Seroprevalence of SARS-CoV-2 and humoral immune responses to mRNA vaccines among people who use drugs - In the light of tailored mitigating strategies (preprint)

Background People who use drugs (PWUD) have increased risk of acquiring SARS-CoV-2 and having severe courses of COVID-19. However, during the first wave of the pandemic, surprisingly few PWUD tested positive for SARS-CoV-2 in Oslo. Aims: To investigate the seroprevalence of SARS-CoV-2, the antibody responses to virus infections and SARS-CoV-2 vaccines, and the vaccination rate among PWUD compared to the general population. Methods: Design: A prospective cohort study. Setting: Data was collected from residents at six institutions for homeless PWUD and users of a low-threshold clinic for opioid agonist treatment. Data was collected at baseline (N=99) and follow-up (N=25) and consisted of questionnaires and blood samples. Data on vaccination was collected from the National Vaccine Register. Serologic methods included detection of antibodies to different virus proteins, detection of neutralizing antibodies to SARS-CoV-2, and antibodies to Spike-FL, receptor-binding domain of the Spike protein and nucleocapsid from SARS-CoV-2. Results Antibodies to SARS-CoV-2 were detected in 4/99 samples from PWUD in the months before vaccines were available. The corresponding frequency for population-based screening was 2.8%.  The levels of serum antibodies to seasonal coronaviruses and EBV in PWUD, were also similar to those measured in population-based screening. The levels of binding and neutralizing antibodies to SARS-CoV-2 measured in samples obtained from PWUD (N=25) after the second vaccine dose were comparable to those observed in healthy controls. Concerning humoral immune responses to COVID-19 vaccination, there was no difference between PWUD and healthy individuals. Eighty-four and eighty-nine per cent had received at least one dose of corona vaccine among PWUD and the general population, respectively. Conclusion Results showed that PWUD did not have increased seroprevalence of SARS-CoV-2 and did not have increased serum antibodies to seasonal coronaviruses and EBV. Vaccine responses were not different from controls demonstrating that vaccination is a viable strategy to confer protection against SARS-CoV-2 in PWUD

Prevalence and predictors of long COVID among non-hospitalised adolescents and young adults: a prospective controlled cohort study (preprint)

The prevalence and predictors of long COVID in young people remain unresolved. We aimed to determine the point prevalence of long COVID in non-hospitalised adolescents and young adults six months after the acute infection, to determine the risk of developing long COVID adjusted for possible confounders, and to explore a broad range of potential risk factors (prespecified outcomes). We conducted a prospective controlled cohort study of 404 SARS-CoV-2-positive and 105 SARS-CoV-2-negative non-hospitalised individuals aged 12–25 years (ClinicalTrial ID: NCT04686734). Data acquisition was completed February 2022. Assessments included pulmonary, cardiac and cognitive functional testing, biomarker analyses, and completion of a questionnaire, and were performed at inclusion (early convalescent stage) and six months follow-up. The WHO case definition of long COVID was applied. The point prevalence of long COVID at six months was 49% and 47% in the SARS-CoV-2-positive and negative group, respectively. SARS-CoV-2-positivity did not predict development of long COVID (relative risk 1.06, 95% CI 0.83 to 1.37). The main predictor was symptom severity at inclusion, which correlated strongly to personality traits. Low physical activity and loneliness were also predictive, while biological markers were not. In conlusion, our study aims were met, and the findings suggest that persistent symptoms were not driven by the infection, but were associated with psychosocial factors.

T cell responses to SARS-CoV-2 vaccination in people with multiple sclerosis differ between disease-modifying therapies (preprint)

Immune responses in people with multiple sclerosis (pwMS) on disease-modifying therapies (DMTs) have been of significant interest throughout the COVID-19 pandemic. Lymphocyte-targeting immunotherapies including anti-CD20 treatments and sphingosine-1-phosphate receptor (S1PR) modulators attenuate antibody responses after vaccination. Evaluation of cellular responses after vaccination is therefore of particular importance in these populations. In this study, we analysed CD4 and CD8 T cell functional responses to SARS-CoV-2 spike peptides in healthy controls and pwMS on five different DMTs by flow cytometry. Although pwMS on anti-CD20 and S1PR therapies had low antibody responses after both 2 and 3 vaccine doses, T cell responses in pwMS on anti-CD20 therapies were preserved after third vaccination, even when additional anti-CD20 treatment was administered between vaccine doses 2 and 3. PwMS taking S1PR modulators had low detectable T cell responses in peripheral blood. CD4 and CD8 T cell responses to SARS-CoV-2 variants of concern Delta and Omicron were lower than to the ancestral Wuhan-Hu-1 variant. Our results indicate the importance of assessing both cellular and humoral responses after vaccination and suggest that even in the absence of robust antibody responses vaccination can generate immune responses in pwMS.

