Vaccines | Free Full-Text | Long-Term Dynamic Changes in Hybrid Immunity over Six Months after Inactivated and Adenoviral Vector Vaccination in Individuals with Previous SARS-CoV-2 Infection
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1. Introduction
The Coronavirus Disease 2019 (COVID-19) pandemic has affected nearly 800 million confirmed cases globally, resulting in seven million reported deaths as of 20 November 2023 [1]. COVID-19 vaccines were developed to mitigate the impact of the disease and reduce its severity. As of 17 November 2023, more than 13.5 billion COVID-19 vaccine doses have been administered worldwide [1]. In Thailand, a national COVID-19 immunization program commenced on 28 February 2021, which primarily employed the CoronaVac (Sinovac Life Sciences Co., LTD, Beijing, China) and ChAdOx1 nCoV-19 (recombinant; Oxford-AstraZeneca) vaccines during its initial phase. CoronaVac, an inactivated whole virus COVID-19 vaccine adjuvanted with aluminum hydroxide, has seen over three billion doses administered across 62 countries, with a primary focus on low or middle-income regions. Clinical trials reported an efficacy ranging from 50 to 84% in protecting against symptomatic COVID-19 [2]. Additionally, the ChAdOx1 nCoV-19 vaccine, a replication-deficient chimpanzee adenoviral vector containing the SARS-CoV-2 spike antigen, accounts for three billion doses distributed across more than 170 countries worldwide, and it has shown efficacy rates of up to 70.4% in preventing symptomatic COVID-19 [3].
Recent findings indicate that individuals recovering from SARS-CoV-2 infection typically develop robust humoral and T cell-mediated immune responses that are associated with disease protection [4]. Long-term monitoring has revealed a modest decline in SARS-CoV-2 antibody levels, and this was observed between 5 to 8 months post-infection [5]. B cell memory to SARS-CoV-2 can remain detectable for up to eight months following natural infection [5,6]. Although the T cell-mediated immune response appears to persist for several months, where it lasts longer than the detectable antibodies in recovered patients, a decrease in memory T cells with an initial half-life of 3 to 5 months has been reported [5,6]. Emerging evidence has suggested that vaccination among individuals with prior SARS-CoV-2 infection could sustain protective immunity and offer additional protection against symptomatic reinfection and severe outcomes [7,8].
The combination of natural immunity and vaccine-induced protection, termed hybrid immunity, presents a notably stronger and broader immune response, thereby surpassing the effects of either infection or vaccination alone [9]. However, existing evidence has largely focused on the early response following mRNA vaccination among previously infected individuals [10,11,12,13]. Few studies have evaluated the impacts of inactivated and vector-based vaccines [14,15,16]. Additionally, most investigations have focused on short-term immunogenicity, while long-term evaluations remain limited.
The surge of omicron variants, marked by substantial mutations, has the potential to increase virus transmissibility and reduce susceptibility to neutralization by antibodies induced through natural infection or vaccination [17]. Additionally, the documented progressive decline in the humoral immune response is well established in vaccinated individuals and those previously infected with SARS-CoV-2 [18,19]. Therefore, it is crucial to comprehend the trajectory and long-term durability of the immune response and its potential for neutralizing the omicron variant. However, the longitudinal data characterizing the extended dynamics of antibodies, neutralization capability, and T cell responses post-immunization with various vaccines—specifically comparing those currently in use, such as inactivated and adenoviral vectored vaccines, in individuals with prior infection—remain unclear.
In this study, we aimed to assess the dynamic changes and decline in humoral and T cell immune responses over a six-month period following the administration of either inactivated (CoronaVac) or adenoviral vector-based vaccines (ChAdOx1 nCoV-19) among individuals previously infected with SARS-CoV-2. Additionally, we quantified the impact of the time interval between infection and vaccination on neutralization against pre-omicron and omicron variants, as well as of T cell responses. These analyses provide insights into the durability of immune responses induced by inactivated and adenoviral vectored vaccines in individuals with prior infection. They also shed light on the neutralization capacity against omicron variants and offer implications for the immunogenicity of the current vaccines deployed against the SARS-CoV-2 pandemic.
