Epidemiological and Immunological Characteristics of the Study Population
To understand immune responses to SARS-CoV-2 in patients with predominantly antibody deficiencies, 20 patients who fulfilled CVID diagnosis according to ESID criteria, 20 patients with SID to B- cell lymphoproliferative disorder (B-CLPD) and 81 healthy controls (HC) were consecutively assessed. The study population characteristics are shown in Table 1. All CVID patients had suffered from recurrent respiratory tract infections and had decreased IgG, IgA and/or IgM levels before IgRT. Six out of 20 CVID patients (30%) had lymphoproliferative manifestations, one with a current clinical course of large granular lymphocytic (LGL) leukemia and 5 cases (25%) had clinical signs of autoimmune disease. Five CVID patients (25%) had lymphopenia, one of which after rituximab treatment years after CVID diagnosis. All but 1 patient were receiving IgRT with improvement in infections, 6 patients (30%) were treated with subcutaneous (SC) IgRT and 13 patients (65%) with intravenous (IV) IgRT.
Among patients diagnosed with SID to B-CLPD, the most frequent cancer was non-Hodgkin's lymphoma (n = 12, 60%), chronic lymphocytic leukemia (n = 5, 25%), monoclonal gammopathy of undetermined significance (n = 2, 10%) and multiple myeloma (n = 1, 5%). Fifteen out of 20 patients (75%) with SID were on IgRT (all IV).
Table 1
Epidemiological features of the three study groups.
|
CVID
No. = 20
|
SID
No. = 20
|
Healthy controls
No. = 81
|
M/F
|
6/14 (30%/70%)
|
5/15 (25%/75%)
|
21/60 (26%/74%)
|
Age (years)
|
49.8 ± 16.4
|
67.9 ± 8.8
|
48.1 ± 15.6
|
IgG at diagnosis (mg/dL)
|
464 ± 276
499 (269–595)
|
720 ± 1106
369 (162–816)
|
NA
|
IgA at diagnosis (mg/dL)
|
41 ± 44
40 (0–51)
|
56 ± 64
25 (12–104)
|
NA
|
IgM at diagnosis (mg/dL)
|
64 ± 112
17 (5–52)
|
32 ± 45
11 (8–43)
|
NA
|
CD4 + T-lymphocytes (/uL)
|
659 ± 288
598(431–870)
|
900 ± 650
725(436–1222)
|
NA
|
CD8 + T T-lymphocytes (/uL)
|
574 ± 414
530(257–682)
|
751 ± 370
709(440–1047)
|
NA
|
IgRT
|
SCIG 6/20 (30%)
IVIG 13/20 (65%)
|
IVIG 15/20 (75%)
|
NA
|
Prior COVID-19 to vaccine
|
5/20 (25%)
|
3/20 (15%)
|
31/81 (38%)
|
M: male; F: female. Results are expressed as No. (%) or mean + ESM, median (IQR). |
SARS-CoV-2 History
CVID patients were vaccinated with the first and second vaccine dose between April to June, 2021 depending on the vaccine brand received. According to the Spanish SARS-CoV-2 vaccine schedule, 17 patients (85%) received two doses of the mRNA-1273 vaccine (Moderna), 2 patients (10%) the mRNA vaccine BNT162b2 (Pfizer/Biontech) and 1 patient (5%) the adenovirus-vectored vaccine ChAdOx1 nCoV-19 (AstraZeneca). Five CVID patients (25%) had already presented COVID-19, with asymptomatic (n = 2), mild (n = 2) and severe (n = 1) clinical courses.
Among patients with SID, 11 cases (55%) received the mRNA-1273 vaccine (Moderna) and the remaining 9 patients (45%) the mRNA vaccine BNT162b2 (Pfizer/Biontech). They received the two doses between January to June, 2021. Three of the 20 SID patients had presented COVID-19, with mild (n = 2) and severe (n = 1) clinical courses.
