Round 1

Reviewer 1 Report

The article titled "Vaccination with two doses of AstraZeneca® (ChAdOx1-S) and Pfizer® (BNT162b2) induces the production of neutralizing antibodies for COVID-19, without causing damage to hematological, biochemical, inflammatory, and oxidative biomarkers" presents results and discussion relevant to the topic of COVID-19 vaccines. The findings and discussion in this article are relevant and reinforce the importance of the development and implementation of antiviral vaccines in public health.

The paragraph "The Food and Drug Administration produced a document to guide the development and licensing of vaccines, requiring a minimum efficacy of 50%. In Brazil, AstraZeneca® (ChAdOx1-S) and Pfizer® (BNT162b2) vaccines were the most administered in the state of Rio Grande do Sul, Brazil" is contextout. Please, it's need improve this part of introduction.

The Figure 6 is not included in the discussion.

Author Response

# REVIEWER 1

Thank you for your comment. We appreciate your approval and your acknowledgment of the importance of our results.

Thank you for your comment. We have implemented the suggested corrections, which are highlighted in the revised manuscript. The relevant supporting studies have been duly cited to substantiate these changes. Many topics of the introduction were restructured.

The Figure 6 is not included in the discussion.

Thank you for your comment. We improved the discussion about the Figure 6.

Author Response File: Author Response.pdf

Reviewer 2 Report

This article raises valid questions about inflammation following Covid vaccines

However, in 2025, it is no longer possible to write that Covid vaccines are safe and effective (see the articles proving this below). The authors must remove this sentence or counterbalance it with the arguments cited below.

The introduction should explain why it is important to study inflammation parameters after vaccination.

It should also mention the consequences of the spike protein's interaction with the renin-angiotensin system via its binding to the ACE2 receptor (Cao Z, Wu Y, Faucon E, Sabatier JM. SARS-CoV-2 & Covid-19: Key-Roles of the ‘Renin-Angiotensin’ System / Vitamin D Impacting Drug and Vaccine Developments. Infect Disord Drug Targets. 2020;20(3):348-349. doi: 10.2174/1871526520999200505174704. PMID: 32370727. https://pubmed.ncbi.nlm.nih.gov/32370727/

Gao X, Zhang S, Gou J, Wen Y, Fan L, Zhou J, Zhou G, Xu G, Zhang Z. Spike-mediated ACE2 down-regulation was involved in the pathogenesis of SARS-CoV-2 infection. J Infect. 2022 Oct;85(4):418-427. doi: 10.1016/j.jinf.2022.06.030. Epub 2022 Jul 3. https://doi.org/10.1016/j.jinf.2022.06.030 )

Similarly, the different results found should be discussed: to what extent are they predictable or unpredictable?

I did not find the results of neutralizing antibody levels in the manuscript, which appear to have been omitted.

Abstract: Covid vaccines are presented as safe and effective

Reminder: AZ has been withdrawn!

Suspension in March 2021 https://www.nih.gov/news-events/news-releases/niaid-statement-astrazeneca-vaccine

Tuesday, March 23, 2021

The European Medicines Agency suspended all use of the product on March 16, 2021, only to reauthorize it for people over 55 a few days later, arguing that the risk-benefit balance of this vaccination still favored continuing the vaccinationcampaign. European Medicines Agency. COVID-19 Vaccine AstraZeneca: benefits still outweigh the risks despite possible link to rare blood clots with low platelets. Eur Med Agency 2021. https://www.ema.europa.eu/en/news/covid-19-vaccine-astrazeneca-benefits-still-outweigh-risks-despite-possible-link-rare-blood-clots

The AZ vaccine causes thrombosis

https://www.who.int/fr/news-room/feature-stories/detail/the-oxford-astrazeneca-covid-19-vaccine-what-you-need-to-know

Complaints have been filed about the effects of the vaccine, particularly blood clots

blood clots along with abnormally low platelet levels. BMJ2023;380:p725

http://dx.doi.org/10.1136/bmj.p725 Published: March 28, 2023

Thrombocytopenia and blood clots

https://www.sciencedirect.com/science/article/pii/S2052297521000482

BMJ. 2021; 374: n1931. Published online 2021 Aug 26. doi: 10.1136/bmj.n1931 PMCID: PMC8388189 PMID: 34446426 Risk of thrombocytopenia and thromboembolism after COVID-19 vaccination and SARS-CoV-2 positive testing: self-controlled case series study

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopenia https://doi.org/10.1038/s41586-021-03744-4

Coagulation disorders (including deaths, see for example the ANSM reports for France https://ansm.sante.fr/actualites/point-de-situation-sur-la-surveillance-des-vaccins-contre-la-covid-19-periode-du-12-03-2021-au-18-03-2021

According to the AZ clinical trial report (https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-005226-28/results), there were 621 serious adverse events for 21,587 subjects who received the vaccine and 136 for 10,793 in the placebo group (vaccinated with the meningococcal vaccine).

