Low humoral and cellular immune responses early after breakthrough infection may contribute to severe COVID-19

Background Little is known about the immune determinants for severe coronavirus disease 2019 (COVID-19) in individuals vaccinated against severe acute respiratory syndrome coronavirus 2. We therefore attempted to identify differences in humoral and cellular immune responses between patients with non-severe and severe breakthrough COVID-19. Methods We prospectively enrolled hospitalized patients with breakthrough COVID-19 (severe and non-severe groups) and uninfected individuals who were vaccinated at a similar time (control group). Severe cases were defined as those who required oxygen therapy while hospitalized. Enzyme-linked immunosorbent assays and flow cytometry were used to evaluate humoral and cellular immune responses, respectively. Results Anti-S1 IgG titers were significantly lower in the severe group than in the non-severe group within 1 week of symptom onset and higher in the non-severe group than in the control group. Compared with the control group, the cellular immune response tended to be diminished in breakthrough cases, particularly in the severe group. In multivariate analysis, advanced age and low anti-S1 IgG titer were associated with severe breakthrough COVID-19. Conclusions Severe breakthrough COVID-19 might be attributed by low humoral and cellular immune responses early after infection. In the vaccinated population, delayed humoral and cellular immune responses may contribute to severe breakthrough COVID-19.


Introduction
Since the coronavirus disease 2019 (COVID-19) pandemic was declared, various types of vaccines against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) have been rapidly developed. Vaccination has been the primary strategy for containing the COVID-19 pandemic, and therefore, several COVID-19 vaccines have been rapidly rolled out.
Based on real-world data together with clinical trials, vaccination was associated with reduced risks of COVID-19-related hospital admission and mortality (1-3) as well as reduced risks of symptomatic and severe COVID-19 (4,5). However, despite the high vaccine efficacy and effectiveness against COVID-19 (6-9), breakthrough COVID-19 cases have continued to emerge (10). While the majority of breakthrough COVID-19 have been mild or moderate (11), severe or fatal cases have not been rare (12)(13)(14). In addition, breakthrough COVID-19 cases have also been reported even after booster doses (15).
However, little is known about the immune response to breakthrough infection. Bergwerk and colleagues reported a correlation between peri-infection antibody titers and breakthrough COVID-19 (10), but a subsequent study found no difference in post-vaccination neutralizing antibody titers between controls and patients with breakthrough infection (16). Another previous study reported that the antibody titers declined after a second vaccination but were sharply elevated in breakthrough COVID-19 cases (17). These previous studies were limited, however, by their focus on healthy healthcare personnel and their analysis of only mild breakthrough COVID-19 cases.
It has become crucial to characterize the immune response in cases of breakthrough COVID-19, especially severe cases, because the vaccinated population has become the mainstay, and the incidence and medical burden of breakthrough COVID-19 continue to persist. This study therefore attempted to identify differences in humoral and cellular immune responses according to severity among hospitalized patients with breakthrough COVID-19.

Study population and design
From May 2021 to January 2022, we prospectively enrolled study participants aged ≥18 years with reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection who were admitted to the biocontainment units of Seoul National University Hospital or Boramae Medical Center. Uninfected vaccinated individuals were also enrolled. The uninfected vaccinated individuals were enrolled from the non-hospitalized general population. Breakthrough COVID-19 was defined by the presence of COVID-19 symptoms and RT-PCR-confirmed diagnosis of COVID-19 more than 14 days after at least one vaccine dose. Patients with breakthrough COVID-19 were divided into a severe group who required oxygen therapy and a non-severe group who did not require supplemental oxygen therapy during hospitalization (18). The uninfected vaccinated individuals served as a control group.
Within 1 week of onset of symptoms, blood samples were collected from patients hospitalized with COVID-19. Additional serial samples were obtained from a portion of the patients. Blood samples were also collected from unvaccinated patients with severe COVID-19 and uninfected vaccine recipients. Data were collected regarding demographic characteristics, vaccination type and status, days from vaccination to symptom onset, Charlson's comorbidity index, underlying diseases, and clinical outcomes. Fully vaccinated patients were defined as those with COVID-19 diagnosis more than 14 days after completion of the recommended vaccination regimen.
Collection of peripheral blood mononuclear cells, antigen stimulation, and flow cytometry After whole blood was drawn into heparin vacutainers (Becton Dickinson, NJ, USA), PBMCs were purified using Ficoll-Hypaque (GE Healthcare Life Sciences, Piscataway, NJ, USA). PBMCs were stored in serum-free cryopreservation medium (Cellbanker 2; Zenoaq, Japan) in liquid nitrogen containers until further use.

