Coronavirus spike protein-specific antibodies indicate frequent infections and reinfections in infancy and among BNT162b2-vaccinated healthcare workers.

The prevalence of seasonal human coronavirus (HCoV) infections in early childhood and adults has not been well analyzed in longitudinal serological studies. Here we analyzed the changes in HCoV (229E, HKU1, NL63, OC43, MERS, and SARS-CoV-2) spike-specific antibody levels in follow-up serum specimens of 140 children at the age of 1, 2, and 3 years, and of 113 healthcare workers vaccinated for Covid-19 with BNT162b2-vaccine. IgG antibody levels against six recombinant HCoV spike subunit 1 (S1) proteins were measured by enzyme immunoassay. We show that by the age of three years the cumulative seropositivity for seasonal HCoVs increased to 38-81% depending on virus type. BNT162b2 vaccinations increased anti-SARS-CoV-2 S1 antibodies, but no increase in seasonal coronavirus antibodies associated with vaccinations. In healthcare workers (HCWs), during a 1-year follow-up, diagnostic antibody rises were seen in 5, 4 and 14% of the cases against 229E, NL63 and OC43 viruses, respectively, correlating well with the circulating HCoVs. In 6% of the HCWs, a diagnostic antibody rise was seen against S1 of HKU1, however, these rises coincided with anti-OC43 S1 antibody rises. Rabbit and guinea pig immune sera against HCoV S1 proteins indicated immunological cross-reactivity within alpha-CoV (229E and NL63) and beta-CoV (HKU1 and OC43) genera.

COVID-19 adenovirus vector vaccine induces higher interferon and pro-inflammatory responses than mRNA vaccines in human PBMCs, macrophages and moDCs

Background During the COVID-19 pandemic multiple vaccines were rapidly developed and widely used throughout the world. At present there is very little information on COVID-19 vaccine interactions with primary human immune cells such as peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages and dendritic cells (moDCs). Methods Human PBMCs, macrophages and moDCs were stimulated with different COVID-19 vaccines, and the expression of interferon (IFN-λ1, IFN-α1), pro-inflammatory (IL-1β, IL-6, IL-8, IL-18, CXCL-4, CXCL-10, TNF-α) and Th1-type cytokine mRNAs (IL-2, IFN-γ) were analyzed by qPCR. In addition, the expression of vaccine induced spike (S) protein and antiviral molecules were studied in primary immune cells and in A549 lung epithelial cells. Results Adenovirus vector (Ad-vector) vaccine AZD1222 induced high levels of IFN-λ1, IFN-α1, CXCL-10, IL-6, and TNF-α mRNAs in PBMCs at early time points of stimulation while the expression of IFN-γ and IL-2 mRNA took place at later times. AZD1222 also induced IFN-λ1, CXCL-10 and IL-6 mRNA expression in monocyte-derived macrophages and DCs in a dose-dependent fashion. AZD1222 also activated the phosphorylation of IRF3 and induced MxA expression. BNT162b2 and mRNA-1273 mRNA vaccines failed to induce or induced very weak cytokine gene expression in all cell models. None of the vaccines enhanced the expression of CXCL-4. AZD1222 and mRNA-1273 vaccines induced high expression of S protein in all studied cells. Conclusions Ad-vector vaccine induces higher IFN and pro-inflammatory responses than the mRNA vaccines in human immune cells. This data shows that AZD1222 readily activates IFN and pro-inflammatory cytokine gene expression in PBMCs, macrophages and DCs, but fails to further enhance CXCL-4 mRNA expression.

Persistent T cell-mediated immune responses against Omicron variants after the third COVID-19 mRNA vaccine dose.