Voices were raised as early as 2020 and 2021 to question the efficacy demonstrated in clinical trials, highlighting biases such as the exclusion of Covid cases within 14 days of the injections

Rapid response to: Covid-19: WHO says rollout of AstraZeneca vaccine should continue, as Europe divides over safety

BMJ 2021; 372 doi: https://doi.org/10.1136/bmj.n728 (Published March 16, 2021)Cite this as: BMJ 2021;372:n728

and

Peter Doshi

Peter Doshi: Pfizer and Moderna’s “95% effective” vaccines—let’s be cautious and first see the full data, November 26, 2020

https://blogs.bmj.com/bmj/2020/11/ 26/peter-doshi-pfizer-and-modernas-95-effective-vaccines-lets-be-cautious-and-first-see-the-full-data/

Doubts about the integrity of the data had been raised as early as 2021

Paul Thacker, BMJ Investigation

Covid-19: Researcher blows the whistle on data integrity issues in Pfizer's vaccine trial

https://www.bmj.com/content/375/bmj.n2635

As early as 2021, the real-world effectiveness of Covid vaccines was much lower than announced:

"Estimated BNT162b2 effectiveness against any SARS-CoV-2 infection was negligible for the first 2 weeks after the first dose, increased to 36.8% (95% confidence interval [CI], 33.2 to 40.2) in the third week after the first dose, and reached its peak at 77.5% (95% CI, 76.4 to 78.6) in the first month after the second dose (Table 2 and Fig. 2A). However, effectiveness declined gradually, starting from the first month after the second dose. The decline accelerated after the fourth month, and effectiveness reached a low level of approximately 20% in months 5 through 7 after the second dose.

Chemaitelly H, Tang P, Hasan MR, AlMukdad S, Yassine HM, Benslimane FM, Al Khatib HA, Coyle P, Ayoub HH, Al Kanaani Z, Al Kuwari E, Jeremijenko A, Kaleeckal AH, Latif AN, Shaik RM, Abdul Rahim HF, Nasrallah GK, Al Kuwari MG, Al Romaihi HE, Butt AA, Al-Thani MH, Al Khal A, Bertollini R, Abu-Raddad LJ. Waning of BNT162b2 Vaccine Protection against SARS-CoV-2 Infection in Qatar. N Engl J Med. 2021 Dec 9;385(24):e83. doi: 10.1056/NEJMoa2114114. Epub 2021 Oct 6. PMID: 34614327; PMCID: PMC8522799. https://pubmed.ncbi.nlm.nih.gov/34614327/

In 2025, real-world studies confirmed this lack of effectiveness of Covid vaccines and even showed that the risk of infection increases with the number of doses:

Lin DY, Gu Y, Xu Y, Zeng D, Wheeler B, Young H, Sunny SK, Moore Z. Effects of Vaccination and Previous Infection on Omicron Infections in Children. N Engl J Med. 2022 Sep 22;387(12): 1141-1143. doi: 10.1056/NEJMc2209371. Epub 2022 Sep 7. PMID: 36069811; PMCID: PMC9511630. https://pubmed.ncbi.nlm.nih.gov/36069811/

Tseng, H.F., Ackerson, B.K., Bruxvoort, K.J. et al. Effectiveness of mRNA-1273 vaccination against SARS-CoV-2 omicron subvariants BA.1, BA.2, BA.2.12.1, BA.4, and BA.5. Nat Commun 14, 189 (2023). https://doi.org/10.1038/s41467-023-35815-7

Nabin K Shrestha, Patrick C Burke, Amy S Nowacki, James F Simon, Amanda Hagen, Steven M Gordon, Effectiveness of the Coronavirus Disease 2019 Bivalent Vaccine, Open Forum Infectious Diseases, Volume 10, Issue 6, June 2023, ofad209, https://doi.org/10.1093/ofid/ofad209

Nabin K Shrestha, Patrick C Burke, Amy S Nowacki, Steven M Gordon, Effectiveness of the 2023–2024 Formulation of the COVID-19 Messenger RNA Vaccine, Clinical Infectious Diseases, Volume 79, Issue 2, August 15, 2024, Pages 405–411, https://doi.org/10.1093/cid/ciae132

Uversky, V.N.; Redwan, E.M.; Makis, W.; Rubio-Casillas, A. IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein. Vaccines 2023, 11, 991. https://doi.org/10.3390/vaccines11050991

Risk of Coronavirus Disease 2019 (COVID-19) among those up-to-date and not up-to-date on COVID-19 vaccination by US CDC criteria

https://doi.org/10.1371/journal.pone.0293449

Chalupka A, Richter L, Chakeri A, El-Khatib Z, Theiler-Schwetz V, Trummer C, Krause R, Willeit P, Benka B, Ioannidis JPA, Pilz S. Effectiveness of a fourth SARS-CoV-2 vaccine dose in previously infected individuals from Austria. Eur J Clin Invest. 2024 Mar;54(3):e14136. doi: 10.1111/eci.14136. Epub 2023 Nov 30. PMID: 38032853; PMCID: PMC11475503. https://pubmed.ncbi.nlm.nih.gov/38032853/

Effectiveness and durability of mRNA-1273 BA.4/BA.5 bivalent vaccine (mRNA-1273.222)

against SARS-CoV-2 BA.4/BA.5 and XBB sublineages

https://www.medrxiv.org/content/10.1101/2023.12.11.23299663v1.full.pdf

Dörr, T., Lacy, J., Ballouz, T. et al. Association of SARS-CoV-2 vaccination status with risk of influenza-like illness and loss of workdays in healthcare workers. Commun Med 5, 347 (2025). https://doi.org/10.1038/s43856-025-01046-8

Given the wording of the special issue for which this article is intended, a focus on results for older people should cite this article (in preprint but from official agencies) dedicated to those over 65, which shows that there are more Covid hospitalizations and Covid deaths among the vaccinated than among the unvaccinated.

