Assessment of Humoral Immunogenicity of ChAdOx1 H5 HA Influenza Vaccine for Dairy Cattle

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a timely and potentially valuable pilot study. However, the strength of the conclusions currently exceeds the evidence provided by the small experimental groups. The following points should be addressed:

1. Clearly frame the work as a pilot immunogenicity study. State the group size of three cows per route in the Abstract and temper terms such as “robust,” “mucosal immunity,” “protection,” and “control of viral shedding.”
2. Reassess the statistical analysis. With only three animals per group, normality testing and extensive repeated-measures ANOVA are not sufficiently convincing. Confirm that the cow—not technical replicates—was the experimental unit, explain data transformation and handling of values below the LOD, and display individual animal trajectories and source data.
3. Clarify the control strategy and study limitations. There was no unvaccinated or empty-vector cattle group. Explain how pre-vaccination samples were used as controls and explicitly acknowledge this limitation. Provide the sample-size rationale and relevant ARRIVE information.
4. Correct and expand the ELISA methodology. The current text appears to describe mouse sera using anti-bovine detection antibodies and does not clearly describe bovine serum analysis. Separate the mouse, bovine serum, and milk protocols and report technical replicates, cutoff calculation, and assay variability.
5. Resolve methodological inconsistencies. Milk collection is variously described as daily, every other day, and three times weekly. Harmonize the schedule and provide vaccine volume, intranasal delivery details, exact sample numbers, missing samples, and any safety or tolerability observations.
6. Clarify the functional antibody results. Figure 4 is described as serum data, whereas the Results also discuss milk neutralization. Present the milk results or remove unsupported statements. Define the 50% neutralization endpoint, controls, replicates, and the antigenic relationship among the Texas, Michigan, Ibis, and American wigeon viruses.
7. Avoid equating milk antibodies with local mucosal immunity. Detection of IgG or IgA in milk following intramuscular vaccination does not by itself demonstrate locally generated mammary mucosal immunity. The conclusions should be restricted to antibody detection in milk unless tissue or cellular evidence is provided.
8. Undertake thorough language and formatting revision. Correct grammatical errors, virus nomenclature, figure legends, inconsistent terminology such as “immunisation site” versus “route,” incomplete references, and the Data Availability Statement.

Also, for the quality of English, ｔhe manuscript is generally understandable; however, substantial English-language editing is required. Please correct grammatical errors, awkward sentence structures, sentence fragments, inconsistent verb tenses, and inconsistent use of British and American spelling throughout the manuscript.

Author Response

Comments 1: Clearly frame the work as a pilot immunogenicity study. State the group size of three cows per route in the Abstract and temper terms such as “robust,” “mucosal immunity,” “protection,” and “control of viral shedding.”

Response 1: Thank you for the reviewing comments. We agree that our original wording was very ambitious and too strong for the preliminary study we are presenting here. Accordingly, we have framed the work as a pilot preliminary humoral immunogenicity study throughout the manuscript. We have also stated the group size (three cows per vaccination) in the Abstract and in Figure legends. We have revised the language, and tempered down words like “robust”, mucosal Immunity, “protection” and “control of viral shedding”.

Comments 2: Reassess the statistical analysis. With only three animals per group, normality testing and extensive repeated-measures ANOVA are not sufficiently convincing. Confirm that the cow—not technical replicates—was the experimental unit, explain data transformation and handling of values below the LOD, and display individual animal trajectories and source data.

Response 2: Thank you for this important comment. We agree that the small group size limits the statistical power of the study and that normality testing is not informative with only three animals per group. Accordingly, we have removed the normality testing from the manuscript.

The experimental unit throughout the study was the individual cow, not technical replicates. This has now been explicitly stated in the Materials and Methods. As the same animals were sampled longitudinally, we retained the two-way repeated-measures ANOVA to justify for the repeated measurements over time. However, we have revised the manuscript to recognise that, given the small sample size, the statistical analyses should be interpreted with caution and that the findings are preliminary. We have modified the discussion section accordingly.

We have also clarified the data transformation (log transformation) applied prior to statistical analysis and described how values below the limit of detection (LOD) were handled in the Material and Methods Section. We have updated the figures to display individual animal antibody titres.

During revision, we identified that the statistical analyses had been performed using a natural logarithm (ln) transformation rather than the intended log₁₀ transformation described in the manuscript. We have repeated the analyses using the log₁₀-transformed data and updated the methods and the figures accordingly. Importantly, this correction did not alter the statistical outcomes or the interpretation of the results. The revised analyses are now presented in the manuscript.

