key: cord-0733483-7wf1cc49
authors: Marfella, Raffaele; D'Onofrio, Nunzia; Sardu, Celestino; Scisciola, Lucia; Maggi, Paolo; Coppola, Nicola; Romano, Ciro; Messina, Vincenzo; Turriziani, Fabrizio; Siniscalchi, Mario; Maniscalco, Mauro; Boccalatte, Marco; Napolitano, Giovanni; Salemme, Luigi; Marfella, Ludovica Vittoria; Basile, Eugenio; Montemurro, Maria Vittoria; Papa, Carmela; Frascaria, Francesco; Papa, Antonio; Russo, Ferdinando; Tirino, Virginia; Papaccio, Gianpaolo; Galdiero, Marilena; Sasso, Ferdinando Carlo; Barbieri, Michelangela; Rizzo, Maria Rosaria; Balestrieri, Maria Luisa; Angelillo, Italo Francesco; Napoli, Claudio; Paolisso, Giuseppe
title: Does poor glycaemic control affect the immunogenicity of the COVID‐19 vaccination in patients with type 2 diabetes: The CAVEAT study
date: 2021-10-01
journal: Diabetes Obes Metab
DOI: 10.1111/dom.14547
sha: c81b00af27ef8cd7cfceda941440ddbbe1438959
doc_id: 733483
cord_uid: 7wf1cc49

nan

Type 2 diabetes (T2D) is associated with an increased risk of infection-related morbidity and mortality. 1 Accordingly, coronavirus disease 2019 (COVID-19) results in increased hospitalization rates and illness severity in people with T2D. 2 Moreover, poor glycaemic control worsens patients' prognosis with COVID-19, raising the risk of mechanical ventilation, shock, and multiple organ failure necessitating ICU treatment. 3 Thus, the absolute burden of infection attributable to poor glycaemic control in this population would be substantial. This association may be explained by the degree of poor glycaemic control, which may be correlated to the worsening of cell-mediated immunity. 3 Although a normal humoral response against severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been evidenced in T2D patients, 4 previous studies have shown that dysregulation of the cellular immune response, especially T lymphocytes, might be highly involved in the pathological process of COVID-19. 5 Although no significant difference was observed in total lymphocyte count or lymphopenia incidence between patients with T2D and those without diabetes, distinguishable differences in the subpopulations of lymphocytes have been observed. 7 The sustained decrease in total T cells and Th and Tc subsets and NK subsets were all more remarkable in people with T2D than in those without diabetes. 6 Therefore, T2D may hamper the immune responses after vaccination against SARS-CoV2.

Previous studies have shown that individuals with diabetes had a consistently lower immunological response to the hepatitis B vaccine, 7 while less consistent results were noted for influenza and varicella-zoster vaccines. 8 In this context, evaluating SARS-CoV-2 vaccine efficacy is critical to reducing the morbidity and mortality associated with COVID-19 in this vulnerable population. Testing vaccines that prevent infection with SARS-CoV-2 in T2D populations with poorly controlled glycaemia, therefore, is important because increased incidences of illness and death from COVID-19 have been associated with hyperglycaemia. Two doses of 30 μg BNT162b (Pfizer-BioNtech) elicited similar binding-antibody responses in people with or without T2D. 9 However, there are no data on neutralizing antibodies and cell-mediated response to BNT162b vaccine in T2D patients, nor data on the immunological vaccine responses related to glycaemic control. 10 The aim of the present study, therefore, was to evaluate cellmediated response to the COVID-19 vaccine with regard to diabetic status and glycaemic control.

We conducted a prospective observational study at vaccination sites in Campania, Italy. A total of 1123 adults, without previous COVID-19 or SARS-CoV-2 asymptomatic status, were screened from December 2020 (Supplementary Figure S1 ). Details of the study design and participant characteristics are provided in the Supplementary Methods and Supplementary Table S1.

Plasma glucose, creatinine, and serum lipids were measured using enzymatic assays in the hospital's Chemistry Department (Supplementary Methods).

To determine the immune status of SARS-CoV-2-vaccinated subjects, a GenScript SARS-CoV-2 Surrogate Virus Neutralization Test (sVNT; cat. no.: L00847-5) was used for neutralizing antibody evaluation. The assay is a blocking ELISA, which mimics this virus receptor-binding process, so that the neutralization capacity of anti-SARS-CoV-2 antibodies directed against the receptor-binding domain can be measured (Supplementary Methods).

