A Serum Multi-Parametric Analysis Identifies an Early Innate Immune Signature Associated to Increased Vaccine-Specific Antibody Production and Seroconversion in Simultaneous COVID-19 mRNA and Cell-Based Quadrivalent Influenza Vaccination

Abstract

In this pilot study, a multi-parametric analysis comparing immune responses in sera of adult healthy subjects (HS) or people with type 2 diabetes mellitus (T2D) undergoing the single or simultaneous administration of mRNA-based COVID-19 and cellular quadrivalent inactivated influenza vaccines was conducted. While SARS-CoV-2 antibodies remains comparable, influenza antibody titers and seroconversion were significantly higher upon simultaneous vaccination. Magnitude of anti-influenza humoral response closely correlated with an early innate immune signature, previously described for the COVID-19 vaccine, composed of IL-15, IL-6, TNF-α, IFN-γ, CXCL-10 and here extended also to acute-phase protein Pentraxin 3. People with T2D receiving simultaneous vaccination showed a protective response comparable to HS correlating with the early induction of IFN-γ/CXCL10 and a significant reduction of the circulating glucose level due to increased oxidation of glucose digestion and consumption. These data, although preliminary and in-need of validation in larger cohorts, might be exploited to optimize future vaccination in people with chronic disorders, including diabetes.

Keywords: COVID-19; influenza virus; innate immunity; metabolism; vaccination.

Figure 1

Scheme of enrollment and collected samples from healthy and type 2 diabetes mellitus (T2D) vaccine recipients. A schematic representation of groups, number of study participants and timeline of sample collection are depicted. “CoV_only” group (in grey) comprised of healthy subjects (HS) (n = 24) to whom only the third booster dose of anti-COVID-19 BNT162b2 mRNA vaccine was administered. “Flu_beforeCoV” group comprised of HS (n = 18) (in light blue) to whom the 2021–2022 seasonal cell-based quadrivalent influenza (Flu) vaccine (Flucelvax) was administered 1 month before immunization with the third booster dose of anti-COVID-19 BNT162b2 mRNA vaccine. “Flu_plusCoV” groups comprised of HS (n = 19) (in blue) and of T2D (n = 6) (in petrol green), to whom 2021–2022 seasonal cell-based quadrivalent flu vaccine (Flucelvax) was simultaneously inoculated in different limbs with the third booster dose of anti-COVID-19 BNT162b2 mRNA vaccine. Demographic and clinical information as well as serum samples from longitudinal peripheral blood withdrawals were collected at the following time points: immediately before (day 0, d0), as well as 1 day and 30 days (d1 and d30, respectively) after the third booster dose of COVID-19 mRNA vaccine in the presence or absence of the flu vaccine. Only for “Flu_beforeCoV” group sera were stored immediately before (day 0, d0) quadrivalent flu vaccine, as well as before (day 30, d30) and 1 and 30 days after (d30 + 1 and d30 + 30, respectively) the third booster dose of the COVID-19 mRNA vaccine.

Figure 2

Anti-influenza and anti-COVID-19 vaccine-specific antibody production in healthy and type 2 diabetes mellitus (T2D) vaccine recipients. Levels of binding class G immunoglobulins recognizing SARS-CoV-2 trimeric spike protein (anti-SARS-CoV-2 S IgG; expressed as BAU/mL) (A) and specific anti-influenza (Flu) antibodies against all the vaccine antigen components included in the Flucelvax Quadrivalent flu vaccine used in the 2021–2022 season (A/Wisconsin, A/Cambodia, B/Phuket and B/Washington; expressed as hemagglutination inhibition, HAI, titers) (B–E) were measured in “CoV_only” group (in grey), “Flu_beforeCoV” (in light blue) as well as “Flu_plusCoV” groups (in blue for healthy subjects, HS; in petrol green for T2D individuals). Serum samples were analyzed before (day 0, d0) and 30 days (d30) after the third booster dose of COVID-19 mRNA vaccine in the presence or absence of flu vaccination. Only for “Flu_beforeCoV” subjects, sera were tested before (day 0, d0) quadrivalent flu vaccine, as well before (day 30, d30) and 30 days after the third booster dose of COVID-19 mRNA vaccine (d30 + 30). p-values calculated by one-way ANOVA test were assigned as follows: * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001, **** ≤ 0.0001.

