Comparison of the clinical features, viral shedding and immune response in vaccine breakthrough infection by the Omicron and Delta variants

On the the of Severe Acute Respiratory as the variant of Infections have quickly worldwide, but understanding of the viral dynamics and the cytokine and cellular immunological response during remain limited. Detailed patient-level data from 174 age-matched patients with sequence conrmed Omicron or Delta infection admitted to the National Centre for Infectious Diseases, Singapore were analyzed in an observational cohort study. Peripheral blood samples for measurement of SARS-CoV-2 immunological parameters were obtained from a subset. Respiratory samples were collected for viral cultures and correlated to corresponding PCR cycle threshold (Ct) values. In cohort kinetics


Introduction
Since the emergence of the Alpha (B.1.1.7) variant in September 2020, the Coronavirus disease 2019 (COVID- 19) pandemic has been shaped by successive waves of variants with increased transmissibility and/or immune evasion compared with the ancestral strain. 1 Delta (B.1.617.2) caused a large outbreak in India from February 2021 and displaced other variants to become globally dominant over the following six months. 2 Omicron (B.1.1.529) was rst reported from Gauteng province, South Africa in November 2021. 3 On 26 November 2021 it was classi ed as the fth variant of concern (VOC) by the World Health Organization (WHO) and has spread rapidly to many countries worldwide.
The Omicron variant is characterized by >30 mutations in the gene encoding for the spike protein. 4 Some of the deletions and mutations identi ed in the Omicron variant (e.g., 69-70del, T95I, G142D/143-145del) are known to result in higher viral binding a nity, higher rates of antibody escape and increased transmissibility. 5,6 As a results of these changes, Omicron is able to avoid neutralization by serum samples from recovered or vaccinated individuals and by a large range of human monoclonal antibodies. [7][8][9][10] Mice experimentally infected with Omicron had less severe clinical signs (less weight loss and severe pneumonia) and lower viral loads in lower and upper respiratory tract compared with Delta. 11 Lower viral loads and no histopathological evidence of bronchopulmonary in ammation were found in Omicron infected hamsters. 12 In a preliminary report, Ferguson et al. recently described a reduction in risk of hospitalization for Omicron relative to Delta, including an apparent difference by vaccination state. 13 However, immunological and clinical data remain limited.
In December 2021, Singapore saw a wave of imported Omicron infections with limited local transmission. As part of the initial public health response all individuals with suspected Omicron infection (S-gene target failure) were admitted to the National Centre for Infectious Diseases (NCID) for isolation. Close contacts were identi ed, isolated in a dedicated facility for 10 days, with PCR testing at the start and end of the quarantine period.
A more granular understanding of viral shedding, as well as in ammatory mediators and cellular immune features of emerging VOCs is essential in guiding healthcare policies globally, including isolation, hospitalization, and vaccination policies. In this cohort study we characterize the clinical features, virological and immunological kinetics of patients with vaccine breakthrough COVID-19 caused by the Omicron variant in comparison with Delta.

Patient Recruitment
Individuals con rmed to have COVID-19 by SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction (RT-PCR), admitted to the National Centre for Infectious Diseases (NCID), Singapore, and fully vaccinated were eligible for inclusion in the study. Fully vaccinated was de ned as infection onset ≥14 days after completion of any primary COVID-19 vaccine series with a WHO Emergency Use Listing (EUL).
Illness onset was de ned as whichever was early from date of symptom onset or rst PCR with SARS-CoV-2 detected.
Two age-matched (+/-10 years) cohorts were selected: Omicron (B.1.1529) or Delta (B.1.617.2) variant infections as assigned by direct whole-genome sequencing (WGS) of RNA extracted from nasopharyngeal swabs by the National Public Health Laboratory (NPHL) at NCID. Pangolin COVID-19 Lineage Assigner and CoVsurver were used to assign sequence lineages. Individuals were included if they were admitted to NCID between 1-18 Dec 2021 for Omicron and 27 April to 11 Aug 2021 for Delta. During these two periods, all individuals with Omicron or Delta infection respectively were admitted to hospital for isolation and evaluation regardless of disease severity, and NPHL attempted WGS for all infections with RT-PCR Cycle threshold (Ct) value <30.

