key: cord-0253114-zh8xipn5
authors: Liu, Y.; Zeng, Q.; Deng, C.; Li, M.; Li, L.; Liu, D.; Mei, J.; Mo, R.; Zhou, Q.; Liu, M.; Peng, S.; Wang, J.; Zhang, H.; Xiao, H.
title: Robust induction of B cell and T cell responses by a third dose of inactivated SARS-CoV-2 vaccine
date: 2021-09-15
journal: nan
DOI: 10.1101/2021.09.12.21263373
sha: 7fd6914169c25c3057810838aab27d707b03e9ff
doc_id: 253114
cord_uid: zh8xipn5

SARS-CoV-2 inactivated vaccines have shown remarkable efficacy in clinical trials, especially in reducing severe illness and casualty. However, the waning of humoral immunity over time has raised concern over the durability of immune memory following vaccination. Thus, we conducted a non-randomized trial among the healthcare professionals (HCWs) to investigate the long-term sustainability of SARS-CoV-2-specific B cells and T cells stimulated by inactivated vaccine and the potential need for a third booster dose for the HCWs. Although neutralizing antibodies elicited by the standard two-dose vaccination schedule dropped from a peak of 31.2 AU/ml to 9.2 AU/ml 5 months after the second vaccination, spike-specific memory B and T cells were still detectable, forming the basis for a quick recall response. As expected, the faded humoral immune response was vigorously elevated to 66.8 AU/ml by 7.2 folds 1 week after the third dose along with abundant spike-specific circulating follicular helper T cells in parallel. Meanwhile, spike-specific CD4+ and CD8+ T cells were also robustly elevated by 5.9 and 2.7 folds respectively. Robust expansion of memory pools by the third dose potentiated greater durability of protective immune responses. Another key finding in this trial was that HCWs with low serological response to 2 doses were not truly no responders but fully equipped with immune memory that could be quickly recalled by a third dose even 5 months after the second vaccination. Collectively, these data provide insights into the generation of long-term immunological memory by the inactivated vaccine, which has implications for future booster strategies that the frontline HCWs, individuals with low serological response to 2 dose of vaccine and immune compromised patients could benefit from a third dose of inactivated vaccine.

The coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to spread across the globe currently 1,2 . The pandemic has brought profound casualties of human life and socioeconomic issues. The establishment of herd immunity by effective and sustainable vaccines represents the best strategy to prevent COVID-19. The rapid spreading of COVID-19 has urged the governments to authorize the emergent use of vaccines against SARS-CoV-2 [3] [4] [5] .

The presence of neutralizing antibodies (NAbs) against SARS-CoV-2 is an indicator of protective immunity after vaccination or infection 6, 7 . NAbs capable of blocking the interaction between the spike protein and its receptor angiotensin converting enzyme 2 (ACE2) are particularly important for protection from COVID-19 8 . Therefore, inducing potent NAbs and long-lasting memory B cells are the primary goal of SARS-CoV-2 vaccines. Two doses of mRNA or inactivated vaccines are capable of inducing potent neutralizing antibody responses 9, 3, 10 . Our previous study further demonstrated that inactivated vaccines elicited SARS-CoV-2 specific memory B cells 11 , which is important for a rapid and robust recall of protective responses against viral infection. However, little is known how long can these immune responses sustain. A rapid decline of neutralizing antibodies has been observed among infected healthcare workers (HCWs) 12 . Neutralizing antibodies also waned over time after the second dose of BNT162b2 or ChAdOx1 13 , which indicates . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ;  https://doi.org/10.1101/2021.09.12.21263373 doi: medRxiv preprint 6 weakened protection from SARS-CoV-2 infection. In addition, the rapid emergence of novel SARS-CoV-2 variants of concern dampens the efficacy of SARS-CoV-2 vaccines, since vaccine-induced antibodies were always less effective in neutralizing emerging variants of concern (VOCs) 14 . Moreover, cross-reactive antibodies decayed even faster than antibodies against wild-type strain 15 .

Besides humoral immune responses, T cells also play a pivotal role in coordinating the adaptive immune responses and as effectors against viral infection. In some cases, patients with inherited or treatment-induced B cell deficiency were able to recover from COVID-19 16, 17 , suggesting a potential role of cellular responses in fighting against SARS-CoV-2 infection. Induction of CD8 + T cell responses was documented in both SARS-CoV-2 infection and vaccination 18, 19 . On the other hand, rapid induction of CD4 + T cells is associated with coordinated humoral and cellular response to a SARS-CoV-2 mRNA vaccine 20 . SARS-CoV-2 specific circulating TFH (cTFH) cells, whose counterparts in lymph nodes reinforce B cell differentiation and humoral response 21 , correlated with neutralizing antibody levels in convalescent COVID-19 patients 22, 23 .

