Long-term systemic and mucosal humoral response in SARS- CoV-2 vaccinated post-Covid-19 infected patients

Long-term systemic and mucosal humoral response in SARS- CoV-2 vaccinated post-Covid-19 infected patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Long-term systemic and mucosal humoral response in SARS- CoV-2 vaccinated post-Covid-19 infected patients Anirban Sengupta, Mohammad Azharuddin, Edén Johanna, Aili Daniel, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2113175/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Longitudinal serum samples, nasopharyngeal/nasal swabs and rectal swab samples were collected from eighty-nine individuals (median age 66 y) with SARS-CoV-2 PCR-positive test results at Linköping University Hospital. Samples were collected from the initial visit and thereafter for up to 2 years of follow-up. The presence of serum IgG and IgA against SARS-CoV-2 antigens (S1-spike, nucleocapsid, and NSP3) was analysed. Nasal and rectal swabs were tested for the presence of mucosal IgA against the outer envelope S1 spike and the nucleocapsid protein. Ninety percent of the participants were seropositive for SARS-CoV-2 recombinant proteins on Day 28 after study entry, and all (100%) were seropositive based on samples collected 2 months or later. Almost all (95%) developed serum SARS-CoV-2-neutralizing antibodies that were measurable from 6 to 24 months. The most common antibody responses (both serum IgG, mainly IgG1, and in nasal mucosa IgA) reacted with the S1-spike protein and the nucleoprotein. In samples collected from nasal tissues, IgA anti-S1 spike protein was mainly observed during 2 months of follow-up. In a subpopulation (18% of tested individuals), rectal IgA swabs showed the presence of anti-S1 spike IgA for 1 month of follow-up among the participants studied. . long-term immunity antibody response spike neutralization immunoglobulin mucosal immunity serological response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction One of the cornerstones of the protective immune response against viral infections is humoral immunity, systemically and on mucosal surfaces. In most viral vaccine trials, in childhood vaccination programs 1 – 3 and in adult vaccination, humoral immunity is evaluated and most often correlated with prevention or reduction in disease severity. This phenomenon is well documented for respiratory tract infections, such as influenza and RSV 4 – 6 . Thus, it is also very likely applicable to the betacoronavirus SARS CoV-2 7–11 . This study aims to determine the longevity of humoral immunity in the blood and respiratory tract among hospitalized COVID-19 patients through long-term follow-up starting at hospital admission. To further investigate the humoral response in different mucosal surfaces, rectal swabs were collected from the patients in addition to blood serum and nasopharyngeal swab samples to investigate their humoral response. Among the study participants, the majority were first infected with SARS-CoV-2, and most participants were vaccinated with the mRNA S1-spike vaccines (Pfizer or Moderna) a few months thereafter. The combination of infection and vaccination induces so-called hybrid immunity containing both cell-mediated immunity and an enhanced humoral immune response. Here, we present an in-depth investigation of the antibody responses detectable in three study sites, namely, in blood/plasma, nasal mucosa and rectal samples. Virus serum neutralization and angiotensin-converting enzyme 2 (ACE2) inhibition are key hallmarks of the induced serum response 12 , 13 . To obtain a comprehensive view of the immune response of our patients, our study encompassed these factors. Another critical factor that influences the immune response against SARS-CoV-2 infection is the age of the patients 14 , 15 . Age-related effects on influenza virus immunity are well known, therefore we included the age-based response comparison in the study population to investigate age-related effects on the immune response against SARS-CoV-2 in our study. In addition to the most important spike protein (S1) of SARS-CoV-2, interest in other nonstructural proteins (nsp) and the nucleoprotein of the virus has recently developed. Very few studies have explored the possibility of inducing an immune response against any proteins other than Spike S1 and the internal viral helical nucleoprotein (NP) 16 – 18 . This study attempted to address the impact of these proteins as the target of interest for the generation of the humoral immune response of the host. Furthermore, the great protective value of the antibody response against the S1-spike protein with virus-neutralizing activity is highlighted by the recently available therapeutic monoclonal antibodies 19 , which show very good efficacy in clinical treatment and prevention 20 , 21 . In short, the systemic, respiratory, and rectal humoral responses against SARS-CoV-2 proteins were investigated in this comprehensive longitudinal study based on clinical patient samples. The generated immune response against Spike S1, nucleocapsid protein and nonstructural protein (nsp) of the virus was explored. Furthermore, the serum neutralization capacity against live virus, especially towards ACE2-binding viral spike protein S1, was also analyzed. For a period of up to 2 years after SARS-CoV-2 diagnosis, the immunoglobulin IgG subtypes were compared in patient groups stratified by sex and age (elderly (70–89 years) vs. adult (22–69 years). The study will shed light on the 2-year post-COVID-19 kinetics of the anti-SARS-CoV-2 humoral condition based on a proportion of infected individuals. 2. Materials And Methods 2.1 Clinical Samples: Eighty-nine patients were admitted to the University Hospital in Linköping, Sweden from summer 2020 to 2022. Fifty-seven percent (57.3%) of the participants were female (n = 51). All involved patient samples from study participants sampled by nasal/oral swabs were PCR-positive for SARS-CoV-2 (age range 22–87 years, median age 66). The patients were asked for blood samples and additional nasopharyngeal, oral, and rectal swabs. Samples were collected on the day of admission and at 2 weeks, 4 weeks, 2 months and 6 months. For 15 individuals, their last samples were collected at a median timepoint of 669.6 days (range 608–717) during follow-up (Table 1 ). Not all participants allowed the collection of blood or rectal or nasal swab samples at each planned timepoint. All participants were SARS-CoV-2 vaccinated from approximately 60 days postdiagnosis. A total of 81.8% of patients were administered the Pfizer BioNTech-Covid-19 or the Moderna mRNA vaccines, 9.09% received the AstraZeneca ChAdOx1nCoV-19/AZD1222 vaccine, and 9.09% received heterologous vaccines combining mRNA and AstraZeneca vaccines. Negative and positive controls were obtained from pre-pandemic blood donors and laboratory staff at Linköping University (n = 33). Nasal swabs and rectal swabs were collected from volunteers and frozen at -80°C until use. Blood plasma was stored at -20°C until use. Table 1 Samples and study timepoints (In total 89 individuals distributed as shown below): Timepoints Day 0 Day 14 Day 28 Day 60 Day 180 Day 710–730 Number/participants (n) 16 38 42 44 75 15 Sex F/M 9/7 17/21 19/23 19/25 32/43 3/12 Age range Median 24–80 68,5 41–81 65 34–81 64 34–78 66 34–85 66 53–77 65 > 70 yrs n = 6 n = 10 n = 10 n = 11 n = 26 n = 4 2.2 Nasopharynx/saliva swab PCR-RT assays . The presence of SARS-CoV-2 RNA was assayed on the day of hospital admission. Quantitative SARS-CoV-2 PCR was performed for up to 40 cycles (Ct). RNA was isolated from the patient samples followed by reverse transcription to generate complementary DNA (cDNA). Real-time PCR was performed on the samples using the forward and reverse primers against the spike S1 protein. Abbott RealTime qPCR-SARS-CoV-2 assays using nasopharyngeal sample swabs were used to determine the presence of viral RNA in nasal samples at the Clinical Microbiological Department of Linköping University Hospital, Sweden. Swabs used to collect mucosal samples for humoral analyses were not always obtained during scheduled sampling time points due to the health condition of the individual study participant. 2.3 SARS-CoV-2 neutralization analyses The population (89 sampled individuals) was tested for the presence of SARS-CoV-2 neutralizing serum activity against viable virus in Vero E6 cells in vitro 22 . Briefly, heat-inactivated (45 min at 56°C) serum samples were diluted threefold with a beginning dilution of 1:10 (75 µl/well) and mixed with 75 µl/well 10 3 TCID 50 /ml SARS-CoV-2 (Wuhan strain) virus. All dilutions were tested in duplicate wells. After incubating for 60 min at 37°C, 100 µl of the serum-virus mixture was transferred to 80–90% semiconfluent Vero E6 cells in each well. The mixtures were incubated for 48–72 h at 37°C with 5% CO 2 . The neutralizing serum titre was calculated with the CPE inhibition rate according to the Reed-Muench calculation (Ref. Reed-Muench, 1921). The serum samples (n = 89) were further analyzed for their capacity to inhibit the interaction between the SARS CoV-2 virus S1 spike protein and the ACE2 cell receptor in vitro (Teca VLV-test, Nacka, Sweden). 2.4 Serology and ELISA antigens used and tested by samples: All participants were invited to be study participants during their hospital visit; thus, all patients were severely to moderately ill due to SARS-CoV-2 infection. Serum was collected, aliquoted, and frozen until use. SARS-CoV-2 S1-spike protein (Wuhan strain 2019) was used as a soluble antigen on 96-well microwell plates (0.5 ug/mL PBS pH 7.2, Toronto University, SGU Toronto ON, Canada/SinoBiologicals, Eschborn, Germany). SARS-CoV-2 nucleoproteins NC-(aa47-174 and aa267-364) and nsp3 (Toronto University, ON, Canada) were used as recombinant viral coating proteins for performing the ELISA. Serum samples from COVID-19 patients and positive and negative controls were diluted in PBS-Tween 20 (0.05%) with 2.5% fat-free milk buffer (Knopf J et al.2022). Serum dilutions and standard serum control samples were added to the coated plate wells and incubated for 90 min at 37°C. Conjugates against anti-human IgG-HRP (BioRad, Richmond, CA) or anti-human IgA-HRP (Nordic BioSite, Täby, Sweden) were added to separate wells with diluted serum samples and incubated for 90 min at 37°C. Finally, the substrate 0.003% H 2 O 2 /o-phenylene diamine (Sigma‒Aldrich, S:t Louis, MA. 0,4 mg/mL) was added and incubated at room temperature for 30 min before 2.5 M H 2 SO 4 was added as a stop solution. The plates were read at OD490. Standard curve samples for determining AU antibody quantitation were used. The antibodies used are HRP-anti-human IgA (BioRad, STAR141P), HRP-Goat-anti-human IgM (ThermoFisher, Invitrogen A18909), HRP-goat-anti-human IgM, (Sigma-Aldrich, MERCK A6907), HRP-goat anti-human IgA (Sigma-Aldrich, AP114P (Fc5m), Goat-anti-human SC (Sigma-Aldrich, I-6635, 0,2 mL) MoAb Clone GA-1. 2.5 Serum IgG subclass analyses : Selected serum samples (60 individuals) were tested for the content of serum IgG-anti-S1 spike protein-specific IgG1 and IgG3. The serum IgG subclasses were tested with isotype-specific murine anti-human IgG subclasses (Sigma‒Aldrich, S:t Louis, MA, anti-human IgG1 clone BAM 09 and anti-human IgG3 clone BAM 08) (Sundqvist V-A et al. 1984). In brief, S1-spike-coated plates were incubated with serum samples diluted 1/100, 1/1000, and 1/10 000 in PBS-T with 2.5% dry milk and incubated for 90 min at 37°C. After washing, 100 µl of diluted monoclonal anti-human IgG1 and IgG3 (Sigma‒Aldrich/Merck, Paris, France, diluted 1/1000 in PBS-T 2.5% dry milk) was added to each well and incubated for 90 min at 37°C. After washing, 100 µl of diluted HRP-labelled anti-mouse IgG (BioRad, Richmond, CA, at a dilution of 1/3000 in PBS-T 2,5% dry-milk) was added and incubated for 90 min at 37°C. After washing, the OPD substrate was used as shown above. Based on studies with negative control serum samples, an OD of 0.2 was set as the cut-off value for positive samples. 