With respect to the population of 81 HC, 70 controls (86.4%) were vaccinated with the first and second dose between January to June, 2021, according to the Spanish SARS-CoV-2 vaccine calendar. Fifty-one HC (72.9%) received the mRNA vaccine BNT162b2 (Pfizer/Biontech), 12 (17.1%) the mRNA-1273 vaccine, and 5 (7.1%) the adenovirus-vectored vaccine ChAdOx1 nCoV-19 (AstraZeneca) and 2 cases (2.9%) received a single dose of the adenovirus-vectored vaccine Ad.26.COV2.S (Janssen). The remaining 11 HC had not received any vaccine at the time of study, 8 out 11 had experienced COVID-19 and 3 were neither vaccinated or with known COVID-19, to determine the specificity of the test. Globally, 31 HC (39.2%) had presented the COVID-19, with asymptomatic (n = 3), mild (n = 23) and severe (n = 5) clinical courses.
SARS-CoV-2 T Cell Responses in PID and SID
Post-vaccine results showed positive cellular response in 18 out of 20 (90%) CVID patients, with median (IQR) IFN-γ levels of 1,694.1 (651.5-1,856.5) mUI/ml; and in 74 out of 81 (96%) HC, with median (IQR) IFN-γ levels of 1,908.5 (1,149.5-2,001) mUI/ml, with significant differences (p = 0.005) (Fig. 1a). Specific anti-SARS-CoV-2 IFN-γ responses in the remaining two CVID patients (PID#6 and PID#11) were borderline, both had lymphopenia. PID#6 displayed panhypogammaglobulinemia with upper respiratory tract infections since she was 6 months-old, chronic gastritis due to H. pylori treated and eradicated in 2013, and mild T CD8 + lymphopenia (195 cell/uL). PID#11 showed a low increase of IFN-γ production (from 0 to 99 mUI/ml) post-vaccine. PID#11 had panhypogammaglobulinemia and lymphoid granulomatosis with interstitial lung involvement in 2016, which was treated with rituximab until 2017 with complete remission. She developed lymphopenia T CD8+ (196 cell/uL) secondary to treatment.
Cellular responses before vaccine administration were measured in 10 CVID patients with median (IQR) increase of IFN-γ levels of 80.5 (0-270) mUI/ml, statistically significant with respect to post-vaccination levels (p < 0.001) (Fig. 1b). Four of these ten patients (PID#1, PID#3, PID#7 and PID#9), who had passed COVID-19 prior to vaccination (one with bilateral pneumonia and the rest with mild symptoms), showed higher specific anti-SARS-CoV-2 IFN-γ production than those without previous disease, as expected 270 (68.7-1,375) mUI/ml versus 1.1 (0–17) mUI/ml. PID#2, whose parents in close contact had had COVID-19 showed baseline borderline results, suggesting asymptomatic COVID-19.
With respect to the 20 SID patients, specific cellular responses showed median (IQR) IFN-γ levels of 1,877.9 (167-1,937) mUI/ml without differences with HC (p = 0.215). Also, no differences were observed between PID and SID groups (p = 0.371). Positive T cell responses were observed in 14 out of 20 (70%) SID patients. Three patients (SID#5, SID#8 and SID#11) displayed borderline specific anti-SARS-CoV-2 IFN-γ responses and no response in the remaining three patients (SID#1, SID#2 and SID#6). SID#5 showed panhypogammaglobulinemia secondary to CLL diagnosed in 2002 without treatment to date. SID#8 had a NHL diagnosed in 2003 in complete remission (CR) and CD8 + T lymphocytopenia. SID#11 had a NHL treated with bendamustine and rituximab (BR) until 2018. With respect to SID patients with no response: SID#1 was diagnosed with pulmonary lymphoma in 2021, and she is currently under radiotherapy combined with chemotherapy. At the time of the study she presented marked B and NK lymphopenia (27 cell/uL and 32 cell/uL). SID#2 had a follicular lymphoma in 2003 treated with fludarabine, cyclophosphamide and rituximab (FCR) with CR since 2008 and showed panhypogammaglobulinemia. SID#6 had CLL diagnosed in 2003 and panhypogammaglobulinemia without anticancer treatment, serious recurrent respiratory infections, UTIs and asthma.