Effectiveness of the 2023 Autumn XBB.1.5 COVID-19 Booster During Summer 2024 in the EU/EEA: A VEBIS Electronic Health Record Network Study

James Humphreys, Nathalie Nicolay, Toon Braeye, Izaak Van Evercooren, Christian Holm Hansen, Ida Rask Moustsen-Helms, Chiara Sacco, Massimo Fabiani, Jesús Castilla, Iván Martínez-Baz, Ausenda Machado, Patricia Soares, Rickard Ljung, Nicklas Pihlström, Esther Kissling, Anthony Nardone, Susana Monge, Sabrina Bacci, Baltazar Nunes, VEBIS-EHR working group

medRxiv 2025.04.30.25326709; doi:https://doi.org/10.1101/2025.04.30.25326709

A study analyzing data from the GcoVS Project on 99 million people showed a significant increase in the following adverse effects within 42 days of receiving a Pfizer, Moderna, or AstraZeneca COVID vaccine: Guillain-Barré syndrome, cerebral venous sinus thrombosis after AZ, acute disseminated encephalomyelitis after Moderna, myocarditis and pericarditis following Pfizer, Moderna, and AstraZeneca vaccines

Faksova K, Walsh D, Jiang Y, Griffin J, Phillips A, Gentile A, Kwong JC, Macartney K, Naus M, Grange Z, Escolano S, Sepulveda G, Shetty A, Pillsbury A, Sullivan C, Naveed Z, Janjua NZ, Giglio N, Perälä J, Nasreen S, Gidding H, Hovi P, Vo T, Cui F, Deng L, Cullen L, Artama M, Lu H, Clothier HJ, Batty K, Paynter J, Petousis-Harris H, Buttery J, Black S, Hviid A. COVID-19 vaccines and adverse events of special interest: A multinational Global Vaccine Data Network (GVDN) cohort study of 99 million vaccinated individuals. Vaccine. 2024 Apr 2;42(9):2200-2211. doi: 10.1016/j.vaccine.2024.01.100. Epub 2024 Feb 12. PMID: 38350768. https://pubmed.ncbi.nlm.nih.gov/38350768/

An analysis of VAERS showed a significant rate of reports of spontaneous abortions and vaginal hemorrhage after Covid vaccination

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9377365/

Moro PL, Olson CK, Zhang B, Marquez P, Strid P. Safety of Booster Doses of Coronavirus Disease 2019 (COVID-19) Vaccine in Pregnancy in the Vaccine Adverse Event Reporting System. Obstet Gynecol. 2022 Sep 1;140(3):421-427. doi: 10.1097/AOG.0000000000004889. Epub 2022 Aug 3. PMID: 35926203; PMCID: PMC9377365.

According to Pfizer's clinical trial on pregnant women (not published in peer-reviewed journals), despite incomplete results and a majority of women being injected late in pregnancy, there are more congenital malformations and adverse effects in newborns of vaccinated women.

To Evaluate the Safety, Tolerability, and Immunogenicity of BNT162b2 Against COVID-19 in Healthy Pregnant Women 18 Years of Age and Older

https://clinicaltrials.gov/study/NCT04754594

Israeli data show an increased risk of autoimmune diseases in children vaccinated against Covid: Freiberg, C., Dotan, A., Arnheim, D. et al. Investigating the association between SARS-CoV-2 infection, COVID-19 vaccination, and autoimmune diseases in a pediatric population: a comprehensive analysis. Pediatr Rheumatol 23, 52 (2025). https://doi.org/10.1186/s12969-025-01093-4

Title

The authors have not proven that the Covid vaccines injected in Brazil in the fall of 2021 induce the synthesis of neutralizing antibodies against the virus circulating at that time, so the word “neutralizing” should be removed from the title.

Introduction

Discuss why there is an increase in inflammation parameters

However, remember that the level of neutralizing antibodies correlates with the severity of COVID-19.

Cellular & Molecular Immunology (2021) 18:318–327; https://doi.org/10.1038/s41423-020-00588-2

Materials and methods

The control group was infected with the virus and will therefore have neutralizing antibodies: how can we interpret the differences in antibody levels?

The time between vaccine injections and blood sample collection must be specified. The dates on which these samples were collected and the average and median time between the last injection and collection should be provided.

What variants are circulating in Brazil in the fall of 2021 (the second dose was injected between August and November 2021)?

In the fall of 2021, it would be the delta variant https://www.mdpi.com/2076-2607/11/12/2938

The vaccines target the original strain from December 2019, which was no longer circulating when the second doses were administered in Brazil (August to November 2021). This needs to be discussed: the antibodies found in fall 2021 may not be able to neutralize the viruses circulating at that time.

Is the SARS-CoV-2 IgG II kit Quant (Abbott®) being used?