 

Comments 3: Clarify the control strategy and study limitations. There was no unvaccinated or empty-vector cattle group. Explain how pre-vaccination samples were used as controls and explicitly acknowledge this limitation. Provide the sample-size rationale and relevant ARRIVE information.

Response 3: Thank you for this important comment. We have clarified the control strategy in the revised manuscript. Considering that is a pilot preliminary humoral immunogenicity study, no unvaccinated or either unrelated vector control group was included. Instead, pre-vaccination samples were used as baseline controls to evaluate vaccine humoral response within an animal over time. This has been described in the Material  and Methods section. We recognise the limitation of the absence of the control group in the study and we have included it in the Discussion section.

We have also included the relevant rationale for the sample size in the Material and methods section. The study was designed to be a preliminary study to evaluate safety and humoral immunogenicity of the vaccine following different immunisation routes. This data generated provides enough information for further immunological and efficacy studies.

Comments 4: Correct and expand the ELISA methodology. The current text appears to describe mouse sera using anti-bovine detection antibodies and does not clearly describe bovine serum analysis. Separate the mouse, bovine serum, and milk protocols and report technical replicates, cutoff calculation, and assay variability.

Response 4: Thank you for this helpful comment. We understand that the original description of the ELISA methodology was unclear and the detection of mouse sera antibodies with anti- bovine detection antibodies protocol could be easily confused. To address this, we have adapted the revised manuscript accordingly and we have modified the Material and Methods section separating the ELISA procedures into two protocols: one describing the assays performed for mouse samples and the other describing the assays performed on bovine milk and sera.

We have also expanded the ELISA methodology section in the Material and Methods to explain in detail the technical replicates as well as the cut-off calculation and assay variability. We described that each sample were analysed in duplicate as technical replicates with the mean value used in statistical analysis. The same negative samples and positive samples were included in each ELISA plate. In Bovine assays, negative samples consisted of a pool of pre-immunised (Day 0) serum or milk samples, whereas in mouse assays, the negative sample consisted of a pool of historical negative sera of unrelated vaccine study. The cut off is defined as mean signal of the negative control wells plus three standard deviations. A positive control included on each ELISA plate was used as a reaction calibrator to ensure inter-plate consistency. For the bovine studies the positive control consisted of bovine sera day 42 whereas mouse sera consisted of three-weeks post immunisation. The coefficient of variation of the positive control was <5%.

Comments 5: Resolve methodological inconsistencies. Milk collection is variously described as daily, every other day, and three times weekly. Harmonize the schedule and provide vaccine volume, intranasal delivery details, exact sample numbers, missing samples, and any safety or tolerability observations.

Response 5: Thank you for highlighting the methodological inconsistencies. We have carefully revised the manuscript to ensure the procedures are described consistently.

At the material & Methods section we have described that the milk collection schedule was three days per week (Monday, Wednesday and Friday) which has been harmonised all over the manuscript to reflect the sampling protocol. We have also clarified the administration volume per route: Intramuscular vaccination was administered in a total volume of 1 ml into the left lateral neck, whereas intranasal vaccination was delivered in a total volume of 2 ml (1 ml per nostril). We have also provided the exact number of blood and milk samples analysed. All serum and milk samples were successfully collected according to the study schedule, and no samples were missing. However, because the haemagglutination inhibition (HI) and virus neutralization assays are labour-intensive and require substantial reagent and sample volumes, a predefined subset of the most informative time points from each animal was selected for analysis. These time points were chosen to represent the baseline, post-prime, and post-boost immune responses. This approach has now been clearly described in the Materials and Methods section, the results section and indicated in the relevant figure legends.

Finally, we have expanded in the Results section, the description of the safety profile of the animals through the study. Animals were monitored daily by trained personnel: no adverse events were detected locally or systemically following vaccinations indicating that vaccination routes were well tolerated.

Comments 6: Clarify the functional antibody results. Figure 4 is described as serum data, whereas the Results also discuss milk neutralization. Present the milk results or remove unsupported statements. Define the 50% neutralization endpoint, controls, replicates, and the antigenic relationship among the Texas, Michigan, Ibis, and American wigeon viruses.

Response 6: Thank you for this valuable comment on the functional antibody results. We have revised the manuscript to ensure consistency through the Material and Methods section and the Results section, including Figure legends.

