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Central obesity, smoking habit, and hypertension are associated with lower antibody titres in response to COVID‐19 mRNA vaccine

Abstract Aims To explore variables associated with the serological response following COVID‐19 mRNA vaccine. Methods Eighty‐six healthcare workers adhering to the vaccination campaign against COVID‐19 were enrolled in January–February 2021. All subjects underwent two COVID‐19 mRNA vaccine inoculations (Pfizer/BioNTech) separated by 3 weeks. Blood samples were collected before the 1st and 1–4 weeks after the second inoculation. Clinical history, demographics, and vaccine side effects were recorded. Baseline anthropometric parameters were measured, and body composition was performed through dual‐energy‐X‐ray absorptiometry. Results Higher waist circumference was associated with lower antibody (Ab) titres (R = −0.324, p = 0.004); smokers had lower levels compared to non‐smokers [1099 (1350) vs. 1921 (1375), p = 0.007], as well as hypertensive versus normotensive [650 ± 1192 vs. 1911 (1364), p = 0.001] and dyslipideamic compared to those with normal serum lipids [534 (972) vs 1872 (1406), p = 0.005]. Multivariate analysis showed that higher waist circumference, smoking, hypertension, and longer time elapsed since second vaccine inoculation were associated with lower Ab titres, independent of BMI, age. and gender. Conclusions Central obesity, hypertension, and smoking are associated with lower Ab titres following COVID‐19 vaccination. Although it is currently impossible to determine whether lower SARS‐CoV‐2 Abs lead to higher likelihood of developing COVID‐19, it is well‐established that neutralizing antibodies correlate with protection against several viruses including SARS‐CoV‐2. Our findings, therefore, call for a vigilant approach, as subjects with central obesity, hypertension, and smoking could benefit from earlier vaccine boosters or different vaccine schedules.

correlate with protection against several viruses including SARS-CoV-2. Our findings, therefore, call for a vigilant approach, as subjects with central obesity, hypertension, and smoking could benefit from earlier vaccine boosters or different vaccine schedules.

K E Y W O R D S
BMI, immunogenicity, infection, SARS CoV-2, vaccination, waist circumference

| INTRODUCTION
Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was described in 2019, the world has faced an unprecedented pandemic causing millions of deaths. Obesity and excess visceral fat were shown to be major risk factors for the development of complications following COVID-19 infection, [1][2][3][4] and, even before vaccines were made available, concerns regarding the possibility that obesity may blunt their efficacy were raised. 5 In December 2021, Polack et al. 6 reported promising results regarding an mRNA vaccine against COVID-19, BNT162b2 (Pfizer, Inc., and BioNTech), which conferred 95% protection against COVID-19 in adult subjects. The trial enrolled approximately 44,000 subjects, and there were 8 and 162 COVID-19 cases, respectively, following the two doses of vaccine or placebo. Despite the population comprising 35% of patients with a BMI of more than 30, and the good efficacy data reported towards COVID-19 infection, evidence regarding the ability to protect against severe COVID-19 is less certain for subpopulations, and long-term data are lacking. Moreover, obesity is not defined by BMI, but by the presence of fat excess, 7 BMI often mistakenly including subjects with fat excess in the normal weight category and vice versa. Body fat measured through bioimpedentiometry (BIA) or dual X-ray absorptiometry (DXA) may represent an alternative, but cut-off values have never been validated, and the necessary equipment may not be promptly available in many clinical practices. The use of waist circumference as a measure of central obesity is, conversely, well established, and the European Association for the Study of Obesity, recommends to screen for obesity complications not only those with a BMI ≥ 25, but also those with central obesity (waist circumference ≥80 cm for women and ≥94 cm for men). To the best of our knowledge, no studies are available to date investigating the real-life immunogenicity following BNT162b2 vaccine in relation to body composition and body fat distribution. In Italy, the BNT162b2 Pfizer BioNtech vaccine has been selected to be administered to healthcare professionals since December 2020.
Our primary aim focused on exploring the association between the response to the COVID-19 vaccine and adiposity parameters such as central obesity; as a secondary aim, we explored its possible association with other cardiometabolic conditions and risk factors.

