Dynamics of immune responses to inactivated COVID-19 vaccination over 8 months in China.

Dear editor,

In this Journal, Lazarus and colleagues recently reported results of a randomized controlled trial of a novel inactivated SARS-CoV-2 vaccine (VLA2001) in healthy adults. They found that the highest dose group showed statistically significantly stronger immunogenicity with similar tolerability and safety. We also investigated dynamics of immune responses to inactivated COVID-19 vaccination over 8 months among healthy adults in China. Studies from Israel, UK, Chile and Denmark on the decay of anti-SARS-CoV-2 antibodies elicited by BNT162b2 (mRNA vaccine) and ChAdOx1-nCoV19 (adenovirus-vectored vaccine) revealed that mRNA vaccines could induce robust antibody response but started a rapid decay shortly after vaccination.2, 3, 4, 5 Modeling of the antibody decay uncovered factors such as age, sex, comorbidity, and the interval between vaccine doses that could influence the dynamics of humoral responses.3, 4, 5 Yet, long-term surveillance study is needed to supplement studies of immune responses for inactivated COVID-19 vaccines. , In addition, the relationship between humoral and cellular immune responses to inactivated vaccine has been rarely investigated.

We conducted a large-scale longitudinal study with 6646 serum samples from 4359 eligible participants, including many healthcare workers, who received a 2-dose immunization of inactivated SARS-CoV-2 vaccine (BBIBP-CorV or CoronaVac) at Yunnan University Affiliated Hospital (74.4% were female; average age 33±11 years [±SD]) to evaluate immunogenicity kinetics(Fig.S1). We used comprehensive immune indexes including anti-SARS-CoV-2 receptor-binding domain (RBD) IgG, anti-RBD-IgM, neutralizing antibodies (NAbs) as well as T cell responses (for a subset of participants) to analyze immunogenicity kinetics according to various demographics and disease states. Baseline characteristics of the participants are well-balanced across groups (Table 1 ). To assess the humoral response to inactivated SARS-CoV-2 vaccines, we measured NAbs titers using competitive inhibition method, while anti-RBD-IgG and anti-RBD-IgM using magnetic particle chemiluminescence immunoassay (MCLIA, Bioscience Co., Tianjin, China). We performed FluoroSpot assay to estimate T cell response (Human IFN-g/IL-2 SARS-CoV-2 FluoroSpotPLUS kit, Mabtech AB, Sweden). The study was approved by the Committee on Medical Ethics of Affiliated Hospital of Yunnan University (Approval number: 2021078), and Informed Consent Forms were signed by all participants.

Table 1

Participants demographic characteristics. The study population includes volunteers who provided blood samples after the first dose and after the second dose (Whole study population). Participants who provided at least one blood sample following the second vaccination were subject to analysis (Population in model).

Characteristics		Whole study population (n=4359)
		Population in model (n=4314)
		Total	Before2Dose	week1–2	week3–4⁎	week5–8*	week9–12	week13–16	week17–20	week21+
Age, median (IQR)		31 (24–41)	34(29-43)	32(27-42)	35(28-45)	30(23-42)	24(22-32)	28(23-38)	31(25-41)	33(29-42)
Age n (%)	18–27	1717 (39.4)	207(20.6)	183(29.7)	459(24.3)	163(43.6)	460(63.6)	556(48.8)	142(36.5)	53(18.7)
	27–38	1235 (28.3)	369(36.8)	215(34.8)	621(32.9)	91(24.3)	161(22.3)	289(25.4)	114(29.3)	132(46.5)
	38–47	748 (17.2)	247(24.6)	141(22.9)	402(21.3)	54(14.4)	67(9.3)	182(16.0)	78(20.1)	56(19.7)
	≥48	659 (15.1)	153(15.3)	78(12.6)	406(21.5)	66(17.7)	35(4.8)	111(9.8)	55(14.1)	43(15.1)
Sex n (%)	male	1117 (25.6)	239(23.8)	172(27.9)	556(29.5)	85(22.7)	170(23.5)	235(20.7)	85(21.9)	54(19.0)
	female	3242 (74.4)	764(76.2)	445(72.1)	1332(70.6)	289(77.3)	553(76.5)	903(79.3)	304(78.1)	230(81.0)
BMI n (%)	≤18.5	536 (12.3)	99(9.9)	62(10.0)	174(9.2)	44(11.8)	129(17.8)	156(13.7)	42(10.8)	34(12.0)
	18.5–23.9	3093 (71.2)	626(62.4)	374(60.6)	1137(60.7)	237(63.5)	478(66.1)	731(64.2)	270(69.4)	193(68.0)
	≥23.9	712 (16.5)	268(26.7)	177(28.9)	562(30.0)	92(24.7)	116(16.1)	251(22.1)	77(17.8)	57(20.0)
Body condition n (%)	health	1818 (43.2)	543(54.1)	382(62.0)	1252(66.3)	135(36.1)	129(17.8)	262(23.0)	136(35.0)	71(25.0)
	comorbidity	2386 (56.8)	460(45.9)	235(38)	636(33.7)	239(63.9)	594(82.2)	876(77.0)	253(65.0)	213(75.0)
Seropositivity N (%)	NAbs	4817(72.5)	140(12.5)	525(84.4)	1802(94.6)	336(89.8)	625(86.3)	922(81.0)	301(77.3)	166(58.5)
	IgG	4753(73.5)	108(9.6)	517(83.1)	179(94.1)	339(90.6)	622(85.9)	939(82.5)	296(76.1)	144(50.7)
	IgM	1465(22.0)	156(13.6)	292(46.9)	875(46.0)	57(15.2)	35(4.8)	29(0.8)	13(3.3)	8(2.8)

n: Number of participants; N:Sample size;

:Some individuals did not provide online survey.