Milk and serum neutralisation results are now included in Figure 4 and described and referenced in the Results text accordingly. We have expanded the Neutralisation Assay protocol, to clarify that the neutralisation titre reported consist of the endpoint reciprocal of the highest sample dilution with a 50% of reduction in the infected cell population (ICP) relative to virus only control. This assay was performed using the standardised WHO protocol and virus strain (Influenza virus A/American wigeon/South Carolina/22-000345-001/2021). It is also included that each plate had two technical replicates per

We understand and appreciate the reviewer’s request regarding the antigenically differences among the influenza strains, and we have expanded the Material and Methods section and the Results section accordingly. All the influenza A virus mentioned and used in the vaccine constructs, recombinant proteins, and functional assays belong to the H5N1 clade 2.3.4.4b. Influenza A/dairy cattle/ Texas/24-008749_001/2024 was used for vaccine design as the relevant antigen from influenza virus isolated from cows. Influenza A/Ibis/Egypt/RLQP-229S/2022, has 6 amino-acid changes from Influenza A/dairy cattle/ Texas/24-008749_001/2024, as an avian influenza virus representative from the same clade. Influenza A/Michigan/90/2024, is the CDC (centre for disease control) reference strain (CDC CL24-650397), and more clinically relevant due its association with H5N1human infections and increased hospitalisations. This strain is the one from which the Haemagglutinin H5 protein is commercially available. Influenza A/Michigan/90/2024 shares 100% HA amino acid identity with Influenza A/dairy cattle/ Texas/24-008749_001/2024. Influenza A /American wigeon/South Carolina/22-000345-001/2021 also shares 100% HA amino acid identity with Influenza A/dairy cattle/ Texas/24-008749_001/2024. This strain is the virus used to evaluate neutralisation titres using a standardised WHO protocol. These details have been incorporated into the revised manuscript to clarify the rational for the selection of each viral strain.

Comments 7: Avoid equating milk antibodies with local mucosal immunity. Detection of IgG or IgA in milk following intramuscular vaccination does not by itself demonstrate locally generated mammary mucosal immunity. The conclusions should be restricted to antibody detection in milk unless tissue or cellular evidence is provided.

Response 7: Thank you for this important comment. We agree that the detection of vaccine-specific antibodies in milk does not, by itself, demonstrate locally generated mammary mucosal immunity. We have therefore revised the manuscript to avoid this interpretation throughout the Abstract, Results, Discussion, and Conclusions. The text now refers to the detection of vaccine-induced IgG and IgA antibodies in milk following vaccination, without implying their local production within the mammary gland. We also acknowledge that further studies investigating local immune cell responses and antibody production within the mammary tissue would be required to determine whether vaccination induces mammary mucosal immunity.

Comments 8: Undertake thorough language and formatting revision. Correct grammatical errors, virus nomenclature, figure legends, inconsistent terminology such as “immunisation site” versus “route,” incomplete references, and the Data Availability Statement.

Response 8: Thank you for this helpful comment. We have carefully revised the manuscript to improve the overall language, grammar and formatting. Virus nomenclature has been standardised and the terminology to be consistent throughout the manuscript. Figure legends have been revised for clarity. We have also corrected typographical and grammatical errors, completed and updated the reference list where necessary and revised the data availability statement to ensure consistency with the journal requirements.

Reviewer 2 Report

Comments and Suggestions for Authors

This study conducted research on an adenovirus-vectored vaccine against the highly pathogenic avian influenza H5N1 virus, which holds significant public health and veterinary importance. The authors developed a candidate vaccine based on a chimpanzee adenovirus (ChAdOx1) and evaluated the humoral immune responses induced in lactating dairy cows via both intramuscular and intranasal administration routes. The study found that intramuscular immunization effectively induced IgG and IgA antibodies in both serum and milk, and the antibodies in serum exhibited neutralizing activity. This research provides valuable data for the development of H5N1 vaccines for dairy cattle and supports the potential application of the ChAdOx1 vector platform in bovids. However, the manuscript has several deficiencies in experimental design, data presentation, and mechanistic exploration:

1. The number of experimental animals is relatively small, with only 3 cows per group in the animal experiments. The statistical significance derived from such a small sample size may arise from individual variability, and the reviewer considers the current results insufficiently reliable.

2. This study claims to assess the "immunogenicity" of the developed vaccine, but it lacks evaluation of vaccine-induced cellular immune responses. One of the major advantages of adenovirus-vectored vaccines is their ability to induce both strong antibody and T‑cell responses simultaneously. The authors only mention the importance of T‑cell responses in the discussion but provide no experimental data to support this.