2. Materials and Methods
2.1. Study Design and Participants
This prospective cohort study was conducted between 14 May and 9 December 2021 at the Center for Excellence in Clinical Virology, Chulalongkorn University, Bangkok. The participants were initially screened by physicians and nurse coordinators during the enrollment process. The inclusion criteria involved individuals previously infected with SARS-CoV-2 (identified through anti-nucleocapsid positivity (IgG) or a history of positive SARS-CoV-2 detection), those aged 18 years and older, and those without comorbidity or well-controlled comorbidities. The exclusion criteria encompassed participants who had received a different type of COVID-19 vaccine and those with severe medical conditions, such as compromised immune systems, malignancies, and autoimmune diseases.
Participants were categorized based on the duration between their initial positive SARS-CoV-2 detection and the first vaccination, and they were divided into short- (2 to 5 months) and long-interval groups (13 to 15 months). The short-interval group comprised participants infected between 2 January and 13 April 2021 during the alpha predominant wave, and they were allocated to receive either a single dose of CoronaVac (referred to as Short+1xCV) or the ChAdOx1 nCoV-19 (referred to as Short+1xChAd) vaccine. Meanwhile, the long-interval group included individuals infected between 3 March and 4 April 2020 during the wild type predominant wave, and they were assigned to receive either two doses of CoronaVac (referred to as Long+2xCV) or the ChAdOx1 nCoV-19 (referred to as Long+2xChAd) vaccine. Within each group, participants were assigned to receive a vaccine based on convenience sampling and vaccine availability. Vaccination began on 14 May 2021, and the final blood sample was collected on 9 December 2021. The study design is illustrated in Figure 1.
Reactogenicity data were self-reported adverse events (AEs) collected seven days after the first vaccine doses. Blood samples were collected on days 0, 14, 28, 60, and 180 (6 months) after the first vaccination for the short-interval group. For the long-interval group, samples were collected on days 0, 14, and 28 after the first dose, and on days 28 and 120–150 (4 to 5 months) after the second dose of the vaccine. The study protocol adhered to the guidelines detailed in the Declaration of Helsinki and the Good Clinical Practice principles. Approvals were obtained from the Research Ethics Committee of the Faculty of Medicine, Chulalongkorn University (IRB numbers 192/64 and 281/64). This study has been registered with the Thai Clinical Trials Registry (TCTR20210319003 and TCTR20210520004). All participants provided written consent.
2.2. Study Vaccines
CoronaVac (Sinovac Life Sciences, Beijing, China) is an inactivated virus vaccine produced by cultivating the SARS-CoV-2 virus (CZ02 strain) in African green monkey kidney cells (Vero Cell). This is followed by inactivation using β-propiolactone and formaldehyde, as well as adsorption with aluminum hydroxide [2]. CoronaVac was administered within intervals of 21–28 days.
The ChAdOx1-vectored vaccine (ChAdOx1 nCoV-19) is a recombinant chimpanzee adenovirus-vectored vaccine that is replication-deficient and expresses the SARS-CoV-2 spike surface glycoprotein [3]. ChAdOx1 nCoV-19 was administered at 8 weeks apart.
2.3. Serological Testing
All serum samples underwent testing for anti-nucleocapsid (N) protein IgG and anti-receptor-binding domain (RBD) IgG antibodies against the ancestral strain using the commercially available automated ARCHITECT system (Abbott Diagnostics, Abbott Park, IL, USA) employing a chemiluminescent microparticle immunoassay (CMIA). The determination of anti-N IgG utilized the SARS-CoV-2 IgG assay (Abbott Diagnostics, Abbott Park, IL, USA) in accordance with the manufacturer’s instructions, with seropositivity defined as ≥1.4. The assessment of anti-RBD IgG employed the SARS-CoV-2 IgG II Quant assay (Abbott Diagnostics, Abbott Park, IL, USA) with a positive threshold set at equal to or greater than 50 AU/mL in accordance with the manufacturer’s guidelines. Conversion to binding antibody units per milliliter (BAU/mL) was conducted by multiplying the numerical AU/mL value by a factor of 0.142. An anti-RBD IgG result equal to or greater than 7.1 BAU/mL was considered positive.
