Inactivated vaccine fueled adaptive immune responses to Omicron in 2‐year COVID‐19 convalescents

Over 3 years, humans have experienced multiple rounds of global transmission of SARS‐CoV‐2 and its variants. In addition, the widely used vaccines against SARS‐CoV‐2 involve multiple strategies of development and inoculation. Thus, the acquired immunity established among humans is complicated, and there is a lack of understanding within a panoramic vision. Here, we provided the special characteristics of the cellular and humoral responses in 2‐year convalescents after inactivated vaccines, in parallel to vaccinated COVID‐19 naïve persons and unvaccinated controls. The decreasing trends of the IgG, IgA, and NAb, but not IgM of the convalescents were reversed by the vaccination. Both cellular and humoral immunity in convalescents after vaccination were higher than the vaccinated COVID‐19 naïve persons. Notably, inoculation with inactivated vaccine fueled the NAb to BA.1, BA.2, BA.4, and BA.5 in 2‐year convalescents, much higher than the NAb during 6 months and 1 year after symptoms onset. And no obvious T cell escaping to the S protein was observed in 2‐year convalescents after inoculation. The study provides insight into the complicated features of human acquired immunity to SARS‐CoV‐2 and variants in the real world, indicating that promoting vaccine inoculation is essential for achieving herd immunity against emerging variants, especially in convalescents.

was observed in 2-year convalescents after inoculation. The study provides insight into the complicated features of human acquired immunity to SARS-CoV-2 and variants in the real world, indicating that promoting vaccine inoculation is essential for achieving herd immunity against emerging variants, especially in convalescents. int/). The herd immunity raised by the vaccination or natural infection has been recognized as the barrier to cut off the transmission of the virus. Thus, the immune memory characteristics of the convalescents and the vaccinees, including cellular and humoral immunity have raised wide concern. 1-3 Cellular immune memory can last up to 1 year after the COVID-19 patient's recovery, 2,3 while for humoral immunity, neutralizing antibodies (NAb) are still detectable 16 months after symptoms onset, but the neutralizing activity of antibodies has marked declined over time. 4 But the immune statuses of convalescents become complicated in consideration of the application of vaccines.
In the past 3 years, to effectively prevent COVID-19 and achieve herd immunity with minimal loss of life, different types of vaccines have been developed and widely used globally, such as mRNA, inactivated, polypeptide subunit, and adenovirus vector vaccine. [5][6][7][8] It was indicated that S-RBD IgG, NAb, and CD4 + T cell responses waned quickly after a single inactivated vaccine dose, and further increased after the second dose. Virus-specific CD4 + T cell responses generated by two doses of vaccination lasted more than 2 months. 7 A significantly reduced neutralizing activity against Omicron was observed in the convalescent and two-dose BBIBP-CorV ® (Sinopharm) vaccination group, which was elevated by a third homologous inactivated vaccine booster. 9 Studies also showed that the Omicron breakthrough infected individuals immunized with inactivated vaccines maintained high level of neutralization against wild type (WT, or prototype) and variants, and the enhancement effects by breakthrough infection were of significant differences between two-dose and three-dose groups. 10 Prior immunization with inactivated prototype strain vaccines substantially restrains pneumonia development, reduces cytokine storms, and facilitates clinical recovery after virus breakthrough. 11 However, considering the complicated inoculation involving two or three doses of inactivated vaccines among COVID-19 convalescents in the first wave during the pandemic, the features of the hybrid humoral and cellular immune memory are of importance to be investigated.
The emergence of different severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, notably Delta and Omicron, has led to successive waves of pandemics, posing enormous public health challenges. 12 Studies showed that Omicron is more transmissible than other variants. [13][14][15][16] The Omicron variant harbors 32 mutations in spike glycoproteins, 17 raising concern about the Omicron escaping from immunity established by WT infection or vaccination. Studies have shown that sera from COVID-19 convalescents or individuals who were vaccinated exhibited substantially diminished neutralizing activity against Omicron. [18][19][20][21][22] It has been reported that mRNA-1273, BNT162b2, Ad26.COV2.S, and NVX-CoV2373 vaccines elicited highly conserved cellular immunity to Omicron. [23][24][25][26] The memory status of T-cell immunity and the crossreactivity to Omicron among inactivated vaccines were still unexplored, especially in terms of the vaccinees recovered from COVID-19.
The knowledge of the immune features and the memory persistence in vaccinated COVID-19 convalescents can guide selection of vaccine strategies of convalescents to elicit protective immunity against continuously evolving variants. Additionally, this information can also be utilized to develop vaccines by comprehending conserved T cell immunity. Thus, in this study, we assessed the antibody and Tcell responses to WT (prototype) and Omicron strains in COVID-19 convalescents who have later inoculated with inactivated vaccines, and healthy individuals after routine two-or three-dose vaccination. healthy unvaccinated controls (HC-Unvac). Twenty-three out of the 51 2-year convalescents were also kept before vaccination during the followed up at 6 months and 1 year after symptoms onset (Table 1).
Written informed consent was obtained from all participants. Sera and peripheral blood mononuclear cells (PBMCs) of whole blood were separated and frozen immediately for subsequent analysis (Supporting Information: Methods). In 2Y-Vac group, genomic DNA was extracted using TIANamp Blood kit (TIANGEN). DNA-based HLA polymorphisms were done with the Luminex multianalyte profiling system (Supporting Information: Figure 7).

