After the vaccination, the elevation of post-vac. IgG (RBD) above 50 AU/mL and that of 4,160 AU/mL were seen in 99.8% and 91.6% of the total subjects, respectively. As the percentage of the latter was slightly lower than the one reported by Gobbi et al. (97.4%) , the difference could be due to the difference of elapsed time after the second vaccine shot; ours was five weeks after the second shot while the data of the study by Gobbi et al. were obtained one week after the second shot. In consistent with the report by Gobbi et al. , the presence of previous SARS-CoV-2 infection defined in this study by the elevation of pre-vac. IgG (N) above the cut-off 1.4 S/C resulted in significantly higher IgG (RBD) titre after the vaccination (Table 2). On the other hand, subjects whose post-vac. IgG (RBD) did not exceed the fifth percentile were those who were with pre-vac. IgG (N) below the cut-off (Fig. 1c). This result indicates that repeated immune stimulation, whether by natural infection or vaccination, would help enhance otherwise low antibody response. In fact, the increase of IgG (RBD) was observed even in the subject under the immune suppressant treatment (from 6.9 AU/mL to 43.0 AU/mL before and after the vaccination). Albach et al. also reported that repeated vaccination was successful in obtaining a positive antibody response in a patient with rheumatoid arthritis who were under immune suppressant treatment .
By the multivariable linear regression analysis shown in Table 2, we confirmed that male gender, subjects with advanced age, and absence of previous infection were significantly associated with low antibody responses, in consistent with the preceding reports [4, 5, 8]. In addition to this, hypertension, dyslipidaemia, CKD, and use of immune suppressant were significantly associated with low antibody responses. The result that the antibody response was low in the subject under the treatment with immune suppressant was theoretically understandable and was actually shown in the report by Albach et al. . Hou et al. suggested that vitamin D deficiency, uremic toxin accumulation and erythropoietin deficiency could affect the immune system in CKD patients . Therefore, the antibody response to the vaccination may have been low in the subjects with CKD (Table 2 and 3) due to the compromised immune function.
As Pellini et al. reported that the antibody response to SARS-CoV-2 BNT162b2 vaccine was not associated with hypertension , our result showed the statistical significance (p = 0.018) on the contrary. In the study by Pellini et al., the geometric means of SARS-CoV-2 S1/S2 IgG with and without hypertension were 172.18 AU/mL and 307.42 AU/mL, respectively, if not statistically significant. As IgG (RBD) assay used in this study detects IgG directed specifically to the receptor binding domain in SARS-CoV-2 S1 subunit, this discrepancy could be due to the different specificity of the IgG assay used in each study.
From the result in Table 2 that showed dyslipidaemia but not BMI was significantly associated with low post-vac. IgG (RBD), it is inferred that the cause for the low antibody response here could be more closely related to regulation of cholesterol rather than obesity in general. Contrary to CKD, hypertension and dyslipidaemia are known to be associated with enhanced autoimmune status [14, 15], so that the low antibody response may be caused by a mechanism other than immune modulation in subjects with these diseases. A mechanism related with interaction between S protein and the host receptor could be one of the speculated ones. SARS-CoV-2 is reported to enter host cells in three ways: receptor-mediated plasma membrane fusion, receptor-mediated endocytosis, or antibody-dependent viral entry . For the attachment of SARS-CoV-2 to the host cells in the former two cases, receptor binding domain in S protein binds to its receptor, angiotensin-converting enzyme II (ACE2). It is reported that S protein alone bound to ACE2 and induced several signalling [17, 18]. It could be inferred, therefore, that some portion of the S protein produced by BNT162b2 binds to ACE2 and endocytosed together with ACE2. As medication for hypertension (e.g., ACE inhibitor, angiotensin receptor blocker) is reported to increase expression of ACE2 , highly expressed ACE2 in patients with hypertension may bind and segregate S protein produced by BNT162b2 more efficiently so that reduced amount of S protein remains available for the antigen presentation. By using S2 peptide of SARS-CoV, another subunit of S protein, Meher et al. showed that S2 peptide was important for the viral entry into the host cell and that the peptide binding status was altered by the membrane cholesterol . Choi et al. suggested that dyslipidaemia might play a role in the severity of COVID-19 infection. . From these reports, it is speculated that the altered status of membrane cholesterol in patients with dyslipidaemia facilitates the entry of S protein into the host cell more efficiently and render the S protein less available for the antigen presentation, just as we postulated for hypertension above. In the subject with both hypertension and dyslipidaemia, post-vac. IgG (RBD) titre was 2,139.7 AU/mL. As shown in Table 3, this titre was lower than the 25th percentile of subject group with hypertension and that with dyslipidaemia, suggesting the additive effect by the two diseases. Whether this is statistically significant, however, remains to be seen.
If we assume that IgG (RBD) titre is correlated with the neutralizing activity to certain extent as mentioned earlier, it would be inferred that the efficacy of the vaccination is weaker in people with hypertension, dyslipidaemia, or CKD as well as those of male gender and of advanced age. If so, we might need to prioritize those people for the additional vaccination. Moreover, the antibody response to the natural infection of SARS-CoV-2 may be attenuated in male gender, people with advanced age, and people with hypertension, dyslipidaemia, or CKD. Li et al. reported that patients who were male, with advanced age, obesity, a history of smoking, hypertension, diabetes, malignancy, coronary heart disease, chronic liver disease, chronic obstructive pulmonary disease (COPD), or CKD were more likely to develop severe COVID-19 symptoms . Takahashi et al. reported that a poor T cell response was correlated with advanced age and was associated with worse disease outcome in male patients . Therefore, the antibody response to the natural infection of SARS-CoV-2 in male gender, people with advanced age, those with hypertension, dyslipidaemia, or CKD may be attenuated and, at least partly, may be more susceptible to the progression of COVID-19.
We unexpectedly saw the elevation of IgG (N) during the vaccination period in four subjects who had been confirmed PCR positive (Fig. 2b), which is interesting because of the similarity with breakthrough infection in those who had been vaccinated with SARS-CoV-2 vaccine reported by Hacisuleyman . The elevation of IgG (N) during the vaccination period could be due to waning immune protection elicited by the previous SARS-CoV-2 infection, breakthrough infection by the variants, or reactivation of persisting virus in the body. Considering that the days from PCR positivity were approximately 100 days in the four subjects (Fig. 2b), it would be too early for the immune response to weaken so that the breakthrough infection of SARS-CoV-2 variants, such as delta which was starting to prevail during that period, is considered to be one of the causes. As shown in Fig. 2c, pre-vac. IgG (RBD) titres in the four subjects were below 2,000 AU/mL, which suggested that the breakthrough infection was not preventable at this antibody level. This is consistent with a report by Bergwerk et al. that the occurrence of breakthrough infections with SARS-CoV-2 was correlated with neutralizing antibody titers during the peri-infection period .
By clarifying the antibody level at which SARS-CoV-2 infection could be preventable and by taking factors associated with the low antibody response into account, we would be able to have a more efficient strategy for the vaccination.