key: cord-0690519-i3br75du
authors: Schultz, B. M.; Melo-Gonzalez, F.; Duarte, L. F.; Galvez, N. M.; Pacheco, G. A.; Soto, J. A.; Berrios, R. V.; Gonzalez, L. A.; Moreno-Tapia, D.; Rivera-Perez, D.; Hoppe-Elsholz, G.; Iturriaga, C.; Vallejos, O. P.; Urzua, M.; Vazquez, Y.; Navarrete, M. S.; Rojas, A.; Grifoni, A.; Sette, A.; Weiskopf, D.; Zeng, G.; Meng, W.; CoronaVac03CL Study Group,; Gonzalez-Aramundiz, J. V.; Gonzalez, P. A.; Abarca, K.; Kalergis, A. M.; Bueno, S. M.
title: A booster dose of an inactivated vaccine increases neutralizing antibodies and T cell responses against SARS-CoV-2
date: 2021-11-17
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.11.16.21266350
sha: 3385f06fff952ff6d17baf0c22a81dd00a6d4620
doc_id: 690519
cord_uid: i3br75du

Numerous vaccines have been generated to decrease the morbidity and mortality of COVID-19. CoronaVac(R) is an inactivated SARS-CoV-2 vaccine approved by the World Health Organization (WHO) to prevent COVID-19 that has safety and immunogenicity profiles described in different clinical trials. We previously reported an increase in levels of neutralizing antibodies two- and four-weeks after administering two doses of CoronaVac(R) in a two-week interval (0-14 day) vaccination schedule, as compared to pre-immune sera in adults in the Chilean population that are participating in phase 3 clinical trial. Here we report the levels of antibodies directed against the Receptor Binding Domain of the SARS-CoV-2 spike protein comparing their neutralizing capacities and the cellular response at five months after the second dose and four weeks after a booster (third) dose in volunteers immunized with two doses of CoronaVac(R)in a four-week interval (0-28 day) vaccination schedule. We observed a decrease in the levels of anti-SARS-CoV-2 antibodies with neutralizing capacities five months after the second dose (GMU 39.0 95% confidence interval (CI)(32.4-47.0), which increased up to 12 times at four weeks after the booster dose (GMU 499.4, 95% CI=370.6-673.0). Equivalent results were observed in adults aged 18-59 years old and individuals [≥]60 years old. In the case of cellular response, we observed that activation of specific CD4+ T cells increases in time and reaches its maximum at four weeks after the booster dose in both groups. Our results support the notion that a booster dose of the SARS-CoV-2 inactivated vaccine increases the levels of neutralizing antibodies and the specific cellular response in adults of both groups, which is likely to boost the protective capacity of these vaccines against COVID-19.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has promoted the rapid development of safe, immunogenic, and effective vaccines against SARS-CoV-2 to be used by the general population, which have successfully reduced the transmission of the disease burden. CoronaVac® is an inactivated SARS-CoV-2 vaccine developed by Sinovac Life Sciences Co., Ltd. (Beijing, China) and is among the current vaccines approved by the WHO to combat COVID-19 [1, 2] . Phase 1 and 2 clinical trials in China demonstrated that this vaccine induces cellular and humoral response upon immunization [3] [4] [5] . Furthermore, an ongoing phase 3 clinical trial in Chile has described that two-and four-weeks after the second dose of CoronaVac® there is an increase in the levels of IgG and neutralizing antibodies in adults aged years old and ≥ 60 years old [5] [6] . In addition, the vaccination promotes the activation of the cellular immune response against SARS-CoV-2 antigens in a 0-14 immunization schedule [5] , being an effective vaccine to prevent COVID-19 [7, 8] .

In Chile, 91.5% of the target population has received the first vaccine dose, and 88.7% were fully vaccinated in October 2021 in a 0-28 vaccination schedule [9] .

