Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

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.

Strong Neutralizing Antibody Responses to SARS-CoV-2 Variants Following a Single Vaccine Dose in Subjects With Previous SARS-CoV-2 Infection.

Background: Previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primes the immune system; thus individuals who have recovered from infection have enhanced immune responses to subsequent vaccination (hybrid immunity). However, it remains unclear how well hybrid immunity induced by severe or mild infection can cross-neutralize emerging variants. We aimed to compare the strength and breadth of antibody responses in vaccinated recovered and uninfected subjects. Methods: We measured spike-specific immunoglobulin (Ig)G and neutralizing antibodies (NAbs) from vaccinated subjects including 320 with hybrid immunity and 20 without previous infection. From 29 subjects with a previous severe or mild infection, we also measured NAb responses against Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529/BA.1) variants following vaccination. Results: A single vaccine dose induced 2-fold higher anti-spike IgG concentrations and up to 4-fold higher neutralizing potency of antibodies in subjects with a previous infection compared with vaccinated subjects without a previous infection. Hybrid immunity was more enhanced after a severe than a mild infection, with sequentially decreasing NAb titers against Alpha, Beta, Delta, and Omicron variants. We found similar IgG concentrations in subjects with a previous infection after 1 or 2 vaccine doses. Conclusions: Hybrid immunity induced strong IgG responses, particularly after severe infection. However, the NAb titers were low against heterologous variants, especially against Omicron.

Impaired immunity and high attack rates caused by SARS-CoV-2 variants among vaccinated long-term care facility residents.

INTRODUCTION: Long-term care facilities (LTCF) residents are at high risk for severe coronavirus disease 2019 (COVID-19), and therefore, COVID-19 vaccinations were prioritized for residents and personnel in Finland at the beginning of 2021. METHODS: We investigated COVID-19 outbreaks in two LTCFs, where residents were once or twice vaccinated. After the outbreaks we measured immunoglobulin G (IgG) antibodies to severe acute respiratory syndrome coronavirus 2 spike glycoprotein, neutralizing antibody (NAb) titers, and cell-mediated immunity markers from residents and healthcare workers (HCWs). RESULTS: In LTFC-1, the outbreak was caused by an Alpha variant (B.1.1.7) and the attack rate (AR) among once vaccinated residents was 23%. In LTCF-2 the outbreak was caused by a Beta variant (B.1.351). Its AR was 47% although all residents had received their second dose 1 month before the outbreak. We observed that vaccination had induced lower IgG concentrations, NAb titers and cell-mediated immune responses in residents compared to HCWs. Only 1/8 residents had NAb to the Beta variant after two vaccine doses. CONCLUSIONS: The vaccinated elderly remain susceptible to breakthrough infections caused by Alpha and Beta variants. The weaker vaccine response in the elderly needs to be addressed in vaccination protocols, while new variants capable of evading vaccine-induced immunity continue to emerge.

Impaired immunity and high attack rates caused by SARS‐CoV‐2 variants among vaccinated long‐term care facility residents

Introduction Long‐term care facilities (LTCF) residents are at high risk for severe coronavirus disease 2019 (COVID‐19), and therefore, COVID‐19 vaccinations were prioritized for residents and personnel in Finland at the beginning of 2021. Methods We investigated COVID‐19 outbreaks in two LTCFs, where residents were once or twice vaccinated. After the outbreaks we measured immunoglobulin G (IgG) antibodies to severe acute respiratory syndrome coronavirus 2 spike glycoprotein, neutralizing antibody (NAb) titers, and cell‐mediated immunity markers from residents and healthcare workers (HCWs). Results In LTFC‐1, the outbreak was caused by an Alpha variant (B.1.1.7) and the attack rate (AR) among once vaccinated residents was 23%. In LTCF‐2 the outbreak was caused by a Beta variant (B.1.351). Its AR was 47% although all residents had received their second dose 1 month before the outbreak. We observed that vaccination had induced lower IgG concentrations, NAb titers and cell‐mediated immune responses in residents compared to HCWs. Only 1/8 residents had NAb to the Beta variant after two vaccine doses. Conclusions The vaccinated elderly remain susceptible to breakthrough infections caused by Alpha and Beta variants. The weaker vaccine response in the elderly needs to be addressed in vaccination protocols, while new variants capable of evading vaccine‐induced immunity continue to emerge. IgG levels to WT SARS‐CoV‐2 spike glycoprotein after vaccination with Comirnaty. (A) Receptor binding domain (RBD) and (B) full‐length spike glycoprotein (SFL) in binding antibody units (BAU)/ml. Dashed lines mark the threshold for positive result per antigen. Statistical significance measured with Wilcoxon rank‐sum test, significance level 0.05.

