Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients

Key Points Question Are there differences in the magnitude and durability of neutralizing antibody (nAb) responses against SARS-CoV-2 variants of concern (VOCs) according to the mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech) vaccines? Findings In this comparative effectiveness study of 637 immunocompromised patients and 204 healthy control participants who received 2 doses of messenger RNA COVID-19 vaccines, nAb responses against the Beta and Delta variants were short lived (3 to 7 months) compared with original, nonvariant SARS-CoV-2 and other variants. Higher nAb titers and longer durability of humoral responses were associated with vaccination with the mRNA-1273 vaccine. Meaning The faster disappearance of the nAb responses in certain groups of immunocompromised patients suggests that boosting vaccine strategies need to be personalized to the underlying disease.


eMethods 1.1 Serologic assays and procedures
The levels of binding IgG anti-Spike (S) and anti-Nucleocapdide (N) antibodies were determined using a Luminex-based assay recently developed in our laboratory and a ratio >6.0 corresponds to the cutoff for diagnostic positivity 1 .Neutralizing antibody responses were assessed using a cell-and virus-free surrogate neutralization assay recently developed in our laboratory 2 .In this assay, neutralizing antibodies block the ability of fluorescent angiotensin converting enzyme 2 (ACE2) molecules from binding to recombinant SARS-CoV-2 Spike protein trimers.The assay achieved 96.7% sensitivity and 100% specificity in cross-validation studies with a gold standard, live virus cell-based assay and could be multiplexed to quantify responses against SARS-CoV-2 VOCs in one test.Only sera with positive binding IgG anti-S antibodies were evaluated for neutralizing antibodies.The neutralizing activity was measured as IC50 dilutions of the serum corresponding to the serum dilution inhibiting 50% of the Spike/ACE2 binding.On the basis of the cross-validation with the live virus cell-based assay, an IC50 >50 corresponds to the cutoff for a positive diagnostic test 2 and thus IC50 titers <50 were considered as a negative response.Therefore, the threshold for neutralizing activity was set at 50: <50: negative neutralizing activity, ≥ 50 to <100: weak neutralizing activity, ≥ 100 to <150: moderate neutralizing activity, ≥ 150: good neutralizing activity.

IgG ratio transformation in unit/ml (WHO units)
In order to transform IgG ratio values into the WHO unit/ml, we used a robust linear regression model (rlm function from MASS package in R) on 298 samples with paired measurements using (log10) unit/ml measurement as response and (log10) ratios as covariate.Then, we applied the resulting model on all (log10) IgG ratio values to transform them into (log10) unit/ml.At 3 months, in the untreated hematological cancers, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.029) and all the other VOCs (Alpha P = 0.040, Beta P = 0.042, Gamma P = 0.045, and Delta P = 0.028).In the treated hematological cancers significant differences in IC50 titers were only observed for the 2019-nCoV (P = 0.045).In the untreated solid cancers, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.004) and all the other VOCs (Alpha P = 0.013, Beta P = 0.029, Gamma P = 0.040, and Delta P = 0.004).Similarly, in the treated solid cancers, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.001) and all the other VOCs (Alpha P = 0.001, Beta P = 0.002, Gamma P = 0.013, and Delta P < 001).
eFigure 5. Levels of neutralizing antibody responses following vaccination with the mRNA-1273 or BNT162b2.
IC50 titers of neutralizing antibodies in healthy control (A), solid organ transplant (B) and autoimmune diseases (C) study populations vaccinated with either the mRNA-1273 or the BNT162b2 vaccines.IC50 dilutions were log10 transformed for analysis.At 1 month, in the healthy controls the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.005), the Alpha (P = 0.006), the Gamma (P = 0.020) and the Delta (P = 0.029).In the solid organ transplants, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.005), the Alpha (P = 0.005), the Gamma (P = 0.020), and Delta P = 0.029).In the autoimmune diseases, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.017), the Alpha (P = 0.009), the Gamma (P = 0.020), and the Delta (P = 0.047).At 3 months, in the healthy controls, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P < 0.001) and all the other VOCs (P < 0.001).In the solid organ transplants, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.005), the Alpha (P = 0.007), and the Gamma (P = 0.022).In the autoimmune diseases, the IC50 titers between the two vaccines were significantly different for the 2019-nCoV (P = 0.012), the Alpha (P = 0.035), the Beta (P = 0.036), the Gamma (P = 0.036), and the Delta (P = 0.035).
Resulting p-values were adjusted for multiple testing using the False Discovery Rate (FDR) approach of Benjamini-Hochberg.

eFigure 2 .
Levels of binding IgG anti-S antibodies at month 1 after the 2 nd vaccine dose. A. Percentage of participants with positive diagnostic for binding IgG anti-S antibodies.B. Titers, i.e. units/ml, of binding IgG anti-S antibodies.C. Titers of binding IgG anti-S antibodies in participants vaccinated with the mRNA-1273 or the BNT162b2 vaccines.All the study populations are shown.Resulting p-values were adjusted for multiple testing using the False Discovery Rate (FDR) approach of Benjamini-Hochberg.The titers in SOT, AD and treated HM were significantly lower (P<0.001)compared to HC eFigure 3. Proportion of participants with neutralizing antibodies responses at month 1 and 3 post-vaccination.Proportion of participants positive for neutralizing antibody at Month 1 and 3 in healthy control (A), solid organ transplant (B) and autoimmune diseases (C).Neutralizing antibody responses were measured against the original 2019nCoV and the different VOCs.Data are expressed as IC50 dilutions.eFigure 4. Levels of neutralizing antibody responses at month 1 and 3 after the 2 nd vaccine dose.IC50 titers of neutralizing antibodies at Month 1 and 3 in healthy control (A), solid organ transplant (B) and autoimmune diseases.The dotted line indicates the threshold positivity of the assay, i.e.IC50 >50 dilutions.IC50 dilutions were log10 transformed for analysis.Resulting p-values were adjusted for multiple testing using the False Discovery Rate (FDR) approach of Benjamini-Hochberg.*P<0.05;**P<0.01;*** P<0.001 eTable 3. Neutralizing antibody responses at Month 1 and 3 post-vaccination