2.6 B-cell IgG epitope mapping on the S1-spike receptor-binding motif (RBM) region in serum IgG To analyze the humoral response to SARS-CoV-2 RBM of the RBD, 3 aa35-mer peptides with 5 amino acids offsets covering the RBM were synthesized based on the reference sequence of SARS-CoV-2 S protein (GenBank: YP_009724390). Four synthetic 35-mer peptides representing the S1-spike cell-receptor-binding motif (RBM) of the Wuhan SARS-CoV-2 virus strain were used 23 . The peptide amino acid sequences are given in the table below. The amino acid sequence (aa no. 438–526) was represented by four peptides (Table 2 ). Synthetic SARSCoV2 peptides. Based on high antigenicity analysis of the three-dimensional predicted structure and B cell epitope information (BepiPred-2.0), eight different antigenic peptides were selected from the amino acid sequence of SARS-CoV-2 (Fig. 3 A and Table 2 ). The synthetic peptide was purified by reverse-phase HPLC (> 98% purity) (Peptide Institute Inc., Osaka, Japan.). The synthetic SARS-CoV-2 peptide was reconstituted at 5 mg/ml in sterile PBS and stored below − 20°C. Table 2 SN3 RLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQS. aa 438–508 SN4 EGFNCYFPLQSYGFQGGGNYNYLYRLFR. aa 486–526 SN5 IYQAGSTPCNGVEGFNCYFPLQSYGGGSLYRLFR aa 457–526 SN6 KVGGNYNYLYRLFRGGSGGFPLQSYGFQ aa 445–508 2.7 Serology of mucosal samples: Oral and nasal swabs were collected with cotton pads and stored frozen in 1 mL of sterile saline solution until use. Mucosal samples from COVID-19 patients and positive and negative controls were diluted with PBS-Triton X-100 5% with 2.5% fat-free dry milk, added to coated SARS-CoV-2 S1 spike plate wells and incubated overnight at RT. Conjugates against anti-human IgA-HRP (Nordic BioSite, Täby, Sweden) were added and incubated for 90 min at 37°C. To study the secretory component on mucosal IgA antibodies, the anti-SC-conjugated HRP (Sigma‒Aldrich, St. Louis, MO) was used. Substrate and plate absorbance were measured as described above. The cut-off was determined by identifying the mean reactivity with the antigens from ten SARS-CoV-2 negative control nasal swabs and multiplying this value by two. 2.8 Statistical analysis. Data were analysed using Prism version 8.2d for macOS (GraphPad Inc. Software LLC). The Mann‒Whitney U test was performed to compare differences in antibody reactivity between sampling time points. The statistical analysis was performed using GraphPad Prism 8 (La Jolla, CA, USA). Comparisons between groups with SARS CoV-2 serum, nasal or rectal swabs were performed using the nonparametric Mann‒Whitney U test with Bonferroni correction, and p < 0.05 was considered significant. Ethical permission : The study was conducted according to the rules of the Declaration of Helsinki. Samples were collected after informed consent and ethical permission was obtained and approved by the Ethical Review Board at (EPM) Linköping County council, Linköping, Sweden. (Reference EPM no: 2020–03888 and 2020–02080). 3. Results 3.1 The adult population has a lower viral load than the older population: Quantitative RT‒PCR Ct values were available from 62 infected individuals prior to vaccination. Later, during follow-up and after recovery, all individuals became vaccinated against SARS-CoV-2, making them enter the hybrid-immunity study participant category. The median qRT‒PCR Ct value Wilcoxon signed-rank sum test and Mann‒Whitney U test pairwise comparison for the sex- and age-separated patient groups are shown in Fig. 1 . The results show significantly higher levels of SARS-CoV-2 virus in vaccinated patients < 70 years old. This difference was also significant with the nonparametric Mann‒Whitney U test comparison. Serum with SARS CoV-2-neutralizing properties was identified in 10/16 individuals at the earliest available serum samples of this study (on the day of the analysed SARS-CoV-2 viral qPCR test). 3.2: Adult populations achieved their maximum viral neutralization and ACE-2 inhibition potential earlier than the older population Virus-neutralization serum titres (> 50% inhibiting capacity) are shown for the study group and study time points (Fig. 2 A-E). In Fig. 2 A, all study serum samples were included per study time point. The data show that a significant neutralizing titre increase occurred between Days 0 and 28 when all participants were included and among the study participants 70 years of age, where a significant neutralizing titer was seen (Fig. 2 C). In the 2-year sampled serum of 15 individuals, a significant neutralizing titre was seen in all study categories for all individuals, the 70-year-old group, and males (Fig. 2 A, 2 B, 2 C and 2 E). When women were stratified into a study group (3 of the 15 study participants at the 2-year timepoint), the change in neutralizing serum titre was not significant at the 2-year follow-up time point. Virus-neutralization serum correlating S1-spike-ACE2-binding inhibition serum titres (> 67% inhibiting capacity) are shown for the study group and study time points (Fig. 3 ). When comparing the study time points, a significantly increased median serum S1-ACE2-inhibiting titre was observed between Days 0 and 28 and between Days 180 and 2 (Fig. 3 A). The time period between Days 14 and 180 was a plateau period with no significant median titre increases. The ACE2 – S1-spike protein inhibition capacity at a serum dilution of 100 was very strong in almost all (98%) study participants from Day 28 and onwards, without significant difference between study groups (Fig. 3 A and B). Figure 3 shows the virus-spike protein and recombinant cell-receptor (ACE2) inhibition assay with the serum of study participants. A significant change in inhibition activity was seen between Day 0 and Day 28 and between Day 180 and 2 years (Fig. 3 A). The strength of inhibition as a percentage of prevented S1-spike binding to the recombinant ACE2 protein is shown in Fig. 3 B. Here, the significant inhibition levels were highest from Day 28 onwards in all tested serum samples. Inhibition did not significantly differ between serum collected from individuals younger than 70 years and individuals older than 70 years (Figs. 3 C and 3 D). 3.3: The antibody response against the spike protein showed an initial 50% seropositivity, which increased to 100% for IgG, with the majority being seropositive for both IgG and IgA at the end of the study In total, 89 patients with COVID-19 infection and SARS-CoV-2 vaccination were included. The kinetics of serum IgG and IgA against the SARS-CoV-1 S1-spike protein were analysed (Fig. 4 A and 4 B). Fifty percent (50%) of study participants showed low levels of anti-S1 spike protein IgG and 31% serum IgA at the initial and inclusion time points (Day 0). All patients (100%) developed detectable IgG and IgA responses during the study period. All study participants (100%) were seropositive at 1 month and remained seropositive for serum IgG at the 6- and 24-month follow-ups. Fifteen study participants who reached the 2-year study time point after study entry all showed the presence of serum IgG and IgA against the S1-spike protein. Serum IgA seropositivity was detectable in all tested samples at 2 months but was reduced to 78.6% at 6 months. 3.4: The antibody response is stronger for the N-terminus of the nucleocapsid protein, with no response observed against the nonstructural SARS-CoV-2 protein IgG against SARS-CoV-2 nucleocapsid proteins (NP-protein aa 47–173) was detected in serum samples from all study participants one month postadmission. However, only 2 study participants showed detectable serum IgG against a more C-terminal part of the nucleoprotein (aa 247–364, not shown). Anti-NP-binding antibodies in the serum were compared with serum IgG anti-S1 spike reactivity (Fig. 4 C and D). Serum IgG and IgA reactivity against one nonstructural protein, the NSP3 protein, was assayed, and all tested sera were shown to be negative or below the limit of detection (not shown). 3.5: Serum IgG subclass reactivity against the S1 spike and nucleocapsid protein. The IgG1 subclass against the S1-spike protein was the dominant IgG subclass detected in the involved patients. An analysis of serum IgG subclass reactivity against the S1-spike protein in samples from 34 individuals with severe or intermediate severe COVID-19 showed no difference in subclass reactivity at 1 and 6 months post-PCR positivity. (Fig. 5 ). 3.6: Identification of the B-cell epitope on the spike receptor binding domain (RBD) reveals an amino acid domain based on the serum antibody reactivity of the study participants : Most individuals who were tested for serum anti-S1 spike and ACE2 cell receptor binding inhibition and underwent SARS-CoV-2 virus-neutralizing assays needed > 14 days to develop efficient virus-neutralizing antibodies. Therefore, we investigated if and when IgG antibody epitope reactivity may develop within the S1-RBD and RBD-RBM regions that would interfere with the virus‒cell-receptor interaction. Based on sera that can neutralize the live SARS-CoV-2 virus in vitro , we identified individuals who did develop serum IgG reactivity against peptides representing the C-terminal RBM and individuals who developed S1 spike-binding but nonneutralizing antibodies without reacting with the synthetic peptides. Serum samples from individuals without neutralizing antibodies did not react by binding to any of the tested RBM peptides (See Fig. 6 A-C). The B-cell epitope mapping of the cell-receptor-binding region of the viral S1 spike protein suggests that a neutralizing epitope is situated in the receptor-binding motif (representing aa 444–479). 3.7: Anti-S1-IgA positivity in nasopharyngeal swab samples in the entire study population observed with an anti-nucleocapsid protein IgA response in only a few participants A subgroup of individuals was tested for IgA reactivity in nasopharyngeal swab samples (n = 58). Positive reactivity against the S1 spike protein was seen at least once in up to 90% of tested individuals, between Days 14 and 180 (Fig. 7 A). No significant difference was seen between age groups. Fifty percent of tested individual nasal samples were initially (Day 0) shown to have positive anti-S1-reactive nasal mucosal IgA against the S1-spike protein. This frequency increased to 100% nasal IgA-positive responses from Days 28 to 60. However, this increase cannot be conclusively attributed to either the vaccination booster effect or a sign of local upper respiratory tract infection. In three of fifteen nasal samples, anti-S1-spike IgA was also seen at the 2-year time point. Few signs of reinfection could be identified in nasal samples during follow-up by local mucosal anti-SARS-CoV-2 virus S1-spike protein antibody analysis. Nevertheless, a few individual nasal swab samples contained low but still positive nasal IgA anti-SARS-CoV-2 reactive anti-nucleocapsid protein reactivity (Fig. 8 A) both at Day 180 and during the 2-year follow-up time point (n = 10 and n = 2, respectively). 3.8: Anti-spike-IgA was detected in the rectal swab samples of a subgroup of the participants : A subgroup of individuals was tested for IgA reactivity in rectal swab samples, and positive reactivity against the S1 spike protein was seen at least once in up to 18% of tested participants between Days 14 and 180 after the initial qPCR-positive diagnostic time-point (Fig. 8 ). No significant differences were seen between age groups or between opposite-sex individuals. No sign of reinfection could be identified in intestinal samples during follow-up by local rectal anti-SARS-CoV-2 virus S1-spike protein antibody analysis. Nevertheless, a few individual rectal swab samples contained low but still positive rectal IgA anti-SARS-CoV-2 reactive anti-S1-spike and/or nucleocapsid protein reactivity (Figs. 8 A and 8 B) at Day 180 but not at 2 years (n = 2 and n = 0, respectively). 