Introduction: The prime-boost COVID-19 mRNA vaccination strategy has proven to be effective against severe COVID-19 disease and death. However, concerns have been raised due to decreasing neutralizing antibody levels after COVID-19 vaccination and due to the emergence of new immuno-evasive SARS-CoV-2 variants that may require additional booster vaccinations. Methods: In this study, we analyzed the humoral and cell-mediated immune responses against the Omicron BA.1 and BA.2 subvariants in Finnish healthcare workers (HCWs) vaccinated with three doses of COVID-19 mRNA vaccines. We used enzyme immunoassay and microneutralization test to analyze the levels of SARS-CoV-2 specific IgG antibodies in the sera of the vaccinees and the in vitro neutralization capacity of the sera. Activation induced marker assay together with flow cytometry and extracellular cytokine analysis was used to determine responses in SARS-CoV-2 spike protein stimulated PBMCs. Results: Here we show that within the HCWs, the third mRNA vaccine dose recalls both humoral and T cell-mediated immune responses and induces high levels of neutralizing antibodies against Omicron BA.1 and BA.2 variants. Three weeks after the third vaccine dose, SARS-CoV-2 wild type spike protein-specific CD4+ and CD8+ T cells are observed in 82% and 71% of HCWs, respectively, and the T cells cross-recognize both Omicron BA.1 and BA.2 spike peptides. Although the levels of neutralizing antibodies against Omicron BA.1 and BA.2 decline 2.5 to 3.8-fold three months after the third dose, memory CD4+ T cell responses are maintained for at least eight months post the second dose and three months post the third vaccine dose. Discussion: We show that after the administration of the third mRNA vaccine dose the levels of both humoral and cell-mediated immune responses are effectively activated, and the levels of the spike-specific antibodies are further elevated compared to the levels after the second vaccine dose. Even though at three months after the third vaccine dose antibody levels in sera decrease at a similar rate as after the second vaccine dose, the levels of spike-specific CD4+ and CD8+ T cells remain relatively stable. Additionally, the T cells retain efficiency in cross-recognizing spike protein peptide pools derived from Omicron BA.1 and BA.2 subvariants. Altogether our results suggest durable cellmediated immunity and protection against SARS-CoV-2.

Comparative analysis of COVID-19 vaccine responses and third booster dose-induced neutralizing antibodies against Delta and Omicron variants.

Two COVID-19 mRNA (of BNT162b2, mRNA-1273) and two adenovirus vector vaccines (ChAdOx1 and Janssen) are licensed in Europe, but optimization of regime and dosing is still ongoing. Here we show in health care workers (n = 328) that two doses of BNT162b2, mRNA-1273, or a combination of ChAdOx1 adenovirus vector and mRNA vaccines administrated with a long 12-week dose interval induce equally high levels of anti-SARS-CoV-2 spike antibodies and neutralizing antibodies against D614 and Delta variant. By contrast, two doses of BNT162b2 with a short 3-week interval induce 2-3-fold lower titers of neutralizing antibodies than those from the 12-week interval, yet a third BNT162b2 or mRNA-1273 booster dose increases the antibody levels 4-fold compared to the levels after the second dose, as well as induces neutralizing antibody against Omicron BA.1 variant. Our data thus indicates that a third COVID-19 mRNA vaccine may induce cross-protective neutralizing antibodies against multiple variants.

Serological Follow-Up Study Indicates High Seasonal Coronavirus Infection and Reinfection Rates in Early Childhood.

Seasonal human coronaviruses (HCoVs) cause respiratory infections, especially in children. Currently, the knowledge on early childhood seasonal coronavirus infections and the duration of antibody levels following the first infections is limited. Here we analyzed serological follow-up samples to estimate the rate of primary infection and reinfection(s) caused by seasonal coronaviruses in early childhood. Serum specimens were collected from 140 children at ages of 13, 24, and 36 months (1, 2, and 3 years), and IgG antibody levels against recombinant HCoV nucleoproteins (N) were measured by enzyme immunoassay (EIA). Altogether, 84% (118/140) of the children were seropositive for at least one seasonal coronavirus N by the age of 3 years. Cumulative seroprevalences for HCoVs 229E, HKU1, NL63, and OC43 increased by age, and they were 45%, 27%, 70%, and 44%, respectively, at the age of 3 years. Increased antibody levels between yearly samples indicated reinfections by 229E, NL63, and OC43 viruses in 20-48% of previously seropositive children by the age of 3 years. Antibody levels declined 54-73% or 31-77% during the year after seropositivity in children initially seropositive at 1 or 2 years of age, respectively, in case there was no reinfection. The correlation of 229E and NL63, and OC43 and HKU1 EIA results, suggested potential cross-reactivity between the N specific antibodies inside the coronavirus genera. The data shows that seasonal coronavirus infections and reinfections are common in early childhood and the antibody levels decline relatively rapidly. IMPORTANCE The rapid spread of COVID-19 requires better knowledge on the rate of coronavirus infections and coronavirus specific antibody responses in different population groups. In this work we analyzed changes in seasonal human coronavirus specific antibodies in young children participating in a prospective 3-year serological follow-up study. We show that based on seropositivity and changes in serum coronavirus antibody levels, coronavirus infections and reinfections are common in early childhood and the antibodies elicited by the infection decline relatively rapidly. These observations provide further information on the characteristics of humoral immune responses of coronavirus infections in children.