The difference in IFN-γ levels between clinical groups (PID, SID, HC) remained statistically significative after adjusting for age, sex, time since last exposition to SARS-CoV-2 antigens (vaccination or natural infection) and past history of natural SARS-CoV-2 infection with logistic regression (LR test, p = 0.016). The inspection of the model showed that this difference depended on the difference between the PID and HC groups (Wald test, p = 0.013), while it was not significative for the contrast between the SID and HC groups (Wald test, p = 0.760).
SARS-CoV-2 specific T Cell Responses in Healthy Donors
As mentioned previously, 74 out of 81 (96%) HC had positive cellular response. Among 7 patients who had not achieved positive IFN-γ production, 3 were not-vaccinated and without known COVID-19, to ensure the specificity of the study. The remaining 4 presented borderline cellular responses, without medical history suggestive of immunodeficiency. HC were divided into three subgroups: SARS-CoV-2 vaccination (HC-vaccine); naturally immunized by infection (HC-nat-immun); or both (HC-hybrid), respectively. Regarding the cellular immune responses, 39 out of 42 (93%) HC-vaccine, all 8 HC-nat immun (100%) and 27 out of 28 (96.4%) of HC-hybrid showed positive IFN-γ levels. Median (IQR) IFN-γ levels are as follows: 1,863 (1,064 − 1,995) mUI/ml for HC-vaccine, 1,601.1 (945.8-1.944,6) mUI/ml for HC-nat-immun and 1,964.5 (1,879.9-2,059.6) mUI/ml for HC-hybrid, respectively (Fig. 2). Specific anti-SARS-CoV-2 IFN-γ levels were higher in hybrid HC subgroups without significant differences with the other subgroups, but a trend was observed (p = 0.097). The greater variability in specific anti-SARS-CoV-2 cellular responses was observed in the HC-vaccine subgroup, despite the narrow time frame (3–6 months) with respect to HC-nat-immun (3–18 months).
SARS-CoV-2 Antibody Responses
SARS-CoV-2 antibody testing displayed adequate B-cell antibody responses in 80% of CVID patients with median (IQR) anti-spike IgG levels of 2,015.6 (51.9-5,611.4) UA/ml. PID#6 was the only patient who failed to produce positive both humoral and cellular responses. As mentioned previously, she had B (31 cells/µl) and T CD8 lymphopenia. All SID patients tested for antibodies (17/20, 85%) developed positive humoral responses with median (IQR) of 465.7 (238.6-1,984.9). All HC tested for humoral response (14/81, 17%) presented adequate humoral responses with a median (IQR) of 4,392.8 (2,528.2–13,384) UA/ml, significantly higher than SID (p = 0.040), and without significant differences between PID and HC (p = 0.123) and PID and SID (p = 0.683) (Fig. 3). Interestingly, no correlation was observed between specific cellular responses and to specific humoral responses to SARS-CoV-2.
SARS-CoV-2 Infection Follow-Up
Our study population was followed-up 4 months after the cellular and humoral assay to evaluate whether high cellular IFN-γ responses correlated with protection against a subsequent exposure to SARS-CoV-2 infection. Three out of 20 CVID patients (PID#1, PID#7 with previous COVID-19 plus 2-dose of vaccine and PID#11, with borderline IFN-γ levels after 2-dose vaccine) were exposed to SARS-CoV-2 during the sixth wave of the coronavirus in Spain, all of them related to mild symptoms. Among SID patients, only one patient (SID#20) experienced asymptomatic COVID-19 after a routine RT-PCR testing that resulted positive. Nine out of 81 (11%) HC referred exposure to SARS-CoV-2, 6 with mild symptoms, 1 with moderate course and the remaining 2 of the HC-vaccine group (HC#39 and HC#51) (2.5%) with bilateral pneumonia despite IFN-γ titers of 2,083 mUI/ml and 1,958 mUI/ml, respectively. None of them required hospitalization and are currently without sequels of the disease.