Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV⁺ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3⁺ T, CD8⁺ T, CD4⁺ T, and FOXP3⁺ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Background: The currently used SARS-CoV-2 mRNA vaccines have proven to induce a strong and protective immune response. However, functional relevance of vaccine-generated antibodies and their temporal progression are still poorly understood. Thus, the central aim of this study is to gain a better understanding of systemic and mucosal humoral immune response after mRNA vaccination with BNT162b2. Methods: We compared antibody production against the S1 subunit and the RBD of the SARS-CoV-2 spike protein in sera of BNT162b2 vaccinees, heterologous ChAdOx1-S/BNT162b2 vaccinees and COVID-19 patients. We monitored the neutralizing humoral response against SARS-CoV-2 wildtype strain and three VOCs over a period of up to eight months after second and after a subsequent third vaccination. Results: In comparison to COVID-19 patients, vaccinees showed higher or similar amounts of S1- and RBD-binding antibodies but similar or lower virus neutralizing titers. Antibodies peaked two weeks after the second dose, followed by a decrease three and eight months later. Neutralizing antibodies (nAbs) poorly correlated with S1-IgG levels but strongly with RBD-IgGAM titers. After second vaccination we observed a reduced vaccine-induced neutralizing capacity against VOCs, especially against the Omicron variant. Compared to the nAb levels after the second vaccination, the neutralizing capacity against wildtype strain and VOCs was significantly enhanced after third vaccination. In saliva samples, relevant levels of RBD antibodies were detected in convalescent samples but not in vaccinees. Conclusions: Our data demonstrate that BNT162b2 vaccinated individuals generate relevant nAbs titers, which begin to decrease within three months after immunization and show lower neutralizing potential against VOCs as compared to the wildtype strain. Large proportion of vaccine-induced S1-IgG might be non-neutralizing whereas RBD-IgGAM appears to be a good surrogate marker to estimate nAb levels. A third vaccination increases the nAb response. Furthermore, the systemic vaccine does not seem to elicit readily detectable mucosal immunity.

Background: Screening newborns for severe combined immunodeficiency (SCID) has become essential, since efficient methods to identify infants with these disorders exist and early stem cell transplantation is life-saving. Method: We performed a three-year screening trial in Stockholm comprised of 89,462 newborn infants. The number of T-cell receptor excision circle (TREC)/kappa-deleting recombination excision circle (KREC)/β-actin (ACTB) copies were quantified simultaneously by real time polymerase chain reaction (PCR) in 3.2 mm punches from dried blood samples taken in the regular neonatal screening program. Results: Five patients with immune deficiencies were identified: two with SCID caused by mutations in the Artemis- and adenosine deaminase gene, respectively, one with ataxia telangiectasia and two with reversible agammagloblinemia, which so far, is of unknown cause. This points to an incidence of SCID at the same level as in other studies (around 1:50,000). In 19 recalled infants, low KREC levels and in one case, also low TREC levels, were caused by immunosuppressive treatment of the mother during pregnancy. The levels normalized within a month in all these infants. The total recall rate was 0.10%, and 40% of the recalled infants were born prematurely (<37 weeks gestation). Among 69 patients with inborn errors of metabolism screened retrospectively, only two, who were severely ill with organic acidemias when the sample was taken, and two with mitochondrial disorders, screened positive.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI− cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI− cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.