Remember that there are standardization issues between serological tests

https://www.sciencedirect.com/science/article/pii/S1386653222002773

The reference method for measuring vaccine-induced neutralizing antibodies is the viral neutralization test and the plaque reduction neutralization test using live viruses.

ELISA tests for binding antibodies, due to the different antigens used by different laboratories, including the complete spike protein, RBD protein, S1 protein, and S2 protein, have again resulted in poor comparability of results.

Wang Y. Standardized neutralizing antibody assays are needed for evaluating COVID-19 vaccines. EBioMedicine. 2021 Nov;73:103677. doi: 10.1016/j.ebiom.2021.103677. Epub 2021 Nov 3. PMID: 34742128; PMCID: PMC8564504.

The Pfizer and AZ vaccines are based on the original 2019 strain, the Abbot test tests for antibodies against this strain, so how relevant is it to measure antibodies against this 2019 strain in 2021? This needs to be discussed.

Use of the Abbott test to evaluate neutralizing antibodies Abbott SARS-CoV-2 IgG II Quant assay

chemiluminescent microparticle immunoassay for IgG against the receptor binding domain (RBD) region of S

https://journals.asm.org/doi/10.1128/spectrum.02811-22

We also later determined that the neutralizing capacity of donor plasma could vary between wild-type and emerging variants of concern (VOCs

The highest correlations were found between the BAU-per-milliliter values determined by the Abbott Quant assay and wild-type VLP log ID₅₀ values (Spearman r = 0.92; 95% CI, 0.86 to 0.95; 55 pairs; P < 0.0001). This was then followed by Abbott Quant assay versus Alpha VLP log ID₅₀ (Spearman r = 0.87; 95% CI, 0.78 to 0.92; 55 pairs; P < 0.0001), Abbott Quant assay versus Delta VLP log ID50 (Spearman r = 0.85; 95% CI, 0.75 to 0.91; 55 pairs; P < 0.0001), Abbott Quant assay versus Beta VLP log ID50 (Spearman r = 0.75; 95% CI, 0.60 to 0.84; 55 pairs; P < 0.0001) and Abbott Quant assay versus Gamma VLP log ID₅₀ (Spearman r = 0.75; 95% CI, 0.60 to 0.85;, 55 pairs; P < 0.0001).

In this series of experiments, the rank-ordered correlations between the Abbott Quant assay and both neutralization assays were wild type > Alpha > Delta > Beta > Gamma. This ordinal drop in correlations might suggest that the Abbott Quant assay has some bias for quantifying wild-type S antigen and reflects the molecular evolution of the S protein.

Results and discussion

Reminder: antibody level measurement results are not provided

Discuss the decrease in CRP: why is it reduced by vaccines?

Why is hemoglobin reduced? Anemia has been reported following Covid vaccines (example: Fattizzo B, Pasquale R, Croci GA, Pettine L, Cassanello G, Barcellini W. Aplastic anemia after SARS-CoV-2 infection or vaccines: case series and literature review. Blood Transfus. 2024 May;22(3) :266-272. doi: 10.2450/BloodTransfus.500. Epub 2023 Sep 4. PMID: 37677091; PMCID: PMC11073627)

https://pubmed.ncbi.nlm.nih.gov/?term=anemia+covid+vaccine

, and platelets? Discuss post-vaccination thrombocytopenia

https://pubmed.ncbi.nlm.nih.gov/?term=thrombopenia+covid+vaccine&page=1

Decrease in neutrophils: provide references that show this

https://pubmed.ncbi.nlm.nih.gov/?term=neutropenia+covid+vaccine

Regarding biological changes:

This article reports changes in biological parameters after Pfizer vaccination

Li J, Hui A, Zhang X, Yang Y, Tang R, Ye H, Ji R, Lin M, Zhu Z, Türeci Ö, Lagkadinou E, Jia S, Pan H, Peng F, Ma Z, Wu Z, Guo X, Shi Y, Muik A, Şahin U, Zhu L, Zhu F. Safety and immunogenicity of the SARS-CoV-2 BNT162b1 mRNA vaccine in younger and older Chinese adults: a randomized, placebo-controlled, double-blind phase 1 study. Nat Med. 2021 Jun;27(6):1062-1070. doi: 10.1038/s41591-021-01330-9. Epub 2021 Apr 22. PMID: 33888900. https://pubmed.ncbi.nlm.nih.gov/33888900/

Also: https://www.nature.com/articles/s41392-022-00919-x

Changes in immune function after vaccination

The immune function of individuals vaccinated eight months after receiving two doses of the COVID-19 vaccine was lower than that of unvaccinated individuals (response by Kenji Yamamoto https://pubmed.ncbi.nlm.nih.gov/35659687/to Norström et al https://doi.org/ 10.1016/S0140-6736(22)00089-7

Also: Front. Immunol. 12:684014. doi: 10.3389/fimmu.2021.684014

Cell Death & Differentiation (2021) 28:2765–2777 https://doi.org/10.1038/s41418-021-00782-3

https://doi.org/10.7554/eLife.84790

The Pfizer vaccination causes a transient fall in lymphocytes for the first three days after vaccination. [Walsh EE, Frenck RW Jr, Falsey AR, et al. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N Engl J Med 2020;383:2439–50. doi:10.1056/NEJMoa2027906 ],