Vaccine-Induced Antibody Responses against SARS-CoV-2 Variants-Of-Concern Six Months after the BNT162b2 COVID-19 mRNA Vaccination.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concern about increased transmissibility, infectivity, and immune evasion from a vaccine and infection-induced immune responses. Although COVID-19 mRNA vaccines have proven to be highly effective against severe COVID-19 disease, the decrease in vaccine efficacy against emerged Beta and Delta variants emphasizes the need for constant monitoring of new virus lineages and studies on the persistence of vaccine-induced neutralizing antibodies. To analyze the dynamics of COVID-19 mRNA vaccine-induced antibody responses, we followed 52 health care workers in Finland for 6 months after receiving two doses of BNT162b2 vaccine with a 3-week interval. We demonstrate that, although anti-S1 antibody levels decrease 2.3-fold compared to peak antibody levels, anti-SARS-CoV-2 antibodies persist for months after BNT162b2 vaccination. Variants D614G, Alpha, and Eta are neutralized by sera of 100% of vaccinees, whereas neutralization of Delta is 3.8-fold reduced and neutralization of Beta is 5.8-fold reduced compared to D614G. Despite this reduction, 85% of sera collected 6 months postvaccination neutralizes Delta variant. IMPORTANCE A decrease in vaccine efficacy against emerging SARS-CoV-2 variants has increased the importance of assessing the persistence of SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies. Our data show that after 6 months post two doses of BNT162b2 vaccine, antibody levels decrease yet remain detectable and capable of neutralizing emerging variants. By monitoring the vaccine-induced antibody responses, vaccination strategies and administration of booster doses can be optimized.

SARS-CoV-2 Isolates Show Impaired Replication in Human Immune Cells but Differential Ability to Replicate and Induce Innate Immunity in Lung Epithelial Cells.

The primary target organ of coronavirus disease 2019 (COVID-19) infection is the respiratory tract. Currently, there is limited information on the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect and regulate innate immunity in human immune cells and lung epithelial cells. Here, we compared the ability of four Finnish isolates of SARS-CoV-2 from COVID-19 patients to replicate and induce interferons (IFNs) and other cytokines in different human cells. All isolates failed to replicate in dendritic cells, macrophages, monocytes, and lymphocytes, and no induction of cytokine gene expression was seen. However, most of the isolates replicated in Calu-3 cells, and they readily induced type I and type III IFN gene expression. The hCoV-19/Finland/FIN-25/2020 isolate, originating from a traveler from Milan in March 2020, showed better ability to replicate and induce IFN and inflammatory responses in Calu-3 cells than other isolates of SARS-CoV-2. Our data increase the knowledge on the pathogenesis and antiviral mechanisms of SARS-CoV-2 infection in human cell systems. IMPORTANCE With the rapid spread of the coronavirus disease 2019 (COVID-19) pandemic, information on the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and regulation of innate immunity in human immune cells and lung epithelial cells is needed. In the present study, we show that SARS-CoV-2 failed to productively infect human immune cells, but different isolates of SARS-CoV-2 showed differential ability to replicate and regulate innate interferon responses in human lung epithelial Calu-3 cells. These findings will open up the way for further studies on the mechanisms of pathogenesis of SARS-CoV-2 in human cells.

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants.

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n = 180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the seronegative vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees' neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides evidence that the second dose of the BNT162b2 vaccine induces cross-neutralization of at least some of the circulating SARS-CoV-2 variants.

A Combination of N and S Antigens With IgA and IgG Measurement Strengthens the Accuracy of SARS-CoV-2 Serodiagnostics.

BACKGROUND: Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. METHODS: We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. RESULTS: The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. CONCLUSIONS: The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.