2.4. Surrogate Virus Neutralization Tests (sVNT) for Pre- and Omicron Variants
Neutralizing activity against pre- and omicron variants induced by different vaccines was assessed using the cPass SAR-CoV-2 neutralizing antibody detection kit according to the manufacturer’s instructions (GenScript Biotech, Piscataway, NJ, USA). For this assay, serum samples obtained one month after the first and second vaccine doses were tested against recombinant SARS-CoV-2 RBD proteins of the wild type, B.1.1.7 (alpha), B.1.351 (beta), B.1.617.2 (delta), and B.1.1.529 (omicron BA.1) variants. Additionally, the sera collected six months after a single dose and 4–5 months after the second dose were tested against B.1.1.529 (omicron BA.1) using previously described methods [20]. Briefly, serum samples, along with positive and negative controls, were diluted at 1:10 and incubated with horseradish peroxidase-conjugated recombinant SARS-CoV-2 RBD proteins at 37 °C for 30 min. Subsequently, the reaction mixture was transferred to ELISA plates coated with human angiotensin-converting enzyme 2 proteins and incubated at 37 °C for 15 min. After washing, tetramethylbenzidine (TMB) solution was added, and the plate was incubated in the dark at room temperature for 15 min. After adding the stop solution, absorbance was promptly measured at 450 nm. Results were calculated as inhibition (%) = (1 − OD value of sample/average OD of negative control) × 100. Values above 30% indicated the presence of neutralizing antibodies.
2.5. Focus Reduction Neutralization Test (FRNT50)
Live SARS-CoV-2 neutralizing antibody titers against omicron BA.2 (EPI_ISL_11698090) subvariants were determined in a subset of serum samples collected one month and four to five months post-second dose using a 50% focus reduction neutralization test (FRNT50). The test involved assessing infected cell counts, as described previously [21]. Heat-inactivated serum samples underwent serial dilutions ranging from 1:10 to 1:7290. These diluted samples were then incubated with a live virus for 1 h at 37 °C. Afterward, the mixtures of the virus and sera were added to Vero cell monolayers in a 96-well plate and incubated for 2 h. The focus reduction percentage for each sample was calculated, and the half-maximal inhibitory concentration (IC50) was determined using PROBIT analysis from the SPSS package v23.0. In instances where no neutralization was observed, the FRNT50 was set at 10, which represents one dilution step below the lower limit of detection (dilution 1:20).
2.6. Quantification of Interferon-Gamma Response
In addition, the specific T-cell response to SARS-CoV-2 was assessed by measuring the total IFN-γ response in the whole blood following the manufacturer’s instructions (QuantiFERON, Qiagen, Hilden, Germany). Approximately 0.8–1.2 mL of heparinized whole blood was transferred to specialized blood collection tubes that contained two SARS-CoV-2 antigen tubes, a Mitogen tube (positive control), and a Nil tube (negative control). The blood collection tubes containing whole heparinized blood sample were then incubated for 24 h at 37 °C. The antigen tubes were coated with either S1 (RBD) peptides targeting CD4+ epitopes (Ag1) or S1+S2 peptides covering CD4+ and CD8+ epitopes (Ag2) from the ancestral strain. Following incubation, the tubes were centrifuged to collect the plasma. The plasma samples were then diluted at a 1:2 ratio with diluent and subjected to IFN-γ detection using an ELISA kit (Qiagen, cat. no. 626410) following the manufacturer’s guidelines. The concentration of IFN-γ was quantified based on an eight-point standard (ranging from 0.125 to 8 IU/mL) and calculated as IU/mL using QuantiFERON RD (v5.03) software. The final IFN-γ values were calculated by subtracting the value obtained from the Nil tube. A positive threshold was considered as IFN-γ (Ag1−Nil or Ag2−Nil) at ≥0.15 IU/mL and at ≥25% of Nil.