High secondary attack rate and persistence of SARS-CoV-2 antibodies in household transmission study participants, Finland 2020-2021.

Background: Household transmission studies offer the opportunity to assess both secondary attack rate (SAR) and persistence of SARS-CoV-2 antibodies over time. Methods: In Spring 2020, we invited confirmed COVID-19 cases and their household members to four visits, where we collected nasopharyngeal and serum samples over 28 days after index case onset. We calculated SAR based on the presence of SARS-CoV-2 neutralizing antibodies (NAb) and assessed the persistence of NAb and IgG antibodies (Ab) against SARS-CoV-2 spike glycoprotein and nucleoprotein. Results: SAR was 45% (39/87), including 35 symptomatic secondary cases. During the initial 28-day follow-up, 62% (80/129) of participants developed NAb. Of those that seroconverted, 90% (63/70), 85% (63/74), and 78% (45/58) still had NAb to early B-lineage SARS-CoV-2 3, 6, and 12 months after the onset of the index case. Anti-spike IgG Ab persisted in 100% (69/69), 97% (72/74), and 93% (55/59) of seroconverted participants after 3, 6, and 12 months, while anti-nucleoprotein IgG Ab levels waned faster, persisting in 99% (68/69), 78% (58/74), and 55% (39/71) of participants, respectively. Conclusion: Following detection of a COVID-19 case in a household, other members had a high risk of becoming infected. NAb to early B-lineage SARS-CoV-2 persisted for at least a year in most cases.

Neutralizing antibodies to SARS-CoV-2 Omicron variant after third mRNA vaccination in health care workers and elderly subjects.

The emergence of SARS-CoV-2 Omicron variant (B.1.1.529) with major spike protein mutations has raised concern over potential neutralization escape and breakthrough infections among vaccinated and previously SARS-CoV-2-infected subjects. We measured cross-protective antibodies against variants in health care workers (HCW, n = 20) and nursing home residents (n = 9) from samples collected at 1-2 months, following the booster (3rd) dose. We also assessed the antibody responses in subjects infected before the Omicron era (n = 38) with subsequent administration of a single mRNA vaccine dose. Following booster vaccination, HCWs had high IgG antibody concentrations to the spike protein and neutralizing antibodies (NAb) were detectable against all variants. IgG concentrations among the elderly remained lower, and some lacked NAbs against the Beta and Omicron variants. NAb titers were significantly reduced against Delta, Beta, and Omicron compared to WT virus regardless of age. Vaccination induced high IgG concentrations and variable titers of cross-reactive NAbs in previously infected subjects, whereas NAb titers against Omicron were barely detectable 1 month postinfection. High IgG concentrations with cross-protective neutralizing activity were detected after three Coronavirus Disease 2019 (COVID-19) vaccine doses in HCWs. However, lower NAb titers seen in the frail elderly suggest inadequate protection against Omicron breakthrough infections, yet protection against severe COVID-19 is expected.

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.

A Highly Sensitive and Specific SARS-CoV-2 Spike- and Nucleoprotein-Based Fluorescent Multiplex Immunoassay (FMIA) to Measure IgG, IgA, and IgM Class Antibodies.

Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organization (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralization test (MNT). We also compared the MNT results of two laboratories. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13 to 150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA, 100% specificity and sensitivity were obtained for a shorter time window (13 to 36 and 13 to 28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralizing antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ = 0.77 to 0.84, P < 2.2 × 10-16) with NAb titers, and the two laboratories' NAb titers displayed a very strong correlation (ρ = 0.95, P < 2.2 × 10-16). Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against the WHO international standard did not, however, improve the comparability of FMIA and EIA results. IMPORTANCE SARS-CoV-2 serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers. Neutralization tests are still considered the gold standard for SARS-CoV-2 serological assays, but our assay can identify samples with neutralizing antibodies with 100% sensitivity and 96% specificity without the need for laborious and slow biosafety level 3 (BSL-3) facility-requiring analyses.

Clinical validation of automated and rapid mariPOC SARS-CoV-2 antigen test.