3. This study also did not conduct animal experiments to verify vaccine protective efficacy. Although challenge experiments in cattle are not easy to perform, it is advisable to use a mouse model to carry out relevant studies.

4. Several core phenomena in this study lack in-depth explanation. For example, although neutralizing antibodies were present in serum, no hemagglutination inhibition (HI) activity was detected (line 355). The authors suggest this may be due to epitopes targeting non-receptor-binding domains, but no experimental verification was performed. Likewise, despite high titers of IgG and IgA in milk, no neutralizing activity was observed (line 368). The authors speculate that these antibodies may function through other mechanisms. Such hypotheses require experimental evidence for support.

5. The study shows that vaccine-induced antibodies can bind to the HA protein of the Egyptian IbIs strain, but this is based solely on binding activity measured by ELISA. The reviewer considers that functional evidence is needed, such as detection of neutralizing antibodies or HI activity against the IbIs strain.

6. Viral strain consistency: The vaccine was constructed using the HA from the A/dairy cattle/Texas strain, the neutralization assay used the A/American wigeon/South Carolina strain, and the ELISA coating antigen used the A/Michigan/90/2024 strain. The authors need to clearly state the scientific rationale for choosing these different strains for the assays and explain the HA sequence homology among them.

7. Insufficient details in the Materials and Methods section: For example, the neutralization assay details: the "Microneutralization assay" section cites a reference but does not specify which cell line was used (MDCK?), the virus titer, or the endpoint criteria for determining neutralization. ELISA limit of detection (LOD): The LOD is mentioned multiple times in the text, but its definition and numerical value are not clearly stated.

Author Response

Comment 1:The number of experimental animals is relatively small, with only 3 cows per group in the animal experiments. The statistical significance derived from such a small sample size may arise from individual variability, and the reviewer considers the current results insufficiently reliable.

Response 1: We appreciate the reviewer’s important comments and we agree that the small sample size is a limitation of the present study. We have revised the manuscript to more clearly state that this work was designed as a preliminary pilot study to evaluate safety and humoral immunogenicity of the ChAdOx1 H5-Texas vaccine in cattle following two different routes of immunisation. The inclusion of three animals per group was intended to generate initial data while minimising animal use, in accordance with the principles of 3Rs, before larger and more exploratory studies. In response to reviewer’s concern, we have used a more conservative statistical analysis and presentation of the data. We have removed statistical analysis comparisons between groups and just report the repeated measures two-way ANOVA to evaluate changes in antibody responses over time. We have changed the Figures and Figure Legends to show the individual differences accordingly. The Discussion has also been updated to acknowledge this limitation of the study.

Comment 2: This study claims to assess the "immunogenicity" of the developed vaccine, but it lacks evaluation of vaccine-induced cellular immune responses. One of the major advantages of adenovirus-vectored vaccines is their ability to induce both strong antibody and T‑cell responses simultaneously. The authors only mention the importance of T‑cell responses in the discussion but provide no experimental data to support this.

Response 2: We thank the reviewer for this valuable comment and agree that the term immunogenicity covers both humoral and cellular immune responses. To address this concern, we have revised the manuscript, including the title to specify that this study evaluates the humoral immunogenicity of the ChAdOx1 H5-Texas vaccine rather than overall immunogenicity.

We fully agree that one of the major advantages of adenoviral-vectored vaccines is their capacity to induce robust T-cell responses, which are likely to contribute to vaccine-induced protection. However, the primary objective of this pilot study was to evaluate the safety of the vaccine and to characterise the humoral antibody response in serum and milk following intramuscular and intranasal immunisation. Accordingly, the study was designed to determine whether vaccine-specific antibodies, particularly in milk, could be induced by these two vaccination strategies, rather than to provide a comprehensive immunological characterisation.

We agree that assessing vaccine-induced cellular immune responses would provide important complementary information. We have therefore acknowledged this limitation in the Discussion and state that future studies will investigate antigen-specific T-cell responses to provide a more complete assessment of the immunogenicity and mechanisms of protection elicited by the ChAdOx1 H5-Texas vaccine.

Comment 3: This study also did not conduct animal experiments to verify vaccine protective efficacy. Although challenge experiments in cattle are not easy to perform, it is advisable to use a mouse model to carry out relevant studies.