2.7. Statistical Analysis
The associations among the categorical variables were assessed using the chi-squared test or Fisher’s exact test, while the differences among the continuous variables were evaluated using one-way ANOVA with a Bonferroni adjustment. The geometric mean ratio (GMR) was calculated using a general linear model univariate analysis with log-transformed data, and it was adjusted for sex and age. A higher GMR indicated a slower decay rate. The fold decrease in neutralizing activity was calculated by comparing the results observed at one month with those at five to six months. Differences between matched paired samples were determined using paired sample t-tests or Wilcoxon matched-pairs signed-rank tests for nonparametric data. The comparisons of the differences between groups were conducted using analysis of covariance (ANCOVA) with a Bonferroni adjustment or Kruskal–Wallis tests with Dunn’s post hoc correction for nonparametric data. Furthermore, the data from vaccinated naïve individuals who received a two-dose regimen of CoronaVac or ChAdOx1, as has been previously reported [22,23,24], were obtained as comparators, and the baseline characteristics are provided in Tables S1 and S2. All statistical analyses were performed using IBM SPSS Statistics v23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism v9.4.1 (GraphPad, San Diego, CA, USA). Statistical significance was considered at a p-value of
4. Discussion
This study examined the durability of hybrid immunity, and it explored both the humoral and T cell responses up to six months after CoronaVac or ChAdOx1 vaccination in previously infected participants with varying intervals between infection and first vaccination. We found both CoronaVac and ChAdOx1 vaccinations in previously infected individuals elicited higher antibody levels and enhanced neutralizing activities against pre-omicron and omicron variants when compared to infection-naïve individuals. This suggests that the enhanced response to inactivated and adenoviral vector-based vaccinations relies on pre-existing immunity, and this is consistent with earlier findings in mRNA vaccination [25,26]. Moreover, vaccinations after a longer interval (13 to 15 months) between infection and first vaccination induces higher neutralizing activity than a shorter interval (2 to 5 months). In addition, binding antibody levels and neutralizing activities against the omicron BA.1 and BA.2 variants persisted above pre-vaccination levels at six months post-vaccination, albeit they also showed a gradual waning. Although the total IFN-γ response declined to pre-vaccination levels by six months post-vaccination, a vaccination after infection benefits an increasing number of individuals with a reactive T cell response.
In the absence of immunization, we observed a sustained, albeit low-titer, presence of anti-RBD IgG for 13–15 months following SARS-CoV-2 infection, which aligns with a previous study [27]. The persistence of the antibody response was associated with a relatively stable number of RBD-specific memory B cells at least one year after infection [28]. Moreover, we found that nearly half of the individuals maintained reactive T cell responses for 13–15 months after infection. Consistent with a previous report, there was indications that a polyfunctionality and proliferation capacity of SARS-CoV-2-specific T cells persists at least 12 months post infection [29].
In line with our findings, several studies have indicated that hybrid immunity, which combines prior infection and mRNA vaccination, elicits robust antibody responses, often by displaying superior outcomes compared to vaccination in infection-naïve individuals [12,13,30,31]. Additionally, our study was extended to identify the effect of the hybrid antigen exposure interval on the magnitude of the immune response. Consistent with previous studies [25,32], a longer interval between infection and vaccination could enhance the level of antibody response and the strength of neutralization against SARS-CoV-2 variants. This finding could be partially explained by the increasing number of RBD-specific memory cells at 6 months compared to 1 month, which remain relatively stable at 12 months after infection [28]. Moreover, memory B cells continue to evolve even after 12 months post-infection, whereby they accumulate somatic mutations that increase neutralizing activity against SARS-CoV-2 mutants [28].