| Peptide pools
Fifteen to 18-mer peptides with 10 amino acids of overlap were synthesized and covered the full length of S, M, and N proteins.
Meanwhile, the peptide pools spanned WT and BA.1 RBD were also designed. PBMCs were cultured for 9 days with S1, S2, M, and N peptide pools stimulation, respectively (Supporting Information: Methods). 3

| SARS-CoV-2-specific T cell responses
Fresh PBMCs after thawing were used to determine the responses to different SARS-CoV-2 peptide pools (S1, S2, M, and N). Then the left PBMCs were cultured for 9 days, and stimulated with four SARS-CoV-2 structural peptide pools, respectively. After cultivation for 9 days, T cell reaction against four peptide pools were tested by Enzyme-linked immunospot (ELISpot) assay (BD) and intracellular cytokine staining (ICS) in 2-year COVID-19 convalescents and health controls, as described in our previous study (Supporting Information:  Two-year convalescents after vaccination exhibited IgG level that were 1.2-fold more than healthy individuals after vaccination ( Figure 1B,C).
We found that over 96% of 2-year convalescents after inactivated vaccines have a positive IgG response to WT, as presented by the double positive for ELISA and MCLIA tests (Supporting Information:  Figure 1A,B and Table 1).
To reveal the dynamic antibody responses within the convalescents, we retrieved the paired sera at the 6-month and 1-year followup visits of the convalescents. The 2-year convalescents had higher IgG antibodies following vaccination in contrast with the 1-year visit, with 50% (9/18) of employees increased more than 1-fold (p = 0.005 for ELISA and p = 0.004 for MCLIA), but with no difference with the 6-month visit ( Figure 1D,E). However, a significant reducing trend was observed for IgM levels among the convalescents from 6 months to 2-year recovery, although the convalescents were vaccinated before the 2-year visit ( Figure 1F,G). We next analyzed whether severity affected the levels of IgG/IgM, and no differences were observed among vaccinated 2-year convalescents with different disease severities (Supporting Information: Figure 1C-F).
We also tested the IgA antibody within the sera of the donors.

| T cell reactivity to SARS-CoV-2 in 2-year convalescents after inactivated vaccines
We also compared the SARS-CoV-2-specific T cell responses among  T-cell response memory to at least one of these four antigens, which was markedly higher than the corresponding proportion (54.3%) of vaccinated healthy controls ( Figure 2F and Supporting Information:  Figures 3A,B and 4G). Moreover, females showed higher antigen-specific T cell than males, but the differences were not statistically significant except for S1 peptide pool (Supporting Information: Figure 5K-N). The interaction between T cell responses and age was not significant (Supporting Information: Figure 6K-N).
To further investigate the SARS-CoV-2-specific CD8 + and CD4 + T cells in participants, we stimulated PBMCs with peptide pools of four antigens (S1, S2, M, and N) and measured cytokine production (IFN-γ, IL-2, and TNF-α) using flow cytometry ( Figure 3A). Though without statistical significance, the median ratios of cytokinesecreting T cells against four peptide pools were higher in the 2- year convalescents than in healthy controls who had been vaccinated ( Figure 3B,C). Furthermore, specific T cells among both groups developed stronger cellular immunity than individuals with neither infection nor vaccination ( Figure 3C).

