Epitopes Displayed in a Cyclic Peptide Scaffold Bind SARS-COV-2 Antibodies.

The SARS-CoV-2 virus that causes COVID-19 is a global health issue. The spread of the virus has resulted in seven million deaths to date. The emergence of new viral strains highlights the importance of continuous surveillance of the SARS-CoV-2 virus by using timely and accurate diagnostic tools. Here, we used a stable cyclic peptide scaffolds to present antigenic sequences derived from the spike protein that are reactive to SARS-CoV-2 antibodies. Using peptide sequences from different domains of SARS-CoV-2 spike proteins, we grafted epitopes on the peptide scaffold sunflower trypsin inhibitor 1 (SFTI-1). These scaffold peptides were then used to develop an ELISA to detect SARS-CoV-2 antibodies in serum. We show that displaying epitopes on the scaffold improves reactivity overall. One of the scaffold peptides (S2_1146-1161_c) has reactivity equal to that of commercial assays, and shows diagnostic potential.

Correlates of protection and viral load trajectories in omicron breakthrough infections in triple vaccinated healthcare workers.

Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.

Factors Associated With Serological Response to SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis Treated With Rituximab.

Importance: B-cell-depleting monoclonal antibodies are widely used for treatment of multiple sclerosis but are associated with an impaired response to vaccines. Objective: To identify factors associated with a favorable vaccine response to tozinameran. Design, Setting, and Participants: This prospective cohort study was conducted in a specialized multiple sclerosis clinic at a university hospital from January 21 to December 1, 2021. Of 75 patients evaluated for participation who received a diagnosis of multiple sclerosis with planned or ongoing treatment with rituximab, 69 were included in the study, and data from 67 were analyzed. Exposures: Sex, age, number of previous rituximab infusions, accumulated dose of rituximab, previous COVID-19 infection, time since last rituximab treatment, CD19+ B-cell count before vaccination, CD4+ T-cell count, and CD8+ T-cell count were considered potential factors associated with the main outcome. Main Outcomes and Measures: Serological vaccine responses were measured by quantitation of anti-spike immunoglobulin G (IgG) antibodies, anti-receptor-binding domain (RBD) IgG antibodies, and their neutralizing capacities. Cellular responses to spike protein-derived SARS-CoV-2 peptide pools were assessed by counting interferon gamma spot-forming units in a FluoroSpot assay. Results: Among 60 patients with ongoing rituximab treatment (49 women [82%]; mean (SD) age, 43 [10] years), the median (range) disease duration was 9 (1-29) years, and the median (range) dose of rituximab was 2750 (500-10 000) mg during a median (range) time of 2.8 (0.5-8.3) years. The median (range) follow-up from the first vaccination dose was 7.3 (4.3-10.0) months. Vaccine responses were determined before vaccination with tozinameran and 6 weeks after vaccination. By using established cutoff values for anti-spike IgG (264 binding antibody units/mL) and anti-RBD IgG (506 binding antibody units/mL), the proportion of patients with a positive response increased with the number of B cells, which was the only factor associated with these outcomes. A cutoff for the B-cell count of at least 40/µL was associated with an optimal serological response. At this cutoff, 26 of 29 patients (90%) had positive test results for anti-spike IgG and 21 of 29 patients (72%) for anti-RBD IgG, and 27 of 29 patients (93%) developed antibodies with greater than 90% inhibition of angiotensin-converting enzyme 2. No factor associated with the cellular response was identified. Depending on the peptide pool, 21 of 25 patients (84%) to 22 of 25 patients (88%) developed a T-cell response with interferon gamma production at the B-cell count cutoff of at least 40/µL. Conclusions and Relevance: This cohort study found that for an optimal vaccine response from tozinameran, rituximab-treated patients with multiple sclerosis may be vaccinated as soon as possible, with rituximab treatment delayed until B-cell counts have reached at least 40/µL. An additional vaccination with tozinameran should be considered at that point.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BACKGROUND: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. METHODS: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFNγ) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. RESULTS: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFNγ and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFNγ in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. CONCLUSION: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

Duration of SARS-CoV-2 Immune Responses Up to Six Months Following Homologous or Heterologous Primary Immunization with ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccines.

Heterologous primary immunization against SARS-CoV-2 is part of applied recommendations. However, little is known about duration of immune responses after heterologous vaccine regimens. To evaluate duration of immune responses after primary vaccination with homologous adeno-vectored ChAdOx1 nCoV-19 vaccine (ChAd) or heterologous ChAd/BNT162b2 mRNA vaccine (BNT), anti-spike-IgG and SARS-CoV-2 VOC-neutralizing antibody responses were measured in 354 healthcare workers (HCW) at 2 weeks, 3 months, 5 months and 6 months after the second vaccine dose. T-cell responses were investigated using a whole blood interferon gamma (IFN-γ) release assay 2 weeks and 3 months post second vaccine dose. Two hundred and ten HCW immunized with homologous BNT were enrolled for comparison of antibody responses. In study participants naïve to SARS-CoV-2 prior to vaccination, heterologous ChAd/BNT resulted in 6-fold higher peak anti-spike IgG antibody titers compared to homologous ChAd vaccination. The half-life of antibody titers was 3.1 months (95% CI 2.8-3.6) following homologous ChAd vaccination and 1.9 months (95% CI 1.7-2.1) after heterologous vaccination, reducing the GMT difference between the groups to 3-fold 6 months post vaccination. Peak T-cell responses were stronger in ChAd/BNT vaccinees, but no significant difference was observed 3 months post vaccination. SARS-CoV-2 infection prior to vaccination resulted in substantially higher peak GMTs and IFN-γ levels and enhanced SARS-CoV-2 specific antibody and T cell responses over time. Heterologous primary SARS-CoV-2 immunization with ChAd and BNT elicits a stronger initial immune response compared to homologous vaccination with ChAd. However, although the differences in humoral responses remain over 6 months, the difference in SARS-CoV-2 specific T cell responses are no longer significant three months after vaccination.

SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection.

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10-23 and 2*10-13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys.

An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses.

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.

Robust humoral and cellular immune responses and low risk for reinfection at least 8 months following asymptomatic to mild COVID-19.

BACKGROUND: Emerging data support detectable immune responses for months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination, but it is not yet established to what degree and for how long protection against reinfection lasts. METHODS: We investigated SARS-CoV-2-specific humoral and cellular immune responses more than 8 months post-asymptomatic, mild and severe infection in a cohort of 1884 healthcare workers (HCW) and 51 hospitalized COVID-19 patients. Possible protection against SARS-CoV-2 reinfection was analyzed by a weekly 3-month polymerase chain reaction (PCR) screening of 252 HCW that had seroconverted 7 months prior to start of screening and 48 HCW that had remained seronegative at multiple time points. RESULTS: All COVID-19 patients and 96% (355/370) of HCW who were anti-spike IgG positive at inclusion remained anti-spike IgG positive at the 8-month follow-up. Circulating SARS-CoV-2-specific memory T cell responses were detected in 88% (45/51) of COVID-19 patients and in 63% (233/370) of seropositive HCW. The cumulative incidence of PCR-confirmed SARS-CoV-2 infection was 1% (3/252) among anti-spike IgG positive HCW (0.13 cases per 100 weeks at risk) compared to 23% (11/48) among anti-spike IgG negative HCW (2.78 cases per 100 weeks at risk), resulting in a protective effect of 95.2% (95% CI 81.9%-99.1%). CONCLUSIONS: The vast majority of anti-spike IgG positive individuals remain anti-spike IgG positive for at least 8 months regardless of initial COVID-19 disease severity. The presence of anti-spike IgG antibodies is associated with a substantially reduced risk of reinfection up to 9 months following asymptomatic to mild COVID-19.

Antibody responses after a single dose of ChAdOx1 nCoV-19 vaccine in healthcare workers previously infected with SARS-CoV-2.

BACKGROUND: Recent reports demonstrate robust serological responses to a single dose of messenger RNA (mRNA) vaccines in individuals previously infected with SARS-CoV-2. Data on immune responses following a single-dose adenovirus-vectored vaccine expressing the SARS-CoV-2 spike protein (ChAdOx1 nCoV-19) in individuals with previous SARS-CoV-2 infection are however limited, and current guidelines recommend a two-dose regimen regardless of preexisting immunity. METHODS: We compared RBD-specific IgG and RBD-ACE2 blocking antibodies against SARS-CoV-2 wild type and variants of concern following two doses of the mRNA vaccine BNT162b2 in SARS-CoV-2 naïve healthcare workers (n=65) and a single dose of the adenovector vaccine ChAdOx1 nCoV-19 in 82 healthcare workers more than (n=45) and less than (n=37) 11 months post mild SARS-CoV-2 infection at time of vaccination. FINDINGS: The post-vaccine levels of RBD-specific IgG and neutralizing antibodies against the SARS-CoV-2 wild type and variants of concern including Delta lineage 1.617.2 were similar or higher in participants receiving a single dose of ChAdOx1 nCoV-19 vaccine post SARS-CoV-2 infection (both more than and less than 11 months post infection) compared to SARS-CoV-2 naïve participants who received two doses of BNT162b2 vaccine. INTERPRETATION: Our data support that a single dose ChAdOx1 nCoV-19 vaccine that is administered up to at least 11 months post SARS-CoV-2 infection serves as an effective immune booster. This provides a possible rationale for a single-dose vaccine regimen. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Reactive oxygen species as an initiator of toxic innate immune responses in retort to SARS-CoV-2 in an ageing population, consider N-acetylcysteine as early therapeutic intervention.

During the current COVID-19 pandemic, a need for evaluation of already available drugs for treatment of the disease is crucial. Hereby, based on literature review from the current pandemic and previous outbreaks with corona viruses we analyze the impact of the virus infection on cell stress responses and redox balance. High levels of mortality are noticed in elderly individuals infected with SARS-CoV2 and during the previous SARS-CoV1 outbreak. Elderly individuals maintain a chronic low level of inflammation which is associated with oxidative stress and inflammatory cytokine production, a condition that increases the severity of viral infections in this population. Coronavirus infections can lead to alterations of redox balance in infected cells through modulation of NAD + biosynthesis, PARP function along with altering proteasome and mitochondrial function in the cell thereby leading to enhanced cell stress responses which further exacerbate inflammation. ROS production can increase IL-6 production and lipid peroxidation resulting in cell damage. Therefore, early treatment with anti-oxidants such as NAC during COVID-19 can be a way to bypass the excessive inflammation and cell damage that lead to severe infection, thus early NAC as intervention should be evaluated in a clinical trial setting.