Statistical analyses
The chi-squared test or Fisher's exact test was used to compare categorical variables, and the Mann-Whitney U test was used to compare continuous variables. To identify risk factors for severe breakthrough COVID-19, variables with a P value of <0.10 in the univariate analysis were included in the multivariable logistic regression analysis. Statistical analyses were performed using SPSS Statistics, version 26.0 (IBM Corp., Armonk, NY, USA). P values <0.05 were considered statistically significant. Data are presented as median with interquartile range (IQR) and as dot plots. All graphs were generated using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA).

Study participants
A total of 108 breakthrough COVID-19 cases admitted within 1 week of the onset of symptoms were enrolled. The non-severe group included 79 (73.1%) patients, and the severe group included 29 (26.9%) patients. In addition, the control group included 22 uninfected individuals who completed the standard doses of SARS-CoV-2 vaccine ( Table 1)

Humoral immune responses against SARS-CoV-2
Anti-S1 IgG titer within 1 week after symptom onset in breakthrough COVID-19 patients was significantly lower in the severe group than in the non-severe group ( Figure 1B).
Serum antibody responses to SARS-CoV-2 RBD WT and RBD Delta were compared between groups ( Figures 1C, D), but two cases from the non-severe group were excluded due to shortage of samples. Titers of both anti-RBD WT IgG and anti-RBD Delta IgG were significantly lower in the severe group than in the non-severe group The kinetics of antibody responses over time beginning at symptom onset were evaluated in 17 severe breakthrough cases, 10 non-severe breakthrough cases, and 7 unvaccinated severe cases ( Figure 2). In non-severe breakthrough cases, the anti-S1 IgG titer was high at the time of symptom onset or elevated during the early phase of infection. However, the anti-S1 IgG titer increased later in some of severe breakthrough cases. In two severe breakthrough cases, the antibody titer began to increase 2 weeks after symptom onset, similar to unvaccinated severe cases. One of these two severe cases had received an immunosuppressant.
* The BMI of the control group was not collected. ** One case was cross-vaccinated; the first vaccine was an adenoviral vector vaccine and the second vaccine was an mRNA vaccine. *** Days from last vaccination to sampling. vaccinated individuals. Clinical data for the study participants whose cellular immune responses were analyzed are provided in Supplementary Table 1. Among severe cases, all PBMCs used in the analysis of cellular immune responses were isolated from blood samples collected before oxygen therapy. Compared with uninfected vaccinated controls, the proportion of AIM + CD4 + T cells specific for whole-spike Ag was significantly lower in both the non-severe breakthrough cases  Figure 3A). The proportion of AIM + CD4 + T cells specific for the WT and Delta Ags tended to be lower in the severe group than in the nonsevere group, although the difference was not statistically significant. In addition, the proportions of cytokine-producing CD4 + T cells tended to be lower in the severe group than in the non-severe group (Supplementary Figure 2). Although the differences were not statistically significant, the proportions of AIM + CD8 + T cells specific for the WT and Delta Ags tended to be lower in the severe group than in the non-severe group ( Figure 3B).

Risk factors for severe breakthrough COVID-19
The univariate analysis identified older age, higher Charlson's comorbidity index, and lower anti-S1 IgG titer in the early phase Humoral immune responses to SARS-CoV-2 within 1 week after symptom onset in patients with breakthrough COVID-19. (A). Anti-S1 IgG antibody titers in the control (vaccinated subjects without infection), severe, and non-severe groups. (B). Anti-S1 IgG antibody titers according to the number of days from symptom onset. (≤1 week after symptom onset) as risk factors for severe breakthrough COVID-19 ( Table 2). The multivariate analysis identified older age (adjusted odds ratio [aOR] 1.07, 95% confidence interval [CI] 1.01-1.12, P = 0.014) and low anti-S1 IgG titer in the early phase (aOR 0.88, 95% CI 0.77-0.99, P = 0.037) as independent risk factors for severe breakthrough COVID-19.