The phase 2 trials of AstraZeneca similarly showed a fall in neutrophils.[Folegatti PM, Ewer KJ, Aley PK, et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomized controlled trial. Lancet 2020;396: 467–78. doi:10.1016/S0140-6736(20)31604-4 ]

Post vaccination neutrophil depletion [Muturi-Kioi V, Lewis D, Launay O, et al. Neutropenia as an Adverse Event following Vaccination: Results from Randomized Clinical Trials in Healthy Adults and Systematic Review. PLoS One 2016;11:e0157385. doi:10.1371/journal.pone.0157385]

and lymphocyte depletion [Munyer TP, Mangi RJ, Dolan T, et al. Depressed lymphocyte function after measles-mumps-rubella vaccination. J Infect Dis 1975;132:75–8. doi:10.1093/infdis/132.1.75] has been shown for other vaccines and the latter has been known about since 1981.[Faguet GB. The effect of killed influenza virus vaccine on the kinetics of normal human lymphocytes. J Infect Dis 1981;143:252–8. doi:10.1093/infdis/143.2.252

Discuss the causes of the decrease in LDH

Discuss the causes of the increase in ferritin

Provide references and hypotheses

Why is CAT (catalase) increased by vaccines, especially by Pfizer?

Discussion

The authors write:

"Voulgaridi et al. (2022) showed that Pfizer was superior in terms of the intensity of neutralizing antibody responses and protection against the SARS-CoV-2 virus [14], which corroborates our results, as there was also a very high production in those vaccinated with Pfizer compared to the control group. However, it is worth reminding that the study control group for neutralizing antibodies is composed of individuals infected with COVID-19, and since the reference value for neutralizing antibodies is above 50 U/mL, it can be observed that in this control group there was production of neutralizing antibodies, and that it also occurred in the group vaccinated with AstraZeneca, which showed no significant difference in relation to the control group, but which also produced neutralizing antibodies. "

Voulgardi et al. also do not measure neutralizing antibodies using the gold standard, which is neutralization on plates.

Only one reference on mice is given concerning the decrease in platelets after AZ and the decrease in blood cell counts. There are references concerning this phenomenon in humans (see above).

The authors write: “that AstraZeneca vaccine did not affect blood clotting, platelet count, and activation markers in rats”!

However, numerous adverse events of the thrombosis type have been reported after AstraZeneca.

Vaccination with ChAdOx1 nCov-19 can result in the rare development of immune thrombotic thrombocytopenia mediated by platelet-activating antibodies against PF4, which clinically mimics autoimmune heparin-induced thrombocytopenia.

Greinacher A, Thiele T, Warkentin TE, Weisser K, Kyrle PA, Eichinger S. Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination. N Engl J Med. 2021 Jun 3;384(22):2092-2101. doi: 10.1056/NEJMoa2104840. Epub 2021 Apr 9. PMID: 33835769; PMCID: PMC8095372.

https://www.sciencedirect.com/science/article/pii/S0006497120417847?via%3Dihub

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

Conclusion: in 2025, we can no longer write that Pfizer and AZ are safe and effective, given the numerous adverse effects reported in clinical trials

Pfizer myocarditis (https://journals.lww.com/ hjhs/fulltext/9900/myocarditis_and_pericarditis_following_the.10.aspx

https://doi.org/10.3390/pediatric14040048

https://jamanetwork.com/journals/jamacardiology/fullarticle/2781601

Montgomery J, Ryan M, Engler R, et al. Myocarditis Following Immunization With mRNA COVID-19 Vaccines in Members of the US Military. JAMA Cardiol. 2021;6(10):1202–1206. doi:10.1001/jamacardio.2021.2833

Department of Defense confirms that heart inflammation is linked to COVID vaccines in a study of US military personnel (aged 20-51)

In a study published June 29 in JAMA Cardiology, researchers described 23 cases of myocarditis in healthy military personnel who developed the condition within four days of receiving Pfizer or Moderna COVID vaccines.

https://www.nature.com/articles/s41467-022-31401-5#Tab2

Age and sex-specific risks of myocarditis and pericarditis following Covid-19 messenger

RNA vaccines, Stéphane Le Vu, Marion Bertrand, Marie-Joelle Jabagi, Jérémie Botton, Jérôme Drouin, Bérangère Baricault, Alain Weill, Rosemary Dray-Spira & Mahmoud Zureik

Nature Communications volume 13, Article number: 3633 (2022)

Jae Yeong Cho and others, COVID-19 vaccination-related myocarditis: a Korean nationwide study, European Heart Journal, 2023;, ehad339, https://doi.org/10.1093/eurheartj/ehad339

https://doi.org/10.1016/j.hrtlng.2023.02.003

Thrombocytopenia Pfizer Moderna = DOI: 10.1002/ajh.26132

Author Response

# REVIEWER 2

The authors have not proven that the COVID vaccines administered in Brazil in the fall of 2021 induce the synthesis of neutralizing antibodies against the virus circulating at that time, so the word “neutralizing” should be removed from the title.