A needle-free subunit vaccine platform inducing mucosal and systemic antibody immunity (preprint)

While the established route for vaccines against the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is intramuscular, it may be preferable to deliver vaccines intranasally to secure mucosal protection at the site of infection. This will limit the spread of the virus, ease administration and likely improve vaccine acceptance. Here, we report on a subunit vaccine platform, where the antigen is genetically fused to engineered human albumin. Upon intranasal delivery the subunit vaccines target the neonatal Fc receptor (FcRn) and induce both local and systemic antigen-specific antibody responses at magnitudes higher than after intramuscular delivery. We provide evidence that such needle-free vaccination induces production of antibodies with neutralizing capacity against SARS-CoV-2 or influenza A. Thus, the vaccine platform is particularly well suited for design of subunit vaccines against these and other infectious respiratory diseases.

Titers of antibodies the receptor-binding domain (RBD) of ancestral SARS-CoV-2 are predictive for levels of neutralizing antibodies to multiple variants (preprint)

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

Multiplexed measurement of binding- and neutralizing antibodies to SARS-CoV-2 variants in 12.000 post-vaccine sera (preprint)

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

Breakthrough infections with the omicron and delta variants of SARS-CoV-2 result in similar re-activation of vaccine-induced immunity (preprint)

Background: The Omicron variant of SARS-CoV-2 is now overtaking the Delta variant in many countries. Results showing that sera from double vaccinated individuals have minimal neutralizing activity against Omicron may indicate that the higher rate of transmission is due to evasion from vaccine-induced immunity. However, there is little information about activation of recall responses to Omicron in vaccinated individuals. Methods: We measured inflammatory mediators, antibodies to the SARS-CoV-2 spike and nucleocapsid proteins, and spike peptide-induced release of interferon gamma in whole blood in 51 vaccinated individuals infected with Omicron, in 14 infected with Delta, and in 18 healthy controls. The median time points for the first and second samples were 7 and 14 days after symptom onset, respectively. Findings: Infection with Omicron or Delta led to a rapid and similar increase in antibodies to the SARS-CoV-2 spike and nucleocapsid proteins and spike peptide-induced interferon gamma in whole blood. Both the Omicron and the Delta infected patients had a mild and transient increase in inflammatory parameters. Interpretation: The results suggest that vaccine-induced immunological memory yields similar coverage for the Omicron and Delta variants.

Immunity in Omicron SARS-CoV-2 breakthrough COVID-19 in vaccinated adults (preprint)

The new SARS-CoV-2 variant of concern (VOC) Omicron has more than 30 mutations in the receptor binding domain (RBD) of the Spike protein enabling viral escape from antibodies in vaccinated individuals and increased transmissibility. It is unclear how vaccine immunity protects against Omicron infection. Here we show that vaccinated participants at a superspreader event had robust recall response of humoral and pre-existing cellular immunity induced by the vaccines, and an emergent de novo T cell response to non-Spike antigens. We compared cases from a Christmas party where 81 of 110 (74%) developed Omicron breakthrough COVID-19, with Delta breakthrough cases and vaccinated non-infected controls. Omicron cases had significantly increased activated SARS-CoV-2 wild type Spike-specific (vaccine) cytotoxic T cells, activated follicular helper (TFH) cells, functional T cell responses, boosted humoral responses, activated anti-Spike plasmablasts and anti-RBD memory B cells compared to controls. Omicron cases had significantly increased de novo memory T cell responses to non-Spike viral antigens compared to Delta breakthrough cases demonstrating development of broad immunity. The rapid release of Spike and RBD-specific IgG+ B cell plasmablasts and memory B cells into circulation suggested affinity maturation of antibodies and that concerted T and B cell immunity may provide durable broad immunity.

Efficacy and safety of a third SARS-CoV-2 vaccination in multiple sclerosis vaccine non-responders (preprint)

Importance: Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to protect against coronavirus disease of 2019 (COVID-19) is recommended for patients with multiple sclerosis (pwMS). However, approximately 80% of all pwMS treated with anti-CD20 therapy (rituximab, ocrelizumab) or fingolimod have low or absent humoral immunity after vaccination with two doses of SARS-CoV-2 mRNA vaccines. The efficacy and safety of a third vaccine dose in this group is largely unknown. Objective: To characterize the humoral immunogenicity and the safety of a third dose of mRNA-COVID-19 vaccine in anti-CD20- or fingolimod-treated pwMS with low or absent humoral immunity (i.e., anti-SARS-CoV-2 IgG <70 arbitrary units (AU) and <5 AU, respectively) after two vaccinations. Design, setting and participants: 130 anti-CD20- or fingolimod-treated pwMS with low or absent humoral immunity despite full vaccination against SARS-CoV-2, received a third dose of SARS-CoV-2 mRNA vaccine. Humoral immunity (i.e., antibody response against SARS-CoV-2) and the frequency and characteristics of side-effects were analyzed in all participants. Exposures: A third vaccine dose against SARS-CoV-2 with BNT162b2- or mRNA-1273-COVID-19 vaccine. Main outcomes and measures: Patient- and treatment-specific variables were acquired using a digital questionnaire, the Norwegian Immunization Registry and hospital journals. Humoral immunity was assessed by measuring SARS-CoV-2 SPIKE receptor-binding domain (RBD) IgG response. Low/absent humoral immunity was assumed in cases of AU<70 after anti-SPIKE protein-based serology 3-5 weeks after revaccination. Results: A third dose of SARS-CoV-2 mRNA vaccine increased anti-SARS-CoV-2 SPIKE RBD IgG levels significantly. The proportion of patients with assumed protective humoral immunity (anti-SARS-CoV-2 SPIKE RBD IgG > 70 AU) were 25% among patients using anti-CD20 therapy and 7% among those treated with fingolimod. No adverse events were registered during the study period. Conclusion and relevance: A third dose of mRNA-COVID-19 vaccine was associated with significantly increased levels of anti-SARS-CoV-2 SPIKE RBD IgG, and hence assumed protective humoral immunity - in anti-CD20- or fingolimod-treated pwMS with low or absent humoral immunity despite full vaccination. The effect of a third vaccine dose was limited and more prominent among those treated with anti-CD20 therapy.

Public T-cell epitopes shared among SARS-CoV-2 variants are presented on prevalent HLA class I alleles (preprint)

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. Here, we investigate CD8 T-cell responses to 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identify nine conserved SARS-CoV-2 specific epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians, to which responding CD8 T cells are detected in 70-100% of convalescents expressing the relevant HLA allele, including two novel epitopes. We find a strong correlation between immunoprevalence and immunodominance. Using a new algorithm, we predict that a vaccine including these epitopes would induce a T cell response in 83% of Caucasians. Significance Statement: Vaccines that induce broad T-cell responses may boost immunity as protection from current vaccines against SARS-CoV-2 is waning. From a manufacturing standpoint, and to deliver the highest possible dose of the most immunogenic antigens, it is rational to limit the number of epitopes to those inducing the strongest immune responses in the highest proportion of individuals in a population. Our data show that the CD8 T cell response to SARS-CoV-2 is more focused than previously believed. We identify nine conserved SARS-CoV-2 specific CD8 T cell epitopes restricted by four of the most prevalent HLA class I alleles in Caucasians and demonstrate that seven of these are endogenously presented.

Seroprevalence of antibodies against SARS-CoV-2 virus in the adult Norwegian population, winter 2020/2021: pre-vaccination period (preprint)

Since early 2020, over 123 million people worldwide have been diagnosed with coronavirus disease (Covid-19), but the true number of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undoubtedly higher. The seroprevalence of antibodies against SARS-CoV-2 can provide crucial epidemiological information about the extent of infections independent of virologically detected case numbers. There is no large population-based SARS-CoV-2 seroprevalence survey from Norway; thus we estimated SARS-CoV-2 seroprevalence in Norway before the introduction of vaccines and described its distribution across demographic groups. In November-December 2020, a total of 110,000 people aged 16 years or older were randomly selected from the National Population Register and invited to complete a questionnaire and provide a dried blood spot (DBS) sample. The response rate was 30%; compliance rate for return of DBS samples was 88%. The national weighted and adjusted seroprevalence was 0.9% (confidence interval 0.7-1.0). Seroprevalence was highest among those aged 16-19 years (1.9%, 0.9-2.9), those born outside the Nordic countries 1.4% (1.0-1.9), and in the counties of Oslo 1.7 % (1.2-2.2) and Vestland 1.4% (0.9-1.8). The ratio of SARS-CoV-2 seroprevalence (0.9) to the cumulative incidence of virologically detected cases by mid-December 2020 (0.8%) was slightly above one. SARS-CoV-2 seroprevalence was low before the introduction of vaccines in Norway and was comparable to virologically detected cases, indicating that most cases in the first 10 months of the pandemic were detected. Preventive measures including contact tracing have been effective, people are complying with social distancing recommendations, and local efforts to contain outbreaks have been essential.

Preexisting Cross-Reactive T Cells are Boosted and Comprise Significant Immunity in COVID-19 Recovered Patients (preprint)

Most SARS-CoV-2 infected individuals develop symptoms that do not require medical management. We hypothesized that pre-existing cross-reactive T cell responses could protect the majority from severe disease. Here we found that CTL and Th cells specific for seasonal human coronaviruses (HCoV) were significantly expanded in recovered COVID-19 donors and that CTL responses were significantly higher than responses to private SARS-CoV-2 peptides not shared with seasonal HCoV. A third of the SARS-CoV-2 peptide:HLA ligandome was matched by highly similar peptide mimics from seasonal HCoV, constituting a common HCoV peptide pool. CTL immunity was significantly skewed to the common HCoV peptide pool in age groups 20-70y, but not >70y-old donors. Over 40% of recovered donors lacked neutralizing antibodies, highlighting the role of T cell immunity in COVID-19. Results suggest a protective pre-acquired T cell immunity to SARS-CoV-2 and identify epitopes that may help boost vaccine responses and ensure broad protection against this family of viruses.Trial Registration: ClinicalTrials.gov: NCT04320732.Funding: This study was funded by The Health-South East Health Authority (Project 29286), the Research Council of Norway (Project 312693), the Oslo University Hospital, the KG Jebsen Foundation (grant 19), the University of Oslo, The Norwegian Cancer Society.Conflict of Interest: The authors declare that we have no competing interests.