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Neutralizing antibodies induced by homologous and heterologous boosters in CoronaVac vaccines in Chile

Objectives To determine the impact of a booster dose on the humoral response in individuals inoculated with a complete schedule of any SARS-CoV-2 vaccine, we evaluated the neutralizing antibody (NAb) titres of homologous or heterologous booster doses over a 90-days period in CoronaVac vaccinees from 3 centres in Santiago, Chile. Methods Individuals previously inoculated with 2 doses of CoronaVac (N = 523) were recruited in the context of the REFUERZO clinical trial (NCT04992182) and received either placebo (N = 129), or a booster dose of CoronaVac (N = 134), BNT162b2 (N = 133), or ChAdOx1 (N = 127). Pseudovirus neutralizing antibody titres (pVNT) were determined at baseline (day 0) as well as at days 14, 30, 60, and 90 after booster dose administration. Results Inoculating a booster dose increases the pVNTs titres at days 14 and 30 in all groups, (13.5- and 12.0-fold increase for the CoronaVac group; 247.0- and 212.3-fold increase for the BTN162b2 group; and 89.1- and 128.1-fold increase for ChAdOx1 at each time point, respectively) with a decline observed at days 60 and 90. However, although pVNTs remained significantly higher for the BTN162b2 and ChAdOx1 groups at days 60 and 90, NAb titres reached baseline levels in the CoronaVac group at 90 days after inoculation. Discussion A single heterologous booster (BTN162b2 or ChAdOx1) in individuals who completed the CoronaVac primary series resulted in an important increase in NAb titres remaining significantly higher at least for 90 days. These data may directly impact middle- and low-income countries currently using CoronaVac as the main vaccination strategy.


Introduction
Waning humoral response, determined as a decline in circulating neutralizing antibodies (NAbs) titres after a period, has been widely reported for individuals receiving any of the SARS-CoV-2 vaccines currently used to face the COVID-19 pandemic [1]. Some countries, including Chile, have implemented a booster dose first focusing on the risk groups that include older adults and immunosuppressed patients, and then to the general population. This booster dose complemented the strong vaccination programme implemented in Chile, which was mainly performed with CoronaVac, an inactivated virus-based vaccine representing one of the most widely inoculated vaccines worldwide [2]. Several studies have shown that CoronaVac elicits lower levels of NAbs than other vaccine platforms with NAbs titres decreasing 3 to 6 months after inoculation [3]. However, studies in China and Chile have reported that inoculation with the third dose of CoronaVac is immunogenic indicating that a homologous booster is sufficient to recall a waned humoral response against SARS-CoV-2 [4,5].
Because boosters with heterologous platforms might provide improved immune responses [6e8], we conducted the clinical trial REFUERZO (NCT04992182), which was intended to compare the reactogenicity, safety, and immunogenicity of homologous versus heterologous (mRNA-based or viral vector-based) booster dose in individuals with a previous two-dose CoronaVac schedule in Chile.
Here, we report results of the immunogenicity arm, which determined NAbs titres in plasma, measured at baseline as well as 14-, 30-, 60-and 90-days after booster inoculation.

Study design and participants
We conducted a phase III, randomized, double-blind, placebocontrolled clinical trial for the inoculation of a booster dose with either the mRNA vaccine BNT162b2 or the recombinant adenoviral vectored vaccine ChAdOx1, compared with a homologous boost with the inactivated virus vaccine CoronaVac, in accordance with the booster vaccine policy implemented by the Chilean National Immunization Program (PNI).
This trial had 4 intervention arms, according to the planning of possible reinforcement schemes of the Chilean Ministry of Health (MINSAL). The intervention assignment was performed through a simple randomization strategy, 1:1, double-blind. Randomization was 4 interventions stratified into 2 groups according to the age criteria of the PNI booster dose policy (55 years or older and younger than 55 years). Intervention 1: participants over 55 years received a booster dose of ChAdOx1 (AstraZeneca). Participants were eligible if they were 18 years or older, had received their second dose of CoronaVac before 15 April 2021, and all participants had given written informed consent. Exclusion criteria were history of laboratory-confirmed COVID-19. The three study sites were in Santiago, Chile (older residents and healthcare workers (HCWs)) from long-stay establishments for the elderly (ELEAMs); HCWs from both Public Emergency Hospital (HUAP) and the MINSAL. Regarding the sampling framework, we defined ELEAMs with a cumulative infection attack rate of COVID-19 lower than 30% between residents and HCWs. Finally, there were instances of dissemination of information and awareness in each ELEAM to publicize the study and motivate participation. In the case of older residents, the information was also extended to their families.