Response 3: We thank the reviewer for this valuable comment and we agree that a challenge experiment is essential for demonstrating vaccine efficacy. We have addressed this limitation and recognise the importance of a challenge study to evaluate protective efficacy in future studies.

Although mouse models are widely used for the preliminary evaluation of influenza vaccine efficacy, we believe they would not adequately address the specific objectives of the present study. This work was designed to investigate the humoral immune response, particularly the induction of vaccine-specific antibodies in the milk of lactating dairy cattle following intramuscular and intranasal vaccination. The anatomy and physiology of the murine mammary gland, lactation, and milk production differ substantially from those of dairy cattle, limiting the value of a mouse model.

Furthermore, H5N1 infection in mice is typically established by lethal intranasal challenge and primarily results in respiratory disease (Pyles 2026, npj vaccines). While such a model can provide valuable information on vaccine-mediated protection against infection, it would not allow assessment of the vaccine's impact on antibody secretion into bovine milk or its potential role in reducing viral shedding through milk during lactation. Natural H5N1 infection in cattle is predominantly a respiratory infection, whereas viral dissemination to the mammary gland is a feature of infection in lactating dairy cows. Therefore, we consider lactating dairy cattle to be the most appropriate model for evaluating the protective efficacy of this vaccination strategy. We have included this justification in the Discussion and recognise that future challenge studies in dairy cattle will be necessary to determine the protective efficacy of the vaccine.

 

Comment 4: Several core phenomena in this study lack in-depth explanation. For example, although neutralizing antibodies were present in serum, no hemagglutination inhibition (HI) activity was detected (line 355). The authors suggest this may be due to epitopes targeting non-receptor-binding domains, but no experimental verification was performed. Likewise, despite high titres of IgG and IgA in milk, no neutralising activity was observed (line 368). The authors speculate that these antibodies may function through other mechanisms. Such hypotheses require experimental evidence for support.

Response 4: We thank the reviewer for this comment and agree that the proposed mechanisms underlying the titres of neutralising antibodies detected were not directly investigated in this study. We have revised the Discussion to clarify this as a potential explanation. We have tempered down the interpretation of serum neutralisation results by stating the presence of neutralising antibodies in the absence of detectable haemagglutination inhibition activity may reflect the recognition of epitopes outside the haemagglutinin receptor binding site.

Although the functional relevance of milk antibodies detected in this study remains unknown, previous work has shown that influenza -specific antibodies may contribute protection through mechanisms beyond virus neutralisation or haemagglutination inhibition, including Fc-mediated effector functions. Whether the milk antibodies induced by ChAdOx1 H5-Texas vaccine have those activities remain to be determined (Sicca 2018 Expert Rev Vaccines; Vanderven 2017 Curr Opin Virol).

Comment 5: The study shows that vaccine-induced antibodies can bind to the HA protein of the Egyptian Ibis strain, but this is based solely on binding activity measured by ELISA. The reviewer considers that functional evidence is needed, such as detection of neutralizing antibodies or HI activity against the IbIs strain.

Response 5: We thank the reviewer for the valuable comment and agree that functional assays such as neutralising assays and haemagglutination assays using the Influenza A/Ibis/Egypt/RLQP-229S/2022 strain, would provide more information about cross-reactive antibody responses. In the present study, ELISA against the Ibis HA protein was included to evaluate antibody binding to a heterologous H5 antigen rather than demonstrate functional cross-protection. We have revised the manuscript and clarify this distinction and tempered down our discussion conclusion accordingly.

Comment 6: Viral strain consistency: The vaccine was constructed using the HA from the A/dairy cattle/Texas strain, the neutralisation assay used the A/American wigeon/South Carolina strain, and the ELISA coating antigen used the A/Michigan/90/2024 strain. The authors need to clearly state the scientific rationale for choosing these different strains for the assays and explain the HA sequence homology among them.

Response 6: We thank the reviewer for this important comment. We have revised the Material and Methods and Results sections to clearly explain the rationale behind the selection of the virus strains used in the different assays.