Figure 3

Seroconversion for influenza vaccine strains in healthy and type 2 diabetes mellitus (T2D) vaccine recipients. A comparison of seroconversion rate based on hemagglutination inhibition (HAI) titers obtained for all the vaccine antigen components included in the Flucelvax Quadrivalent influenza (flu) vaccine used in the 2021–2022 season (A/Wisconsin, A/Cambodia, B/Phuket and B/Washington) was reported for all the study groups. In particular, the seroconversion rate was calculated as a fold increase of HAI titers (A–D) and as a percentage (%) of seroconverted individuals (E–H) in “CoV_only” group (in grey) and “Flu_plusCoV” groups (in blue for healthy subjects, HS, and in petrol green for T2D individuals) and comparing HAI titers measured before (day 0, d0) and 30 days (d30) after the third booster dose of COVID-19 mRNA vaccine in presence or absence of flu vaccine (d30/d0). For the “Flu_beforeCoV” group (in light blue), the seroconversion rate was calculated comparing the HAI titers obtained before (day 0, d0) and 30 or 60 days after the quadrivalent flu vaccine (d30/d0 and d30 + 30/d0, respectively). p-values calculated by one-way ANOVA test were assigned as follows: * ≤ 0.05.

Figure 4

Early vaccine-induced cytokine and chemokine profile in healthy and type 2 diabetes mellitus (T2D) vaccine recipients. Level of cytokines (IL-15, IL-6, TNF-α, IFN-γ) (A–D), of the chemokine CXCL-10 (E) and of the acute-phase protein Pentraxin 3 (PTX3) (F) were measured using multiplex magnetic bead panel in serum samples longitudinally collected from the “CoV_only” group (in grey) and the “Flu_plusCoV” groups (in blue for healthy subjects, HS, and in petrol green for T2D individuals) immediately before (day 0, d0), as well as 1 day and 30 days (d1 and d30, respectively) after the third booster dose of COVID-19 mRNA vaccine in the presence or absence of the flu vaccine. Only for the “Flu_beforeCoV” group (in light blue) was the analysis performed on sera stored before (day 0, d0) the quadrivalent flu vaccine, as well as before (day 30, d30) and 1 and 30 days after (d30 + 1 and d30 + 30, respectively) the third booster dose of COVID-19 mRNA vaccine. p-values calculated by one-way ANOVA test were assigned as follows: * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001, **** ≤ 0.0001.

Figure 5

Regulation of circulating metabolites involved in glucose/pyruvate pathways in healthy and type 2 diabetes mellitus (T2D) individuals receiving influenza and COVID-19 vaccination. Changes in glucose (A), lactate (B), formate (C) and citrate (D) levels were measured in serum samples longitudinally collected in fasting condition from the “CoVonly” group (in grey) and “FluplusCoV” groups (in blue for healthy subjects, HS; in petrol green for T2D individuals) immediately before (day 0, d0), as well as 1 and 30 days (d1 and d30, respectively) after the third booster dose of the COVID-19 mRNA vaccine in presence or absence of the flu vaccine. Only for the “FlubeforeCoV” group (in light blue) was the analysis performed on sera stored before (day 0, d0) the quadrivalent flu vaccine, as well as before (day 30, d30) and 1 and 30 days after (d30 + 1 and d30 + 30, respectively) the third booster dose of COVID-19 mRNA vaccine. Values are shown as percentage (%) of the metabolite level relative to total metabolites. p-values calculated by one-way ANOVA test were assigned as follows: * ≤ 0.05; ** ≤ 0.01; *** ≤ 0.001, **** ≤ 0.0001.

Figure 6

Correlation of anti-influenza and anti-COVID-19 vaccine-specific antibody production with early innate signature in healthy and type 2 diabetes mellitus (T2D) vaccine recipients. Correlations were built between levels of binding class G immunoglobulins recognizing anti-SARS-CoV-2 trimeric spike protein (anti-SARS-CoV-2 S) and specific anti-influenza (flu) antibodies against Flucelvax 2021/2022 vaccine antigen components (A/Wisconsin, A/Cambodia, B/Phuket and B/Washington) and serum level of cytokines (IL-15, IL-6, TNF-α, IFN-γ), the chemokine CXCL-10 and the acute-phase protein Pentraxin 3 (PTX3). Innate factors and vaccine antibodies were measured 1 day (d1) and 30 days (d30), respectively, after the third booster dose of COVID-19 mRNA vaccine (CoV_only) (A), in presence of previous flu vaccination (d30 + 30 for Flu_beforeCoV) (B) or co-administered with the flu vaccine (Flu_plusCoV) in both healthy subjects (HS) and T2D (C,D). Correlations were obtained by deriving a Pearson r correlation coefficient (in the legend positive correlation is indicated in shades of blue, while negative correlation is indicated in shades of red).