Patients with confirmed diagnosis of MM and who were being treated in the University Hospital were invited to participate. Patients previously vaccinated against SARS-CoV2 with mRNA vaccines (BNT162b2 and mRNA1273) or the adenovector vaccine AZD1222 vaccine were eligible if they were aged 18 to 65 and had an Eastern Cooperative Oncology (ECOG) performance status score between 0 and 2 (on a 5-point scale, where higher scores represent increased disability). Individuals were excluded if they had active disease, central nervous system involvement, primary amyloidosis, or showed inadequate hematologic, hepatic, renal, or cardiac function. The enrolled patients and controls should have at least received a single dose of one of the following three COVID-19 vaccines authorized for emergency or full use during the study period (2020–2021): the mRNA vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna), and the adenoviral vector vaccine AZD1222 (AstraZeneca-Oxford).

Healthy controls were recruited through convenience sampling from hospital staff and patient companions. The following exclusion criteria were applied specifically to the control group: immunosuppressive conditions (including autoimmune diseases, immunomodulator use, or HIV infection), active infection at recruitment, history of hematologic malignancies, and prior organ or stem cell transplantation. All controls received their SARS-CoV-2 vaccinations according to the national guidelines during the same epidemiological period as patients, ensuring comparable exposure to circulating variants.

Patients and controls provided both written and verbal informed consent for trial participation. Patient clinicodemographic characteristics and outcomes were retrospectively gathered from their electronic medical records. No interventions were conducted as part of this study. This research adhered to institutional guidelines and received approval from the Institutional Review Board (IN23–00,003) and the School of Medicine Ethics Committee. Additionally, the study followed Good Clinical Practice standards Practices.

This study included 33 patients with MM and 28 healthy controls. Blood samples were obtained from each patient and control; the sera were recovered by centrifugation and stored at −80 °C until use. Total IgG antibodies directed against the RBD of the SARS-CoV-2 spike protein were quantified using the SARS-CoV-2 IgG II Quant assay (Abbott). Additionally, qualitative detection of IgG directed against the nucleocapsid protein was performed using the SARS-CoV-2 IgG assay (Abbott), in accordance with the manufacturer's established protocols. The cutoff for RBD IgG antibodies was ≥250 AU/mL according to previous reports [5] and the cutoff for the nucleocapsid antibodies index was ≥1.40 S/C, according to the manufacturer’s guidelines.

A complete blood count and routine serological assessment of MM patients, including total proteins, gamma globulin, and serum-free chains, were performed along with the serum sample for IgG antibody determination with the results being obtained from electronic health records following the attainment of informed consent from all participants and formal approval from the Institutional Review Board.

Statistical analyses were performed using Graph Pad Prism-8 (La Jolla, GraphPad Software). Normality was analyzed using Shapiro-Wilk test. The differences between the group means were evaluated using one-way ANOVA followed by Tukey's post hoc test, or Kruskal-Wallis followed by Dunn's post hoc test for multiple comparisons. P-values <0.05 was considered statistically significant.

Patients were treated with bortezomib, lenalidomide and dexamethasone (VRD) induction therapy. Nineteen patients (57.6 %) underwent autologous stem cell transplant (ASCT) with the antibody levels being obtained at least six months post-transplant. Maintenance treatment included lenalidomide 10 mg/day or bortezomib 1.3 mg/m2 every other week. All patients were in very good partial response or better at the time of study.

The evaluation of serum immunoglobulins and light chains demonstrated a notable predominance of IgG, accounting for 71.4 % (95 % confidence interval [95 % CI]: 52.9–84.9 %) of the cases analyzed. Remarkably, even at the lower limit of the confidence interval, IgG constituted the most prevalent isotype, appearing in more than 50 % of the cases. In the analysis of light chains, kappa was observed to have a slightly higher prevalence, represented in 53.6 % (95 % CI: 35.8–70.5 %), compared to lambda, which accounted for 46.4 %. However, this difference lacked statistical significance.

A distinct trend was evident in the immunoglobulin A (IgA) cases, where lambda light chains observed in 75 % of these cases (6 out of 8), predominated. Nonetheless, the limited sample size of IgA patients led to a considerable degree of uncertainty in this estimate (95 % CI:10.0–40.1 %).

Patient characteristics, vaccination status, anti-SARS-CoV-2 antibody levels, and laboratory findings are summarized in Table 1. Notably, only one patient tested negative for IgG spike-RBD antibodies, with a 2.2 level of AU/mL. Additionally, 17 patients (51.5 %) were positive for IgG nucleocapsid antibodies. Regarding vaccination status, 13 patients (39.4 %) had received a single vaccine dose. Of these, 12 (92.3 %) were vaccinated with BNT162b2, while only one (7.7 %) received AZD1222. The only patient who was negative for spike RBD antibodies received one dose of the BNT162b2 vaccine. In respect the administration of multiple doses of the vaccine, no patients reported the use of two doses. Twelve patients (36.4 %) reported they had received three doses, consisting of various combinations of BNT162b2 (B), AZD1222 (A), and mRNA-1273 (M). Of these, six (50 %) received three doses of AZD1222, two (16.7 %) received the BBA combination, and the remaining patients received the following regimens: BAA (8.3 %), ABB (8.3 %), BBB (8.3 %), and MMM (8.3 %). Seven patients (21.2 %) had received four doses of the vaccine: six (85.7 %) received four doses of AZD1222, and one patient (14.3 %) received four doses of mRNA-1273. Finally, one patient had received five doses, with the vaccination scheme being BBMMB.