Clinical Chart Review and Sample Collection
Clinical information was extracted from the medical record using a standardized data collection form adapted from the International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) case record form as previously described. 14

SARS-CoV-2-speci c T cells by Intracellular Cytokine Staining
To pro le the SARS-CoV-2 speci c T effector subsets in COVID-19 patients, intracellular cytokine staining was carried out as previously described 20 with modi cation. Brie y, PBMCs collected from whole blood of vaccine breakthrough cases and healthy vaccinated controls were stimulated with phorbol 12myristate 13-acetate (PMA 100ng/mL, Sigma Aldrich) and ionomycin (1µg/mL, Sigma Aldrich), or pooled SARS-CoV-2 PepTivator S, S1, M and N peptides (0.6nmol/mL each) (Miltenyi Biotec) for six hours. Brefeldin A and Monesin (1× , ThermoFisher Scienti c) were added at two hours post-stimulation. Cells were stained for surface markers in the dark at room temperature for 30 minutes (Supplemental Table 2, Row 1-21), followed by xation and permeabilization for 30 mins with Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher Scienti c). Permeabilized cells were then stained for intracellular cytokines in the dark at room temperature for 30 minutes (Supplemental Table 2, . Cells were then washed with PBS and centrifuged at 800 x g for 5 minutes before transferring to respective polystyrene FACS tubes containing 5 mL (4.8 x 10 3 beads) of CountBright Absolute Counting Beads (Invitrogen Cat#C36950, Lot 2324193). Cells were acquired with the CytekTM Aurora cytometer running SpectroFlo® Version 2.2.0.3 with automated unmixing and analyzed using FlowJo v10.8.0.
Spike protein ow cytometry-based assay (SFB assay) for antibody detection The SFB assay was performed as previously described. 21,22 S protein-expressing cells were seeded at 1.5 x 10 5 cells per well in 96 well V-bottom plates. Cells were incubated with human serum (diluted 1:100 in 10% FBS) followed by a secondary incubation with a double stain, comprising Alexa Fluor 647conjugated anti-human IgG (1:500 dilution) and propidium iodide (PI; 1:2500 dilution). Cells were acquired using a BD Biosciences LSR4 laser and analyzed using FlowJo (Tree Star). The assay was performed as two independent experiments with technical duplicates each time.
Memory B cell ELISpot SARS-CoV-2 RBD-speci c memory B cell numbers were counted using ELISpot. MultiScreenHTS IP Filter Plate, 0.45 µm plates (Merck Millipore) were coated overnight at 4°C with puri ed anti-human-IgG (MT91/145, Mabtech) prepared at 15μg/ml in PBS 1X. Plates were washed 4 times with PBS 1X and blocked at least 30 minutes at room temperature with RPMI 10% FBS. Fresh PBMCs were plated directly into ELISpot plate (100,000 and 400,000) to determine RBD-speci c plasmablast numbers. In parallel, 1 x 10 6 PBMCs were resuspended in 1ml RPMI + 10% FBS + 1 μg/ml R848 + 10 ng/ml IL-2, and incubated at 37°C, 5% CO 2 for 4-5 days to differentiate memory B cells into antibody-secreting cells. After incubation, cells were counted, and 100,000 or 400,000 live cells were taken for ELISpot plating to determine RBDspeci c memory B cell numbers. Total IgG secreting cells were determined by plating 1,500 or 3,000 live cells. In both cases (plasmablasts or memory B cells), cells were incubated 18-22h at 37°C, 5% CO 2 in the ELISpot plate before detection. A combination of RBD-WASP/anti-WASP-ALP or anti-IgGbiotinylated/streptavidin-ALP (Mabtech) were used to detect RBD-speci c or total IgG secreting cells respectively. Plates were then read on an IRIS ELISpot reader (Mabtech). Spots were calculated based on the average of two wells.

