Memory B Cells Induced by Sputnik V Vaccination Produce SARS-CoV-2 Neutralizing Antibodies Upon Ex Vivo Restimulation

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    Evaluation Summary:

    In this study Byazrova and colleagues provide an assessment of antibody and B cell responses in a small cohort of naïve and previously infected individuals after Sputnik V immunisation. This research will be of interest to those in the fields of vaccinology and immunology, providing some insight into B cell responses following Sputnik V vaccination.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The development of effective vaccines against SARS-CoV-2 remains a global health priority. Despite extensive use, the effects of Sputnik V on B cell immunity need to be explored in detail. We performed comprehensive profiling of humoral and B cell responses in a cohort of vaccinated subjects (n = 22), and demonstrate that Sputnik vaccination results in robust B cell immunity.

We show that B memory cell (MBC) and antibody responses to Sputnik V were heavily dependent on whether the vaccinee had a history of SARS-CoV-2 infection or not. 85 days after the first dose of the vaccine, ex vivo stimulated MBCs from the vast majority of Sputnik V vaccinees produced antibodies that robustly neutralized the Wuhan Spike-pseudotyped lentivirus. MBC-derived antibodies from all previously infected and some of the naïve vaccine recipients could also cross-neutralize Beta (B.1.351) variant of SARS-CoV-2.

Virus-neutralizing activity of MBC-derived antibodies correlated well with that of the serum antibodies, suggesting the interplay between the MBC and long-lived plasma cell responses. Thus, our in-depth analysis of MBC responses in Sputnik V vaccinees complements traditional serological approaches and may provide important outlook into future B cell responses upon re-encounter with the emerging variants of SARS-CoV-2.

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  1. Author Response:

    Thank you for your detailed and very stimulating analysis of our manuscript “Memory B cell and humoral responses elicited by Sputnik V in naïve and COVID-19-recovered vaccine recipients.” We fully agree with most of the concerns voiced by the reviewers and feel all of them can be properly addressed.

    All the reviewers noted a small cohort size as a major weakness of our study. Although we agree that a larger sampling would likely strengthen our conclusions, it must be noted that over a dozen of B cell parameters were measured across four time points in our work. With this in mind, some of the tests, such as B cell ELISpot and virus neutralization assays using antibodies from stimulated cells, etc, are known to be very time- and resource-consuming – which naturally limits the number of samples that can be processed. We chose to provide a truly comprehensive analysis of B cell biology following Sputnik V vaccination by using a set of very challenging and informative cellular assays, and not merely semi-automatic antiserum screening systems which can be easily performed en masse.

    Next, all the reviewers noted the issue with separating all the vaccinees into naïve and previously infected subgroups, which follows from the data presented in the Supplementary Figure 2. This is indeed an unfortunate technical/labeling mistake, as the data for the patient 19 have been swapped with those of a different donor. This has now been corrected.

    As suggested by the Reviewer #1, additional experiments have been performed, and virus neutralization tests using antibodies from stimulated cells were run against both the ancestral virus and the Beta variant. All other minor comments have been gratefully taken into account.

    We thank the Reviewers for constructive criticism, which prompted us to conduct additional experiments and clarify the findings.

    Thank you for your time and consideration.

  2. Evaluation Summary:

    In this study Byazrova and colleagues provide an assessment of antibody and B cell responses in a small cohort of naïve and previously infected individuals after Sputnik V immunisation. This research will be of interest to those in the fields of vaccinology and immunology, providing some insight into B cell responses following Sputnik V vaccination.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. Reviewer #1 (Public Review):

    The work by Byazrova and colleagues examines B cell responses following Sputnik V vaccination in naïve individuals, as well as individuals who were previously infected with SARS-CoV-2. A strength of the work is that there have been few very studies examining immune responses following Sputnik V vaccination, this paper goes beyond simply measuring the neutralising antibody response and encompasses a detailed investigation into plasma cell and memory B cell responses. Similar to findings examining responses to other SARS-CoV-2 vaccines, they show that individuals with a prior history of SARS-CoV-2 infection mount a stronger neutralising antibody response following a single dose of vaccination than naïve individuals. Following 2 doses of vaccine, antibody responses were generally still superior in previously infected individuals, including neutralising antibodies against the ancestral and Beta variants.