We analyzed the seroprevalence of SARS-CoV-2-specific IgM or IgG among 2,4705 participants prior to the massive vaccination campaign in China (Table S1). We observed very low IgG seroprevalence among local hospital patients and healthcare workers (0.11–0.62%) and significantly elevated seroprevalence among healthcare workers who have worked as temporary support team at Wuhan during the early pandemic outbreak (2.35%). This data indicates a very low local natural infection rate before vaccination campaign.

We then assessed antibody levels for the vaccine study population cohort spanning from receiving the first dose to over 200 days after the second vaccination (Table 1). After the first dose of inactivated vaccine, a minority of participants showed significant increase of anti-RBD-IgG, anti-RBD-IgM, and NAbs (108 [9.6%], 156 [13.9%], and 140 [12.5%], respectively). A second dose elicited a sharp increase in antibody concentrations among most people (Fig. 1 A, 1B and 1C). The average NAbs, anti-RBD-IgG, and anti-RBD-IgM concentration increased 12, 45, and 4 folds, respectively, after 1-2 weeks of the second dose compared to the first dose. The concentration and seropositive rate of NAbs and anti-RBD-IgG peaked at the 4th week after the second dose (236.3 IU/mL, 94.6% and 47.6 S/CO, 94.1%, respectively) (Fig. 1A and 1B). From the 5th week, NAbs levels decreased sustainably and culminated in a 4-fold decrease in NAbs level reaching 58.5% seropositivity at the 21st week. Anti-RBD-IgG levels significantly decreased by a factor of 9.3 with a 50.7% seropositivity at the 21th week. Anti-RBD-IgM level also dropped by a factor of 11.3 with 4.8% seropositivity during 9-12th weeks, but the decline from the 9th week to the end of study was much slower, with an overall decrease by a factor of 1.3. Importantly, we noted anti-RBD-IgG and NAbs kinetics were consistent in their degree of immunogenicity (R = 0.89, P <2×10−16, Fig. S2).

Fig. 1

Antibody kinetics and T-cells responses following vaccination. (A) shows the neutralizing antibody levels following vaccination. The cutoff level of positive neutralizing concentration is 50 IU/mL. (B) shows the IgG levels following vaccination. The cutoff level of positive antibody concentration is 5 S/CO. (C) shows the IgM levels following vaccination. The cutoff level of positive antibody concentration is 5 S/CO. Each point represents a serum sample. The error-bar indicates 95% confidence interval (CI) of geometrical mean concentrations(GMTs). (D) Distribution of IFN-γ and IL2 levels from activated T-cells upon recognition of S peptides. Samples were collected at the 30th day after a second vaccination. Data reported as the median and interquartile range (box), whiskers represent 1.5 times the interquartile range.

Although most people showed a significant waning antibodies after two-dose inactivated vaccine immunization, cellular responses developed in majority of individuals, especially Th1 cell responses (Fig. 1D), suggesting that a second vaccination could effectively promote SARS-CoV-2-specific T cells immunity. Moreover, humoral immune response represented by antibody levels positively correlated with Th1 responses represented by IFN-γ and IL2 secreting cells (Fig. S3).

We found that age, sex, BMI, health condition, vaccine products, the days since the second-dose vaccination were significantly related to the antibody waning kinetics by linear regression analysis (Fig. S4-S7). Therefore, we estimated the dynamics of anti-RBD-IgG and NAbs over 27 weeks after the second dose and associated changes of SARS-CoV-2 specific antibodies with demographic characteristics of participants by linear mixed models (Table. S2 and S3). Mixed model analysis revealed that individuals with diabetes, obesity (BMI ≥ 23.9), older age (≥ 48 years-old), and male sex significantly associated with lower NAbs and anti-RBD-IgG concentrations. Conversely, vaccine product CoronaVac was associated with higher NAbs and anti-RBD-IgG concentrations compared to BBIBP-CorV (Table S4). We also observed an age-by-sex interaction in affecting antibodies titers, suggesting age-dependent antibody kinetics vary differently condition on sex.

Our analysis showed that antibody levels decline at different rates depending on age, sex, BMI, diabetes, vaccine products, and the time since the second-dose vaccination. Although antibody levels drop sharply, the cellular immunity was activated in most people and T cell immune memory induced by inactivated vaccines could last over 6 months post vaccination. The finding provides valuable insights of humoral response dynamics and advice to vaccination strategy with inactivated COVID-19 vaccine.

Funding

This study was funded and supported by Yunnan Provincial Science and Technology Department (202102AA100051 and 202003AC100010, China), National Natural Science Foundation of China (81960116; 82060368, China), Reserve Talents Project for Young and Middle-Aged Academic and Technical Leaders of Yunnan Province (202205AC160023), Yunnan Natural Science Foundation (202001AT070085, China). Spring City Plan:the High-level Talent Promotion and Training Project of Kunming.

Declaration of Competing Interest

Z.Z. served as a PI in a phase 4 clinical study sponsored by Sinovac Biotech Ltd. The funder has no role in study design, implementation and manuscript writing in this study.