Although neutralizing antibody titers present in the serum of vaccinated people are thought to be highly predictive of immune protection [10] , these titers decrease in time [6, 11, 12] . Besides this, vaccine-induce antibodies have lower levels of neutralization against highly transmissible variants of the virus as compared to the original vaccine strain, potentially decreasing the effectiveness of these vaccines as new variants emerge [13, 14] . For these reasons, the use of booster doses was All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted  https://doi.org /10.1101/2021.11.16.21266350 doi: medRxiv preprint approved in adults in August 2021 in Chile, in high-risk populations and subjects with more than five months after the second dose applied in a 0-28-day vaccination schedule [15] . Notably, a previous study performed in adults between 18-59 years old demonstrates that a booster dose of CoronaVac®, applied after six months to individuals previously receiving two doses of this vaccine, increases the levels of antibodies 3-5-fold as compared to those levels observed four weeks after the second dose [12] . Here, we further extend these results by reporting the levels of neutralizing antibodies and specific T cells against SARS-CoV-2 in adults ≥ 18 years old who participated in phase 3 clinical trial carried out in Chile, who were vaccinated in a 0-28-day vaccination schedule with a booster (third) dose five months after the second dose. [16] , and local regulations. Informed consent was obtained from all volunteers upon enrollment.

Volunteers receive two doses of CoronaVac® (3 µg or 600SU of inactivated SARS-All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CoV-2 inactivated along with alum adjuvant) in a four-week interval (0-28-day immunization schedule) and then a booster dose five months after the second dose. A complete inclusion and exclusion criteria list has been reported. On November 11 st 2021, one hundred and eighty-six volunteers in the immunogenicity branch received the booster dose, and the antibodies against RBD with neutralizing capacities were quantified in 77 volunteers who had completed all their previous visits in one of the centers of the study (Figure 1A ). Blood samples were obtained from all the volunteers before administration of the first dose (preimmune), two weeks after the second dose, four weeks after the second dose, twenty weeks (or five months) after the 2 nd dose, and four weeks after the booster (third) dose ( Figure 1B) .

To assess the presence of antibodies against RBD with neutralizing capacities, blood samples from 77 volunteers that had completed all their study visits, including one month after the booster dose of CoronaVac®, were measured.

The neutralizing capacities of circulating antibodies were evaluated by a surrogate virus neutralization test (sVNT) (Genscript Cat#L00847-A). Samples were serially two-fold diluted starting at a 4-fold until reaching a 512-fold dilution. Assays were performed according to the instructions of the manufacturer and as reported previously [5] . Neutralizing antibody titers were determined as the last fold dilution with a cut-off over 30% of inhibition. Samples with a percentage of inhibition ≤ 30 at lowest dilution (1:4) were assigned as seronegative with a titer of 2. A sample was considered seropositive when its titer is higher than the pre-immune titer. The All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. [17] . Data were analyzed using a sigmoidal curve model with log concentration transformed, and the final concentration for each sample was the average of the product of the interpolated IU from the standard curve and the sample dilution factor required to achieve the OD450 value that falls within the linear range. Samples with undetermined concentration at the lowest dilution tested by T cells was evaluated by flow cytometry. Assays were performed according to the instructions of the manufacturer and as reported previously [5] . Further details All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

Statistical differences for the immunogenicity results considered one-way ANOVAs mixed-effects analysis for comparisons between the booster dose and the other visits performed on the logarithms of the data. The significance level was set at 0.05 for all the analyses. All data were analyzed with GraphPad Prism 9.0.1.

One hundred and twenty-nine volunteers from the immunogenicity branch, who received the booster dose of the CoronaVac®, were included in this study.

The first dose of the vaccine was inoculated from January -March of 2021, and the second dose was inoculated 28 days after the first one. Of them, we evaluated the neutralization capacity of circulating antibodies in 77 volunteers at five different time points indicated previously by sVNT and 33 of the same volunteers by ELISPOT and flow cytometry ( Figure 1B) .