Fieldwork
HCWs from the PNI conducted the fieldwork on 14 ELEAMs and MINSAL. HCWs from the quality and patient safety unit of the HUAP conducted their fieldwork. Both fieldwork teams had overseen the immunization campaign against SARS-CoV-2 of their respective study sites.
At the baseline visit (day 0), volunteers participated in the written informed consent process and answered a health survey on personal history of sociodemographic characteristics and comorbidities. The history of previous infection and immunization were obtained from the databases of MINSAL. In this visit, a 4-mL blood sample was collected with the Venojet system, and the blind vaccine was applied to the participants.
During follow-up visits (days 14, 30, 60, and 90), the participants answered a brief survey about exposures to COVID-19, and then a blood sample of 4 ml was collected as indicated above. At least 2 follow-up visits were performed for each centre to increase the possibility that participants would be available at the time of the follow-up visit because of the shift system of HCWs.
Finally, a follow-up closure visit was made after all determinations of neutralizing antibody titres were obtained. The fieldwork teams visited the participants and confidentially delivered the results.

Randomization and blinding
We made 2 randomization lists for each age group in a 1:1 ratio of the 4 interventions. An electronic randomization system in software R Study was used to define the intervention, kept in envelopes sealed in the study sites. The design was double blinded for the HCWs who administered the vaccine, the participants, and the laboratory staff. Only the team in charge of preparing the vaccine had access to the information on the type of vaccine at the time of preparation.
Intervention 3: Each 0.3 mL dose of vaccine contains 30 mg of SARS-CoV-2 spike protein nucleoside-modified mRNA encapsulated in lipid nanoparticles. Intervention 4 (comparison group): Each dose of 0.5 ml of 0.9% saline. All vaccines were administrated intramuscularly.
All blood samples were collected by qualified nursing staff, through the Venojet system, in a tube without anticoagulant, transported to the Laboratory of Molecular and Cellular Virology at Universidad de Chile and stored at 4 C. NAbs titres were measured with an HIV-1-based SARS-CoV-2 pseudotype expressing the spike protein of the Wuhan reference strain (Wuhan-Hu-1) [9]. Results are presented as the value of 50% pseudovirus neutralization titres (pVNT 50 ).

Primary outcome and sample size
The primary outcome was the humoral immune response towards the spike protein of SARS-CoV-2 Wuhan-Hu-1, measured as the change in the half-maximal inhibitory dilution (ID50) generated by the intramuscular administration of vaccines. The sample size was determined to detect differences between the mean ID50 at baseline and 30 days after the inoculation, with a statistical power of 80%, based on a t-test for paired samples.

Statistical analysis
We considered a random intercept model [10] to account for the correlation in the ID50 measurements across time within the same subject. Specifically, we assumed a linear mixed model with Gaussian error terms and Gaussian random intercept terms for the logarithm of the ID50. We did not assume a parametric form for the evolution of the ID50 measurements across time for each treatment group by considering treatment-specific intercept coefficients and the visit-treatment-specific slope coefficients. A Bayesian version of the model was considered [11]. Independent Gaussian prior distributions were considered for the treatment-specific intercept coefficients and for the visit-treatment-specific slope coefficients. Following Gelman [12], independent uniform prior distributions were considered for the standard deviation of the random intercepts and errors. Markov chain Monte Carlo techniques were employed for exploring the posterior distributions of interest.
For the model 120,000 scans of a single Markov chain cycle were completed. The full chain was subsampled every 10 steps after a burn-in period of 20,000 samples, to give a reduced chain of length 10,000. We used the rjags package [13] for R version 4.1.1 [14]. Standard tests, as implemented in the coda R package [15], suggested convergence of the chains. Observations outside the measurement range were treated as censored observations. Both missing and censored observations were treated as random variables. We reported the posterior inferences for the marginal means of the ID50 for each treatment group and visit. The random intercept model is employed as a tool for taking into account the correlation of repeated measurements for the same subjects across time, which is necessary to properly estimate the means of ID50 for each time point and for the correct estimation of the associated uncertainty measures. A random intercept model for log-normal responses generates regression coefficients with subject-specific interpretations. However, their results can be employed for estimating the population-average (not subject-specific) means of ID50 for each time point, which is done in the present article.
The statistical significance was based on a Bayesian counterpart to a frequentist p value, referred to as posterior contour probability [16]. In particular, we computed simultaneous credible regions for the differences between the means of the different time points, based on the order statistics approach described by Besag et al. [17], to obtain contour posterior probabilities for the null hypothesis of no differences.

Role of the funding source
The funders of this study had no role in the study design, in the collection, analysis, and interpretation of data, in the writing of this manuscript, or in the decision to submit the paper for publication.