Nevertheless, T cell responses induced by inactivated vaccines are much less well characterized than humoral immunity. One previous study revealed that the inactivated vaccine could elicit T cells response after 2 dose vaccination 24 , but the phenotype and sustainability of antigen-specific CD4 + . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ;  https://doi.org/10.1101/2021.09.12.21263373 doi: medRxiv preprint 7 and CD8 + T cells remained unclear yet.

In this study, we sought to investigate the sustainability of immune memory stimulated by the inactivated vaccine and profile humoral and cellular responses to a third dose among HCWs. Although NAbs declined substantially, SARS-CoV-2-specific memory B, CD4 + , and CD8 + T cells persisted in the peripheral blood 5 months after the second vaccination, even in participants who were seronegative after 2 doses of the inactivated vaccines. The third vaccination robustly recalled both humoral and cellular immune responses in all participants.

In this study, we conducted a non-randomized trial and recruited participants from a prospective cohort at the First Affiliated Hospital of Sun Yat-sen University (FAH-SYSU) in Guangzhou, China. As we described previously 11 , 63 HCWs received the inactivated SARS-CoV-2 vaccine BBIBP-CorV (BBIBP-CorV, Sinopharm, Beijing) in the morning (9am-11am) or afternoon (15pm-17pm) on day (d) 0 and d28, respectively. Fifty of the 63 HCWs were volunteered to receive a third booster shot of the inactivated vaccine 6 months after the prime vaccination (d180). They were assigned to the morning or afternoon group as to their previous vaccinating time accordingly. Demographic characteristics of the HCWs were summarized in S. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint Table 1 . Blood samples were collected on d180 before the booster dose and d187, d194 and d208 after the booster dose. Convalescent patients who had recovered from SARS-CoV-2 infection were recruited as the positive control (S Table 2 ). All studies were approved by the Institutional Review Board of FAH-SYSU and written consent was obtained from all participants. The prospective cohort and the trial were registered to Chinese Clinical Trial Registry (ChiCTR2100042222 and ChiCTR2100048665).

Blood samples were collected into the heparinized tubes and processed right after sample collection. Peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation. Briefly, blood samples were diluted with PBS at 1:1 ratio and loaded on top of Lymphoprep™ (StemCell) in the Falcon tubes. The falcon tubes were then centrifuged at 1500 rpm for 30 mins. The medium layer was collected and washed with PBS twice.

PBMCs were cryopreserved in Bambanker (StemCell) immediately.

A one-step competitive immunoassay was used to detect the serum concentration of neutralizing antibodies (NAbs) for SARS-CoV-2 in the sera through Chemiluminescent immunoassay using iFlash 2019-nCoV NAb kit (YHLO Biotech Co, Ltd). As for this assay, receptor binding domain (RBD) of . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.12.21263373 doi: medRxiv preprint the SARS-CoV-2 was coated on magnetic beads. Acridinium ester-labeled ACE2 was designed to compete with SARS-CoV-2 NAbs in the sera for the RBD. SARS-CoV-2 NAb titers were calculated by an iFlash3000

Chemiluminescence Immunoassay Analyzer (YHLO Biotech Co, Ltd). The surrogate neutralization assay detects antibodies that are able to compete with the binding of the extracellular domain of the SARS-CoV-2 receptor ACE2 to a recombinant RBD of the SARS-CoV-2 S1 protein coated on the microparticles, which indicates the neutralization activity of sera as tested before 25 .

Neutralizing activity is determined in arbitrary units (AU) and the cut-off is 10 AU/ml.

IFN-γ ELISpot assays were performed as described previously 26 Unstimulated cells were used as negative control while anti-CD3/CD28 dynabeads (Thermo Fisher) stimulated cells were set as the positive control.

Plates were incubated with IFN-γ detection antibody (1μg/ml, MabTech, #3420-6-250), followed by Avidin-HRP (1μg/ml, Vector, #A-2004-5) and

visualized using the ACE substrate. Antigen-specific T cell responses were . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 

PBMCs were cultured in RPMI 1640 supplemented with 10% FBS and 1%

penicillin and streptomycin (Thermo Fisher) at 37 °C overnight. The cells were then cultured with or without the peptide pool of SARS-CoV-2 spike protein (2 Stimulation with anti-CD3/CD28 dynabeads was included as positive controls.

Any sample with a low response to anti-CD3/CD28 stimulation was excluded as a quality control for the samples. Data were acquired by flow cytometry.