In a normal 0-28-day schedule, the peak in the neutralizing capacity of the antibodies is reached at two weeks after the second dose (GMT 25.8, 95%

CI=19.5-34.2) (Supp. Figure 1) , decreasing at four weeks after the second dose (GMT 16.6, 95% CI=13.1-21.0). However, this neutralizing capacity present an important decreased five months after the second dose (GMT 3.5, 95% CI=3.0-4.1), which is in line with previous reports where the immunity against SARS-CoV-2 wanes six months after infection or vaccination [19, 20] . As expected, after the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint booster dose, the neutralizing capacity of the antibodies increased even more than the one reported two weeks after the second dose. When we expressed the neutralizing capacity in arbitrary units of WHO (Figure 2 ) we observed that four weeks after the booster dose the neutralizing capacity increased more than 12-fold (GMU 499.4, 95% CI=370.6-673.0), as compared to the response at five months after the second dose (GMU 39.0 ± 32.4-47.0) and more than 2-fold as compared to the two weeks after the second dose (GMU 168.0 ± 126.8-222.5) (Figure 2A) .

In adults between 18-59 years old, the neutralizing capacity of circulating antibodies reach its maximum four weeks after the booster dose (GMU 918.8 ± 623.4-1354) increasing more than 18-fold as compared to five months after the second dose (48.9 ± 37.6-63.5) and more than 4-fold as compared with two weeks after the second dose (GMU 220.2 ± 150.7-321.7) (Figure 2B ). Seropositivity in this group reach 100% four weeks after the second dose ( Table 1) . 53.2% of the total volunteer analyzed here were adults ≥ 60 years. As seen in Figure 2C , the neutralizing capacity of circulating antibodies in this population also reached its peak at two weeks after the second dose (GMU 134.1 ± 89.2-201.6), decreasing at four weeks after the second dose (GMU 104.1 ± 71.8-151.0), and reaching its minimum at five months after the second dose (GMU 32.4 ± 25.1-41.8). In this group, we also observed an increase of more than 9-fold (GMU 300.5 ± 203.5-443.6) in the neutralizing capacity as compared to the response observed five months after the second dose (GMU 32.4). The seropositivity rate reached 49.4% in the total vaccine group and 35.7% in adults ≥ 60 years at five months after the second dose, which increased to 97.4% and 95.2%, respectively, four weeks after All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint the booster dose ( Table 1) . The seropositivity rate achieved at four weeks after the booster dose was the highest when compared with the other visits in the study in the total vaccinated group and in both groups analyzed.

Here we also report cellular responses following the booster dose of CoronaVac®, which is the first report of T cell responses in subjects vaccinated with a third dose of CoronaVac® to our knowledge. We did observe a significantly further increase in CD4 + T cell activation in both age groups following the third booster dose by flow cytometry (Figure 3 ) but we did not see a further increase in IFN-γ production upon stimulation with S and R MPs by ELISPOT at that time point (Supp. Figure 2 ). In addition, CD4 + T cell activation was still significantly increased 5 months after the 2 nd dose in both age groups, suggesting that the 0-28 schedule can stimulate CD4 + cell responses over time. Moreover, we observed a significant increase in CD8 + AIM + T cells following the third dose as compared to the time point 2 weeks following the second booster but not as compared to the preimmune, whereas we did not observe a significant increase in IFN-γ upon stimulation with CD8 MPs at any time point, suggesting that CoronaVac promotes a reduced CD8 + T cell responses, even after a third dose. Thus, although humoral responses decrease over time following vaccination with CoronaVac®, CD4 + T cell responses stay significantly increased as compared to the pre-immune and the booster dose increases at least their activation.