Thank you for your comment. We changed the manuscript title for “Vaccination with two doses of AstraZeneca® (ChAdOx1-S) and Pfizer® (BNT162b2) induces the production of immunoglobulin G to COVID-19, without causing damage to hematological, biochemical, inflammatory and oxidative biomarkers” aiming to remove the expression “neutralizing antibodies”. All the manuscript were carefully revised aiming to replace this expression.

Introduction

Remember that vaccines are based on the spike protein: the vaccine spike can also interact with ACE2 and SRA and thus cause inflammation (Cao Z, Wu Y, Faucon E, Sabatier JM. SARS-CoV-2 & Covid-19: Key-Roles of the ‘Renin-Angiotensin’ System / Vitamin D Impacting Drug and Vaccine Developments. Infect Disord Drug Targets. 2020;20(3):348-349. doi: 10.2174/1871526520999200505174704. PMID: 32370727. https://pubmed.ncbi.nlm.nih.gov/32370727/ Gao X, Zhang S, Gou J, Wen Y, Fan L, Zhou J, Zhou G, Xu G, Zhang Z. Spike-mediated ACE2 down-regulation was involved in the pathogenesis of SARS-CoV-2 infection. J Infect. 2022 Oct;85(4):418-427. doi: 10.1016/j.jinf.2022.06.030. Epub 2022 Jul 3. https://doi.org/10.1016/j.jinf.2022.06.030 )

Thank you for your comment. This topic has been addressed in the Introduction, and the suggested reference has been cited.

Discuss why there is an increase in inflammation parameters. However, remember that the level of neutralizing antibodies correlates with the severity of COVID-19. Cellular & Molecular Immunology (2021) 18:318–327; https://doi.org/10.1038/s41423-020-00588-2

Thank you for your comment. This topic was better explored in your text and the suggested reference was cited.

Material and methods

The control group was infected with the virus and will therefore have neutralizing antibodies: how can we interpret the differences in antibody levels?

Thank you for your comment. The control group in this study comprised individuals with prior SARS-CoV-2 infection. This design choice aligns with the study’s objective, which was to compare differences in immune response between the vaccinated groups and the control group, rather than to assess the mere presence or absence of antibodies in individuals without previous exposure. Including an uninfected group would likely result in undetectable antibody levels, precluding meaningful quantitative or qualitative analyses. Accordingly, the results should be interpreted as differences in response magnitude among previously infected individuals, rather than as a comparison between exposed and unexposed populations. We believe that this aspect of the methodology is now more clearly explained in the current version of the manuscript.

Thank you for your comment. The methodology section was carefully revised to ensure that these details are clearly reported.

What variants are circulating in Brazil in fall 2021 (the second dose was administered between August and November 2021) In fall 2021, it would be the delta variant https://www.mdpi.com/2076-2607/11/12/2938

Thank you for your comment. The methodology section was carefully revised to ensure that these details are clearly reported, and the suggested reference has been properly cited.

Thank you for your comment. The vaccines evaluated in this study were developed against the original SARS-CoV-2 strain identified in December 2019. Nevertheless, previous studies have demonstrated that both the AstraZeneca (ChAdOx1 nCoV-19) and Pfizer-BioNTech (BNT162b2) vaccines reduce susceptibility to infection by the Delta variant, with higher effectiveness observed for the Pfizer vaccine after the second dose. The following references were cited to support this statement:

Pattni K et al. BMC Infectious Diseases, 2022. doi:10.1186/s12879-022-07239-z.

Pormohammad A et al. Vaccines, 2022;10(1):23. doi:10.3390/vaccines10010023.

Is the SARS-CoV-2 IgG II kit Quant (Abbott®) really being used? Remember that there are standardization issues between serological tests https://www.sciencedirect.com/science/article/pii/S1386653222002773

The reference method for measuring vaccine-induced neutralizing antibodies is the viral neutralization test and the plaque reduction neutralization test using live viruses. ELISA tests for binding antibodies, due to the different antigens used by different laboratories, including the complete spike protein, RBD protein, S1 protein, and S2 protein, have again resulted in poor comparability of results. Wang Y. Standardized neutralizing antibody assays are needed for evaluating COVID-19 vaccines. EBioMedicine. 2021 Nov;73:103677. doi: 10.1016/j.ebiom.2021.103677. Epub 2021 Nov 3. PMID: 34742128; PMCID: PMC8564504.

Thank you for your comment. The SARS-CoV-2 IgG II Quant assay (Abbott®) was indeed used in this study as a tool to assess the humoral immune response by measuring IgG antibodies directed against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. We fully acknowledge that the reference methods for evaluating vaccine-induced neutralizing antibodies are the viral neutralization test and the plaque reduction neutralization test using live viruses.

We also recognize the lack of full standardization among serological assays. Variations in antigenic targets (such as the full-length spike protein, RBD, S1, or S2 subunits) and methodological protocols across laboratories limit the direct comparability of results obtained using binding antibody assays. Nevertheless, despite these limitations, RBD-based ELISA assays are widely used in clinical and translational research to quantify post-infection or post-vaccination humoral responses.

