Robust and Functional Immune Memory Up to 9 Months After SARS-CoV-2 Infection: A Southeast Asian Longitudinal Cohort

  1. Hoa Thi My Vo
  2. Alvino Maestri
  3. Heidi Auerswald
  4. Sopheak Sorn
  5. Sokchea Lay
  6. Heng Seng
  7. Sotheary Sann
  8. Nisa Ya
  9. Polidy Pean
  10. Philippe Dussart
  11. Olivier Schwartz
  12. Sovann Ly
  13. Timothée Bruel
  14. Sowath Ly
  15. Veasna Duong
  16. Erik A. Karlsson
  17. Tineke Cantaert

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Abstract

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4 + and CD8 + T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG + . CD4 + and CD8 + T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4 + T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.

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  1. Version published to 10.3389/fimmu.2022.817905
  2. ScreenIT

    SciScore for 10.1101/2021.08.12.455901: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Study population: Ethical approval for the study was obtained from the National Ethics Committee of Health Research of Cambodia.
    Consent: Written informed consent was obtained from all participants prior to inclusion in the study.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Virus neutralization assay: The detection of neutralizing antibodies was achieved by foci reduction neutralization test (FRNT) similar as described before (80) and adapted to SARS-CoV-2.
    SARS-CoV-2
    suggested: None
    Infection was visualized 16-18h after inoculation by staining of infected cells with a SARS-CoV-2-specific antibody (rabbit, antibodies-online GmbH), targeting the S2 subunit of the viral spike protein, and afterwards with antibody anti-rabbit IgG HRP conjugate (goat; antibodies-online GmbH).
    antibodies-online GmbH
    suggested: None
    anti-rabbit IgG
    suggested: None
    antibodies-online GmbH).
    suggested: None
    The cells were washed with PBS and stained with Zombie Aqua viability dye (BioLegend) for 20 minutes on ice and then stained anti-APC C3/C3b/iC3b antibody (Cedarlane) for 30 minutes on ice.
    anti-APC C3/C3b/iC3b
    suggested: None
    Anti-CD107a and Monensin (Biolegend) 1:1000 dilution were added to the suspension and incubated at 37°C, 5% CO2 for 6 hours.
    Anti-CD107a
    suggested: None
    Then the cells were washed and stained with anti-IgG antibody, for 30 minutes on ice.
    anti-IgG
    suggested: None
    After that, the cells were washed and stained with master mix containing of anti-CD3, anti-CD19, anti-CD27, anti-CD38, anti-IgD, anti-IgM and anti-IgA antibodies for 30 minutes on ice Antibodies are listed in Table S2.
    anti-CD19
    suggested: None
    anti-CD27
    suggested: None
    anti-CD38
    suggested: None
    anti-IgD ,
    suggested: (GeneTex Cat# GTX75526, RRID:AB_379139)
    anti-IgM
    suggested: None
    anti-IgA
    suggested: None
    Surface (CD3, CD4 and CD8) and intracellular markers (IFN-γ, IL-2, IL-4, IL-6 and IL-17) were detected via the subsequent addition of directly conjugated antibodies incubating for 30 minutes at 4°C.
    CD3
    suggested: (Thermo Fisher Scientific Cat# 8822-6853-41, RRID:AB_2575278)
    CD4
    suggested: (Thermo Fisher Scientific Cat# 8822-6853-41, RRID:AB_2575278)
    IFN-γ
    suggested: (Bio-Rad Cat# M6000007NY, RRID:AB_2784537)
    IL-2
    suggested: None
    IL-17
    suggested: None
    CD8
    suggested: None
    IL-4, IL-6
    suggested: None
    Surface (CD3, CD4 and CD8) and intracellular markers (IFN-γ, IL-2, IL-4, IL-6 and IL-17) were detected via the subsequent addition of directly conjugated antibodies incubating for 30 minutes at 4°C.
    CD3
    suggested: (Thermo Fisher Scientific Cat# 8822-6853-41, RRID:AB_2575278)
    CD4
    suggested: (Thermo Fisher Scientific Cat# 8822-6853-41, RRID:AB_2575278)
    IFN-γ
    suggested: (Bio-Rad Cat# M6000007NY, RRID:AB_2784537)
    IL-2
    suggested: None
    IL-17
    suggested: None
    CD8
    suggested: None
    IL-4, IL-6
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Spike-expressing Raji cells and Raji control cells were cultured at 37°C, 5% CO2 in RPMI medium while 293T-spike cells and 293T control cells were cultured in DMEM medium.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Phagocytosis activity was scored by the integrate mean fluorescence intensity (iMFI) value (% positive fluorescence THP-1 cells x MFI of the positive fluorescence THP-1 cells).
    THP-1
    suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)
    First, 293T-spike cells were incubated with heated-inactivated patient plasma diluted in complete DMEM medium (1:50) at 37°C, 5% CO2 for 30 minutes.
    293T-spike
    suggested: None
    Software and Algorithms
    SentencesResources
    The amount of neutralizing antibodies is expressed as the reciprocal serum dilution that induces 50% reduction of infection (FRNT50) compared to the positive control (virus only) and is calculated by log probit regression analysis (SPSS for Windows, Version 16.0, SPSS Inc., Chicago, IL, USA).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)
    Data were analyzed with FlowJo software version 10.7.1 (FlowJo LLC).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Calculations, figures and statistics were made using Prism 9 (GraphPad Software) or RStudio (Version 1.2.1335).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Spearman correlation plot was calculated and visualized with the following packages: FactoMineR, factoextra (https://cran.r-project.org/web/ packages/factoextra/index.html) and corrplot (https://github.com/taiyun/corrplot) in R (Version 3.6.1) and RStudio (Version 1.2.1335).
    FactoMineR
    suggested: (FactoMineR, RRID:SCR_014602)

    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: We detected the following sentences addressing limitations in the study:
    One limitation is the uncertainty of the exact timing of exposure/infection, as infections were identified by screening at entry into Cambodia rather than in a direct surveillance or community cohort. Studies assessing long-term immunity to SARS-CoV-2 in Asian populations are scarce (16, 32–34, 41). In addition, studies on cross-reactivity of the humoral and cellular compartment with other hCoVs have mainly focused on European and US populations (42–45). Historically, the population in East Asia seems to be more exposed to coronavirus-like viruses as only East Asian population show genetic adaptation to coronaviruses (46). The main natural reservoir of SARS-related coronaviruses is believed to be Horseshoe bats (genus Rhinolophus), which are endemic to Southeast Asia and China (47–49). Whether possible cross-reactivity to other coronavirus-like viruses or hCoVs may have influenced the adaptive immune response to SARS-CoV-2 in Southeast Asian populations remained to be investigated. As expected, anti-S IgM, IgG and IgA titers declined over time and anti-S IgG becomes the major isotype at late convalescence (24, 50–52). In this study, IgA titers were the most affected over time. The formation of anti-S IgA is shown to be dependent on local lung inflammation (53–55) hence titers decline the strongest in asymptomatic/mild patients. Titers of neutralizing antibodies are reported to reach their maximum within the first month after infection and then decay, but mostly remain detecta...

    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 42. 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.

  3. Version published to 10.1101/2021.08.12.455901v1 on bioRxiv