A total of 28 healthy controls were included for the assessment of antibody response after a single dose of the mRNA vaccines BNT162b2 and mRNA1273. The healthy control group consisted of 28 participants, with nine males (32.1 %) and a mean age of 47 years (standard deviation ± 22.0). All controls received mRNA vaccines as their most recent dose, with Pfizer-BioNTech (BNT162b2) being the most common (64.3 %; n = 18), followed by Moderna (mRNA-1273 - 35.7 %; n = 10). Serological analysis revealed robust immunogenicity, with 92.9 % (n = 26) showing positive anti-spike-RBD IgG responses. The median antibody titer was 8201 AU/mL (interquartile range [IQR]: 1354–19,219), indicating substantial variation in individual humoral responses. Only two participants (7.1 %) had negative antibody results, potentially reflecting individual differences in immune reactivity to vaccination.

The IgG levels of anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) were compared between healthy controls (with a single dose of the vaccine when they were included in the study) versus MM patients (with a single dose and ≥3 doses). A significant difference was found between the study groups in the non-parametric analysis (p-value <0.05); however, in the multiple comparison analysis no differences were identified between the study groups (p-value >0.05) (Figure 1a). Additionally, the group receiving three or more doses showed significantly elevated levels of anti-nucleocapsid antibodies above the cutoff point of 1.4 S/C (p-value <0.001), thus suggesting an unreported prior subclinical infection (Figure 1b). No significant differences were observed between the groups in other laboratory findings. The patients were divided into groups based on their vaccination schemes and anti-nucleocapsid serological status. The group of MM patients that received three or more doses and with anti-nucleocapsid antibodies showed higher levels of IgG against spike RBD compared to those who had lower values of anti-nucleocapsid antibodies (p-value = 0.0024) (Figure 1c).

Figure 1.

Evaluation of anti-spike receptor-binding domain (RBD) immunoglobulin G (IgG) concentrations in patients with multiple myeloma (MM). a) Serological assessment of levels of anti-spike RBD IgG in healthy controls and MM patients, and b) anti-nucleocapsid IgG of patients who received 1 dose of anti-SARS-CoV-2 vaccine and three or more doses; c) Comparison based on the vaccination doses and anti-nucleocapsid IgG positive or negative status; d) Assessment of monocytes and e) Lymphocytes in the complete blood count of peripheral blood according quartile classification of anti-spike RBD IgG antibody levels (Q1 ≤ 2523.2 AU/mL, Q2 = 2523.2–10,427.9 AU/mL, Q3 = 10,428.0–23,954.0 AU/mL, and Q4 ≥23,955.0 AU/mL); f) Analysis of patients with a subclinical infection and normal (N) or abnormal (A) kappa/lambda serum free chain ratio. The differences between the groups means were evaluated using one-way ANOVA followed by Tukey's post hoc test, or Kruskal-Wallis followed by Dunn's post hoc test for multiple comparisons. P-value <0.05 was considered statistically significant.

* P-value < 0.05, ** P-value < 0.01, and *** P-value < 0.001.

In a logistic regression model, this study aimed to predict whether high levels of Anti-spike RBD IgG were associated with the presence of anti-nucleocapsid antibodies. Although a significant relationship was found between these variables (p-value = 0.0007), the practical impact was minimal, as indicated by the odds ratio (1.000) and a small coefficient B (−0.000138). Therefore, anti-spike RBD levels are not a strong predictor of subclinical infection in this patient cohort. Additionally, non-significant correlations were observed between serum anti-spike RBD IgG levels and various laboratory parameters, including the lymphocyte count (Pearson's: r = 0.0310; p-value = 0.8650), monocyte count (Spearman's: r = −0.0930; p-value = 0.6120), total serum proteins (Pearson's: r = 0.1790; p-value = 0.3280), and serum gamma globulins (Spearman's: r = 0.1980; p-value = 0.2860).

The levels of anti-spike RBD antibodies were classified in MM patients into quartiles to assess potential differences in laboratory findings. The quartile ranges were as follows: Q1 ≤2523.2 AU/mL, Q2 = 2523.3 –10,427.9 AU/mL, Q3 = 10,428.0–23,954.0 AU/mL, and Q4 ≥23,955.0 AU/mL. MM patients with anti-spike RBD antibodies in Q3 had an elevated number of monocytes compared to Q2 (p-value <0.05) and Q4 (p-value = 0.0216) (Figure 1d). No other significant changes were observed regarding lymphocytes (Figure 1e) or other laboratory findings. Additionally, based on the normal Serum-Free Kappa/Lambda (κ/λ) light chain ratio reference range (0.26–1.65), patients were grouped to assess whether abnormal ratios were associated with significant changes in the anti-spike RBD IgG levels in patients with a subclinical infection (Figure 1f). Among patients exhibiting an abnormal κ/λ ratio, those with detectable Anti-nucleocapsid IgG demonstrated significantly higher RBD IgG titers (p-value <0.01). These data suggest that despite the presence of underlying monoclonal plasma cell proliferation, these individuals maintained a robust humoral response following subclinical SARS-CoV-2 infection. There are no significant differences in any other laboratory findings between the groups with an abnormal κ/λ ratio compared with those who had a normal ratio.

Additionally, the antibody response was evaluated in the patients who had undergone ASCT compared to those who had not. No statistically significant differences were observed in anti-spike RBD IgG levels (p-value = 0.8709) or anti-nucleoprotein antibodies (p-value = 0.1011).