    A weakness of the study is the size of the cohort, which limits the conclusions that can be drawn from the data. The last figure of the manuscript attempts to group the patients based on their B cell responses and finds that 3 naïve patients cluster with the previously infected individuals and these patients have been termed 'high-responders'. However, I note that patient 19 is within this cluster, and I am wondering if they are truly a naïve individual. ELISA data presented in supplementary figure 2 shows that this patient possessed anti-RBD IgG antibodies, although lacked anti-N antibodies. Studies have shown that anti-nucleocapsid antibodies wane faster than those directed against spike, so perhaps the authors need to consider that this patient was not truly naïve prior to vaccination.

    It would also be of interest to readers if an analysis of neutralising antibody titres against the delta variant were investigated. This variant is now the dominant in most regions including countries where Sputnik V is in use and therefore the protection afforded by Sputnik against this variant is pertinent.

    I think it is also important to include historic control samples when performing pVNT assays to control of off-target effects, particularly when using undiluted sera or media from stimulated cells. Were any of these controls performed? Additionally, why only test against ancestral virus when using antibodies from stimulated cells? Inclusion of variants in these experiments could provide additional information about affinity maturation over time and the impact on recognition of different variants.

  4. Reviewer #2 (Public Review):

    In this manuscript, Byazrova and colleagues assessed aspects of the humoral immune response to the Sputnik V COVID-19 vaccine, deployed extensively in Russia and selected countries globally. A cohort of 22 individuals were recruited, encompassing 5 who had previously been infected with SARS-Cov-2, and longitudinally followed over the course of a two immunisation schedule. In naïve individuals, two doses was sufficient to raise robust titres of binding and neutralising antibodies against the SARS-CoV-2 spike, whilst convalescent individuals experienced a rapid recall of humoral immunity after a single shot that surpassed levels obtained in fully immunised naïve individuals. Antibody levels were stably maintained out to ~85 days post immunisation and a degree of neutralisation escape against the Beta variant of concern were reported.

    A strength of the study is the in-depth phenotypic characterisation of the plasmablast and memory B cell responses to immunisation, which were quantified using flow cytometric and ELISpot approaches. In terms of weaknesses, the small cohort size limits the robustness of the conclusions, and the lack of a direct comparison to other available vaccines does not allow any conclusions to be made about the comparative benefits or limitations of the Sputnik V platform over alternative platforms. In addition, a characterisation of anti-vector responses would have been informative to guide deployment of Adenoviral vectored vaccines in the future.

    Overall, this study complements suggests the broad immunogenic properties of Sputnik V largely mirror that of other COVID-19 vaccines deployed to date. The findings support the idea that a single immunisation of any vaccine is sufficient to boost protection in previously infected individuals, while two or three immunisations are required in naïve individuals to reach similar serologic outcomes.

  5. Reviewer #3 (Public Review):

    Byazrova et al. examined the effect of previous infection on Sputnik V vaccination elicited anti-SARS-CoV-2 spike antibodies and neutralization. They found that neutralization is enhanced. Interestingly, the authors also observed higher numbers SARS-CoV-2 spike RBD specific antibody secreting and memory B cells in vaccinated previously infected participants. In these participants, there were prominent B cell responses like anti-spike RBD IgG secretion and neutralization capacity of antibodies derived from in vitro stimulation which were absent in the vaccinated only participants. This may show qualitative differences between vaccinated only and previously infected and vaccinated participants.