In the present study, the assay was employed exclusively for research purposes to evaluate the magnitude of the immune response and not for diagnostic or definitive neutralization assessment. These methodological limitations are now explicitly acknowledged in the revised manuscript, and two additional references have been added to support this discussion, as listed below:

Wang Y. Standardized neutralizing antibody assays are needed for evaluating COVID-19 vaccines. EBioMedicine, 2021;73:103677. doi:10.1016/j.ebiom.2021.103677.

van Elslande J et al. Performance of antibody assays for SARS-CoV-2 in a multicenter evaluation. Clinical Biochemistry, 2022. doi:10.1016/j.clinbiochem.2022.02.009.

The Pfizer and AZ vaccines are based on the original 2019 strain, and the Abbot test measures antibodies against this strain. How is it relevant to measure antibodies against this 2019 strain in 2021? This needs to be discussed. Use of the Abbott test to evaluate neutralizing antibodies Abbott SARS-CoV-2 IgG II Quant assay chemiluminescent microparticle immunoassay for IgG against the receptor binding domain (RBD) region of S https://journals.asm.org/doi/10.1128/spectrum.02811-22 We also later determined that the neutralizing capacity of donor plasma could vary between wild-type and emerging variants of concern (VOCs The highest correlations were found between the BAU-per-milliliter values determined by the Abbott Quant assay and wild-type VLP log ID50 values (Spearman r = 0.92; 95% CI, 0.86 to 0.95; 55 pairs; P < 0.0001). This was then followed by Abbott Quant assay versus Alpha VLP log ID50 (Spearman r = 0.87; 95% CI, 0.78 to 0.92; 55 pairs; P < 0.0001), Abbott Quant assay versus Delta VLP log ID50 (Spearman r = 0.85; 95% CI, 0.75 to 0.91; 55 pairs; P < 0.0001), Abbott Quant assay versus Beta VLP log ID50 (Spearman r = 0.75; 95% CI, 0.60 to 0.84; 55 pairs; P < 0.0001) and Abbott Quant assay versus Gamma VLP log ID50 (Spearman r = 0.75; 95% CI, 0.60 to 0.85;, 55 pairs; P < 0.0001). In this series of experiments, the rank-ordered correlations between the Abbott Quant assay and both neutralization assays were wild type > Alpha > Delta > Beta > Gamma. This ordinal drop in correlations might suggest that the Abbott Quant assay has some bias for quantifying wild-type S antigen and reflects the molecular evolution of the S protein.

Thank you for your comment. The vaccines used at the time were based on the ancestral SARS-CoV-2 spike (S) protein, and the antibody responses elicited by vaccination primarily targeted this antigen. The observed decrease in correlations (wild-type > Alpha > Delta > Beta > Gamma) reflects the increasing antigenic distance of emerging variants and indicates that assays based on the 2019 strain show a progressively reduced correlation with neutralizing activity against more divergent variants circulating in 2021.

Importantly, this does not invalidate the assay but highlights its biological context: since the vaccines evaluated were designed against the ancestral virus, measuring antibody titers against the original S antigen remained informative for assessing the magnitude of the vaccine-induced humoral response in 2021. This approach allows comparison of immune responses elicited by the vaccines in use at that time, while acknowledging the reduced predictive value of binding antibody levels for neutralization against later variants.

The suggested article has been added to the revised manuscript (reference 29: doi:10.1128/spectrum.02811-22).

Results and discussion

Reminder: antibody level measurement results are not provided

Thank you for your comment. The IgG antibody results are presented in Section 3.4 (Biochemical Parameters), Figure 3C.

Discuss the decrease in CRP: why is it reduced by vaccines?

Thank you for your comment. Our discussion was improved in aiming to better explore this topic. The reference: Medicina 2025, 61, (9), 1510, was cited.

Why is hemoglobin reduced? Anemia has been reported following Covid vaccines (example: Fattizzo B, Pasquale R, Croci GA, Pettine L, Cassanello G, Barcellini W. Aplastic anemia after SARS-CoV-2 infection or vaccines: case series and literature review. Blood Transfus. 2024 May;22(3):266-272. doi: 10.2450/BloodTransfus.500. Epub 2023 Sep 4. PMID: 37677091; PMCID: PMC11073627)

Thank you for your comment. This topic has been discussed in greater detail, and the suggested reference has been cited.

Discuss post-vaccination thrombocytopenia. The authors write: “that AstraZeneca vaccine did not affect blood clotting, platelet count, and activation markers in rats”! However, numerous adverse events of the thrombosis type have been reported after AstraZeneca. Vaccination with ChAdOx1 nCov-19 can result in the rare development of immune thrombotic thrombocytopenia mediated by platelet-activating antibodies against PF4, which clinically mimics autoimmune heparin-induced thrombocytopenia.

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

Thank you for your comment. We have revised the manuscript to explicitly discuss this distinction, emphasizing that such thrombotic events are rare, immune-mediated adverse reactions observed in specific clinical contexts and are not necessarily detected in experimental animal models or reflected in routine hematological parameters. The suggested references have been added to support this discussion (doi:10.1056/NEJMoa2104840 and doi:10.1182/blood-2006-06-032524), and the corresponding revisions are highlighted in the manuscript.

Decrease in neutrophils: provide references that show this. The phase 2 trials of AstraZeneca similarly showed a fall in neutrophils.