| Vaccination procedure and blood collection
All patients were subjected to two COVID-19 vaccine inoculations, separated by 21 days (Comirnaty, Pfizer-BioNTech). Before the first inoculation, all patients underwent a blood draw that was handled according to local standards of practice. A second blood draw was collected between 1 and 4 weeks after the second inoculation, 28-49 days after the first inoculation. Samples were centrifuged and plasma kept at −80°C until further analysis.

| Biochemical measures
Routine biochemical tests were handled according to standard operating procedures. Anti-SARS-Cov-2 antibodies were measured through a commercially available assay (Elecsys® Anti-SARS-CoV-2 assay; Roche Diagnostics), which detects total antibodies against the SARS-CoV-2 spike (S) antigen in a sandwich electrochemiluminescence assay. 8

| Anthropometric and body composition assessment
Anthropometric parameters were measured at baseline. Body weight was measured using a balance-beam scale (Seca GmbH & Co). Height was rounded to the closest 0.5 cm. BMI was calculated as weight in kilograms divided by squared height in metres (kg/m 2 ). Waist circumference was measured midway between the lower rib and the iliac crest, hip circumference at the level of the widest circumference over the great trochanters to the closest 1.0 cm. The measurements were performed with the means of an anelastic tape by trained professionals. Body composition was measured through DXA (Hologic 4500) as previously reported. 9

| Statistics
The Statistical Package for Social Sciences (SPSS), v.20 was used for statistical analysis. Results are presented as mean, standard deviation (SD), or median, interquartile range (IQR) according to their distribution. Normality was assessed with the Kolmogorov-Smirnov test.
Variables not normally distributed were log-transformed. To build a multivariate linear regression model with Ab titres as the dependent variable, we used an enter method approach (all the independent variables included in the same regression equation) and investigated the following variables/models: (1) multivariate analysis including age and BMI together with variables with significant univariate association (p ≤ .05) analysed one by one as regressors (age + BMI + Waist circumference; age + BMI + waist to hip ratio; age + BMI + time; age + BMI + hypertension; age + BMI + dyslipidaemia; age + BMI + smoking habit). (2) Multivariate analysis including age, BMI, and time since second inoculation together with variables with significant association (p ≤ .05) at multivariate model 1, analysed one by one as regressors (age + BMI + time since second inoculation + waist circumference; age + BMI + time since second inoculation + hypertension; age + BMI + time since second inoculation + dyslipidaemia; age + BMI + time since second inoculation + smoking habit). suggesting the absence of multicollinearity between included variables. 10 The results were considered statistically significant when p < 0.05.

| Study population
Eighty-six subjects were enrolled in the present study in January and A small panel of routine biochemical tests was normal for all participants and is summarized in Table 1.
However, central obesity was observed in 60.9% (n = 53) of subjects out of 78 for whom waist circumference measurements had been recorded. The cutoff to determine central obesity was 80 cm for women, and 94 cm for men. 11