  6. SciScore for 10.1101/2021.10.13.21264894: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: Written informed consent was obtained from each of the study participants before performing any study procedures.
    IRB: The study protocol was reviewed and approved by the Medical Ethical Committee of Institute of Immunology (#12-1, December 29, 2020).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To obtain MBC-derived antibody-secreting cells (ASCs), B cells were stimulated with 25 ng/mL interleukin-21 (IL-21; PeproTech, Cat. No. 200-21) in the presence of mitomycin-treated feeder A549 cells stably expressing CD40L (A549-CD40L, 1 × 105 cells/well) for 7 days at a density of 5 × 103 B cells/well in 96-well plates at 37°C in 5% CO2.
    A549-CD40L, 1
    suggested: None
    To determine the concentration of IgG, a serial dilution of anti-SARS-CoV-2 RBD-specific human monoclonal antibody iB12 was included on each plate, a calibration curve was built and IgG levels were calculated (μg/mL).
    anti-SARS-CoV-2
    suggested: None
    Flow cytometry: Freshly isolated PBMCs were stained with the following antibodies: CD3 FITC (clone TB3), CD16 FITC (clone LNK16), CD19 PE (clone LT19), CD27 PECy5.5 (clone LT27), CD38 PECy7 (clone LT38) (all were produced in-house earlier (Khvastunova et al., 2015); CD14 FITC (clone MEM-15, Exbio, Cat. No. ED7028); anti-human IgG APC (clone M1310G05, Biolegend Cat. No. 410720) and anti-human IgM APC-Fire750 (clone MHM-88, Biolegend Cat. No. 314546).
    CD27
    suggested: None
    CD38
    suggested: None
    anti-human IgG
    suggested: (BioLegend Cat# 410720, RRID:AB_2721576)
    anti-human IgM
    suggested: (BioLegend Cat# 314546, RRID:AB_2800834)
    To capture the total immunoglobulin (IgG, IgM or IgA) produced by ASCs, wells were coated with 10 μg/ml of rabbit anti-human IgG or IgM (R&D Systems, Cat. No. SELB002, SELB003), or goat anti-human IgA antibodies (SouthernBiotech, Cat. No. 2050-01).
    total immunoglobulin (IgG
    suggested: None
    IgA
    suggested: (SouthernBiotech Cat# 2050-01, RRID:AB_2795701)
    anti-human IgA
    suggested: (SouthernBiotech Cat# 2050-01, RRID:AB_2795701)
    Isotype-specific ASCs were detected using IgG- or IgM-specific biotinylated rabbit antibodies (R&D Systems, Cat. No. SELB002, SELB003) or IgA-specific biotinylated goat antibodies (SouthernBiotech, Cat. No. 2052-08).
    SELB003
    suggested: None
    IgA-specific biotinylated goat
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To obtain MBC-derived antibody-secreting cells (ASCs), B cells were stimulated with 25 ng/mL interleukin-21 (IL-21; PeproTech, Cat. No. 200-21) in the presence of mitomycin-treated feeder A549 cells stably expressing CD40L (A549-CD40L, 1 × 105 cells/well) for 7 days at a density of 5 × 103 B cells/well in 96-well plates at 37°C in 5% CO2.
    A549
    suggested: None
    HEK293T cells were transfected with plasmids psPax2 (kind gift from Dr. Didier Trono), pLV-eGFP (was a gift from Pantelis Tsoulfas, (Addgene, Cat. No. 36083)), and the pCAGGS-SΔ19 plasmid encoding wild-type or B1.351 S protein (see below).
    HEK293T
    suggested: None
    Viral yield was quantified using titration on HEK293T-hACE2 cells.
    HEK293T-hACE2
    suggested: RRID:CVCL_A7UK)
    Recombinant DNA
    SentencesResources
    HEK293T cells were transfected with plasmids psPax2 (kind gift from Dr. Didier Trono), pLV-eGFP (was a gift from Pantelis Tsoulfas, (Addgene, Cat. No. 36083)), and the pCAGGS-SΔ19 plasmid encoding wild-type or B1.351 S protein (see below).
    psPax2
    suggested: RRID:Addgene_12260)
    pLV-eGFP
    suggested: RRID:Addgene_36083)
    pCAGGS-SΔ19
    suggested: None
    To obtain pCAGGS-SΔ19_B.1.351, sets of complementary mutagenic primers (27 nt each) centered at the desired site were used to sequentially introduce the individual mutations (L18F, D80A, D215G, del241-243, R246I, K417N, E484K, N501Y, D614G, A701V) into the coding sequence of SΔ19.
    pCAGGS-SΔ19_B.1.351
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analyzed using FlowJo Software (version 10.6.1.,
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Spots were counted using ImmunoSpot® software.
    ImmunoSpot®
    suggested: None
    Statistical analysis: Statistical analysis was performed using Graph Pad Prism (version 8.4.3 GraphPad Software, La Jolla California).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    A normalized non-linear regression was performed using GraphPad Prism software (Sigmoidal, 4PL).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Heatmap generation and principal component analysis were performed with Clustvis (Metsalu, Vilo 2015) using normalized data.
    Clustvis
    suggested: (ClustVis, RRID:SCR_017133)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).


    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.


    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.


    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.


    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.


    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.