SARS-CoV-2-specific memory B cells were detected as we described previously 11 . First, biotinylated antigens were multimerized with fluorescence . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

All flow cytometry samples were analyzed using cryopreserved cells which were thawed and suspended in RPMI 1640 media supplemented with 2% FBS.

All samples were analyzed by flow cytometry with Cytek TM AURORA. FlowJo (Tree Star, USA) software was used for FACS data analysis. Details of antibodies used in this study are listed in S. Table. 3.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 

To investigate the immune response and the subsequent duration of immune protection by the inactivated SARS-CoV-2 vaccine BBIBP-CorV, we conducted a non-randomized trial and recruited HCWs from a prospective cohort. Fifty HCWs from this cohort were volunteered to participate in this non-randomized trial. They were assigned to receive a third dose of the inactivated vaccine 6 months after the primary vaccination (Fig. 1A) . No severe side effects related to vaccination were recorded during the trial (S. Table 1 ).

Among the 50 participants, 36 of them were tested negative for neutralizing activities by Chemiluminescent immunoassay 6 months after the primary vaccination. Nine HCWs who were seronegative on d56 after two doses of vaccine remained to be negative and 27 of the positive HCWs turned negative.

Of the 9 negative HCWs, 7 of them were from the afternoon group and 2 were from the morning group. The average concentration of NAbs dropped by 70% from d56 to d180 (Fig. 1B) . Consistent with our previous study 11 , stronger . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint NAbs was maintained in the morning vaccination group when compared to the afternoon group after 6 months.

Blood samples were collected from multiple time points before and after the third dose of vaccine longitudinally and NAbs was measured by Chemiluminescent immunoassay. We found that serological response was induced within 1 week after the boost dose. The concentration of NAbs was increased by 7.2 folds from 9.3 AU/ml on d180 to 66.9 AU/ml on d187. The concentration of NAbs continued to rise and reach the peak by 10.6 folds at around 2 weeks after the booster shot (Fig. 1C) . The enhanced neutralizing antibody response was still observed at the early response after the third dose.

There was a trend that the morning group benefited more from the vaccination.

The difference between the two groups diminished over time (Fig. 1D ).

However, we did not observe different levels of NAbs between female and male (Fig. 1E ).

To further profile the antibody spectrum induced by the inactivated vaccin, we measured antibodies against SARS-CoV-2 nucleocapsid, spike, spike S1, spike S2, RBD, spike N terminal domain (NTD) and envelope in the serum samples from d194 by ELISA. Supprisingly, antibodies against against these SARS-CoV-2 structure proteins were detected in all of the HCWs (S. Fig. 1 ). In addition to spike-targeted antibodies, inactivated vaccine is capable of inducing a broad specturm of antibodies against SARS-CoV-2 and might provide greater protection than expected.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 

Antigen-specific memory B cells play an important role in the recall response upon reinfection or boost by vaccination. To study the durability of SARS-CoV-2-specific memory B cells and their response to the booster vaccine, we first measured spike-and RBD-specific B cells by flow cytometry.

We found that spike-and RBD-specific memory B cells were detected 6 months after vaccination. The percentages of spike-and RBD-specific memory B cells were increased by 1.7 and 2 folds respectively by the third dose of vaccine ( Fig. 2A-2C) . As compared to data on d56, there was a decreasing trend for the percentage SARS-CoV-2-specific memory B cells over time.

However, the percentage viral specific memory B cells were largely maintained on d180 (Fig. 2D, 2E) . As for the spike-and RBD-specific memory B cells, the majority of the cells could be mapped into IgG + or IgM + B cells. IgA + B cells accounted for a minimal percentage of these SARS-CoV-2-specific memory B cells (Fig. 2F, 2G) . Spike-or RBD-specific memory B cells were further detected by ELISpot and similar results were found. The third dose inactivated vaccine boosted spike-or RBD-specific memory B cells from 8 u.f.c/10 6

PBMCs to 17 u.f.c/10 6 PBMCs and 4 u.f.c/10 6 PBMCs to 10.7 u.f.c/10 6 PBMCs respectively as by ELISpot (Fig. 2H-2J ). As we phenotyping the total B cell population by flow cytometry, we found that percentage of total and naïve B cells were not affected by the third dose of vaccine. Double negative . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (IgD -CD27 -) B cells were increased. However, IgG + B cells remained unchanged after the booster dose (S. Fig. 2, 3) .