Isolation of peripheral blood mononuclear cells was performed as previously described 14 (Supplementary Methods).

Continuous variables are summarized as mean and standard deviation, and categorical variables as absolute and relative frequencies. For continuous variables, the differences among the groups were evaluated using Student's t-test or analysis of variance (ANOVA) for normally distributed data and the Kruskal-Wallis test for non-normally distributed data. When differences were found among the groups, Bonferroni correction was used to make pairwise comparisons. The chi-squared test was used to compare categorical variables. Normality of distribution was tested with the Shapiro-Wilk test.

Repeated-measures ANOVA was used for comparison of neutralizing antibodies at 21 and 52 days. Regression analysis was performed to estimate the relationships among baseline glycated haemoglobin (HbA1c) levels and immunological response 21 days after the second dose. P values ≤ 0.05 were taken to indicate statistical significance. All calculations were performed using SPSS 23 software (SPSS Inc, Chicago, Illinois).

The primary endpoint of the study was to assess neutralizing antibody and T-cell responses in participants with and without diabetes.

The secondary endpoint was to assess neutralizing antibody and Tcell responses in diabetes patients with good and poor glycaemic control.

The sample size required to evaluate the humoral and cellular immune response in participants with and without diabetes was determined.

Using a Student's t-test for independent samples, it was calculated that a sample of 210 participants (70 in each group) would have 90% power to assess an effect size of 0.45 with an alpha error of 5%.

For study eligibility, we assessed individuals aged 18 to 60 years had achieved good glycaemic control (HbA1c 6.6% ± 0.4%). However, 35 participants with poor glycaemic control at baseline (HbA1c 8.2% ± 0.8%) did not achieve good glycaemic control (HbA1c 8.3% ± 1.1%).

Participants with poor glycaemic control at baseline (according to HbA1c level on vaccination day) were divided into two groups based on HbA1c levels evaluated 52 days after the second vaccination dose.

The first follow-up evaluation was conducted after 23.8 ± 4.8 days in participants without diabetes, after 23.6 ± 4.5 days in T2D patients with good glycaemic control, and after 24.6 ± 4.3 days in T2D patients with poor glycaemic control. To assess antibody response in non-T2D and T2D patients, we performed a recently validated sVNT assay that detects circulating antibodies directed against the spike protein receptor-binding domain (RBD), based on antibody-mediated blockade of the interaction between the ACE2 receptor protein and the RBD. 12 This assay allowed analysis of in vitro neutralization activity and was used instead of more sophisticated methods that need to be performed under higher biosafety levels. The degree to which test plasma inhibited binding of the anti-IgG-horseradish peroxidase (HRP)-RBD to ACE2 receptors, compared to control, was determined by optical density reading, with ≥20% inhibition defined as a positive response. We found a high specificity of 89.2% (95% confidence interval [CI] 86.9-89.9) and a sensitivity of 70.3% (95% CI 64.9-74.8) for the sVNT. Results showed that neutralizing responses were undetectable the day before vaccination ( Figure 1A ). By contrast, potent neutralization responses were observed in all the participants 21 days after the second dose of the vaccine; on Day 21 after the second vaccine dose, T2D patients with HbA1c >7% showed significantly reduced virus-neutralizing antibody capacity than normoglycaemic subjects and T2D patients with good glycaemic control (P < 0.05).

Moreover, in response to S-specific peptide pools, we observed a higher CD4 + T/cytokine response involving type 1 helper T cells in normoglycaemic participants and T2D patients with good glycaemic control than in T2D patients with poor glycaemic control ( Figure 1A ).

and IL-13) to SARS-CoV-2 Prot_S peptide pools were observed only at very low levels after the second vaccine dose among the study participants (data not shown). 13 Among the observed cytokine, the tumor necrosis factor α responses were greater than the interleukin-2 and the interferon-γ responses.

