenza challenge, ferrets were monitored for weight loss, increased body temperature and clinical disease. Replication of the influenza virus used for challenge was then quantified using either a TCID₅₀ assay or via quantitative PCR (qPCR). Furthermore, lung pathology in the alveolar and bronchial spaces was scored in H&E-stained tissue sections. Collectively, the data presented in the manuscript demonstrated a dose-dependent immune response to the CpG-adjuvanted TIV vaccine, with the high-dose vaccine conferring superior protection against influenza virus challenge compared to vaccination with the low-dose CpG-TIV or commercial TIV vaccines.

 

Specific Comments:

1. Details of the vaccine and immunization of the ferrets is presently lacking. Standard of care (SOC) seasonal influenza vaccines have 15ug of HA for each of the vaccine components. To this end, how was the vaccine inoculum quantified and what were the specific dosages administered to each ferret? Relatedly, the route of vaccine immunization was not specified.

 

2. Details of the ELISA were also lacking in the manuscript. The manuscript indicated on Line 141“coating antigen concentration was 1.33 ug/mL”, but this is rather vague. Can the authors provide further details of their ELISA method since this would influence data interpretation. Relatedly, the authors utilized a detection antibody that is denoted as Goat anti-Ferret IgG H&L (HRP). Such reagents are unlikely to be IgG (Fc) “class-specific”, and instead they also react with IgM and IgA. Therefore, referring to the existing ELISA data as serum Ig reactivity would be more accurate.

 

3. Serum was collected at 6 days post-infection (6 dpi) according to the detailed methods. Were these samples also evaluated in the ELISA or HI assays? Specifically, did the authors detect (or could they) evidence for immunological memory (boost in antibody titer) in the Commercial Vaccine or TIV-Low Dose Groups? What about the TIV-High Dose Group? (Detection of a boost in antibody titer would support productive infection)

 

4. Specifics of the challenge dose used for H1N1, H3N2 or IBV infection were not provided. Consequently, comparison of these data with other published vaccine/challenge models is not possible at present.

 

5. The manuscript initially refers to Figure 1 on Line 171; which supposedly presents data on the weight loss, increased body temperature, and clinical symptoms of “model” (PBS vaccinated) naive ferrets following IAV or IBV challenge. Additionally, on Line 172 the text indicates “high titers of virus load were detected in nasal washes on Day 1, 3, and 5 after challenge”. This figure is missing and instead Figure 1 is titled “Results of serum binding antibody IgG detection after viral challenge.” Notably, afterwards in the main text of the manuscript there are no references to specific Figures. Additionally, only Table 6 is referenced in the main text (on Line 189 and again on Line 482).

 

6. Regarding quantification of infectious virus in the homogenized nasal washes or lung tissue, can the authors clarify how they normalized their data, which were generated in a TCID₅₀ assay format, into a PFU/mL or PFU/g value? Likewise, the authors also used qPCR as an alternative approach to measure viral replication. Can additional specifics of this method be provided?

 

7. The usage of the “drift variant” H3N2 that was mismatched to the H3N2 (IVR-228) vaccine strain was emphasized as showing “cross-protection”. As such, it was unfortunate that HI titers to the A/Hong Kong/4801/2014 strain were not assessed prior to and following challenge. Relatedly, did immunization with the CpG-adjuvanted TIV vaccine elicit HI titers to “future” H3N2 strains?

 

Concluding Remarks for the Authors: This reviewer found the manuscript to be straight-forward and the data clearly support elicitation of a vaccine-specific immune response in the TIV-High Dose Group. However, the immune readouts detailed in this manuscript were all “standard” (antibody binding, HI titer, weight loss and clinical disease). Owing to the increased utility of ferrets for biomedical research, this reviewer calls specific attention to the growing portfolio of reagents and assay methodologies that facilitate measurement of T/B cellular immunity. Incorporation of such techniques as future readouts of protective immunity used to compare broadly-protective influenza vaccines in the ferret model is certain to reveal additional correlates of protection beyond HI titers.  

Comments on the Quality of English Language

    

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the Vaccines manuscript "Evaluation of Immunogenicity and Cross-Protective Efficacy of a CpG-Adjuvanted Trivalent Inactivated Influenza Vaccine in Ferrets" present their work evaluating vaccine efficacy in a ferret model.  Specifically, they tested the delivery of trivalent inactivated influenza (TIV) adjuvanted with CpG (CpG-TIV) for its ability to induce immunity in ferrets when delivered 3 weeks apart.  The vaccine was delivered as either a high- or low-dose formulation and was given either with or without adjuvant.  Three weeks after the second dose, serum was obtained, antibodies were tested, and ferrets were challenged.  Clinical signs were monitored after challenge as was nasal viral load and lung pathology.  Their results show that CpG-TIV has higher dose-dependent antibodies than unadjuvanted vaccine, the high-dose vaccine recipients had lower viral shedding, and protection was even observed against heterologous (H3N2) viruses.  This supports inclusion of CpG as a vaccine adjuvant.  Below are comments I would like the authors to address during revision.

1. Is it really necessary to list all of the reagents and equipment in the manner presented in the manuscript.  I am not against it, but I have never read a manuscript where it was presented in this way.
2. Why is the Normal Group included twice in Table 6?
3. Is Figure 1 showing the results presented in Table 6, but in graphical form?  If so, please choose one way or the other to present your findings.
4. Figure 2 and Table 7 have the same critique.
5. Why is the y-axis in Figure 2 on a linear scale rather than a log scale?
6. Overall, please choose whether you want to present data as either a graph or a table, it is not appropriate to present both in the same manuscript.
7. Did the authors confirm that HAI and IgG titers against A/Hong Kong/4801/2014 were low and/or absent before doing the heterologous challenge?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1) Details of the experimental vaccine were provided in the response letter, but were not inserted into the revised manuscript.

 

2) HI titer data for the heterologous H3N2 challenge strain (HK/14) is reported in the main text, but these data were not included in a figure or table format.

3) Figure 1 of the revised manuscript is ELISA data and not key clinical disease parameters that validate the challenge model. Hence the author's response to Comment 5 is not accurate.

4) Text starting on Line 277-280 reads more like a figure legend.

Comments on the Quality of English Language

    

Author Response

We sincerely appreciate the reviewer’s valuable suggestions. Comprehensive point-by-point replies addressing every comment are attached in the uploaded response letter document for your thorough review. All corresponding modifications in the revised manuscript have been highlighted.

Author Response File: Author Response.pdf