Booster dose of BNT162b2 after two doses of CoronaVac improves neutralization of SARS-CoV-2 Omicron variant.

Background: The emergence of the new SARS-CoV-2 Omicron variant, which is known to have a large number of mutations when compared to other variants, brought to light the concern about vaccine escape, especially from the neutralization by antibodies induced by vaccination.
Methods: Based on viral microneutralization assays, we evaluated in 90 individuals the impact on antibody neutralization induction, against Omicron variant, by a booster dose of BNT162b2 mRNA vaccine after the CoronaVac primary vaccination scheme.
Results: Here we show that the percentage of seroconverted individuals 30 and 60 days after CoronaVac scheme was 16.6% and 10%, respectively. After booster dose administration, the seroconvertion rate increased to 76.6%. The neutralization mean titer against Omicron in the CoronaVac protocol decreased over time, but after the booster dose, the mean titer increased 43.1 times. Conclusions: These results indicate a positive impact of this vaccine combination in the serological immune response against SARS-CoV-2 Omicron variant.

Introduction

The vaccines against COVID-19 presented an important impact reducing the number of cases, hospitalization and deaths worldwide. However, in some countries the access to vaccination is far from the ideal, where the spread of SARS-CoV-2 continues at high rates favoring the emergence of variants. Indeed, since the beginning of the pandemics, the world has faced a continuous occurrence of variants, which is a natural process for a highly transmissible virus like SARS-CoV-2. Among these variants, the increase of transmission rate, augmented severity of cases and possibility to escape from immune response, stimulated by vaccination or previous infection, are the main concerns surrounding the called variants of concern (VOCs)[1,2]. The Omicron variant (B.1.1.529) has called attention due to its high number of mutations across the whole genome when compared to other VOCs. The Omicron variant accumulates at least 47 mutations in its whole genome (the highest number among variants), of which more than half are present in the Spike protein, the main target of the serological immune response induced by the majority of COVID-19 vaccines[3-5].

In a global panorama, Brazil stands out as one of the epicenters for viral dissemination, presenting high numbers of cases and deaths. Along with USA and India, Brazil ranks in the top three of the most impacted countries, with more than 25 million cases and 625,000 deaths until February 7, 2022[6]. On the other hand, historically, Brazil has a solid immunization program, which leaded to a huge SARS-CoV-2 vaccination adherence when compared to other countries, reaching more than 352 million doses administrated in the population, and almost 152 million people fully vaccinated. Among the distributed vaccines in Brazil, CoronaVac, the most used vaccine worldwide, was approved by our regulatory agency in January of 2021. This vaccine uses an inactivated virus technology and was the most administrated vaccine in Brazil until the middle of 2021, inoculated especially in elderly and healthcare workers individuals[7,8]. The scheme consisted in a two doses protocol, with 2–4 weeks between doses. It is estimated that, until January of 2022, around 85 million doses of CoronaVac were administrated in the Brazilian population. With the introduction of some VOCs in the country, especially Delta (B.1.617.2) and Omicron variants, the Brazilian government adopted the distribution of a booster dose of the mRNA BNT162b2 vaccine (BioNTech/Pfizer) to enhance immune protection against COVID-19[9].

In this context, our study reports the positive impact of the booster dose with BNT162b2 vaccine in a Brazilian cohort, under the CoronaVac two doses scheme, upon the stimulation of neutralizing antibodies against Omicron variant. Here we show that the combination of these vaccines not only improves the number of seroconverted individuals, but also increases the titer of neutralizing antibody in the vaccinated population.

Methods

Cohort definition and serum samples collection

To evaluate the presence of neutralizing antibodies induced by vaccination, the selected participants were submitted to a vaccination scheme consisting of the inoculation with two 0.5 ml shots of CoronaVac (600 SU per dose) in the original protocol, receiving a 0.3 ml booster of BNT162b2 (30 µg of spike mRNA per dose). This is a part of a major study evaluating the CoronaVac vaccination protocol that it is published as a preprint at SSRN[10]. A total of 90 individuals, randomly selected, were included in the study. All of them were health care workers from two hospitals in Belo Horizonte (Minas Gerais, Brazil) that received CoronaVac primary vaccination protocol in the beginning of 2021. The cohort general characteristics are summarized in Table 1, while the full detailed data, for each included individual, can be found in Supplementary Table 1. They were divided equally into three different groups (30 clinical samples per group): the D30 group, where serum samples were collected 30 days after CoronaVac second dose; the D60 group, composed by samples collected 60 days after second dose; and D270 group, a time point where samples were collected 270 days after CoronaVac second dose and also after BNT162b2 booster dose (30 days after the mRNA vaccine administration).

Table 1

General characteristics of the cohort included in the study.

Age
18–30	21
31–50	56
51–62	13
Gender
Male	22
Female	68
Comorbiditiesa
Present	24
Absent	66
Prior infection
Yes	11
No	79

aAsthma, chronic rhinitis, chronic sinusitis, diabetes, dyslipidemia, hyperthyroidism, hypothyroidism, obesity, systemic arterial hypertension, tobacco smoker.

Ethical approval was given by the Ethical Review Committee (CAAE 2898621.9.0000.5091). The included participants provided their written informed consent to participate in this study.

Cell culture

To perform the in vitro neutralization assays, Vero (ATCC CCL-81) cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin in a water-jacked incubator, at 37 °C and 5% CO2. According to ATCC control quality, mycoplasma contamination was not detected.

Viral microneutralization assay

In order to evaluate the antibody neutralization levels against SARS-CoV-2 Omicron variant, in samples collected from a cohort vaccinated with the CoronaVac two doses protocol, followed by the BNT162b2 mRNA vaccine booster dose, a viral microneutralization (VNT) was performed. In a day before, 104 Vero cells were distributed, per well, in clear flat bottom 96-well plates and incubated for 24 h at 37 °C and 5% CO2, reaching 90–95% of confluence after incubation. Serum samples, collected from the cohort participants, were heat-inactivated for 30 min, at 56 °C. To analyze the neutralization capacity of antibodies induced by vaccination, using microdilution 96-well plates, the inactivated samples were serially diluted in fresh DMEM media supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin and 2% FBS. For each serum sample, eight dilutions were tested (1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280 e 1:2480). The fresh diluted samples were incubated with the live SARS-CoV-2 Omicron variant (HIAE—W.A) in a constant concentration of 50 TCID50/ml, at 37 °C, for 1 h. After this period, the diluted samples, mixed with virus, were distributed in the Vero CCL-81 cell plates and maintained in the humidified incubator at 37 °C and 5% CO2 for 72 h. Thereafter, media containing serum and viruses was discarded, cells were fixed with 10% formaldehyde, for 20 min, and stained with crystal violet. The neutralization capacity was determined by the presence or absence of cytopathic effect across the dilutions, and for each sample, it was obtained the reciprocal dilution value where 50% of cytopathic effect was avoided (VNT50). All samples, at all dilutions, were tested in triplicate and VNT50 was calculated by the use of the Spearman-Karber algorithm[10,11]. To validate the assay, a serum sample already known to neutralize Omicron and a sample collected before the pandemics were used as positive and negative control, respectively.

Statistics and reproducibility

For each time point evaluated during the viral microneutralization assay, 30 biologically independent serum samples were analyzed. The statistical analysis was obtained by one-way ANOVA and Tukey’s multiple comparisons test, considering statistically significant when p value < 0.05.