eFigure 6 .
Proportion of participants with different levels of neutralizing antibody titers.IC50 titers were stratified as follows: <50 negative response; >50<100 weak response; >100<150 moderate response; <150 high response.The proportion of participants with different magnitude of IC50 titers was evaluated within each study population vaccinated with either the mRNA-1273 or the BNT162b2 vaccines.eFigure 7. Proportion of participants with neutralizing antibodies responses at month 1, 3 and 6 after the 2 nd Vaccination.Healthy control (A), solid organ transplant (B) and autoimmune diseases (C) study populations are shown.Participants were combined for the analysis within each group.eFigure 8. Estimates of the duration in time of binding response at month 6 ater the 2 nd dose in the SC participants.278 SC received BNT162b2 whereas 68 SC received mRNA-1273.The binding Abs duration in time (in weeks) was estimated by linear regression models using time as continuous covariate (the number of days corresponding to 1, 3 and 6 months after the second dose of vaccine).eTable 5. Univariable linear regression models of binding and neutralizing antibodies at Month 1, 3 and 6 since the 2nd dose of vaccine Analysis performed on binding and neutralizing antibodies log10-transformed values.eFigure 9. Estimates of the duration in time of neutralizing response at month 6 since the 2 nd dose in the HC participants.101 HC received BNT162b2 whereas 43 HC received mRNA-1273.The neutralization Abs duration in time (in weeks) against the Alpha, Beta and Gamma VOC was estimated by linear regression models using time as continuous covariate (the number of days corresponding to 1, 3 and 6 months after the second dose of vaccine).eFigure 10.Estimates of the duration in time of neutralizing response at month 6 since the 2 nd dose in the HM participants.49 HM received BNT162b2 whereas 30 HM received mRNA-1273.The neutralization Abs duration in time (in weeks) against the Alpha, Beta and Gamma VOCs was estimated by linear regression models using time as continuous covariate (the number of days corresponding to 1, 3 and 6 months after the second dose of vaccine).eFigure 11.Percentage of participants reporting local and systemic Reactions Reported at V2 visit after Injection of BNT162b2 or mRNA-1273.Data on local and systemic reactions were collected from 838 participants at visit V2, week 1 after the second vaccine.Solicited injection-site (local) reactions are shown in Panel A. Pain at the injection site was assessed according to the following scale: mild, does not interfere with activity; moderate, interferes with activity; severe, prevents daily activity; and grade 4, emergency department visit or hospitalization.Redness and swelling were measured according to the following scale: mild, 2.0 to 5.0 cm in diameter; moderate, >5.0 to 10.0 cm in diameter; severe, >10.0 cm in diameter; and grade 4, necrosis or exfoliative dermatitis (for redness) and necrosis (for swelling).Systemic events and medication use are shown in Panel B. Fever was assessed according to the following scale: mild; temperature 38.0 to 38.4°C, moderate; temperature >38.4 to 38.9°C severe; temperature >38.9 to 40.0°C, grade 4; temperature >40.0°C.Additional scales were as follows: fatigue, headache, chills, new or worsened muscle pain, new or worsened joint pain (mild: does not interfere with activity; moderate: some interference with activity; or severe: prevents daily activity), vomiting (mild: 1 to 2 times in 24 hours; moderate: >2 times in 24 hours; or severe: requires intravenous hydration), and diarrhea (mild: 2 to 3 loose stools in 24 hours; moderate: 4 to 5 loose stools in 24 hours; or severe: 6 or more loose stools in 24 hours); grade 4 for all events indicated an emergency department visit or hospitalization.

eFigure 1. Recruitment of participants, Laboratory testing, and Follow-up.
This is a prospective longitudinal study of immunocompromised patients and of health care workers as group of control.Participants received two doses of BNT162b2 or mRNA-1273 vaccines.Between January 14 and December 18, 2021, the participants were monitored for 6 months after the 2nd dose of vaccine.Seroconversion to SARS-CoV-2 Spike (S) protein and neutralizing antibodies were tested before vaccination and longitudinally at Week 1, Month 1, 3, and 6 following the 2nd vaccine dose.All participants underwent to 3-4 serologic assays.
databases.The rationale for the observational study were explicitly stated.There are no direct comparative data on the effectiveness of Covid-19 mRNA vaccines in immunocompromised patients and healthy participants.The research questions and hypotheses addressed are relevant and the add value of this study to the pandemic in immunocompromised individuals is important.The research methodology and serological assays have been standardized and automated to guarantee reliable, reproducible and homogeneous results with the minimum of technical error.A narrative description is included in the methods section.The study design and data-analysis were appropriate with adequate numbers of patients to yield sufficient statistical power for the primary analyses.The study design is also appropriate to address the study hypotheses/questions and included two groups of participants vaccinated with BNT162b2 or mRNA-1273.Standardized reporting data system and careful interpretation of results were

eTable 1. Baseline characteristics and type of vaccine of the study groups
HC: healthy controls; SC: solid cancers; HM: haematological malignances; AD: autoimmune diseases; SOT: solid organ transplants eTable 2.

Influence of immunosuppressive treatments on binding and neutralizing antibodies at Month 1 post vaccination eLegend Figure 3.
For example. at 1 month after vaccination, the IC50 titers against 2019nCoV were significantly lower in participants with solid organ transplants (median 16.5, 95% CI 8.5-68.