All the influenza A virus mentioned and used in the vaccine constructs, recombinant proteins, and functional assays belong to the H5N1 clade 2.3.4.4b. Influenza A/dairy cattle/ Texas/24-008749_001/2024 was used for vaccine design as the relevant antigen from influenza virus isolated from cows. Influenza A/Ibis/Egypt/RLQP-229S/2022, has 6 amino-acid changes from Influenza A/dairy cattle/ Texas/24-008749_001/2024, as an avian representative from the same clade. Influenza A/Michigan/90/2024, is the CDC (centre for disease control) reference strain (CDC CL24-650397), and more clinically relevant due its association with H5N1human infections and increased hospitalisations. This strain is the one from which the Haemagglutinin H5 protein is commercially available. Influenza A/Michigan/90/2024 shares 100% HA amino acid identity with Influenza A/dairy cattle/ Texas/24-008749_001/2024. Influenza A /American wigeon/South Carolina/22-000345-001/2021 also shares 100% HA amino acid identity with Influenza A/dairy cattle/ Texas/24-008749_001/2024. This strain is the virus used to evaluate neutralisation titres using a standardised WHO protocol. These details have been incorporated into the revised manuscript to clarify the rational for the selection of each viral strain.

 

Comment 7: Insufficient details in the Materials and Methods section: For example, the neutralization assay details: the "Microneutralization assay" section cites a reference but does not specify which cell line was used (MDCK?), the virus titre, or the endpoint criteria for determining neutralization. ELISA limit of detection (LOD): The LOD is mentioned multiple times in the text, but its definition and numerical value are not clearly stated.

Response 7: We thank the reviewer for highlighting these omissions. The Materials and Methods section has been expanded to provide a more detailed description of the microneutralization assay. Specifically, we now describe the cell line used, the virus strain and input dose, the assay procedure, and the definition of the neutralisation endpoint. Neutralising antibody titres (NT50) are reported as the reciprocal of the highest sample dilution producing a ≥50% reduction in infected cells relative to the virus-only control. We have also clarified that each sample was analysed in duplicate and that appropriate assay controls were included.

In addition, we have revised the ELISA methodology to clearly define the limit of detection (LOD). The method used to calculate the LOD and the numerical threshold applied for data analysis are now explicitly described in the Materials and Methods section and referenced consistently throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Assessment of immunogenicity of ChAdOx1 H5 HA influenza 2 vaccine for dairy cattle 3

Barbara Dema 1,*, Marta Ulaszewska 1, Alice Lilley 3, Abi Lofts 3, Roo Bhasin 1, Ruth Harvey 3, Piyada Supasa 5, 4 Matěj Hlaváč 1, Susan J Morris 1, Richard Booth 4, Alexander M P Byrne 3, Nicola Lewis 2,3, Alex McSloy 2 and 5 Sarah C. Gilbert 1,6

 

This is overall a well written paper. I have only raised some minor comments and suggestions.

Since only 3 animals were per group, the statistical findings can’t be regarded as very reliable, due to low statistical power; 5 to  per group would have been statistically preferable. Hence this  study should be regarded as exploratory or preliminary in nature, and I would suggest that needs to be mentioned in the text.

 

 

Language

Line 167:  Serum and Milk

Suggestion:  Serum and milk

 

Line 196: ..of Casein Line 199: … in Casein

Suggestion: … of casein …in casein

 

Line 198: .. with Gelatin

Suggestion: ..with  gelatin

 

Line 202: Thermofisher scientific

Suggestion: Thermofisher Scientific

 

 

Figure 2 , Figure 3, Fig. A1, A2: mean and standard deviations are shown

Suggestion: mean and 95% confidence intervals would be preferable

Comment: When referring to means, presumably these are geometric mean titres ? perhaps indicate such.

 

Fig.  2 & 3 & 4;  log 10

Suggestion: log₁₀

 

Page 12 Abbreviations

Comment: no need to capitalise

Caesium Chloride, Gut-Associated Lymphoid Tissue, Hemagglutinin 5 Neuraminidase 1

Hemagglutination Inhibition assay, Infected cell Population, Infection Units

Mucosa-Associated Lymphoid Tissue, Optical Density, Phosphate Buffer Saline

Rift Valley Fever, Viral Particles

 

Suggestion:

Caesium chloride, Gut-associated lymphoid tissue, Hemagglutinin 5 neuraminidase 1

Hemagglutination inhibition assay, Infected cell population, Infection units

Mucosa-associated lymphoid tissue, Optical density, Phosphate buffered saline

Rift Valley fever, Viral particles

 

Line 487: the Intramuscular immunisation

Suggestion:   the intramuscular immunisation

Author Response

Comment 1:Since only 3 animals were per group, the statistical findings can’t be regarded as very reliable, due to low statistical power; 5 to  per group would have been statistically preferable. Hence this  study should be regarded as exploratory or preliminary in nature, and I would suggest that needs to be mentioned in the text.