Results
The clinical trial REFUERZO (NCT04992182) included 534 participants with a prior two-dose schedule of CoronaVac (90e120 days after the second dose of CoronaVac) recruited in July 2021 from 3 centres in Santiago, Chile. From these, 523 volunteers were included in the study (Fig. 1). Missing observations were treated as missing at random and imputed as part of the Bayesian analysis, implemented via Markov chain Monte Carlo simulation. Details regarding cohort demographics are shown in Table 1.
Participants were randomly assigned to the following groups: ( Fig. 2). On the day 90 follow-up, 135 participants failed to respond. Among them, we found 4 deaths not associated with vaccine administration, 15 ELEAM residents who left the residence, 18 HCWs who changed jobs, and 98 participants who were not available at the time of the follow-up visits (off duty or outside of the institution). Of note, we made at least 2 visits to each place.
The posterior mean (95% CI) for the intraclass correlation for the natural logarithm of the ID50 was 0.4986 (0.4943; 0.5004), suggesting a significant positive correlation among the measurements of the same subjects and justifying the use of a random intercept model. For all vaccines, the contour posterior probabilities for the null hypothesis of no differences among the means across time were <0.001, indicating significant differences among them. Analyses of NAbs titres using the pseudo typed virus (pVTN) showed that the placebo group only presented an increase in NAbs geometric means titres at days 60 and 90, which is consistent with the BTN162b2 booster received at day 30 (Fig. 3a). A homologous booster dose induced 13.6-fold increase in the pVNT from 243.7 (95% CI, 189.3e308.7) to 3298.5 (95% CI, 2559.9e4192.2) at day 14 after inoculation (Table 2 and Table 3). NAbs titres in the CoronaVac group presented a slight decrease in the pVNT to 2935.7 (95% CI, 2284.5e3712.5) at day 30 but strongly declined to 725.2 (95% CI, 560.2e923.4) by day 60. These NAbs titres further decreased to 492.8 (95% CI, 371.2e639.0) by day 90 reaching only 2.03-fold higher than the baseline (Fig. 3b). For the BTN162b2 group, we observed a 249.1-fold increase in pVNT from 207.4 (95% CI, 161.1e264.6) to 51,306.6 (95% CI, 38,818.8e66 109.9) at 14 days after inoculation of the booster dose (Fig. 3c). NAbs titres were  Fig. 3d). Despite this waning, NAbs titres in the ChAdOx1 group remained 30.9-fold higher than that at baseline (Fig. 3d).

Discussion
CoronaVac was one of the most widely inoculated COVID-19 vaccine as an initial protocol but also as a booster in some countries. However, previous studies have raised concerns about the ability of this inactivated virus vaccine to trigger a potent humoral response as it elicits lower NAbs titres than mRNA vaccines [18]. Moreover, these low NAbs titres were shown to decrease 3 to 6 months after completion of the initial two-dose schedule raising the necessity of booster doses, particularly for high-risk groups. Despite these apparent low levels of NAbs elicited by CoronaVac, a nation-wide study conducted in Chile for the two-dose schedule showed an effectiveness of 65.9% (IC 95%, 65.2e66.6) in preventing infections; 87.5% (IC 95%, 86.7e88.2) in preventing hospitalizations and 86.3% (IC 95%, 84.5e87.9) in preventing COVID-19-related deaths [19]. This effectiveness was achieved under a predominant circulation of the variant of concern Gamma and the variant of interest Lambda, which were characterized by the presence of mutations allowing escape to NAbs [19,20]. A more recent nationwide study from Chile evaluating the effectiveness of the Corona-Vac booster dose revealed a 78.8% (IC 95%, 76.8e80.6) in preventing symptomatic infections, whereas it was 96.5% (IC 95%, 96.2e96.7) for the BNT162b2 booster and 93.2% (IC 95%, 92.9e93.6) for ChA-dOx1 [21]. The vaccine effectiveness against hospitalization, intensive care unit admission, and COVID-19 related deaths was 86.3%, 92.2%, and 86.7%, respectively, for a three-dose schedule with CoronaVac; 96.1%, 96.2%, and 96.8% for BNT162b2 booster; and 97.7%, 98.9%, and 98.1% for ChAdOx1 booster [21]. Because the booster dose in Chile has been mostly administered with BTN162b2 (<55 years old) and to a lesser extent with ChAdOx1 (>55 years old), our data of NAbs titres may provide an explanation to the success of the booster dose programme, which occurred when the Delta variant was predominant. The differences in the effectiveness of the booster schemes followed in Chile combined with the analysis of NAbs titres described in this work provide further evidence for the use of NAb titres as a correlate of protection against the development of symptomatic infection, hospitalization, and death.
One of the limitations of this study is that we have not evaluated the impact of booster doses on cellular immune responses, which has been recently shown in recipients of 2 or 3 doses of CoronaVac  Tables 2 and 3 for summary statistics and comparisons. [3,22]. Moreover, we have not investigated the impact of any of the subvariants of Omicron on the neutralizing capacity of antibodies elicited by the homologous and heterologous booster doses analysed in our cohort. In this sense, 2 recent reports suggested that antibodies elicited by the two-dose schedule of CoronaVac did not provide neutralizing ability against the Omicron BA.1 subvariant and that a heterologous booster with mRNA vaccines should be preferred over an homologous booster [23e25]. Our data are in line with these observations as we showed that a heterologous booster in CoronaVac vaccines provided a stronger and lasting humoral response. Our data may have direct implications for those middleand low-income countries currently using CoronaVac as the main vaccination strategy.

Transparency declaration
The authors declare that they have no conflicts of interest.