COVID-19 diagnostics was quickly ramped up worldwide early 2020 based on the detection of viral RNA. However, based on the scientific knowledge for pre-existing coronaviruses, it was expected that the SARS-CoV-2 RNA will be detected from symptomatic and at significant rates also from asymptomatic individuals due to persistence of non-infectious RNA. To increase the efficacy of diagnostics, surveillance, screening and pandemic control, rapid methods, such as antigen tests, are needed for decentralized testing and to assess infectiousness. A novel automated mariPOC SARS-CoV-2 test was developed for the detection of conserved structural viral nucleocapsid proteins. The test utilizes sophisticated optical laser technology for two-photon excitation and individual detection of immunoassay solid-phase particles. We validated the new method against qRT-PCR. Sensitivity of the test was 100.0% (13/13) directly from nasopharyngeal swab specimens and 84.4% (38/45) from swab specimens in undefined transport mediums. Specificity of the test was 100.0% (201/201). The test's limit of detection was 2.7 TCID50/test. It showed no cross-reactions. Our study shows that the new test can detect infectious individuals already in 20 min with clinical sensitivity close to qRT-PCR. The mariPOC is a versatile platform for syndromic testing and for high capacity infection control screening of infectious individuals.

Persistence of neutralizing antibodies a year after SARS-CoV-2 infection in humans.

Most subjects develop antibodies to SARS-CoV-2 following infection. In order to estimate the duration of immunity induced by SARS-CoV-2 it is important to understand for how long antibodies persist after infection in humans. Here, we assessed the persistence of serum antibodies following WT SARS-CoV-2 infection at 8 and 13 months after diagnosis in 367 individuals. The SARS-CoV-2 spike IgG (S-IgG) and nucleoprotein IgG (N-IgG) concentrations and the proportion of subjects with neutralizing antibodies (NAb) were assessed. Moreover, the NAb titers among a smaller subset of participants (n = 78) against a WT virus (B) and variants of concern (VOCs): Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) were determined. We found that NAb against the WT virus persisted in 89% and S-IgG in 97% of subjects for at least 13 months after infection. Only 36% had N-IgG by 13 months. The mean S-IgG concentrations declined from 8 to 13 months by less than one third; N-IgG concentrations declined by two-thirds. Subjects with severe infection had markedly higher IgG and NAb levels and are expected to remain seropositive for longer. Significantly lower NAb titers against the variants compared to the WT virus, especially after a mild disease, suggests reduced protection against VOCs.

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.

Comparison of the clinical characteristics and outcomes of hospitalized adult COVID-19 and influenza patients - a prospective observational study.

BACKGROUND: We compared the clinical characteristics, findings, and outcomes of hospitalized patients with coronavirus disease 2019 (COVID-19) or influenza to detect relevant differences. METHODS: From December 2019 to April 2020, we recruited all eligible hospitalized adults with respiratory infection to a prospective observational study at a tertiary care hospital in Finland. Influenza and SARS-CoV-2 infections were confirmed by RT-PCR. Follow-up lasted for 3 months from admission. RESULTS: We included 61 patients, of whom 28 were COVID-19 and 33 influenza patients with median ages of 53 and 56 years. Majority of both COVID-19 and influenza patients were men (61% vs. 67%) and had at least one comorbidity (68% vs. 85%). Pulmonary diseases and current smoking were less common among COVID-19 than influenza patients (5 [18%] vs. 15 [45%], p=.03 and 1 [4%] vs. 10 [30%], p=.008). In chest X-ray at admission, ground-glass opacities (GGOs) and consolidations were more frequent among COVID-19 than influenza patients (19 [68%] and 7 [21%], p<.001). Severe disease and intensive care unit (ICU) admission occurred more often among COVID-19 than influenza patients (26 [93%] vs. 19 [58%], p=.003 and 8 [29%] vs. 2 [6%], p=.034). COVID-19 patients were hospitalized longer than influenza patients (six days [IQR 4-21] vs. 3 [2-4], p<.001). CONCLUSIONS: Bilateral GGOs and consolidations in chest X-ray may help to differentiate COVID-19 from influenza. Hospitalized COVID-19 patients had more severe disease, required longer hospitalization and were admitted to ICU more often than influenza patients, which has important implications for public health policies.

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020.

The first case of coronavirus disease (COVID-19) in Finland was confirmed on 29 January 2020. No secondary cases were detected. We describe the clinical picture and laboratory findings 3-23 days since the first symptoms. The SARS-CoV-2/Finland/1/2020 virus strain was isolated, the genome showing a single nucleotide substitution to the reference strain from Wuhan. Neutralising antibody response appeared within 9 days along with specific IgM and IgG response, targeting particularly nucleocapsid and spike proteins.