Thank you for your comment. We have revised the manuscript to explicitly discuss this observation and have added the suggested references to support this finding (references 31: doi:10.1016/S0140-6736(20)31604-4 and 32: doi:10.1371/journal.pone.0157385). The corresponding changes are highlighted in the revised version of the manuscript.

Increase in lymphocytes: provide references that show this.

Thank you for your comment. We have revised the manuscript to explicitly discuss this finding and have added the suggested reference to support this observation (reference 33: doi:10.3390/vaccines10091550). The corresponding changes are highlighted in the revised manuscript.

Discuss the causes of the decrease in LDH

Thank you for your comment. The observed decrease in lactate dehydrogenase (LDH) levels over time may reflect the resolution of systemic inflammation and tissue damage following the acute phase of SARS-CoV-2 infection. We have revised the manuscript to expand the discussion on the potential mechanisms underlying LDH reduction and have added supporting literature to contextualize this finding (references 42: doi:10.1186/s12968-023-00985-2 and 43: doi:10.14740/jocmr4821). The corresponding revisions are highlighted in the manuscript.

Discuss the causes of the increase in ferritin

Thank you for your comment. This issue has been more clearly discussed in the revised manuscript, and the corresponding changes are highlighted in the text.

Provide references and hypotheses Why is CAT (catalase) increased by vaccines, especially by Pfizer?

Thank you for your comment. This topic has been more clearly discussed in the revised manuscript, and the suggested references have been added to support the proposed hypotheses. The corresponding changes are highlighted in the text. Please see references added: doi 10.1016/j.vaccine.2023.04.049 and doi 10.1155/2016/5698931.

The authors write: "Voulgaridi et al. (2022) showed that Pfizer was superior in terms of the intensity of neutralizing antibody responses and protection against the SARS-CoV-2 virus [14], which corroborates our results, as there was also a very high production in those vaccinated with Pfizer compared to the control group. However, it is worth reminding that the study control group for neutralizing antibodies is composed of individuals infected with COVID-19, and since the reference value for neutralizing antibodies is above 50 U/mL, it can be observed that in this control group there was production of neutralizing antibodies, and that it also occurred in the group vaccinated with AstraZeneca, which showed no significant difference in relation to the control group, but which also produced neutralizing antibodies. " Voulgardi et al. also do not measure neutralizing antibodies using the gold standard, which is plaque neutralization.

Thank you for your comment. We agree that both our study and the work by Voulgaridi et al. did not assess neutralizing antibodies using plaque reduction neutralization tests, which remain the gold standard for evaluating neutralizing activity. This methodological limitation has now been explicitly acknowledged in the revised manuscript.

To avoid overinterpretation, we revised the text to emphasize that the assays employed measure binding antibodies directed against the spike protein and should be interpreted as indicators of the magnitude of the humoral immune response rather than as direct measurements of viral neutralization. We further clarified that the control group, composed of previously infected individuals, also exhibited antibody levels above the reference threshold, indicating antibody production in both infected and vaccinated groups, including those vaccinated with AstraZeneca.

In addition, we included a complementary reference that evaluated anti-spike IgG antibody levels using a comparable approach (reference 23: doi:10.15407/microbiolj86.02.075). All revisions are highlighted in the manuscript.

Conclusion

In 2025, it can no longer be said that Pfizer and AZ are safe and effective, given the numerous adverse effects reported in clinical trials.

Thank you for your comment. We agree that the safety and effectiveness of COVID-19 vaccines should be continuously monitored as new evidence emerges. However, we would like to clarify that the aim of the present study was not to assess the overall clinical safety or real-world effectiveness of the Pfizer or AstraZeneca vaccines. Instead, our objective was to evaluate specific laboratory and biochemical outcomes following vaccination.

Within this scope, our results indicate the induction of IgG antibodies, reflecting an immunological response to vaccination, while no clinically relevant alterations were observed in the analyzed biochemical, inflammatory, or oxidative stress parameters. These findings should therefore be interpreted in the context of laboratory response assessment rather than as a comprehensive evaluation of vaccine safety or efficacy.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

As highlighted in the first round of review, it can no longer be claimed in 2025 that Covid vaccines are safe and effective (given the contradictory studies on efficacy and numerous reports of adverse effects).

Furthermore, the authors' results do not prove the efficacy or safety of Covid vaccines.

The sentence “Therefore, AstraZeneca® and Pfizer® vaccines were considered efficient and safe in the population studied” should therefore be removed from the abstract.

Furthermore, the authors' results do not prove the efficacy or safety of Covid vaccines.

The sentence “Therefore, AstraZeneca® and Pfizer® vaccines were considered efficient and safe in the population studied” should therefore be removed from the abstract.

Author Response

We thank the reviewer for highlighting this important point and for reminding us of the need for caution in the interpretation of our findings. As suggested, the sentence stating that “AstraZeneca® and Pfizer® vaccines were considered efficient and safe in the population studied” has been removed from the abstract, and other related statements were adjusted in the conclusion.

Review Reports

Risk of Coronavirus Disease 2019 (COVID-19) among those up-to-date and not up-to-date on COVID-19 vaccination by US CDC criteria