Interestingly, the mean percentage of neutralizing antibody and CD4 + T/cytokine responses between Days 21 and 52 after the second vaccine dose were inversely related to baseline HbA1c levels ( Figure 1B) . Therefore, to exclude bias related to the difference in immunological responses to mRNA and virus-modified vaccines, we evaluated the virus-neutralizing antibody and CD4 cytokine response involving type 1 helper T cells concerning vaccine type in all groups. Figure S2 , no differences among immunological responses induced by the different vaccines were found.

The final follow-up evaluation was conducted after 52 ± 6 days in participants without diabetes, after 50 ± 4 days in T2D patients with good glycaemic control, and after 51 ± 5 days in T2D patients with poor glycaemic control. According to Standards of Medical Care in Diabetes 2021, 11 HbA1c levels were evaluated after 75 ± 4 days in patients without diabetes, 73 ± 5 days in T2D patients with good glycaemic control, and 73 ± 6 days in T2D patients with poor glycaemic control (Supplementary Table) . Therefore, patients with poor glycaemic control at baseline (HbA1c on vaccination day) were divided into two groups based on the HbA1c levels evaluated 52 days after the second vaccination dose. At the final follow-up evaluation, virus-neutralizing antibodies and antigen-specific CD4 + T-cell responses remained higher in participants without diabetes and T2D patients with good glycaemic control than in T2D patients with poor F I G U R E 1 A, Neutralizing antibodies and CD4 + T cells producing tumour necrosis factor-α (TNF-α), interleukin-2 (IL-2) and interferon-γ (IFNγ) in participants without diabetes, type 2 diabetes (T2D) patients with good glycaemic control, and T2D patients with poor glycaemic control on vaccination day, and 21 days and 52 days after the second vaccination dose (boxplots show the median, 25th, and 75th percentiles, range, and extreme values). *P < 0.05 vs. T2D with good glycaemic control group. §P < 0.05 vs nondiabetic group. B, Regression analysis between glycated haemoglobin (HbA1c) levels at baseline and mean of CD4+ T-cell responses at 21 days after the second vaccine dose F I G U R E 2 A, Neutralization antibodies and CD4+ T cells producing tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and interferon-γ (IFNγ) in type 2 diabetes (T2D) patients with poor glycaemic control at baseline (vaccination day) and with glycated haemoglobin (HbA1c) <7% (HbA1c 8.1 ± 0.7 to 6.6% ± 0.4%) and HbA1c >7% (HbA1c 8.2 ± 0.8 to 8.3% ± 1.1%) at follow-up (52 days after the second vaccination dose). Boxplots show the median, 25th and 75th percentiles, range, and extreme values). *P < 0.05 vs. T2D patients with HbA1c <7% at follow-up. B, Regression analysis of HbA1c level changes (vaccination day and 52 days after second vaccination dose) and CD4+ T-cell response changes (21 and 52 days after the second vaccine dose) glycaemic control ( Figure 2 ). As shown in Figure 2 , on Day 52 after the second vaccination dose, the 25th and 75th percentile ranges of virus-neutralizing antibodies and antigen-specific CD4 + T-cell responses were much higher, suggesting extreme variability in immunological responses in T2D patients with poor baseline glycaemic control. In fact, 57 of the T2D patients with poor glycaemic control had HbA1c levels <7% (HbA1c 6.6% ± 0.4%) 52 days after the second vaccine dose. Remarkably, the virus-neutralizing antibodies and antigen-specific CD4 + T-cell responses improved significantly compared to the responses observed 21 days after the second vaccine dose (Figure 2A) . Interestingly, the HbA1c reduction of 1.5% ± 0.3% increased the neutralizing antibody titres and CD4 cytokine response by almost 15% ( Figure 2B) . Finally, 35 patients with poor glycaemic control at baseline (HbA1c 8.2% ± 0.8%) did not have good glycaemic control 52 days after the second vaccination dose (HbA1c 8.3% ± 1.1%). Interestingly, the virus-neutralizing antibodies and antigen-specific CD4-cell responses remained impaired in these patients ( Figure 2A) . Finally, regression analysis showed that reduction of HbA1c levels 52 days after vaccination were associated with neutralizing antibody titres and CD4 cytokine increases ( Figure 2B ).

There were no differences in the immunological responses induced by the different vaccines (data not shown).