Statistical Analysis
For descriptive analysis, data are presented as median (interquartile range [IQR]) for continuous parameters and frequency (percentage) for categorical variables. Fisher's exact test was used to compare categorical variables, while for continuous variables, t-test was used for normal data and Mann-Whitney U test for non-normal data. All statistical tests were 2-sided and P values <0.05 were considered statistically signi cant. For asymptomatic patients, day one of illness was assigned to the day of con rmatory diagnosis by RT-PCR. For symptomatic patients, day of symptom onset was denoted as day one of illness.
For serial Ct values, we tted a generalized additive mixed model (GAMM) with a random intercept by patient. A Ct value of 45 was imputed where the PCR result was not detected. We compared this with previously reported serial Ct values of Delta infected patients. 23 We plotted Ct values with marginal effect of day of illness by variant type and 95% con dence intervals (CI) from the GAMM.
For immunological analysis, to compare between multiple groups, Kruskal-Wallis tests with post hoc tests using Dunn's multiple comparison tests were used to identify signi cant differences. For whole blood immunophenotyping, gated cells were manually exported using FlowJo v10.8.0. Samples were then used for Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) analysis using cytofkit2 R Packages with RStudio v3.5.2. 24 Thirteen healthy vaccinated controls, 15 Delta vaccine breakthrough patients and 17 Omicron breakthrough patients were each concatenated to their respective groups and 150,000 cells were analyzed using the ceil method. Principal Component Analysis (PCA) was performed on the systemic cytokine data using Singular Value Decomposition (SVD) method in ClustVis. 25
The Omicron breakthrough cases had high anti-SARS-CoV-2 S antigen SFB binding, with median IgG SFB binding at 39.45% (IQR 31.3 -52.84%). Patients who received >2 vaccination doses had higher median IgG SFB binding, but otherwise there was no signi cant difference in IgG SFB responses between Omicron breakthrough cases and vaccinated healthy controls or Delta breakthrough infections (Supplementary Figure 3).

Systemic cytokine response
We assessed the immune soluble mediators in acute plasma samples from a subset of Omicron breakthrough cases (n=17) with a 45-plex microbead-based immunoassay (ThermoFisher) and compared their systemic cytokine responses with age-matched vaccinated healthy controls and Delta breakthrough infections. Omicron breakthrough cases had systemic cytokine pro les similar to their age-matched Delta breakthrough infections. Both Omicron and Delta breakthrough infections showed cytokine pro les that clustered closely to their age-matched vaccinated healthy controls (Figure 2a). Levels of previously identi ed severity-associated cytokines 14 , HGF, IL-12p70, IL-1RA, IL-6, MIP-1a, PDGF-BB and VEGF-A were not signi cantly elevated in the Omicron breakthrough infections compared with vaccinated healthy controls (Figure 2b). Nevertheless, these patients had higher concentrations of IP-10 (also called CXCL10), an in ammatory marker induced by IFN-gamma 26 , compared with healthy vaccinated controls ( Figure 2b). In addition, Omicron breakthrough has a lower level of proin ammatory VEGF-A than the Delta infection.

Whole blood immunophenotyping
Unbiased dimensionality reduction and clustering analysis by Uniform Manifold Approximation and Projection (UMAP) identi ed three immune cell clusters, T cells, natural killer cells and neutrophils, with variation in Omicron breakthrough cases compared to vaccinated healthy controls and Delta vaccine breakthrough infections (Figure 3a). Further analysis revealed a reduced cell count for CD4 and γδ1 T cells (Figure 4b), and an increased cell count for total natural killer (NK) cells and CD56 dim NK cells in patients with acute Omicron breakthrough infection than vaccinated healthy controls (Supplemental Figure 4-6). No signi cant changes were observed for dendritic cells, total monocytes and eosinophils ( Figure 3b) between Omicron breakthrough cases and vaccinated healthy controls. In comparison to Delta infections, Omicron infections had higher counts of intermediate monocytes, dendritic cells and NK cells, and a lower count of γδ1 T cells. There was a difference in the composition of neutrophil subsets between Omicron and Delta breakthrough infections, where a higher level of mature neutrophils accompanied a lower immature neutrophil cell count in Omicron breakthrough cases (Figure 3b).
Together, our data suggest milder alterations in the immune cell compartments during Omicron breakthrough infections when compared to vaccinated healthy controls and Delta vaccine breakthrough infections.

Memory B and T cell responses
To further de ne their immunological status at the time of breakthrough infection, we looked at the levels of memory B and T cell responses in these Omicron patients. Using a B cell ELISpot targeting the SARS-CoV-2 receptor-binding region, we examined the frequency of SARS-CoV-2-speci c memory B cells. There was no signi cant difference between Omicron and vaccinated healthy controls in the frequency of RBDspeci c memory B cells (Figure 4a), but we observed a signi cantly higher frequency of RBD-speci c memory B cells in Omicron cases compared with Delta cases. The antibody-secreting plasmablast responses were much higher in vaccine breakthrough cases than vaccinated healthy controls, with no signi cant difference between Delta and Omicron infections (Figure 4a). SARS-CoV-2-speci c T cell responses were also analyzed in these Omicron breakthrough patients. PBMCs were stimulated with a pool of SARS-CoV-2-derived epitope peptides, followed by intracellular staining for cytokines. Although there was no signi cant difference in CD4 Th 1 , Th 2 , Th 17, and CD8 responses between Omicron breakthrough cases and vaccinated healthy controls, Omicron patients had lower frequencies of CD4+ TNF-a+ cells (Figure 4b, Supplemental Figure 7).