Although there was a short report showing T cell responses to SARS-CoV-2 antigens after 2 doses of the inactivated vaccine 24 , it is not clear how long they sustain. Blood samples were acquired from the participants 6 months after the primary vaccination and cellular response was first measured by IFN-γ ELISpot using the spike peptide pool. As expected, antigen-specific T cells were recorded in all of the participants, although low frequencies were detected in some of the participants. However, the SARS-CoV-2-specific CD8 + T cells could be potentially underestimated for the use of the spike peptide pool alone. T cell response was enhanced by a third dose vaccine by 2.3 folds as measured by ELISpot (Fig. 3A, 3B) . Further, SARS-CoV-2-specific CD8 + T cells (CD69 + 4-1BB + ) were robustly primed following the booster shot 2.7 folds (Fig. 3C, 3D) . Phenotypic markers indicated that the majority of the SARS-CoV-2-specific memory CD8 + T cells were CD45RA + CCR7terminally differentiated effector memory cells (T EMRA ). CD45RA -CCR7effector memory T cells (T EM ) accounted for only one-fourth of the total antigen-specific CD8 + T cells (Fig. 3E) , which is similar to that from recovered COVID-19 patients 26 .

However, the subsets of total CD8 + T cells were not affected (S. Fig. 4, 5) . We . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. also detected SARS-CoV-2-specific CD3 + CD4 -CD8 -T cells in inactivated SARS-CoV-2 vaccinated individuals, which were further induced to a greater extent when compared to CD8 + T cells by the booster shot (Fig. 3F, 3G) . The induction of CD3 + CD4 -CD8 -T cells might be correlated with vaccine efficacy 30 . Fig. 4, 5) .

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 

The weakened or negative serological response has been noted in immune-compromised individuals after vaccination or SARS-CoV-2 infection.

The third dose of mRNA vaccine could help to enhance immune responses in these patients 31,32 . Consistent with these studies, we found that NAb was detected in all of the 9 participants with a negative serological response after 2 doses of vaccine. The level of NAb in all of the 9 participants increased rapidly within 1 week and reach the peak 2 weeks after the booster shot (Fig. 5A) .

However, the serological response was boosted to a less extent when compared to the participants with seropositive response to 2 doses of vaccine a less extent 2 weeks after the booster shot ( Fig. 5D-5F ).

Next, we were to investigate T cell responses to the booster dose in seronegative individuals after 2 doses of vaccine. Interestingly, spike-specific T cells were detected even before the third dose of vaccine as measured by

ELISpot. The third dose of vaccine enhanced SARS-CoV-2-specific T cell response consistently (Fig. 5G) . The induction of SARS-CoV-2-specific CD4 + and CD8 + T cells by the third dose of vaccine was further confirmed by measuring AIM + T cells (OX40 + 4-1BB + for CD4 + T cells and CD69 + 4-1BB + for CD8 + T cells) by flow cytometry. Both spike-specific CD4 + T cells and CD8 + T cells were expanded by the third dose of vaccine (Fig. 5H, 5I) . We also observed the expansion of SARS-CoV-2-specific cTFH cells by the third dose of vaccine (Fig. 5J) . Taken together, a third dose of inactivated SARS-CoV-2 vaccine could induce detected B cell and T cell response in individuals with negative serological response to 2 doses of vaccine.

In this study, we investigated the duration of B cell and T cell immunity and their response to a third dose of the inactivated SARS-CoV-2 vaccine BBIBP-CorV in a non-randomized trial. Data in this study showed that neutralizing antibodies gradually decreased after the second dose of vaccine during the 6-month observation. SARS-CoV-2-specific B cells and T cells were detected and persisted in the peripheral blood 6 months after the primary . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Recently, a third dose of inactivated vaccine CoronaVac elicited a rapid and . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

patients could be long-lasting up to 13 months as have been documented in this study (S. Fig. 7) . SARS-CoV-2-specific CD4 + and CD8 + T cells induced by BBIBP-CorV were detected in all the HCWs who received 2 doses of vaccine, and that last up to 6 months at a minimum period in this study. In accordance with previous data from mRNA vaccine 20 , SARS-CoV-2-specific CD4 + T cells . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. T cells. HCWs with low serological response to 2 doses were not truly "no responders" but fully equipped with immune memory that could be quickly . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Comparisons were done by Wilcoxon rank sum test. Ns: not significant.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Representative FACS plots for spike-or RBD-specific BCR were shown. (B, C) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.12.21263373 doi: medRxiv preprint

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The work is supported by The Talent Program of the First Affiliated Hospital,

Yat-sen Univetsity (Y61229).

The authors have no conflicts of interest to disclose.. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021 CD4 + T cells. ****p<0.0001. Caparisons were done by Student's paired-T test.. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were collected on day (d) 180, d187, d194, d201 and analyzed longitudinally.(C) Spike-specific BCR was detected by flow cytometry. ns: not significant . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.12.21263373 doi: medRxiv preprint