To date, the immunological responses to COVID-19 vaccines in T2D patients are poorly elucidated. In the present study, we evaluated whether diabetes and hyperglycaemia affect the ability to mount appropriate virus-neutralizing antibodies and antigen-specific CD4-cell responses after administration of COVID-19 vaccines.

We observed that the COVID-19 vaccines induced a weak immunity in T2D patients with poor glycaemic control compared with normoglycaemic individuals and T2D patients with good glycaemic control. At 21 days after the first vaccine dose, neutralizing antibody titres and CD4 cytokine responses involving type 1 helper T cells were lower in T2D patients with HbA1c levels >7% than in individuals with HbA1c levels ≤7%, evaluated at baseline before the first vaccine dose.

Taken together, our data support the evidence of an efficient immunological response in T2D patients with good glycaemic control, as the prevalence of positivity for virus-neutralizing antibodies and antigen-specific CD4-cell responses was similar, with regard to timing and titres, to that of patients without diabetes. We hypothesized that poor glycaemic control during the vaccination period might impair immunological responses. Previous studies showed that T2D is a chronic disease that leads to persistent immune dysregulation. 14 Whether hyperglycaemia modulates the antibody response to a viral infection is still a matter of discussion. 10 Several defects in immunity have been associated with hyperglycaemia and insulin resistance, including reduced lymphocyte proliferative response, impaired monocyte/macrophage, and neutrophil function, abnormal delayed-type hypersensitivity reaction, and complement activation dysfunction. 15 Individuals with diabetes have a consistently decreased immunological response to the hepatitis B vaccine. 7 immunological responses is relatively low despite achieving statistical significance. Therefore, further studies are needed to confirm our observations. Nevertheless, our data may be helpful to consider the glycaemic control properly during the vaccination period.

In conclusion, our data highlight two critical points: first, hyperglycaemia at the time of vaccination worsens the immunological response and, second, achieving adequate glycaemic control during the post-vaccination period improves the immunological response.

Hence, we predict that stringent glycaemic control may restore the natural predisposal to a good immune response to the SARS-CoV-2 vaccine. Glycaemic control should therefore be the standard of care during pandemics, increasing the contribution of diabetologists to the success of vaccine programmes. In addition, we need to focus on hyperglycaemia which can play a role in clinical COVID-19 outcomes and vaccine efficiency. The present study allows cautious optimism regarding adequate immunization in individuals with diabetes after COVID-19 vaccines, suggesting that achievement of tight glycaemic control during the vaccination period normalizes natural protection against SARS-CoV-2. Open access funding enabled and organized by Projekt DEAL.

The peer review history for this article is available at https://publons. com/publon/10.1111/dom.14547.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. 

Diabetes and infection: assessing the association with glycaemic control in population-based studies

Impact of diabetes mellitus on clinical outcomes in patients affected by Covid-19

Outcomes in patients with hyperglycemia affected by COVID-19: can we do more on glycemic control? Diabetes Care

Antibody response to multiple antigens of SARS-CoV-2 in patients with diabetes: an observational cohort study

Distinguishable immunologic characteristics of COVID-19 patients with comorbid type 2 diabetes compared with nondiabetic individuals

T cells are reduced and rendered unresponsive by hyperglycemia and chronic TNFalpha in mouse models of obesity and metabolic disease

Immune reactivity during COVID-19: implications for treatment

Immune response of hepatitis B vaccine among persons with diabetes: a systematic review of the literature

Immunogenicity, safety, and effectiveness of seasonal influenza vaccination in patients with diabetes mellitus: a systematic review

SARS-CoV-2/COVID-19 vaccines and glycemic control: call for data

American Diabetes Association Classification and Diagnosis of Diabetes. Standards of medical care in diabetes-2020

Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation

Safety and immunogenicity of SARS-CoV-2 mRNA-1273 vaccine in older adults

A SARS-CoV-2 surrogate virus neutralization test (sVNT) based on antibody mediated blockage of ACE2-spike protein-protein interaction

Changes of regulatory T cells and of proinflammatory and immunosuppressive cytokines in patients with type 2 diabetes mellitus: a systematic review and meta-analysis

The effects of type 2 diabetes mellitus on organ metabolism and the immune system

Monocytopenia, monocyte morphological anomalies and hyperinflammation characterise severe COVID-19 in type 2 diabetes

Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine