MATERIALS AND METHODS
The study was approved by the Ethics Committee of the Hospital Geral Dr.
César Cals, through CAAE 39691420.7.0000.5049. We included 64
participants of both sexes, ≥19 years of age, who had received two doses
of CoronaVac between January to February 2021, with an interval of 28
days between doses, and a booster dose of BNT162b2 vaccine in October
2021, 8 months after the second dose of CoronaVac. Blood collections and
serological tests were performed at Fundação Oswaldo Cruz (Fiocruz,
Ceará, Brazil), after an informed consent. We performed a longitudinal
analysis of the humoral response (IgM for the SARS-CoV-2 spike protein
(S) and IgG for the S and nucleocapsid (N) proteins) in samples
collected before the third dose (B/3D), 30 (30d/3D), 60 (60d/3D), 90
(90d/3D) and 180 (180d/3D) days after the third dose. The presence of
antibodies was measured by using Abbott Architect i2000SR
(Abbott®). The cut-off value was 1.0 index value (S/C)
for S IgM antibodies, 50AU/ml for S IgG, and 1.4 index value for N IgG.
The volunteers were monitored for SARS-CoV-2 infection by PCR over time.
The GraphPad Prism version 5.0 (https://www.graphpad.com) was used
for statistical analyses. The data were described
as the median and interquartile range (IQR) or percentage. ANOVA was
used to analyze the antibody titers over time and Mann-Whitney test in
group comparisons. The matrix of antibodies was imported into the
Morpheus program (https://software.broadinstitute.org/morpheus/)
and the results were illustrated as a 3D dendrogram (heat map).
Differences with p<0.05 were considered statistically
significant.
RESULTS AND DISCUSSION
Although all participants completed the vaccination schedule, two
participants were unable to give a blood sample in 180d/3D. The cohort
had a greater representation of female participants, with 81.54% female
and 26.09% male. The average age of the cohort was 33.48 (95% CI: 19 -
69 years).
We evaluated the antibody levels for S protein (IgM and IgG) and N
protein (IgG) before and after the booster shot (Figure 1, Appendix
table 1). The IgM and IgG anti-spike were stimulated mainly in 30D/3D
with a significant decline over time (p<0.0001). However, the
IgG anti-N was stimulated predominantly in 90/3D and 180/3D.
After the BNT162b2, 35 (54.69%) of the participants had a positive PCR
result, of which in 3/64 (4.69%) in 60d/3D, 13/64 (20.31%) in 90d/3D
and 19/62 (30.65%) in 180d/3D. The main symptoms reported were cough
(73.08%), runny nose (65.38%), sore throat (61.57%), headache
(61.54%), body pain (61.54%), low back pain (26.92%), diarrhea and
abdominal pain (19.23%). To evaluate the impact of a SARS-COV-2
infection post-booster dose on the antibody response, the volunteers
were divided into two groups, those who had positive PCR after the
booster shot and who did not have (Figure 2). There was no statistically
significant difference between the groups in terms of S IgM levels
(Figure 2, panel A, Appendix table 2). Otherwise, the N IgG levels were
50 and 35 times higher in the positive PCR group in 90/3D and 180/3D,
respectively (p<0.0001) (Figure 2, panel B, Appendix table 2).
The S IgG titers were 1.5 times elevated in the positive PCR group, in
180/3D (p= 0.0167) (Figure 2, panel C, Appendix table 2).
We found that the BNT162b2 booster shot elicits IgM and IgG antibodies
against Spike protein. BNT162b2 vaccine-elicited IgM antibodies also
were reported by Ruggiero and colleagues (Ruggiero et al., 2022). They
described that S-IgG and S-IgM positive sera were more efficient in
virus-neutralizing activity, compared to the S-IgM negative. This
finding highlights a possible crucial role of IgM in the development of
anti-SARS-CoV-2 humoral response, after vaccination.
The administration of CoronaVac in a two-dose schedule followed by a
booster dose of BNT162b2 could not contain a SARS-CoV-2 infection in the
current cohort, during the omicron wave in Brazil. The omicron peak
outbreak in Brazil was between November and March 2022 (Duong et al.,
2022; Ricardo Valverde, 2022). The cohort received the booster dose of
the BNT162b2 vaccine in October 2021, eight months after the second dose
of CoronaVac, and 1 month before the omicron had emerged in Brazil.
However, 55% of the participants were infected by SARS-CoV-2 following
the booster shot. The S protein is exposed on the surface of virions and
mediates the entry into host cells. It is the primary target of the most
approved vaccines for COVID-19 (Müller et al., 2022). Nonetheless,
Omicron has 50 mutations in its genetic code, of which 32 are in the
gene encoding the Spike protein, facilitating the Omicron transmission
(Saxena et al., 2022), immune evasion, and replication (Duong et al.,
2022) explaining the infection in vaccinated individuals.
The highest S IgM and S IgG levels were founded 30 days after the
booster dose. On the other hand, the higher N IgG levels were noted 90
and 180 days after the booster shot, when the most of participants were
infected. Since mRNA vaccines do not induce a response to the N protein
(Duong et al., 2022; Ricardo Valverde, 2022), the N IgG appears to be
induced mostly by the infection. Interestingly, the infection boosted
the N IgG antibodies by up to 50-fold. The viral N protein is a highly
conserved nucleoprotein, with functions associated with RNA
transcription and viral replication, and is abundantly expressed during
infection (Zeng et al., 2020). Thus, the high replication ability of
Omicron could have allowed a repeated exposure of the N protein to the
immune cells. The N protein is highly immunogenic, explaining the high
levels of N antibodies found (Galipeau et al., 2020).
The BNT162b2 boosted the S IgG levels, however, waned 60 days after the
booster dose. Since not all vaccine-stimulated-B lymphocytes are
maintained as memory cells (Palm e Henry, 2019), the decline of
antibodies following vaccination is expected. Success vaccination
depends on the induction of long-term immunological memory (Siegrist,
2003). The infected participants exhibited S IgG levels 1.5 times higher
than uninfected, 180 days after the booster shot. So, this finding
suggests an immune memory for at least 6 months after the booster dose
(Gray et al., 2021).
Limitations of this study are the absence of cellular immunity and
neutralizing antibodies assays and relatively small sample size.