4. Discussion Highly pathogenic respiratory tract viral infections, such as influenza A/B, respiratory syncytial virus (RSV), and measles virus, show that humoral immune responses can correlate well with protective immunity. However, as with the current pandemic SARS-CoV-2 virus Omicron variants, reinfection is possible, especially in the upper respiratory tract, after having developed efficient neutralizing antibodies against earlier SARS-CoV-2 variants and the current vaccine strain. Instead, efficient protection from severe illness is often provided by the virus-neutralizing humoral response with supportive cell-mediated immune responses (CD4 + and CD8 + T cells), which together constitute the immunological memory response. Most currently used vaccines induce protective immunity and are currently associated with neutralizing antibody levels in the serum and plasma. Important results obtained with anti-S1 monoclonal antibody treatment for severely infected SARS-CoV-2 patients support the protective value of antibodies 19 – 21 . However, humoral first-line defense can also be found in mucosal tissues, the nasal/oral and respiratory tract mucosa, and gastrointestinal mucosal tissues that provides the mucosal immunity. In these tissues, this defense is most often represented by local mucosal B-cell synthesis and the secretion of secretory mucosal dimeric (or tri/quadrimeric) IgA (S-IgA) 24 , 25 . Therefore, the sampling and analysis of nasal swabs and rectal/intestinal (stool) samples may provide important information regarding the duration and presence of protective local mucosal immunity that complements the neutralizing and protective serum-associated IgG/IgA antibody responses. In this study, we investigate the hybrid immunity induced by initial natural SARS-CoV-2 infection, followed by mRNA SARS-CoV-2 vaccination 26 27,28 . Identifying the correlations of protective immunity is important, and the best and most often-defined correlate of protection in almost any respiratory infectious illness to date has been antibodies 29 . Antibodies have been shown to neutralize infectivity by attaching to viral surface proteins and trigger complement activation to provide antibody-dependent, cell-mediated cytotoxicity against infected viruses and cells. However, some evidence suggests that SARS-CoV-2 will not be sensitive to the same correlates of protection 30 – 32 . The challenge will be to obtain local mucosal secretory antibodies capable of recognizing the new circulating S1-envelope protein mutant SARS-CoV-2 strains, especially close to the nasal mucosal surfaces, as seen in a subpopulation of participants in this study (Figs. 6 , 7 and 8 ). The possibly positive result of obtaining SARS-CoV-2-specific immunity via natural infection may be a broader immune response to several viral proteins not limited to those included in the vaccines. Thus, immune memory B- and T-lymphocytes against both viral surface proteins and several hopefully more conserved intraviral proteins, such as nucleocapsid proteins (NCs) and nonstructural proteins (nsp:s), will be obtained. Nevertheless, the longevity, characteristics and magnitude of these immune responses remain to be elucidated in COVID-19 patients with mild and/or severe disease and in vaccinated individuals of different age groups and sexes. Serological analyses in previous studies have shown that the immune response against SARS-CoV-2 S1 Spike protein is one of the most long-lasting responses when compared with those of viral proteins 11 , 33 34 35 . In this study, all involved patients developed detectable anti-S1- and NP-binding IgG and IgA responses during the natural infection and in response to additional SARS CoV-2 vaccination, and these responses persisted for the entire 2-year study period. All followed study participants were S1-spike seropositive and virus-neutralization capable at 6 months. In a subgroup, the neutralization capacity remained for the entire 22-month to 2-year period. There are links between nasal IgA response, mucosal virus neutralization and less severe infection 36 – 39 . All samples showed the presence of both serum IgG (IgG1) and IgA and local mucosal nasopharyngeal IgA against the outer surface proteins of SARS-CoV-2 for up to 2 years of follow-up. Rectal anti-S1-spike binding antibodies were only detectable during the initial 28 days after the initial positive nasal PCR assay. In fifteen individuals (of which 3 were female) for whom a 2-year follow-up sample was available, the presence of high amounts of SARS-CoV-2 neutralizing antibodies and anti-S1 specific serum IgG1 were detected. Serum IgG and IgA responses against the nucleocapsid protein were weak at this late time point, suggesting the absence of detectable reinfection that induced an enhanced humoral immune response. Most likely, the observed antibody responses at this later stage of hybrid immunity (infection followed by vaccination) were due to vaccination with the mRNA S1-spike proteins in the majority of adult to elderly (> 70–85 years) study participants. Anti-SARS-CoV-2 specific antibodies in infection and vaccination has previously been followed and described during shorter time studies 25,36,40−42 . Importantly, the serum IgG against neutralizing B-cell epitopes on viral proteins should remain elevated, hopefully at a protective level. Nevertheless, clear antibody titre or antibody quantity limits to prevent infection or reduce transmissibility have not yet been established. Similarly, serum IgG against the nucleoprotein was also frequently detectable among the infected patients. However, serum IgG/IgA against nucleocapsid proteins (NCs) has been described as both negative and positive for human health, as described in different patient categories. In one study, higher amounts of these immunoglobulins correlated with the antibody-dependent enhancement (ADE) of infection, despite a lack of clear mechanistic process 16 . In other studies, these antibodies have been part of the cure/health-promoting immune response. A possible explanation for the positive interpretations could be the clearance of circulating viral proteins that otherwise may cause the formation of immune complexes or result in inflammation at local sites where the NC proteins aggregate 43 . Approximately 50% and 20% of hospital admissions for pneumonia in young children and elderly individuals, respectively, are estimated to be caused by viral infections. Prior to 2020, these admissions were mainly due to respiratory syncytial virus or influenza viruses 44 . Since then, the dominant cause of respiratory viral infection has been betacoronavirus SARS-CoV-2 (in Scandinavia). Respiratory infectious viruses (historically dominated by approximately eight to ten different virus families) will consequently remain a serious challenge, despite decades of intensive research for developing antiviral drugs and vaccines against these viruses. Stratifying our patient population into subgroups allowed us to compare the impact of natural infection followed by vaccination with two doses supporting the SARS-CoV-2-specific humoral immune response. The >70-year-old group showed reduced expression of the anti-S1 spike IgG response in the long term compared to the younger group (Fig. 4 A). Box 1: Highlights of two years SARS-CoV-2 followup study findings: • A higher virus qRT‒PCR load in 70 -year-old group attained the highest neutralization potential more slowly, 28 days post-infection or later (Fig. 2A, B). Males had a higher median neutralization titre at the 22-24 month timepoint (nonsignificant difference from females). • S1-Spike-ACE2 binding inhibition serum titres showed no significant change between 14 and 180 days. The maximum significant increase was observed during the first 14 days (from the primary infection) and during 180 days of follow - up (after vaccination). • All study groups showed 100% S1-spike protein inhibition capacity 28 days after infection (Figure 3A and B) , with no difference between the 70 -year-old groups (Figures 3C and 3D). • The participants remained seropositive for Spike S1 IgG until the last study point (Fig. 4A). A total of 78.6% of them remained seropositive for Spike S1- IgA at the 6-month time point (Fig. 4B). The serum anti-S1 spike IgG1 subclass dominates in seropositive individuals. • Anti- nucleocapsid IgG is more efficient in targeting the N-terminus of the protein ( aa 47 -173) than its C - terminal part (aa 247-364) (Fig. 4C, 4D). • The B-cell epitope mapping of the cell-receptor-binding region of the viral S1 spike protein suggests that a neutralizing epitope is situated in the receptor-binding motif (representing aa 444-479) (Fig. 5). • In nasal mucosal samples , the anti- spike S1 IgA response increased from 50% of the tested individuals at Day 0 to 100% within Days 28 to 60, after which the response tapered off. Only a few samples were shown to be positive at the 180 -day timepoint (Fig. 6). The anti -NP IgA response in the same nasal mucosal samples was evident from Days 14-28 , after which nasal IgA gradually disappeared (Fig. 8A). • In rectal mucosal samples , IgA-positive reactivity against Spike S1 and NP protein was observed in a small group of individuals (18% of the study population) only within the first three timepoints (Day 0, 14 and 28) and not at the timepoints beyond 2 months (Fig. 7B, 8B) Of the studied participants, a small percentage showed a positive IgA response in rectal swabs (18%). This result is unique, as only very few studies have previously reported test data from intestinal organs. Furthermore, in the few previously reported studies, nondetectable anti-SARS-CoV-2 antibody levels were shown 45 , 46 . In a selected population of eighteen individuals, epitope mapping showed the presence of anti-S1 spike serum IgG, with 10 samples having virus-neutralizing reactivity in vitro and eight with no neutralizing reactivity. The study indicates that 8 of 10 individuals with neutralizing activity had anti-RBM-epitope reactive serum IgG B-cell epitope aa 444–479 (Fig. 6 C), while 8/8 of the individuals without neutralizing activity in vitro lacked this reaction pattern. Similar B-cell epitope results have also been presented by other studies 47 , 48 . Thus, when anti-RBD/RBM-directed antibodies develop, they correspond to a shift to virus-neutralizing functional activity. Our results support data presented in previous studies 23 , 48 . The anti-S1-IgG1 response was detected in all patient groups, which contrasts findings from other studies in which serum IgG3, together with IgG1, was also detected in samples from individuals with more severe disease and symptoms. The reasons for these dissimilarities may depend on several factors, such as differences in patient categories and the timeline of the study (as IgG3 has a shorter biological half-life than the IgG1 subclass) in association with disease symptoms 14 , 49 . Furthermore, other studies have shown that serious illness may even suppress specific B-cell responses completely or below the level of detection. Except for the abovementioned reasoning, the humoral subclass IgG-reaction pattern seems similar to those of several other viral infections, such as Influenza A and B, RSV, HHV-6, and CMV, while subclasses antiviral IgG2 and IgG4 are often rare. Instead, these latter subclasses are more commonly seen in samples from patients with frequently reactivated viral infections 50 , 51 , and they are even more prevalent in bacterial infections. This finding confirms a previous study in which IgG1 levels were shown to be higher in patients who were able to fight off the infection and had better outcomes 14 , 52 , 53 . To mention the limitations of the study, not all study participants provided study samples at each test timepoint. Ethical permission allows only serological analyses. The most frequent samples given were blood samples and nasopharynx samples. Rectal samples were least frequently provided. No cell-mediated immune responses were analysed because ethical permission was limited to the analysis of immunoglobulin responses against SARS-CoV-2 proteins. Box 1 summarizes by highlighting in a nutshell all the key findings from this longitudinal study of clinical covid patient samples for two years. In conclusion, this study suggests that both systemic and mucosal immunity are evident in the included patient groups. Over a period of 6 months, the majority (98.7%) of naturally infected COVID-19 patients in this study population developed lasting detectable levels of serum IgG and IgA responses against the SARS-CoV-2 S1-spike protein and the nucleocapsid protein but not against nonstructural protein 3. Interestingly, samples from all study participants (15/15 individuals) collected at the latest available time point (between 22 months and 2 years after hospital admission) showed potent serum virus-neutralizing properties in vitro . Declarations ACKNOWLEDGEMENTS We are most grateful for the invaluable support with SARS-CoV-2 (Wuhan strain) reagents to Dr. Ackloo S. SGU Toronto, ON, Canada. All involved researchers confirm no competing interests. We wish to thank research nurses Karin Sjöberg and Mona Hansson at the Clinical research unit at Region Östergötland for their invaluable help with the clinical coordination, monitoring and sampling. Funding ALF, Hanberger H, Region Östergötland, and Linköping University. The KACST fund: (KING ABDULAZIZ CITY FOR SCIENCE AND TECHNOLOGY “KACST” ). The MIIC stipend fund from Linköping University for postodoctoral fellowship of AS. Knut and Alice Wal- lenberg Foundation, Wallenberg Center for Molecular Medicine. The Structural Genomics Consortium is a registered charity [1097737] that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Ge- nomics Institute [OGI-196];EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510]; Janssen, Merck KGaA (aka EMD in Canada and US); Pfizer; Takeda Stiftelsen för Strategisk Forskning (FFL15-0026).; Ethical permissions: The study was performed according with the Helsinki Declaration guidelines and was approved by the Ethical Committee, Linköping County Council board, Linköping, Sweden. (Approval No. 2020-02080, 2020-03888). 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Distinct systemic and mucosal immune responses during acute SARS-CoV-2 infection. Nat. Immunol. 22 , 1428–1439 (2021). Roncati, L. et al. Type 3 hypersensitivity in COVID-19 vasculitis. Clin. Immunol. 