By Jiping Liu, et al.

Abstract

Large-scale COVID-19 vaccinations are currently underway in many countries in response to the COVID-19 pandemic. Here, we report, besides generation of neutralizing antibodies, consistent alterations in hemoglobin A1c, serum sodium and potassium levels, coagulation profiles, and renal functions in healthy volunteers after vaccination with an inactivated SARS-CoV-2 vaccine. Similar changes had also been reported in COVID-19 patients, suggesting that vaccination mimicked an infection. Single-cell mRNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) before and 28 days after the first inoculation also revealed consistent alterations in gene expression of many different immune cell types. Reduction of CD8+ T cells and increase in classic monocyte contents were exemplary. Moreover, scRNA-seq revealed increased NF-κB signaling and reduced type I interferon responses, which were confirmed by biological assays and also had been reported to occur after SARS-CoV-2 infection with aggravating symptoms. Altogether, our study recommends additional caution when vaccinating people with pre-existing clinical conditions, including diabetes, electrolyte imbalances, renal dysfunction, and coagulation disorders.

Introduction

The COVID-19 pandemic has profoundly affected humanity. The development of COVID-19 vaccines in various forms has been underway in an unprecedented and accelerated manner. Despite some uncertainties regarding potential consequences, large-scale vaccinations are taking place in many countries. There have been different COVID-19 vaccines developed, including inactivated viral particles, mRNA vaccines, adenoviral-based vaccines, and etc.1,2,3,4,5. Historically, vaccine research has been focused on whether or not vaccination could generate neutralizing antibodies to protect against viral infections, whereas short-term and long-term influences of the various newly developed vaccines to human pathophysiology and other perspectives of the human immune system have not been fully investigated.

With the development of large-scale single-cell mRNA sequencing (scRNA-seq) technology, systematic investigation of people’s immune system function with precision became possible, primarily through scRNA-seq of peripheral blood mononuclear cells (PBMCs). During the COVID-19 pandemic, a large body of studies using scRNA-seq of PBMCs had revealed detailed changes in gene expression in different immune cell subtypes including different types of T and B cells, NK cells, monocytes, dendritic cells, etc. during and after infection, results from which indicated greatly reduced CD4+ and CD8+ T-cell numbers and T-cell exhaustion upon SARS-CoV-2 infection. Reduced peripheral mucosal-associated invariable T (MAIT) cell numbers and their migration in and out of the lung had also been observed. Highly activated inflammatory immune responses, including Interferon-gamma (IFN-γ), interleukin-6 (IL-6), and NF-κB responses, had been reported in COVID-19 patients6,7,8,9,10,11,12. Many studies had revealed immune state differences between people with severe versus mild symptoms, in that strong type I interferon (IFN-α/β) responses were beneficial after COVID-19 infection and attenuated IFN-α/β responses were associated with the development of severe symptoms13. In contrast, stronger NF-κB inflammatory responses were associated with more severe symptoms14. In addition, increased γδ-T cell and reduced neutrophil contents were reported to be associated with milder symptoms15.

Upon SARS-CoV-2 infections, many people developed various degrees of respiratory syndromes, and some with gastrointestinal conditions. It had been reported that blood coagulation disorders, vasculature issues, electrolytes imbalances, renal disorders, metabolic disorders, etc. were major clinical complications with COVID-1916,17. The manner in which vaccination would mimic an infection has not been fully evaluated. In this study, we enrolled healthy volunteers who were to be vaccinated with an inactivated SARS-CoV-2 vaccine (Vero Cell)3, to participate in antibody and neutralizing antibody testings, as well as detailed clinical laboratory measurements before and at different times after vaccination (two-dose regimens with slightly different schedules were applied). To our surprise, we observed quite consistent pathophysiological changes regarding electrolyte contents, coagulation profiles, renal function as well as cholesterol and glucose metabolic-related features, as if these people had experienced an infection with SARS-CoV-2. In addition, PBMCs scRNA-seq results also indicated consistent reductions in CD8+ T cells and increases in monocyte contents, as well as enhanced NF-κB inflammatory signaling, which also mimicked responses after infection. Surprisingly, type I interferon responses, which had been linked to reduced damages after SARS-CoV-2 infection and milder symptoms, appeared to be reduced after vaccination, at least by 28 days post the 1st inoculation. This might suggest that in the short-term (1 month) after vaccination, a person’s immune system is in a non-privileged state, and may require more protection.