Consistent with a previous report [13], our results showed that a two-dose vaccination with inactivated vaccines or adenoviral vectors in recovered individuals provides cross-neutralization against both pre-omicron variants and the omicron variant, even though most of the participants had been infected with the wild type and alpha variant. This was supported by a gradual increase in memory B cell cross-binding to SARS-CoV-2 variants observed after vaccination in recovered individuals [33]. Furthermore, the inconsistent results regarding neutralization against omicron, which was observed through surrogate neutralizing assays and live virus neutralization following CoronaVac vaccination, might be explained by the distinct structural stabilization structure of spike proteins generated by inactivated (whole virus) and genetic vaccines (adenoviral-vectored vaccine) [34]. Moreover, the vaccine induces antibodies targeting other SARS-CoV-2 components, thereby potentially aiding in live virus neutralization, such as antibodies against the nucleoprotein [27].
The decline of the immune response over time and the ongoing evolution of virus variants raise concerns about the longevity of the protective immune response. Our results indicate that hybrid immunity provides a slower decline rate of antibody response and maintains broad neutralizing activities against omicron variants for up to six months. This result was supported by a real-world study [35], thus indicating that vaccination in previously infected individuals appears to enhance vaccine effectiveness, i.e., exceeding 90%, and extend immunity, and this is with no observed decline after more than 1 year post-infection or 6 months post-vaccination. Despite individuals who were previously infected and received two doses of CoronaVac showing a slower decline compared to ChAdOx1, their binding antibody levels and neutralizing activities remained at a low titer six months after vaccination [36]. Conversely, a single dose of ChAdOx1 exhibited robust antibody and neutralizing activity that was consistent with those reported in previous studies [14,37], while a second dose did not clearly enhance the immune response in individuals with prior infection. Similar results were found in mRNA vaccination, indicating that immunization with a single dose significantly boosted the expansion of pre-existing memory B cells in individuals with prior infection, while minimal changes in antibody response was observed after a second dose [11]. However, an alternative explanation could be the influence of anti-ChAd vector antibodies, which were reported to be well maintained for at least six months after vaccination [3]. Although a second dose of ChAd did not clearly increase antibody response, the advantage of a two-dose vaccination in terms of neutralization and slower decline was observed. This is consistent with reports that three antigen exposures, such as two vaccinations in convalescent individuals, could increase antibody avidity, thereby resulting in highly potent neutralization capacity against immune escape variants, including omicron, and maintenance for at least seven months [13].
The superiority of the T cell response in hybrid immunity was reported in prior infections following mRNA vaccination, and it was associated with an enhanced T follicular helper cell polarization of spike protein-specific CD4+ T cells, which likely enhanced IFN-γ production [30]. Our results have shown that vaccination with CoronaVac or ChAdOx1 could induce a high level of total T cell IFN-γ response in individuals with prior infection, indicating that spike-specific memory T cells are relatively sustained for more than six months after infection [38,39]. Although the increase in the median IFN-γ response was transient and returned to near pre-vaccination levels by 6 months post-vaccination, an increase in the number of individuals with a reactive T cell response was observed.
This study has several limitations. Initially, the number of participants at the six-month follow-up was relatively small, thus cautioning against broad generalizations of the results. Another limitation is the lack of data on hybrid immunity in vaccinated individuals who subsequently experience breakthrough infection. Our longitudinal cohort lacks infection-naïve individuals; however, we mitigated this limitation by obtaining data from our previous studies [22,23,24] for comparison, where we employed the same immunoassay to determine the humoral immune response. Additionally, the infecting sequence data were unavailable for participants with prior infection. Nonetheless, it was observed that the majority of recovered participants classified into the long-interval and short-interval groups were infected during the predominant periods of the wild type and alpha variants, respectively. Moreover, a previous study [40] found that the Abbott Diagnostics SARS-CoV-2 IgG assay was not very sensitive in detecting anti-N antibodies in individuals who had previously been infected with SARS-CoV-2 for an extended period. Therefore, caution is needed when interpreting the results. Furthermore, the results of neutralizing activity against pre-omicron variants, which were determined using the RBD-human ACE2 binding inhibition assay, reached the upper limit of detection. Additionally, our study only investigated spike-specific T cell responses, thus warranting further investigation to explore T cell responses against non-spike epitopes.
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