Discussion
Since the beginning of January 2022, Singapore has reported an increasing number of locally acquired COVID-19 infections, of which the majority are now due to the Omicron variant. 27 At the time of data acquisition for this study, all suspected Omicron infected patients (S gene target failure detected) were hospitalized at NCID and as such, our cohort captures the full spectrum of disease severity. In our study the majority of patients with Omicron infection had mild illness with upper respiratory tract infections similar to reports elsewhere. 28 We also found a lower proportion of Omicron infected patients with pneumonia compared with Delta in an age-matched cohort.
The Imperial College COVID-19 response team estimated a 15-20% reduction in hospitalization compared with those infected with Delta 13 . In our study population, the majority of patients did not require hospitalization based on severity of illness. Mild illness partially re ects the population affected in this cohort -younger patients with few comorbidities; most were returning travelers. This may also be related to the predilection of Omicron for upper versus lower respiratory tract. 29 COVID-19 vaccination is associated with more asymptomatic infections and better clinical outcomes with Delta breakthrough infection. 30 We were unable to determine the consequences of Omicron infection in unvaccinated individuals.
Although few studies had reported reduced neutralization of Omicron post-vaccination 7,8 , Dejnirattisai et al. 9 demonstrated higher neutralizing antibody titers against the Omicron variant with booster vaccination doses. Patients who received booster vaccination doses were more frequently asymptomatic and trended towards less systemic in ammation. While there was no signi cant statistical difference between vaccination booster status and development of pneumonia found on multivariate analysis, this could be due to the small number of patients in this study.
While several studies are being conducted to continually assess the clinical severity, it is also critical to understand the immunopathological characteristics of infection caused by Omicron. We evaluated indepth systemic immune response following Omicron breakthrough infections. Vaccine breakthrough infections with the Omicron variant had a low concentration of proin ammatory cytokines, chemokines, and growth factors at the acute phase of infection, in contrast to the high levels reported in severe COVID-19 disease. 14 Instead, a more robust IFN-γ response, which is critical for viral clearance 31 , was detected in these acute Omicron breakthrough infections. These ndings corroborated our clinical observations that patients infected with the Omicron variant are mostly asymptomatic or with mild diseases and with a lower viral load. In addition, this emphasizes the importance of vaccination in preventing severe COVID-19 disease with Omicron.
Immunophenotyping of peripheral blood from Omicron breakthrough cases revealed relatively minimal changes in the myeloid cell compartment. Monocytopenia and dysregulated myelopoiesis are typical hallmarks of severe disease and acute respiratory distress syndrome in COVID-19. [32][33][34][35][36] We did not detect signi cant differences in the number of total monocytes and their subsets between patients with Omicron breakthrough and vaccinated healthy individuals, indicating a minimal migration of the monocyte populations into the lung or other sites of in ammation. 37 The lower frequency of immature neutrophils in the circulation of these patients, which are released following emergency myelopoiesis in response to ongoing in ammation 38,39 , further indicates that Omicron breakthrough infections cause a milder in ammatory response.
In addition to circulating antibodies, memory B cells may also provide an additional line of defense against infection. 40 We previously observed that memory B cell levels were decreased in Delta vaccine breakthrough patients, suggesting that lower levels of memory B cells may have contributed to a failure to protect against infection. 41 In contrast, we observed here that Omicron vaccine breakthrough infections showed a similar frequency of RBD-speci c memory B cells as vaccinated healthy individuals. The reasons for this difference remain unclear. However, it is notable that Omicron infection has been suggested to have a shorter incubation period than Delta infection 42,43 -the faster viral infection and replication speed may mean that memory B cells do not have su cient time to reactivate into antibodysecreting cells to contribute toward preventing infection.
Given the rapid spread of Omicron across the world, there were concerns of increased infectivity compared with Delta. Chen et al. demonstrated signi cantly increased binding free a nity of Omicron, suggesting about 2.8 times infectivity compared with Delta. 44 we showed that initial and lowest median PCR Ct values were higher in Omicron than Delta vaccine breakthrough infections. Our study showed that the lowest Ct values (peak viral load) occurred at similar times for Omicron and Delta vaccine breakthrough infections -after onset of symptoms. These suggest that the apparent increased transmissibility and infectivity of Omicron infections might not be related to asymptomatic infection or higher viral loads.
There are several limitations to our study. Firstly, due to the high vaccine uptake in Singapore and requirements for vaccination to travel, we were not able to study Omicron infection in unvaccinated individuals. Secondly, while RT-PCR screening of travelers and quarantined individuals was not dependent on symptomatic illness, abortive infections may have been missed. Local transmission of Omicron may have occurred undetected in Singapore over the course of this study; however, this is likely to have been limited in early December 2021 as we did not nd S gene target failure in looking back at returned travelers with COVID-19 prior to Omicron being reported in South Africa. Thirdly, initial PCR testing was not standardized in a centralized laboratory. It was performed using various commercially available assays at each center. Ct values are only a surrogate measure of viral load and shedding and might be in uenced by several factors. We attempted to evaluate the viability of shed virus via viral culture, however, sample size was small. Finally, this is an observational study and comparison between Delta and Omicron infected cohorts is subjective to additional confounding (such as time from most recent vaccine administered which may affect level of immune waning) that may have affected outcomes.  Intensive care unit admission and/or death 0 0 - † Non-mRNA vaccines received included ChAdOx1_nCoV-19 (COVISHIELD), COVAXIN, ChAdOx1-S (Oxford/AstraZeneca) and

Conclusion
Sinopharm BBIP-CorV vaccines; ‡ Worst laboratory value defined as the highest recorded admission, except for lymphocyte count in which worst laboratory value is the lowest