Results

Longitudinal follow-up of anti-SARS-CoV-2 antibody and neutralizing antibody productions after inoculation of inactivated SARS-CoV-2 vaccine

A total of 11 healthy adult volunteers of both sexes, aged 24–47 years, with a BMI of 21.5–30.0 kg/m², were enrolled in this study (Fig. 1a and Supplementary Tables S1 and S2). SARS-CoV-2 vaccine (Vero Cell), inactivated (Beijing Institute of Biological Products Co. Ltd), was administered intramuscularly into the deltoid. Volunteers were divided into two cohorts; five participants (cohort A) were vaccinated with a full dose (4 µg) of inactivated SARS-CoV-2 Vaccine (Vero Cell) on days 1 and 14, and six participants (cohort B) received a full dose of the vaccine on days 1 and 28 (Fig. 1a). One of the volunteers in group B was tested positive for anti-SARS-CoV-2 IgM and IgG right before vaccination, suggestive of potential prior infections. However, there was no record of previous positivity by nucleic acid (NA) diagnosis for COVID-19 (marked green in Fig. 1a). For all follow-up examinations, data from this individual was marked green to track any possible influences from potential prior infections.

figure1
Fig. 1: Schematic workflow and SARS-CoV-2 antibody/neutralizing antibody detection after vaccination.

Adverse events were monitored daily during the first 7 days after each inoculation and then self-recorded by the participants on diary cards in the following weeks. Overall, adverse reactions were mild (grades 1 or 2) and transient (Supplementary Table S3). Blood samples were collected on days 0, 7, 14, 28, 42, 56, and 90, and urine samples were collected on days 0, 14, 28, 42, and 90. Plasma samples were subjected to anti-SARS-CoV-2 IgM/IgG testing using multiple diagnostic kits, results from the most sensitive kit were used for quantification (Fig. 1b, c). Testing results from cohort A demonstrated that prior to the 2nd inoculation 0% of the participants developed anti-SARS-CoV-2 IgG, but by day 28, which was 2 weeks post the 2nd inoculation, 100% of the participants were tested positive (Fig. 1b). Overall, IgM showed up earlier than IgG, which was expected. IgG and IgM positivity decreased by day 42 and remained at relatively low levels by day 90 in cohort A. For cohort B, no one developed IgG until after 2nd inoculation. Yet by day 42, IgG positivity reached 100% (Fig. 1c) and sustained until day 56, suggesting that the vaccination protocol for cohort B was more efficacious. By day 90, IgG positivity also reduced to 50%, indicating antibody production did not sustain for a long time. We further carried out tests for SARS-CoV-2 neutralizing antibodies18 (Fig. 1d), and results also indicated that two inoculations 28 days apart (cohort B) resulted in higher protective antibody titers as compared to two inoculations with 14 days apart (cohort A). On the other hand, it appeared that anti-SARS-CoV-2 neutralizing antibody titers were overall lower than those in COVID-19 convalescent individuals as reported before3 (Fig. 1d). By 90 days, neutralizing antibody titers dramatically decreased in all volunteers (Fig. 1d). Interestingly, the individual who was antibody positive prior to vaccination was not more prone to generating neutralizing antibodies as compared to the rest of the participants, suggesting that prior potential infection might not have occurred or may not generate long-lasting protection in the perspective of neutralizing antibody production.

Alterations in clinical laboratory measurements after vaccination

Clinical laboratory routine tests including infection-related indices, hematologic parameters, coagulation function, blood glucose, serum lipids, cardiac function-related enzymes, electrolytes, liver, and renal function-related biomarkers, were measured to reveal safety features of the vaccine (Fig. 2a and Supplementary Tables S4 and S5). White blood cell count was significantly, yet only slightly, increased after vaccination on day 7. No differences were detectable at the following time points (Fig. 2b). To our surprise, quite consistent increases in HbA1c levels were observed in healthy volunteers, regardless of whether they belonged to cohort A or B. By day 28 post the 1st inoculation, three out of 11 individuals reached the prediabetic range (Fig. 2c). By days 42 and 90, medium HbA1c levels appeared to revert back, yet were still significantly higher than those before vaccination. Previous work has demonstrated that diabetic patients with uncontrolled blood glucose levels are more prone to develop severe forms of COVID-1919. High blood glucose levels/glycolysis had been shown to promote SARS-CoV-2 replication in human monocytes via the production of mitochondrial reactive oxygen species and activation of HIF1A20, therefore presenting a disadvantageous feature.

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