| Safety
All adverse events following the vaccine inoculation were recorded with the means of a structured interview, and 65.9% (n = 56) of participants complained of some adverse events following the first inoculation. Of these, 50 reported of pain or pruritus in the site of inoculation, 10 of headache, fatigue, or malaise, three of low-grade fever, two of dyspnoea, and five of other minor adverse events. parameters such as higher waist circumference, waist-to-hip ratio, BMI, or body fat were not associated with more adverse events (data not shown).
Interestingly, obesity identified as a BMI ≥ 30 kg/m 2 was not associated with a lower SARS-CoV-2 antibody titres (p = 0.524; data not shown). Furthermore, subjects with a smoking habit had lower Ab levels compared to those who were not current smokers [1099 (1350) versus 1921 (1375), respectively, p = 0.007; Figure 1B], and the same was for those who were hypertensive compared to those who were not [650 ± 1192 versus 1911 (1364), respectively, p = 0.001; Figure 1C], and those with dyslipidaemia compared to those who had a normal lipid profile and were not on lipid lowering drugs [534 (972) versus 1872 (1406), respectively, p = 0.005; Figure 1D].
Regression analysis showed that the presence of central obesity (as waist circumference measurement or waist-to-hip ratio) was associated with lower antibody concentration following the vaccine, as were hypertension, dyslipidaemia, and smoking habit (Table 2). Moreover, the time since the second vaccine inoculation at which the Ab titres were evaluated was significantly associated with a decline in serum SARS-CoV-2 Ab ( Table 2). The presence of side effects following the first or second inoculation was not associated to different Ab titres, neither was flu vaccination in the preceding 12 months (Table 2).
Each parameter showing a significant univariate association with Ab titres were included in a multivariate analysis together with age and BMI, factors possibly affecting the association, analysed one by one as regressors (

| DISCUSSION
Herein, we report that central obesity, independent of BMI is associated with lower Ab titres following a COVID-19 mRNA vaccine. This could be due to a number of reasons, one of which is the metabolic derangements that often come with visceral adiposity, together with the immune dysfunction that has been reported in patients with obesity. 12,13 In fact, strong evidence supports the fact that obesity is also associated with poor seroconversion upon some vaccine administration, 14 together with increased risk of infection even when the seroconversion seems robust. 15 A recent study has shown that higher BMI is associated with lower Ab titres in response to COVID-19 vaccine in Italian healthcare workers. 16 Although pointing in the same direction, our findings slightly differ from this study, as we could not find any significant association between BMI and SARS-CoV-2 Ab titres T A B L E 1 Descriptive characteristics of study population Mean ± SD or median (IQR) or N (%)

Age (years)
29 (17) Weight ( We also report that smoking and hypertension were strongly associated with lower Ab titres. Interestingly, it was previously reported that Ab titres following influenza vaccination decline more rapidly in smokers, through an unknown mechanism. 19 More generally, the habit of smoking is associated with a dysfunctional immune system, linked with both autoimmune disease and reduced response to F I G U R E 1 Serological response in relation to selected cardiometabolic conditions and risk factors. (A) Scattered dot-plot representing the correlation between anti-SARS-CoV-2 antibody titres and waist circumference. p is from a univariate linear regression analysis, with Ab titres as the dependent variable and waist circumference as the independent variable; (B) smokers; (C) hypertensive subjects; and (D) dyslipideamic subjects showed lower Ab titres compared to non-smokers, normotensive, and those with a normal lipid profile, respectively. *p < 0.05, **p < 0.01, **p < 0.001, ***p < 0.000  infections. 20 Similarly, hypertension and an inappropriate response to vaccinations might have a common root into a dysfunctional immune system according to recent evidence. 21 Moreover, similar to central obesity, the presence of hypertension was previously found to be related to worse COVID-19 outcomes, 22 Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CI, confidence interval; CRP, C-reactive protein.

T A B L E 2 Univariate and multivariate linear regression analyses for anti-SARS-CoV-2 antibody titre following vaccination
8 of 10protection against several viruses including SARS-CoV-2, 28,29 and the finding that central obesity, hypertension, and smoking are associated with lower Ab concentration shortly after the vaccination warrants further attention, as this may mean that these subjects respond in a different way to the same vaccination and may require different vaccine booster schedules over time.
Our study also features some strengths. This is, to the best of our knowledge, the first study reporting data on the immunogenicity of a With the general population now being vaccinated, more and more subjects with central and general obesity will receive the vaccine, and very soon booster schedules will need to be planned. The fact that the Ab response is lower in certain subjects shortly after the second inoculation must lead to a highly vigilant approach, as medium and long-term data will become available only when the schedule will have been necessarily set already.