217 , (2020). Ruuskanen, O., Lahti, E., Jennings, L. C. & Murdoch, D. R. Viral pneumonia. Lancet (London, England) 377 , 1264–1275 (2011). Signorini, L. et al. Longitudinal, virological, and serological assessment of hospitalized COVID-19 patients. J. Neurovirol. 28 , 113–122 (2022). Wang, W. et al. Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA 323 , 1843–1844 (2020). Yoshida, S. et al. SARS-CoV-2-induced humoral immunity through B cell epitope analysis in COVID-19 infected individuals. Sci. Rep. 11 , (2021). Jiang, M. et al. Epitope Profiling Reveals the Critical Antigenic Determinants in SARS-CoV-2 RBD-Based Antigen. Front. Immunol. 12 , (2021). Kober, C. et al. IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools. PLoS One 17 , (2022). Gilljam, G. et al. Sensitive analytic ELISAs for subclass herpes virus IgG. J. Virol. Methods 10 , 203–214 (1985). Sundqvist, V. A., Linde, A. & Wahren, B. Virus-specific immunoglobulin G subclasses in herpes simplex and varicella-zoster virus infections. J. Clin. Microbiol. 20 , 94–98 (1984). Patil, H. P. et al. Antibody (IgA, IgG, and IgG Subtype) Responses to SARS-CoV-2 in Severe and Nonsevere COVID-19 Patients. Viral Immunol. 34 , 201–209 (2021). Dogan, M. et al. SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus. Commun. Biol. 2021 41 4 , 1–13 (2021). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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14:29:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2113175/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2113175/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":27475932,"identity":"278c0543-fa1b-40b2-aa5c-6d2abf14f03b","added_by":"auto","created_at":"2022-10-07 15:24:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eQuantitative SARS-CoV-2 virus RNA RT‒PCR analysis of nasopharyngeal samples. Each dot represents an individual sample among the study groups. The median value and 95% CI/group are shown.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJ.Fig120220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/e761f5277d291eaa335775f8.png"},{"id":27475929,"identity":"52f05a64-c39f-4ebd-b53b-d9fb1f06b2de","added_by":"auto","created_at":"2022-10-07 15:24:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Kinetics of virus-neutralizing serum reactivity against the SARS-CoV-2 (Wuhan) virus in vitro from eighty-nine Covid-19qPCR+ individuals over a median period of 669 days (range 608-717) postinfection. All individuals received the SARS-CoV-2 vaccine postinfection starting on Day 60 or later. Boxplots showing median serum dilution anti-SARS-CoV-2 virus 50% neutralizing reactivity and 95% CI range per timepoint.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB and C\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Kinetics of virus-neutralizing serum antibodies by age group (B) \u0026lt;70 years (22-69 years) and (C) \u0026gt;70 (70–87 years). Boxplots showing median serum dilution anti-SARS-CoV-2 virus 50% neutralizing reactivity and 95% CI range per timepoint.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eD and E.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Kinetics of serum-neutralizing antibodies related to sex. (D) Females and (E) males. The dotted line indicates the limit of positive virus-neutralization serum titre (values below are considered nonspecific inhibiting serum titres). Boxplots showing median serum dilution anti-SARS-CoV-2 virus 50% neutralizing reactivity and 95% CI range per timepoint.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJ.Fig2AE20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/84904e96ec7a3c277d74ec51.png"},{"id":27475934,"identity":"83053ee3-6892-47dd-9e80-84f489cf7281","added_by":"auto","created_at":"2022-10-07 15:24:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) Kinetics of S1-spike protein and ACE2-inhibition serum reactivity in vitro. The median serum dilution showed a \u0026gt;65% inhibiting capacity of S1-spike and ACE2-binding in the serum samples of the entire study cohort.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e S1-ACE2-inhibiting capacity of 100-fold diluted serum as a percentage. Covid-19 patient samples were divided into\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Covid-19 patients \u0026lt; 70 years age serum titre with cell-receptor (ACE2) inhibiting capacity,\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eD)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Covid-19 patients \u0026gt;70 years of age during up to 670 days (range 608-717) from hospital admission. The median 65% inhibiting serum dilution with 95% CI is shown in the bars. Boxplots showing median reactivity and range per timepoint. The dots show individual serum titres. Inhibiting titres below 10 are considered negative or non-neutralizing.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn Figure 3B, the percent inhibition of the interaction between S1-spike and ACE2 receptor was considered negative if below 65% (according to assay recommendation).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJ.Fig3AD20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/8e6e4dd52083fdc1a0975bc2.png"},{"id":27476517,"identity":"6daefeb3-2d5f-4a8e-8d2a-af01095e9bce","added_by":"auto","created_at":"2022-10-07 15:34:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA and B\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Comparison of IgG reactivity in the serum of study participants (n=89) against recombinant S1 Spike antigen (A) (B), Serum IgG median titre responses to S1 protein/timepoint and (B) median IgG serum titre responses against the\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e nucleocapsid protein\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e during a median 670-day follow-up period. The median and 95% CI for each follow-up time point are shown.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC and D\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Comparison of serum IgA reactivity in COVID-19 patients (n=89) against recombinant S1 spike (C) serum IgA responses against nucleocapsid protein (NP aa47-173) protein (D) IgA responses during a median 670 (range 608-717) day follow-up period. The median and 95% CI for each follow-up time point are shown. The limit of positive reaction is indicated by the dotted line in the figures.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJ.Fig4AD20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/a153f10139f16f144384aa42.png"},{"id":27475930,"identity":"d55bb817-e39e-4769-8cd5-61fa0a8811e6","added_by":"auto","created_at":"2022-10-07 15:24:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":26949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA and B\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Comparison of serum IgG subclass 1 and 3 reactivity in selected study participants at two study timepoints (n=11 and n=34) against recombinant S1 spike protein (S1) protein at 1 month (A) and at 6 months (B) after qPCR-positivity. Median reactivity and 95% CI shown/time point.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC and D\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Comparison of serum IgG subclass 1 and 3 reactivity in selected study participants (n=11 and n=34) against recombinant nucleocapsid protein (NP aa 47-179) protein at 1 month (C) and 6 months (D) after qPCR positivity.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJ.Fig5AD20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/fe543bee80717190b4e48ad4.png"},{"id":27476236,"identity":"68692602-376e-4335-9ad6-85eacbc55418","added_by":"auto","created_at":"2022-10-07 15:29:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIdentification of a B-cell epitope potentially discriminating between SARS-CoV-2 patient serum IgG involved in the identification of anti-S1 spike receptor-binding motif and virus neutralization.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) illustrates how serum from two SARS-CoV-2-positive groups (one group with SARS-CoV-2 neutralizing properties in vitro (n=9) and a second group with no/low SARS-CoV-2 neutralizing capacity (n=8)) binds to recombinant S1 spike protein coating.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) shows a comparison of serum IgG reactivity with a coating of recombinant S1 spike receptor-binding domain antigen (aa 444-479).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) shows a comparison of serum IgG reactivity with a synthetic peptide epitope from the receptor-binding motif of the S1 spike receptor binding domain. The data show significantly weaker serum IgG binding to the RBM peptide in the study group that lacks or shows poor SARS-CoV-2-neutralizing properties.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJFig6AC20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/36a22021cedbd7c5e3266b72.png"},{"id":27476240,"identity":"28cac82a-6ce0-4ae5-a282-f7128d5be530","added_by":"auto","created_at":"2022-10-07 15:29:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). Nasal swab IgA ELISA reactivity. Figure 7A. IgA ELISA reactivity against the SARS-CoV-2 surface recombinant S1 spike protein in nasal swab samples was observed in study participants (median IgA titre/µg total IgA). Box-plot with median reactivity and 95% CI per timepoint. Individual data are shown as spots.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). Median and range IgA ELISA reactivity against the SARS-CoV-2 surface recombinant S1 spike protein in nasal swab samples over the study period in the COVID-19 study participants (median IgA titre/µg total IgA). Median reactivity and 95% CI per timepoint.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e). Median and range IgA ELISA reactivity against the SARS-CoV-2 nucleocapsid protein in nasal swab samples over the study period in the study participants (median IgA titre/µg total IgA). Median reactivity and 95% CI per timepoint.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJFig7AC20220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/cda468258ee1edb0da8c74ca.png"},{"id":27476239,"identity":"eb0ed2b0-f334-4233-b06b-b8d4725eecd1","added_by":"auto","created_at":"2022-10-07 15:29:25","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA and B\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) IgA ELISA reactivity against SARS-CoV-2 surface recombinant S1 spike protein in rectal swab samples was observed in eighteen study participants (median IgA titre/µg total IgA). (A) Boxplots showing median reactivity and 95% CI per timepoint. Individual data shown as spots. (B) Kinetics of median (and range) nasal IgA reactivity against the S1-spike recombinant protein.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eC)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Median and range IgA ELISA reactivity against the SARS-CoV-2 nucleocapsid protein in rectal swab samples throughout the study period in the study participants (median IgA titre/µg total IgA). Median reactivity and 95% CI per timepoint.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"HinkulaJFig820220902.png","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/c5bad814e29c984220a94c31.png"},{"id":33442679,"identity":"a8328d88-62e4-4764-bda6-1e7ea745c6b6","added_by":"auto","created_at":"2023-02-25 10:59:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1533465,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2113175/v1/de74ad4f-41ac-4ea1-bacb-3d5c86936de2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-term systemic and mucosal humoral response in SARS- CoV-2 vaccinated post-Covid-19 infected patients","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOne of the cornerstones of the protective immune response against viral infections is humoral immunity, systemically and on mucosal surfaces. In most viral vaccine trials, in childhood vaccination programs \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and in adult vaccination, humoral immunity is evaluated and most often correlated with prevention or reduction in disease severity. This phenomenon is well documented for respiratory tract infections, such as influenza and RSV \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Thus, it is also very likely applicable to the betacoronavirus SARS CoV-2 \u003csup\u003e7\u0026ndash;11\u003c/sup\u003e. This study aims to determine the longevity of humoral immunity in the blood and respiratory tract among hospitalized COVID-19 patients through long-term follow-up starting at hospital admission. To further investigate the humoral response in different mucosal surfaces, rectal swabs were collected from the patients in addition to blood serum and nasopharyngeal swab samples to investigate their humoral response.\u003c/p\u003e \u003cp\u003eAmong the study participants, the majority were first infected with SARS-CoV-2, and most participants were vaccinated with the mRNA S1-spike vaccines (Pfizer or Moderna) a few months thereafter. The combination of infection and vaccination induces so-called hybrid immunity containing both cell-mediated immunity and an enhanced humoral immune response. Here, we present an in-depth investigation of the antibody responses detectable in three study sites, namely, in blood/plasma, nasal mucosa and rectal samples.\u003c/p\u003e \u003cp\u003eVirus serum neutralization and angiotensin-converting enzyme 2 (ACE2) inhibition are key hallmarks of the induced serum response \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. To obtain a comprehensive view of the immune response of our patients, our study encompassed these factors. Another critical factor that influences the immune response against SARS-CoV-2 infection is the age of the patients \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Age-related effects on influenza virus immunity are well known, therefore we included the age-based response comparison in the study population to investigate age-related effects on the immune response against SARS-CoV-2 in our study.\u003c/p\u003e \u003cp\u003eIn addition to the most important spike protein (S1) of SARS-CoV-2, interest in other nonstructural proteins (nsp) and the nucleoprotein of the virus has recently developed. Very few studies have explored the possibility of inducing an immune response against any proteins other than Spike S1 and the internal viral helical nucleoprotein (NP) \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This study attempted to address the impact of these proteins as the target of interest for the generation of the humoral immune response of the host. Furthermore, the great protective value of the antibody response against the S1-spike protein with virus-neutralizing activity is highlighted by the recently available therapeutic monoclonal antibodies \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, which show very good efficacy in clinical treatment and prevention \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn short, the systemic, respiratory, and rectal humoral responses against SARS-CoV-2 proteins were investigated in this comprehensive longitudinal study based on clinical patient samples. The generated immune response against Spike S1, nucleocapsid protein and nonstructural protein (nsp) of the virus was explored. Furthermore, the serum neutralization capacity against live virus, especially towards ACE2-binding viral spike protein S1, was also analyzed. For a period of up to 2 years after SARS-CoV-2 diagnosis, the immunoglobulin IgG subtypes were compared in patient groups stratified by sex and age (elderly (70\u0026ndash;89 years) vs. adult (22\u0026ndash;69 years). The study will shed light on the 2-year post-COVID-19 kinetics of the anti-SARS-CoV-2 humoral condition based on a proportion of infected individuals.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Clinical Samples:\u003c/h2\u003e\n \u003cp\u003eEighty-nine patients were admitted to the University Hospital in Link\u0026ouml;ping, Sweden from summer 2020 to 2022. Fifty-seven percent (57.3%) of the participants were female (n\u0026thinsp;=\u0026thinsp;51). All involved patient samples from study participants sampled by nasal/oral swabs were PCR-positive for SARS-CoV-2 (age range 22\u0026ndash;87 years, median age 66). The patients were asked for blood samples and additional nasopharyngeal, oral, and rectal swabs. Samples were collected on the day of admission and at 2 weeks, 4 weeks, 2 months and 6 months. For 15 individuals, their last samples were collected at a median timepoint of 669.6 days (range 608\u0026ndash;717) during follow-up (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Not all participants allowed the collection of blood or rectal or nasal swab samples at each planned timepoint. All participants were SARS-CoV-2 vaccinated from approximately 60 days postdiagnosis. A total of 81.8% of patients were administered the Pfizer BioNTech-Covid-19 or the Moderna mRNA vaccines, 9.09% received the AstraZeneca ChAdOx1nCoV-19/AZD1222 vaccine, and 9.09% received heterologous vaccines combining mRNA and AstraZeneca vaccines. Negative and positive controls were obtained from pre-pandemic blood donors and laboratory staff at Link\u0026ouml;ping University (n\u0026thinsp;=\u0026thinsp;33). Nasal swabs and rectal swabs were collected from volunteers and frozen at -80\u0026deg;C until use. Blood plasma was stored at -20\u0026deg;C until use.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSamples and study timepoints (In total 89 individuals distributed as shown below):\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTimepoints\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 0\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 14\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 28\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 60\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 180\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 710\u0026ndash;730\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber/participants (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex F/M\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9/7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17/21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32/43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3/12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge range\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24\u0026ndash;80\u003c/p\u003e\n \u003cp\u003e68,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u0026ndash;81\u003c/p\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026ndash;81\u003c/p\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026ndash;78\u003c/p\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u0026ndash;85\u003c/p\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53\u0026ndash;77\u003c/p\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026gt;\u0026thinsp;70 yrs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u0026thinsp;=\u0026thinsp;4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\u003cbr\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e\u003cstrong\u003e2.2 Nasopharynx/saliva swab PCR-RT assays\u003c/strong\u003e.\u003c/h2\u003e\n \u003cp\u003eThe presence of SARS-CoV-2 RNA was assayed on the day of hospital admission. Quantitative SARS-CoV-2 PCR was performed for up to 40 cycles (Ct). RNA was isolated from the patient samples followed by reverse transcription to generate complementary DNA (cDNA). Real-time PCR was performed on the samples using the forward and reverse primers against the spike S1 protein. Abbott RealTime qPCR-SARS-CoV-2 assays using nasopharyngeal sample swabs were used to determine the presence of viral RNA in nasal samples at the Clinical Microbiological Department of Link\u0026ouml;ping University Hospital, Sweden.\u003c/p\u003e\n \u003cp\u003eSwabs used to collect mucosal samples for humoral analyses were not always obtained during scheduled sampling time points due to the health condition of the individual study participant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 SARS-CoV-2 neutralization analyses\u003c/h2\u003e\n \u003cp\u003eThe population (89 sampled individuals) was tested for the presence of SARS-CoV-2 neutralizing serum activity against viable virus in Vero E6 cells \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, heat-inactivated (45 min at 56\u0026deg;C) serum samples were diluted threefold with a beginning dilution of 1:10 (75 \u0026micro;l/well) and mixed with 75 \u0026micro;l/well 10\u003csup\u003e3\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/ml SARS-CoV-2 (Wuhan strain) virus. All dilutions were tested in duplicate wells. After incubating for 60 min at 37\u0026deg;C, 100 \u0026micro;l of the serum-virus mixture was transferred to 80\u0026ndash;90% semiconfluent Vero E6 cells in each well. The mixtures were incubated for 48\u0026ndash;72 h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The neutralizing serum titre was calculated with the CPE inhibition rate according to the Reed-Muench calculation (Ref. Reed-Muench, 1921). The serum samples (n\u0026thinsp;=\u0026thinsp;89) were further analyzed for their capacity to inhibit the interaction between the SARS CoV-2 virus S1 spike protein and the ACE2 cell receptor \u003cem\u003ein vitro\u003c/em\u003e (Teca VLV-test, Nacka, Sweden).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Serology and ELISA antigens used and tested by samples:\u003c/h2\u003e\n \u003cp\u003eAll participants were invited to be study participants during their hospital visit; thus, all patients were severely to moderately ill due to SARS-CoV-2 infection. Serum was collected, aliquoted, and frozen until use. SARS-CoV-2 S1-spike protein (Wuhan strain 2019) was used as a soluble antigen on 96-well microwell plates (0.5 ug/mL PBS pH 7.2, Toronto University, SGU Toronto ON, Canada/SinoBiologicals, Eschborn, Germany). SARS-CoV-2 nucleoproteins NC-(aa47-174 and aa267-364) and nsp3 (Toronto University, ON, Canada) were used as recombinant viral coating proteins for performing the ELISA. Serum samples from COVID-19 patients and positive and negative controls were diluted in PBS-Tween 20 (0.05%) with 2.5% fat-free milk buffer (Knopf J et al.2022). Serum dilutions and standard serum control samples were added to the coated plate wells and incubated for 90 min at 37\u0026deg;C. Conjugates against anti-human IgG-HRP (BioRad, Richmond, CA) or anti-human IgA-HRP (Nordic BioSite, T\u0026auml;by, Sweden) were added to separate wells with diluted serum samples and incubated for 90 min at 37\u0026deg;C. Finally, the substrate 0.003% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/o-phenylene diamine (Sigma‒Aldrich, S:t Louis, MA. 0,4 mg/mL) was added and incubated at room temperature for 30 min before 2.5 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added as a stop solution. The plates were read at OD490. Standard curve samples for determining AU antibody quantitation were used. The antibodies used are HRP-anti-human IgA (BioRad, STAR141P), HRP-Goat-anti-human IgM (ThermoFisher, Invitrogen A18909), HRP-goat-anti-human IgM, (Sigma-Aldrich, MERCK A6907), HRP-goat anti-human IgA (Sigma-Aldrich, AP114P (Fc5m), Goat-anti-human SC (Sigma-Aldrich, I-6635, 0,2 mL) MoAb Clone GA-1.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.5 Serum IgG subclass analyses\u003c/strong\u003e: Selected serum samples (60 individuals) were tested for the content of serum IgG-anti-S1 spike protein-specific IgG1 and IgG3. The serum IgG subclasses were tested with isotype-specific murine anti-human IgG subclasses (Sigma‒Aldrich, S:t Louis, MA, anti-human IgG1 clone BAM 09 and anti-human IgG3 clone BAM 08) (Sundqvist V-A et al. 1984). In brief, S1-spike-coated plates were incubated with serum samples diluted 1/100, 1/1000, and 1/10 000 in PBS-T with 2.5% dry milk and incubated for 90 min at 37\u0026deg;C. After washing, 100 \u0026micro;l of diluted monoclonal anti-human IgG1 and IgG3 (Sigma‒Aldrich/Merck, Paris, France, diluted 1/1000 in PBS-T 2.5% dry milk) was added to each well and incubated for 90 min at 37\u0026deg;C. After washing, 100 \u0026micro;l of diluted HRP-labelled anti-mouse IgG (BioRad, Richmond, CA, at a dilution of 1/3000 in PBS-T 2,5% dry-milk) was added and incubated for 90 min at 37\u0026deg;C. After washing, the OPD substrate was used as shown above. Based on studies with negative control serum samples, an OD of 0.2 was set as the cut-off value for positive samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.6 B-cell IgG epitope mapping on the S1-spike receptor-binding motif (RBM) region in serum IgG\u003c/h2\u003e\n \u003cp\u003eTo analyze the humoral response to SARS-CoV-2 RBM of the RBD, 3 aa35-mer peptides with 5 amino acids offsets covering the RBM were synthesized based on the reference sequence of SARS-CoV-2 S protein (GenBank: YP_009724390). Four synthetic 35-mer peptides representing the S1-spike cell-receptor-binding motif (RBM) of the Wuhan SARS-CoV-2 virus strain were used \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The peptide amino acid sequences are given in the table below. The amino acid sequence (aa no. 438\u0026ndash;526) was represented by four peptides (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Synthetic SARSCoV2 peptides. Based on high antigenicity analysis of the three-dimensional predicted structure and B cell epitope information (BepiPred-2.0), eight different antigenic peptides were selected from the amino acid sequence of SARS-CoV-2 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The synthetic peptide was purified by reverse-phase HPLC (\u0026gt;\u0026thinsp;98% purity) (Peptide Institute Inc., Osaka, Japan.). The synthetic SARS-CoV-2 peptide was reconstituted at 5 mg/ml in sterile PBS and stored below \u0026minus;\u0026thinsp;20\u0026deg;C.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/caption\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSN3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQS. aa 438\u0026ndash;508\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSN4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEGFNCYFPLQSYGFQGGGNYNYLYRLFR. \u003cstrong\u003eaa 486\u0026ndash;526\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSN5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIYQAGSTPCNGVEGFNCYFPLQSYGGGSLYRLFR aa 457\u0026ndash;526\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSN6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKVGGNYNYLYRLFRGGSGGFPLQSYGFQ \u003cstrong\u003eaa 445\u0026ndash;508\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.7 Serology of mucosal samples:\u003c/h2\u003e\n \u003cp\u003eOral and nasal swabs were collected with cotton pads and stored frozen in 1 mL of sterile saline solution until use. Mucosal samples from COVID-19 patients and positive and negative controls were diluted with PBS-Triton X-100 5% with 2.5% fat-free dry milk, added to coated SARS-CoV-2 S1 spike plate wells and incubated overnight at RT. Conjugates against anti-human IgA-HRP (Nordic BioSite, T\u0026auml;by, Sweden) were added and incubated for 90 min at 37\u0026deg;C. To study the secretory component on mucosal IgA antibodies, the anti-SC-conjugated HRP (Sigma‒Aldrich, St. Louis, MO) was used. Substrate and plate absorbance were measured as described above. The cut-off was determined by identifying the mean reactivity with the antigens from ten SARS-CoV-2 negative control nasal swabs and multiplying this value by two.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.8 Statistical analysis.\u003c/h2\u003e\n \u003cp\u003eData were analysed using Prism version 8.2d for macOS (GraphPad Inc. Software LLC).\u003c/p\u003e\n \u003cp\u003eThe Mann‒Whitney U test was performed to compare differences in antibody reactivity between sampling time points. The statistical analysis was performed using GraphPad Prism 8 (La Jolla, CA, USA). Comparisons between groups with SARS CoV-2 serum, nasal or rectal swabs were performed using the nonparametric Mann‒Whitney U test with Bonferroni correction, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical permission\u003c/strong\u003e: The study was conducted according to the rules of the Declaration of Helsinki. Samples were collected after informed consent and ethical permission was obtained and approved by the Ethical Review Board at (EPM) Link\u0026ouml;ping County council, Link\u0026ouml;ping, Sweden. (Reference EPM no: 2020\u0026ndash;03888 and 2020\u0026ndash;02080).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The adult population has a lower viral load than the older population:\u003c/h2\u003e \u003cp\u003eQuantitative RT‒PCR Ct values were available from 62 infected individuals prior to vaccination. Later, during follow-up and after recovery, all individuals became vaccinated against SARS-CoV-2, making them enter the hybrid-immunity study participant category. The median qRT‒PCR Ct value Wilcoxon signed-rank sum test and Mann‒Whitney U test pairwise comparison for the sex- and age-separated patient groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results show significantly higher levels of SARS-CoV-2 virus in vaccinated patients\u0026thinsp;\u0026lt;\u0026thinsp;70 years old. This difference was also significant with the nonparametric Mann‒Whitney U test comparison.\u003c/p\u003e \u003cp\u003eSerum with SARS CoV-2-neutralizing properties was identified in 10/16 individuals at the earliest available serum samples of this study (on the day of the analysed SARS-CoV-2 viral qPCR test).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2: Adult populations achieved their maximum viral neutralization and ACE-2 inhibition potential earlier than the older population\u003c/b\u003e \u003c/p\u003e \u003cp\u003eVirus-neutralization serum titres (\u0026gt;\u0026thinsp;50% inhibiting capacity) are shown for the study group and study time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). In Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, all study serum samples were included per study time point. The data show that a significant neutralizing titre increase occurred between Days 0 and 28 when all participants were included and among the study participants\u0026thinsp;\u0026lt;\u0026thinsp;70 years of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Thereafter, a plateau was reached between Day 28 and Day 180; the median titre appeared to increase, significantly among people\u0026thinsp;\u0026gt;\u0026thinsp;70 years of age, where a significant neutralizing titer was seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In the 2-year sampled serum of 15 individuals, a significant neutralizing titre was seen in all study categories for all individuals, the \u0026lt;\u0026thinsp;70-year old group, the \u0026gt;\u0026thinsp;70-year-old group, and males (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). When women were stratified into a study group (3 of the 15 study participants at the 2-year timepoint), the change in neutralizing serum titre was not significant at the 2-year follow-up time point.\u003c/p\u003e \u003cp\u003eVirus-neutralization serum correlating S1-spike-ACE2-binding inhibition serum titres (\u0026gt;\u0026thinsp;67% inhibiting capacity) are shown for the study group and study time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When comparing the study time points, a significantly increased median serum S1-ACE2-inhibiting titre was observed between Days 0 and 28 and between Days 180 and \u003cb\u003e2\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The time period between Days 14 and 180 was a plateau period with no significant median titre increases.\u003c/p\u003e \u003cp\u003eThe ACE2 \u0026ndash; S1-spike protein inhibition capacity at a serum dilution of 100 was very strong in almost all (98%) study participants from Day 28 and onwards, without significant difference between study groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the virus-spike protein and recombinant cell-receptor (ACE2) inhibition assay with the serum of study participants. A significant change in inhibition activity was seen between Day 0 and Day 28 and between Day 180 and 2 years (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The strength of inhibition as a percentage of prevented S1-spike binding to the recombinant ACE2 protein is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. Here, the significant inhibition levels were highest from Day 28 onwards in all tested serum samples.\u003c/p\u003e \u003cp\u003eInhibition did not significantly differ between serum collected from individuals younger than 70 years and individuals older than 70 years (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3: The antibody response against the spike protein showed an initial 50% seropositivity, which increased to 100% for IgG, with the majority being seropositive for both IgG and IgA at the end of the study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn total, 89 patients with COVID-19 infection and SARS-CoV-2 vaccination were included. The kinetics of serum IgG and IgA against the SARS-CoV-1 S1-spike protein were analysed (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFifty percent (50%) of study participants showed low levels of anti-S1 spike protein IgG and 31% serum IgA at the initial and inclusion time points (Day 0). All patients (100%) developed detectable IgG and IgA responses during the study period. All study participants (100%) were seropositive at 1 month and remained seropositive for serum IgG at the 6- and 24-month follow-ups. Fifteen study participants who reached the 2-year study time point after study entry all showed the presence of serum IgG and IgA against the S1-spike protein. Serum IgA seropositivity was detectable in all tested samples at 2 months but was reduced to 78.6% at 6 months.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4: The antibody response is stronger for the N-terminus of the nucleocapsid protein, with no response observed against the nonstructural SARS-CoV-2 protein\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIgG against SARS-CoV-2 nucleocapsid proteins (NP-protein aa 47\u0026ndash;173) was detected in serum samples from all study participants one month postadmission. However, only 2 study participants showed detectable serum IgG against a more C-terminal part of the nucleoprotein (aa 247\u0026ndash;364, not shown). Anti-NP-binding antibodies in the serum were compared with serum IgG anti-S1 spike reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Serum IgG and IgA reactivity against one nonstructural protein, the NSP3 protein, was assayed, and all tested sera were shown to be negative or below the limit of detection (not shown).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5: Serum IgG subclass reactivity against the S1 spike and nucleocapsid protein.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe IgG1 subclass against the S1-spike protein was the dominant IgG subclass detected in the involved patients. An analysis of serum IgG subclass reactivity against the S1-spike protein in samples from 34 individuals with severe or intermediate severe COVID-19 showed no difference in subclass reactivity at 1 and 6 months post-PCR positivity. (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6: Identification of the B-cell epitope on the spike receptor binding domain (RBD) reveals an amino acid domain based on the serum antibody reactivity of the study participants\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eMost individuals who were tested for serum anti-S1 spike and ACE2 cell receptor binding inhibition and underwent SARS-CoV-2 virus-neutralizing assays needed\u0026thinsp;\u0026gt;\u0026thinsp;14 days to develop efficient virus-neutralizing antibodies. Therefore, we investigated if and when IgG antibody epitope reactivity may develop within the S1-RBD and RBD-RBM regions that would interfere with the virus‒cell-receptor interaction. Based on sera that can neutralize the live SARS-CoV-2 virus \u003cem\u003ein vitro\u003c/em\u003e, we identified individuals who did develop serum IgG reactivity against peptides representing the C-terminal RBM and individuals who developed S1 spike-binding but nonneutralizing antibodies without reacting with the synthetic peptides. Serum samples from individuals without neutralizing antibodies did not react by binding to any of the tested RBM peptides (See Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003eThe B-cell epitope mapping of the cell-receptor-binding region of the viral S1 spike protein suggests that a neutralizing epitope is situated in the receptor-binding motif (representing aa 444\u0026ndash;479).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7: Anti-S1-IgA positivity in nasopharyngeal swab samples in the entire study population observed with an anti-nucleocapsid protein IgA response in only a few participants\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA subgroup of individuals was tested for IgA reactivity in nasopharyngeal swab samples (n\u0026thinsp;=\u0026thinsp;58). Positive reactivity against the S1 spike protein was seen at least once in up to 90% of tested individuals, between Days 14 and 180 (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). No significant difference was seen between age groups. Fifty percent of tested individual nasal samples were initially (Day 0) shown to have positive anti-S1-reactive nasal mucosal IgA against the S1-spike protein. This frequency increased to 100% nasal IgA-positive responses from Days 28 to 60. However, this increase cannot be conclusively attributed to either the vaccination booster effect or a sign of local upper respiratory tract infection. In three of fifteen nasal samples, anti-S1-spike IgA was also seen at the 2-year time point. Few signs of reinfection could be identified in nasal samples during follow-up by local mucosal anti-SARS-CoV-2 virus S1-spike protein antibody analysis. Nevertheless, a few individual nasal swab samples contained low but still positive nasal IgA anti-SARS-CoV-2 reactive anti-nucleocapsid protein reactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) both at Day 180 and during the 2-year follow-up time point (n\u0026thinsp;=\u0026thinsp;10 and n\u0026thinsp;=\u0026thinsp;2, respectively).