Response 1: We thank the reviewer for this important comment and agree that the statistical power of this study is limited. We have revised the manuscript to clearly define this work as a preliminary pilot study designed to evaluate the safety and humoral immunogenicity of the ChAdOx1- H5-Texas vaccine in dairy cattle. 

Comment 2: Language

Line 167:  Serum and Milk

Suggestion:  Serum and milk

Line 196: ..of Casein Line 199: … in Casein

Suggestion: … of casein …in casein

Line 198: .. with Gelatin

Suggestion: ..with  gelatin

Line 202: Thermofisher scientific

Suggestion: Thermofisher Scientific

Response 2: We thank the reviewer for these language and formatting suggestions. We have revised the manuscript accordingly and corrected inconsistencies through the text, including the examples suggested by the reviewer.

Comment 3: Figure 2 , Figure 3, Fig. A1, A2: mean and standard deviations are shown

Suggestion: mean and 95% confidence intervals would be preferable

Comment: When referring to means, presumably these are geometric mean titres ? perhaps indicate such.

Response 3: We thank the reviewer for this helpful suggestion. Given the small sample size of this preliminary study, we have included individual animal values in the figures to provide clearer representation of the inter-animal variability. Antibody binding and neutralising titres were analysed after log-transformation. Neutralising titres are presented with geometric mean titres with 95% confidence intervals as suggested by the reviewer. Figure legends have been updated to explain all these changes.

Comment 4: Fig.  2 & 3 & 4;  log 10

Suggestion: log₁₀

Response 4: Thank you for these formatting suggestions, we have updated the manuscript to include the suggestions.

Comment 5: Page 12 Abbreviations

Comment: no need to capitalise

Caesium Chloride, Gut-Associated Lymphoid Tissue, Hemagglutinin 5 Neuraminidase 1

Hemagglutination Inhibition assay, Infected cell Population, Infection Units

Mucosa-Associated Lymphoid Tissue, Optical Density, Phosphate Buffer Saline

Rift Valley Fever, Viral Particles

Suggestion:

Caesium chloride, Gut-associated lymphoid tissue, Hemagglutinin 5 neuraminidase 1

Hemagglutination inhibition assay, Infected cell population, Infection units

Mucosa-associated lymphoid tissue, Optical density, Phosphate buffered saline

Rift Valley fever, Viral particles

Response 5:Thank you for these formatting suggestions, we have updated the manuscript to include the suggestions.

Comment 6: Line 487: the Intramuscular immunisation

Suggestion:   the intramuscular immunisation

Response 6: Thank you for these formatting suggestions, we have updated the manuscript to include the suggestions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The MS has improved considerably. The authors have appropriately reframed the work as a preliminary study of humoral immunogenicity, clarified the experimental unit and sampling provedures, provided individual longitudinal data, expanded the assay methods, and acknowledged the absence of a control group, the small sample size, and the lack of a challenge experiment. Most of my major concerns have therefore been addressed.

A few issues shoule still be corrected before acceptance:

1. Please clarify the repeated-measures statistical model. Vaccination route should be identified as the between-subject factor, time as the within-subject factor, and individual cow as the repeated-measures subject. Please report the route × time interaction and explain why some figure legends report a separate p-value for “cows.” Please also provide the statistical result, or state that no formal comparison was conducted, for the milk neutralisation data in Figure 4B.

2. The safety findings should be presented descriptively. Because the study included only six cows and no detailed predefined safety endpoints are reported, please state that no vaccine-related adverse events were observed during the study rather than implying that a general safety profile was established.

3. The final Conclusion remains somewhat stronger than supported by an immunogenicity study without viral challenge. Please revise “a promising vaccine candidate for the prevention of H5N1 infection” to “a promising candidate for further evaluation of protective efficacy against H5N1 infection.”

4. Please specify the exact sampling days used for the predefined HI and neutralisation subsets. The isolated milk neutralisation result should be described cautiously as transient activity detected in one intranasally vaccinated cow.

5. Several citations should be verified. Reference 8 does not appear to directly support the vaccination claim in the Introduction; Reference 11 is a phase 1 study in human adults and does not support protective efficacy in sheep, goats, and cattle; and Reference 26 does not appear appropriate for the statement concerning human milk IgA. Please also verify the citation supporting the proposed origin of milk plasma-cell precursors.