| DISCUSSION
Since its emergence in 2019, the SARS-CoV-2 virus has continued to evolve into different mutant strains, posing continuous challenges to the control of COVID-19. 27,28 To achieve the goal of herd immunity, a variety of vaccines have been introduced worldwide. 29 One of the main vaccines promoted in China is the inactivated vaccines, which has a high inoculation coverage among the population up to date. 30  which was also concordant with other cohort studies. 1,2,31,32 Recently, it was reported that NAb against SARS-CoV-2 could persist for one and a half years among COVID-19 convalescents. 4,33 However, most of the studies indicated a decreasing trend of the antibody levels with time among the convalescents. 1 Our studies indicated that the decreasing of IgA antibodies in followup sera from 6 months to 1 year after symptoms onset, and observed inactivated vaccines could also induce an increase of IgA antibody level at 2 years postinfection.
The persistence of T-cell immunity stimulated by natural infection may play an important role in alleviating severe disease in the reinfection. 40 Here we found that T cell immune memory was much higher in the 2-year convalescents after vaccination than the vaccinated even boosted healthy controls, demonstrating that T cell immune memory by natural infection could persist up to 2 years after symptoms onset.
Several studies showed that mRNA vaccines could stimulated a certain level of virus-specific CD4 + and CD8 + T-cell immunity, 8,41,42 while T-cell immunity induced by inactivated vaccines is far from being well-defined.
However, unlike inactivated vaccines, mRNA vaccines are designed primarily to focus on the S protein, the cellular immunity stimulated by this vaccine only targets the S protein. 8  This may indicate a special cross-reactive antibody-inducing feature by natural infection and the necessity for vaccination among the convalescents. Previous studies determined that cellular immunity elicited by the mRNA vaccine could crossrecognize the Omicron. 25,26,43 We herein showed that the cellular immunity stimulated by inactivated vaccine also has a cross-response to Omicron. As it turns out, the vaccination of inactivated vaccine is beneficial to the population against the WT and its variants, and it is an effective method to realize herd immunity. Based on our data in HC-3 doses group and 2Y-2 doses group, we did not observe significant influence of gender and age on the antibody levels, which may be related to small sample size. Our current study has several limitations. First, despite our best attempts to recruit participants, the sample size was relatively limited.
However, the statistical differences were significant in the study, which could support the conclusions. Second, we did not perform PCR testing on the healthy controls to exclude asymptomatic infection, but the infection of the donors could be excluded considering no local COVID-19 cases reported during our recruiting period and this was also supported by our earlier epidemiological analysis. 46 In addition, the lack of comorbidities may causes limitations for the group comparison. However, previous study indicated that after CoronaVac vaccination, no significant difference was found between spike IgG antibody positive and negative groups in terms of most comorbidities. 47 Third, the detection of the neutralizing ability may be inaccurate when we use the cELISA kit with the antigen S-RBD since other regions may also be neutralizing.
However, previous studies suggested that the cELISA assay had a strong correlation with conventional virus neutralization test (cVNT), and had shown robust internal validity parameters for both cats, mouse, and human. 48,49 In summary, we provide the special characteristics of the cellular and humoral immune responses in 2-year convalescents after the inoculation of inactivated vaccines. The decreasing trends of the IgG, IgA, and neutralizing antibodies, but not IgM of the convalescents were reversed by the vaccination. And both cross-reactive cellular and humoral immunity to Omicron variants in convalescents after vaccination were higher than in the vaccinated healthy controls.
Thus, promoting vaccine inoculation is an essential way to achieve herd immunity during the ongoing COVID-19 pandemic, even for the convalescents.