\u003c/p\u003e \u003cp\u003e\u003cb\u003e3.8: Anti-spike-IgA was detected in the rectal swab samples of a subgroup of the participants\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eA subgroup of individuals was tested for IgA reactivity in rectal swab samples, and positive reactivity against the S1 spike protein was seen at least once in up to 18% of tested participants between Days 14 and 180 after the initial qPCR-positive diagnostic time-point (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e8\u003c/span\u003e). No significant differences were seen between age groups or between opposite-sex individuals. No sign of reinfection could be identified in intestinal samples during follow-up by local rectal anti-SARS-CoV-2 virus S1-spike protein antibody analysis. Nevertheless, a few individual rectal swab samples contained low but still positive rectal IgA anti-SARS-CoV-2 reactive anti-S1-spike and/or nucleocapsid protein reactivity (Figs.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) at Day 180 but not at 2 years (n\u0026thinsp;=\u0026thinsp;2 and n\u0026thinsp;=\u0026thinsp;0, respectively).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHighly pathogenic respiratory tract viral infections, such as influenza A/B, respiratory syncytial virus (RSV), and measles virus, show that humoral immune responses can correlate well with protective immunity. However, as with the current pandemic SARS-CoV-2 virus Omicron variants, reinfection is possible, especially in the upper respiratory tract, after having developed efficient neutralizing antibodies against earlier SARS-CoV-2 variants and the current vaccine strain. Instead, efficient protection from severe illness is often provided by the virus-neutralizing humoral response with supportive cell-mediated immune responses (CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells), which together constitute the immunological memory response.\u003c/p\u003e\n\u003cp\u003eMost currently used vaccines induce protective immunity and are currently associated with neutralizing antibody levels in the serum and plasma. Important results obtained with anti-S1 monoclonal antibody treatment for severely infected SARS-CoV-2 patients support the protective value of antibodies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHowever, humoral first-line defense can also be found in mucosal tissues, the nasal/oral and respiratory tract mucosa, and gastrointestinal mucosal tissues that provides the mucosal immunity. In these tissues, this defense is most often represented by local mucosal B-cell synthesis and the secretion of secretory mucosal dimeric (or tri/quadrimeric) IgA (S-IgA) \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Therefore, the sampling and analysis of nasal swabs and rectal/intestinal (stool) samples may provide important information regarding the duration and presence of protective local mucosal immunity that complements the neutralizing and protective serum-associated IgG/IgA antibody responses. In this study, we investigate the hybrid immunity induced by initial natural SARS-CoV-2 infection, followed by mRNA SARS-CoV-2 vaccination \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e 27,28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIdentifying the correlations of protective immunity is important, and the best and most often-defined correlate of protection in almost any respiratory infectious illness to date has been antibodies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Antibodies have been shown to neutralize infectivity by attaching to viral surface proteins and trigger complement activation to provide antibody-dependent, cell-mediated cytotoxicity against infected viruses and cells. However, some evidence suggests that SARS-CoV-2 will not be sensitive to the same correlates of protection \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The challenge will be to obtain local mucosal secretory antibodies capable of recognizing the new circulating S1-envelope protein mutant SARS-CoV-2 strains, especially close to the nasal mucosal surfaces, as seen in a subpopulation of participants in this study (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The possibly positive result of obtaining SARS-CoV-2-specific immunity via natural infection may be a broader immune response to several viral proteins not limited to those included in the vaccines. Thus, immune memory B- and T-lymphocytes against both viral surface proteins and several hopefully more conserved intraviral proteins, such as nucleocapsid proteins (NCs) and nonstructural proteins (nsp:s), will be obtained. Nevertheless, the longevity, characteristics and magnitude of these immune responses remain to be elucidated in COVID-19 patients with mild and/or severe disease and in vaccinated individuals of different age groups and sexes.\u003c/p\u003e\n\u003cp\u003eSerological analyses in previous studies have shown that the immune response against SARS-CoV-2 S1 Spike protein is one of the most long-lasting responses when compared with those of viral proteins \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e 34 35\u003c/sup\u003e. In this study, all involved patients developed detectable anti-S1- and NP-binding IgG and IgA responses during the natural infection and in response to additional SARS CoV-2 vaccination, and these responses persisted for the entire 2-year study period. All followed study participants were S1-spike seropositive and virus-neutralization capable at 6 months. In a subgroup, the neutralization capacity remained for the entire 22-month to 2-year period.\u003c/p\u003e\n\u003cp\u003eThere are links between nasal IgA response, mucosal virus neutralization and less severe infection \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. All samples showed the presence of both serum IgG (IgG1) and IgA and local mucosal nasopharyngeal IgA against the outer surface proteins of SARS-CoV-2 for up to 2 years of follow-up. Rectal anti-S1-spike binding antibodies were only detectable during the initial 28 days after the initial positive nasal PCR assay. In fifteen individuals (of which 3 were female) for whom a 2-year follow-up sample was available, the presence of high amounts of SARS-CoV-2 neutralizing antibodies and anti-S1 specific serum IgG1 were detected. Serum IgG and IgA responses against the nucleocapsid protein were weak at this late time point, suggesting the absence of detectable reinfection that induced an enhanced humoral immune response. Most likely, the observed antibody responses at this later stage of hybrid immunity (infection followed by vaccination) were due to vaccination with the mRNA S1-spike proteins in the majority of adult to elderly (\u0026gt;\u0026thinsp;70\u0026ndash;85 years) study participants. Anti-SARS-CoV-2 specific antibodies in infection and vaccination has previously been followed and described during shorter time studies \u003csup\u003e25,36,40\u0026minus;42\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eImportantly, the serum IgG against neutralizing B-cell epitopes on viral proteins should remain elevated, hopefully at a protective level. Nevertheless, clear antibody titre or antibody quantity limits to prevent infection or reduce transmissibility have not yet been established. Similarly, serum IgG against the nucleoprotein was also frequently detectable among the infected patients. However, serum IgG/IgA against nucleocapsid proteins (NCs) has been described as both negative and positive for human health, as described in different patient categories. In one study, higher amounts of these immunoglobulins correlated with the antibody-dependent enhancement (ADE) of infection, despite a lack of clear mechanistic process \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In other studies, these antibodies have been part of the cure/health-promoting immune response. A possible explanation for the positive interpretations could be the clearance of circulating viral proteins that otherwise may cause the formation of immune complexes or result in inflammation at local sites where the NC proteins aggregate \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eApproximately 50% and 20% of hospital admissions for pneumonia in young children and elderly individuals, respectively, are estimated to be caused by viral infections. Prior to 2020, these admissions were mainly due to respiratory syncytial virus or influenza viruses \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Since then, the dominant cause of respiratory viral infection has been betacoronavirus SARS-CoV-2 (in Scandinavia). Respiratory infectious viruses (historically dominated by approximately eight to ten different virus families) will consequently remain a serious challenge, despite decades of intensive research for developing antiviral drugs and vaccines against these viruses.\u003c/p\u003e\n\u003cp\u003eStratifying our patient population into subgroups allowed us to compare the impact of natural infection followed by vaccination with two doses supporting the SARS-CoV-2-specific humoral immune response. The \u0026gt;70-year-old group showed reduced expression of the anti-S1 spike IgG response in the long term compared to the younger group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n\u003ctable style=\"width: 100%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cdiv style='margin:0in;margin-bottom:.0001pt;font-size:16px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:16px;font-family:\"Calibri\",sans-serif;margin:0in;margin-bottom:.0001pt;'\u003e\u003cstrong\u003e\u003cu\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eBox 1: Highlights of two years SARS-CoV-2 followup study findings:\u003c/span\u003e\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv style='margin:0in;margin-bottom:.0001pt;font-size:16px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; A higher\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;virus qRT‒PCR load in \u0026lt;70\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-year-old\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eindividuals (Fig. 1)\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;correlates\u003c/span\u003e\u003cspan\u003e\u0026nbsp;with\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ea\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ehigher neutralization titre during\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003efirst 28 days of infection\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e,\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;with a subsequent plateau of the response.\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; The \u0026gt;70\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-year-old\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;group attained the highest neutralization potential more slowly, 28 days post-infection or later (Fig. 2A, B). Males\u003c/span\u003e\u003cspan\u003e\u0026nbsp;had a\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;higher median neutralization titre at\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e22-24 month timepoint (nonsignificant difference from females).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; S1-Spike-ACE2 binding inhibition serum titres showed no significant change between 14\u0026nbsp;\u003c/span\u003e\u003cspan\u003eand\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;180 days. The maximum significant increase\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;was\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;observed during\u0026nbsp;\u003c/span\u003e\u003cspan\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003efirst 14 days (from the primary infection) and during 180 days of follow\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eup (after vaccination).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; All study groups showed 100% S1-spike protein inhibition capacity 28\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003edays\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eafter\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;infection (Figure 3A and B)\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e,\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;with no difference between\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026lt;/\u0026gt; 70\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-year-old\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;groups (Figures 3C and 3D).\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; The participants\u0026nbsp;\u003c/span\u003e\u003cspan\u003eremained\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;seropositive for Spike S1 IgG until the last study point (Fig. 4A).