Discussion
Compared with non-severe breakthrough COVID-19 patients, those with severe breakthrough COVID-19 exhibited significantly lower humoral immune responses and a trend toward lower cellular immune responses in the early phase of infection. In addition, we identified low anti-S1 IgG titer in the early phase as an independent risk factor for severe breakthrough COVID-19. Although previous studies identified immunologic factors associated with the occurrence of breakthrough COVID-19 (10, 23), our study analyzed immunologic factors that influence the severity of breakthrough infection.
We also investigated the relationship between various demographic characteristics and the severity of breakthrough infection and identified older age as an independent risk factor for severe breakthrough COVID-19. In a retrospective study, Suleyman and colleagues identified older age, together with underlying conditions such as cardiovascular disease and immunocompromised status, as a risk factor for hospitalization in breakthrough COVID-19 cases (24), which is consistent with our findings. Older age and multiple comorbidities are decisive risk factors for poor outcomes in unvaccinated patients infected with SARS-CoV-2 (25,26). This suggests that certain critical factors associated with a poor outcome prior to vaccination may continue to be crucial predictors for severe breakthrough COVID-19.
The anti-S1 IgG titer differed between breakthrough COVID-19 patients and uninfected vaccinated controls. Although the interval between the last vaccination and sampling was shorter in the uninfected vaccinated control group than in the breakthrough infection groups (meaning that the control group exhibited less waning of antibodies over time), the anti-S1 IgG titer was higher in the breakthrough infection groups than in the uninfected vaccinated control group within 1 week after symptom onset. This finding suggests that memory B-cell responses to SARS-CoV-2 infection occurred within several days after symptom onset in patients with breakthrough infection. A previous study found that anti-S1 RBD IgG titer post-infection was significantly higher in mildly symptomatic patients than in those with no breakthrough infection or asymptomatic patients (27). This finding is also indicative of early immune boosting by symptomatic breakthrough infection.
The lower anti-S1 IgG antibody titer in the severe group compared with the non-severe group in the early phase after infection suggests that a poor memory B-cell response to SARS-CoV-2 infection might be related to progression of severe disease. Brosh-Nissimov and colleagues also reported that the anti-spike IgG titer was lower in fully vaccinated hospitalized COVID-19 patients with a poor outcome, but the difference was not statistically significant (28). In our study, the difference in anti-S1 IgG antibody titer between the non-severe and severe groups was prominent at 5-7 days after symptom onset. This suggests that a delay in antibody response of up to 1 week after symptom onset might be associated with severe COVID-19 after vaccination. A previous study found that peak viral load followed by a delayed increase in antibody response coincided with viral clearance after breakthrough infection, indicating a protective role of vaccination against severe COVID-19 (29). In addition, antibody kinetics assessed using serial samples revealed that memory B-cell responses were poor in the severe group; in two severe breakthrough cases, we did not observe any memory B-cell response until the antibody titer began to increase 2 weeks after symptom onset, similar to unvaccinated cases (30). These findings suggest that an adequate memory B-cell response in the early phase of breakthrough COVID-19 infection is critical to prevent progression to severe disease.
The cellular immune response is considered an important host factor affecting the severity of COVID-19. Hypofunction and a low number of T cells in the early phase have been associated with severe COVID-19 (31). In the present study, the severe group tended to exhibit a lower cellular immune response than the nonsevere group, suggesting that a lower cellular immune response is associated with progression of severe breakthrough COVID-19.
Paniskaki and colleagues found that alpha variant-reactive CD4 + and CD8 + T-cell responses were very poor at disease onset in patients with moderate-to-critical breakthrough infection compared with uninfected vaccinated controls (32). Similarly, in the present study, cellular immune responses were lower in the breakthrough infection groups than in the uninfected vaccinated control group. This conflicts with the results of a previous study reporting that breakthrough cases exhibited a lower memory B-cell response but similar T-cell response compared with uninfected participants (33). These discrepancies might be due to differences in other host factors, as our study included hospitalized breakthrough COVID-19 cases with underlying medical conditions. This study has several limitations. First, we could not obtain baseline samples before breakthrough COVID-19 to measure the immunologic response achieved purely by vaccination. Therefore, the immune responses after breakthrough COVID-19 in this study should be interpreted in consideration of contributions by both the vaccine-induced immune responses and immune boosting by natural infection. Second, the sample size for the investigation of cellular immune responses was relatively small, which precluded demonstration of statistical significance. Third, the sampling time was not consistent for each patient, even though patients in the early stage of infection were enrolled. Finally, the infecting virus was not analyzed; however, it is likely that most patients were infected with the Delta variant, considering that this variant was dominant during the study period.
In conclusion, we found that humoral and cellular immune responses in the early phase of infection were low in patients with severe breakthrough COVID-19 compared with non-severe patients. In the vaccinated population, delayed humoral and cellular immune responses, possibly due to poor memory B-or T-cell responses, may contribute to severe COVID-19.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The studies involving human participants were reviewed and approved by the Institutional Review Boards of Seoul National University Hospital (IRB no. 2104-182-1215) and Boramae Medical Center (IRB no. 20-2021-54). The patients/participants provided their written informed consent to participate in this study.

Author contributions
HS, WP and S-WP conceptualized the study. CL, PG, and CK analyzed the data and drafted the initial manuscript.   management, analysis, and interpretation of the data; in the preparation, review, or approval of the manuscript; or in the decision to submit the manuscript for publication.