6. Moderate English-language editing is still needed for grammar, subject–verb agreement, and clarity.

Author Response

Comment 1: Please clarify the repeated-measures statistical model. Vaccination route should be identified as the between-subject factor, time as the within-subject factor, and individual cow as the repeated-measures subject. Please report the route × time interaction and explain why some figure legends report a separate p-value for “cows.” Please also provide the statistical result, or state that no formal comparison was conducted, for the milk neutralisation data in Figure 4B.

 

Response 1: We thank the reviewer for this helpful comment. We have revised the Materials and Methods to clearly describe the repeated-measures statistical model, specifying vaccination route as the between-subject factor, time as the within-subject factor, and individual cow as the repeated-measures subject. The statistical model evaluates the main effects of time, vaccination route, and their interaction. We have also removed the separate p-value. Given the exploratory nature of this pilot study and the limited sample size, we have simplified the statistical reporting in the figure legends and now present individual animal responses without emphasising formal statistical significance. We have also clarified on the Material and Methods section that the interpretation of the results is merely descriptive.

Comment 2: The safety findings should be presented descriptively. Because the study included only six cows and no detailed predefined safety endpoints are reported, please state that no vaccine-related adverse events were observed during the study rather than implying that a general safety profile was established.

Response 2: We thank the reviewer for this helpful comment. We have revised the manuscript to present the safety observations descriptively and now state that no vaccine-related adverse events were observed during the study. We have removed statements implying that a comprehensive safety profile was established.

Comment 3: The final Conclusion remains somewhat stronger than supported by an immunogenicity study without viral challenge. Please revise “a promising vaccine candidate for the prevention of H5N1 infection” to “a promising candidate for further evaluation of protective efficacy against H5N1 infection.”

Response 3: Thank you for the reviewer’s suggestion. We have toned down the conclusion and included in the text that ChAdOx1 H5-Texas is a promising vaccine candidate for further evaluation of protective efficacy against H5N1 infection in dairy cows.

Comment 4:  Please specify the exact sampling days used for the predefined HI and neutralisation subsets. The isolated milk neutralisation result should be described cautiously as transient activity detected in one intranasally vaccinated cow.

Response 4: We thank the reviewer for this valuable comment. We have revised the Materials and Methods and results section to specify the exact sampling days included in the haemagglutination inhibition and microneutralisation assays. In addition, we have revised the Results section to describe the milk neutralisation findings more cautiously. We now state that neutralising activity was largely absent in milk samples, with only a transient response detected in a single intranasally vaccinated cow, and we avoid any further conclusions from this observation.

Comment 5: Several citations should be verified. Reference 8 does not appear to directly support the vaccination claim in the Introduction; Reference 11 is a phase 1 study in human adults and does not support protective efficacy in sheep, goats, and cattle; and Reference 26 does not appear appropriate for the statement concerning human milk IgA. Please also verify the citation supporting the proposed origin of milk plasma-cell precursors.

Response 5: We thank the reviewer for the detailed revision of the references. We have reviewed all of the citations mentioned and corrected those that did not appropriately support the corresponding statements.

Reference 8 (Shi et al.) was cited to support the concept that systemic intramuscular vaccination can induce immune responses capable of protecting the mammary gland against H5N1 infection. We agree that our original wording could be interpreted as implying a mechanistic role for mammary-associated lymphoid tissue that was not directly investigated in the cited study. We have therefore revised the Introduction to clarify this point.

Reference 11, we agree that the reference included is the Phase 1 clinical trial of the vaccine in human adults. The correct reference is now included into the Discussion section .

Reference 26, now reference 27 from Ulfman et al. Front Nutr 2018. We agree that in the original manuscript it was not correctly positioned on the text, and it could be misleading and confusing. We have revised the Discussion section to clarify this part on the reference of human immunoglobulins in milk, IgA and IgG.

The actual reference 26, Rainard et al. 2022 is the correct reference to support the origin of plasma cell precursor in milk instead of Ulfman et al. 2018. We have changed this reference on the text.

Comment 6: Moderate English-language editing is still needed for grammar, subject–verb agreement, and clarity.

Response 6: We thank the reviewer for the profound revision with regards language and grammar edits. We have gone through the revised manuscript and edited some grammar, subject–verb agreement as well as clarified some problematic sentences. 

Reviewer 2 Report

Comments and Suggestions for Authors

Although the authors did not provide additional experimental evidence, they essentially described it in the discussion and improved the methodological description. I agree to accept this paper.

Author Response

We thank the reviewer for their positive assessment of our revised manuscript. Thank you for recommending our manuscript for acceptance.