\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eA total of\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e78.6% of them\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eremained\u003c/span\u003e\u003cspan\u003e\u0026nbsp;seropositive for Spike S1- IgA at\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e6-month time point (Fig. 4B).\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eThe serum\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;anti-S1 spike IgG1 subclass dominates in seropositive individuals.\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; Anti-\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003enucleocapsid\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;IgG\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eis\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;more efficient in targeting the N-terminus of the protein (\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eaa 47\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-173) than its C\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eterminal part (aa 247-364) (Fig. 4C, 4D).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; The B-cell epitope mapping of the cell-receptor-binding region of the viral S1 spike protein\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003esuggests\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;that a neutralizing epitope is situated in the receptor-binding motif (representing aa 444-479) (Fig. 5).\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; In\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003enasal\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;mucosal samples\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e,\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;the\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;anti-\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003espike\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;S1 IgA response increased from 50% of the tested individuals at Day 0 to 100% within\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eDays\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e28 to 60, after which\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;response tapered off. Only\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;a\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;few samples\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ewere\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;shown to be positive at\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e180\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-day\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003etimepoint (Fig. 6).\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eThe anti\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e-NP IgA response in the same nasal mucosal samples\u0026nbsp;\u003c/span\u003e\u003cspan\u003ewas\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;evident from Days 14-28\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e,\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;after which nasal IgA\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003egradually\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;disappeared (Fig. 8A).\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026bull; In \u003cstrong\u003erectal mucosal samples\u003c/strong\u003e, IgA-positive reactivity against Spike S1 and NP protein was observed in a small group of individuals (18% of the study population) only within\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003ethe\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003efirst three timepoints (Day 0, 14 and 28) and not\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003eat\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;the timepoints beyond 2 months (Fig. 7B, 8B)\u003c/span\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cp style='margin-right:0in;margin-left:0in;font-size:16px;font-family:\"Calibri\",sans-serif;margin:0in;margin-bottom:.0001pt;'\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOf the studied participants, a small percentage showed a positive IgA response in rectal swabs (18%). This result is unique, as only very few studies have previously reported test data from intestinal organs. Furthermore, in the few previously reported studies, nondetectable anti-SARS-CoV-2 antibody levels were shown \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn a selected population of eighteen individuals, epitope mapping showed the presence of anti-S1 spike serum IgG, with 10 samples having virus-neutralizing reactivity \u003cem\u003ein vitro\u003c/em\u003e and eight with no neutralizing reactivity. The study indicates that 8 of 10 individuals with neutralizing activity had anti-RBM-epitope reactive serum IgG B-cell epitope aa 444\u0026ndash;479 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC), while 8/8 of the individuals without neutralizing activity \u003cem\u003ein vitro\u003c/em\u003e lacked this reaction pattern. Similar B-cell epitope results have also been presented by other studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Thus, when anti-RBD/RBM-directed antibodies develop, they correspond to a shift to virus-neutralizing functional activity. Our results support data presented in previous studies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe anti-S1-IgG1 response was detected in all patient groups, which contrasts findings from other studies in which serum IgG3, together with IgG1, was also detected in samples from individuals with more severe disease and symptoms. The reasons for these dissimilarities may depend on several factors, such as differences in patient categories and the timeline of the study (as IgG3 has a shorter biological half-life than the IgG1 subclass) in association with disease symptoms \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Furthermore, other studies have shown that serious illness may even suppress specific B-cell responses completely or below the level of detection. Except for the abovementioned reasoning, the humoral subclass IgG-reaction pattern seems similar to those of several other viral infections, such as Influenza A and B, RSV, HHV-6, and CMV, while subclasses antiviral IgG2 and IgG4 are often rare. Instead, these latter subclasses are more commonly seen in samples from patients with frequently reactivated viral infections \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and they are even more prevalent in bacterial infections. This finding confirms a previous study in which IgG1 levels were shown to be higher in patients who were able to fight off the infection and had better outcomes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo mention the limitations of the study, not all study participants provided study samples at each test timepoint. Ethical permission allows only serological analyses. The most frequent samples given were blood samples and nasopharynx samples. Rectal samples were least frequently provided. No cell-mediated immune responses were analysed because ethical permission was limited to the analysis of immunoglobulin responses against SARS-CoV-2 proteins.\u003c/p\u003e\n\u003cp\u003eBox 1 summarizes by highlighting in a nutshell all the key findings from this longitudinal study of clinical covid patient samples for two years. In conclusion, this study suggests that both systemic and mucosal immunity are evident in the included patient groups. Over a period of 6 months, the majority (98.7%) of naturally infected COVID-19 patients in this study population developed lasting detectable levels of serum IgG and IgA responses against the SARS-CoV-2 S1-spike protein and the nucleocapsid protein but not against nonstructural protein 3. Interestingly, samples from all study participants (15/15 individuals) collected at the latest available time point (between 22 months and 2 years after hospital admission) showed potent serum virus-neutralizing properties \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are most grateful for the invaluable support with SARS-CoV-2 (Wuhan strain) reagents to Dr. Ackloo S. SGU Toronto, ON, Canada. All involved researchers confirm no competing interests. We wish to thank research nurses Karin Sjöberg and Mona Hansson at the Clinical research unit at Region Östergötland for their invaluable help with the clinical coordination, monitoring and sampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e ALF, Hanberger H, Region Östergötland, and Linköping University.\u0026nbsp;The KACST fund: (KING ABDULAZIZ CITY FOR SCIENCE AND TECHNOLOGY “KACST”\u0026nbsp;).\u0026nbsp;The MIIC stipend fund from Linköping University for postodoctoral fellowship of AS. Knut and Alice Wal- lenberg Foundation, Wallenberg Center for Molecular Medicine. The Structural Genomics Consortium is a registered charity [1097737] that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Ge- nomics Institute [OGI-196];EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510]; Janssen, Merck KGaA (aka EMD in Canada and US); Pfizer; Takeda Stiftelsen för Strategisk Forskning (FFL15-0026).;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical permissions:\u0026nbsp;The study was performed according with the Helsinki Declaration guidelines and was approved by the Ethical Committee, Linköping County Council board, Linköping, Sweden. (Approval No. 2020-02080, 2020-03888). Written informed consent was obtained by participants. The permission allow only serological studies based on blood and mucosal (nasopharyngeal/rectal) samples thus cell-mediated studies were not possible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNo conflict of interest\u003c/strong\u003e. All authors confirm No conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eB\u0026ouml;ttiger, M. Polio immunity to killed vaccine: an 18-year follow-up. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 443\u0026ndash;445 (1990).\u003c/li\u003e\n\u003cli\u003eB\u0026ouml;ttiger, M. \u0026amp; Larsson, B. Swedish inactivated polio vaccine: laboratory standardization and clinical experience over a 30-year period. \u003cem\u003eBiologicals\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 267\u0026ndash;275 (1992).\u003c/li\u003e\n\u003cli\u003eHaralambieva, I. H., Kennedy, R. B., Ovsyannikova, I. G., Schaid, D. J. \u0026amp; Poland, G. A. 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Biol. 2021 41\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1\u0026ndash;13 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"long-term immunity, antibody response, spike neutralization, immunoglobulin, mucosal immunity, serological response","lastPublishedDoi":"10.21203/rs.3.rs-2113175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2113175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLongitudinal serum samples, nasopharyngeal/nasal swabs and rectal swab samples were collected from eighty-nine individuals (median age 66 y) with SARS-CoV-2 PCR-positive test results at Link\u0026ouml;ping University Hospital. Samples were collected from the initial visit and thereafter for up to 2 years of follow-up. The presence of serum IgG and IgA against SARS-CoV-2 antigens (S1-spike, nucleocapsid, and NSP3) was analysed. Nasal and rectal swabs were tested for the presence of mucosal IgA against the outer envelope S1 spike and the nucleocapsid protein.\u003c/p\u003e \u003cp\u003eNinety percent of the participants were seropositive for SARS-CoV-2 recombinant proteins on Day 28 after study entry, and all (100%) were seropositive based on samples collected 2 months or later. Almost all (95%) developed serum SARS-CoV-2-neutralizing antibodies that were measurable from 6 to 24 months. The most common antibody responses (both serum IgG, mainly IgG1, and in nasal mucosa IgA) reacted with the S1-spike protein and the nucleoprotein. In samples collected from nasal tissues, IgA anti-S1 spike protein was mainly observed during 2 months of follow-up. In a subpopulation (18% of tested individuals), rectal IgA swabs showed the presence of anti-S1 spike IgA for 1 month of follow-up among the participants studied.\u003c/p\u003e \u003cp\u003e.\u003c/p\u003e","manuscriptTitle":"Long-term systemic and mucosal humoral response in SARS- CoV-2 vaccinated post-Covid-19 infected patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-07 15:24:22","doi":"10.21203/rs.3.rs-2113175/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48b0341a-d1c6-4b2a-9f07-767e35f07e51","owner":[],"postedDate":"October 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-25T10:59:12+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-07 15:24:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2113175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2113175","identity":"rs-2113175","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}