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Although there was an adequate neutralization titer of anti-SARS-CoV-2 antibodies after two doses of CoronaVac® in the 0-28 schedule, with a 65.9% of effectiveness of preventing COVID-19 [8] , the GMT waned in time, which was observed five months after the second dose. Due to this decrease in neutralizing capacity, a booster dose of CoronaVac® was evaluated in a clinical study in China, showing promising results in humoral immune responses [12] . The evaluation of the neutralization capacities reported here shows that after the booster dose, the neutralizing titers and seroconversion rates increase in the whole group even higher than two weeks after the second dose where was observed the peak in neutralization. As the neutralizing antibody titers correlate with protection against SARS-CoV-2 infection [10] , these results likely imply a better outcome and protection against illness, as reported in previous studies performed in Israel that showed a decrease in the transmission and the severe disease by COVID-19 twelve or more days after booster inoculation [21] . Another study, performed with a booster dose of CoronaVac®, showed that an additional dose induced a good neutralization against SARS-CoV-2 WT strain and against variants four weeks after the booster dose, generating a long-lasting humoral response that was due to an enhancement of the memory immune response generated by B cells [22] . Adults ≥ 60 years old produced lower levels of antibodies with neutralizing capacities than the whole group during this study, which was also described in Bueno et al. [5] . This result is in line with previous data reported for a population vaccinated in Chile [6] , a study among hospital workers who received two doses of CoronaVac® [23] , and with the mRNA-1273 vaccine [24] . In this sense, our results are equivalent to those described in a phase 1/2 of the clinical trial with All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org /10.1101 /10. /2021 CoronaVac®, showing that the neutralizing antibody titers in this group decrease at five months after the second dose and that a booster dose is required 6-8 months after the first vaccination to rapidly increased and steadily the neutralizing antibody titers [25] .

In the case of cellular response, other studies have shown that Pfizer BNT162b2 and mRNA-1273 induce durable CD4 + T cell activation and cytokine production up to six months following vaccination but it remains to be elucidated whether CD4 + AIM + T cells and cytokine production further increase following a booster dose of these vaccines [26, 27] . In contrast to these vaccines, CoronaVac® delivers not only the Spike protein but other viral antigens, which may explain why vaccinated individuals still display CD4 + AIM + T cells five months after the second dose, without even a third dose.

Our report shows that the booster dose with CoronaVac® in a 0-28 schedule induces a higher production of antibodies with neutralizing capacities, which are higher than the levels observed with 2-and 4-weeks after the first doses, generating an increased humoral response even in adults ≥ 60 years old. Besides this, our results suggest that a third dose of CoronaVac® supports CD4 + T cell activation, which may confer either protection or enhanced immune responses against the virus and prevent severe disease following SARS-CoV-2 exposure.

This study presents some limitations, such as the reduced sample size for the assays. The assessment of total antibody response against Spike proteins and other SARS-CoV-2 proteins would also add additional information about the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint humoral immune response against SARS-CoV-2 after the booster dose. Due to the limit of quantification of the technique, samples with undetermined concentration at the lowest dilution tested (1:4) were assigned the lower limit of quantification (16.4 IU) and other neutralization assays, such as conventional neutralization test, would confirm our results with the surrogate neutralization test used in this study. 

ZG and MW are SINOVAC employees and contributed to the conceptualization of the study (clinical protocol and eCRF design) and did not participate in the analysis or interpretation of the data presented in the manuscript. All other authors declare no conflict of interest. A.S. is a consultant for Gritstone, Flow Pharma, Arcturus, All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint Immunoscape, CellCarta, OxfordImmunotech and Avalia. Jolla Institute for Immunology (LJI) has filed for patent protection for various aspects of T cell epitope and vaccine design work. All other authors declare no conflict of interest.

The authors declare this study received the investigational product (placebo and vaccines) from the company SINOVAC Biotech. SINOVAC employees contributed to the conceptualization of the study (clinical protocol and eCRF design) but did not participate in either the analysis or interpretation of the data shown in this manuscript.

We Group are listed in the Supplementary Appendix (SA). We would also like to thank the members of the independent data safety monitoring committee (members in the SA) for their oversight, and the subjects enrolled in the study for their All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. differences to compare all times against 3 rd dose + four weeks. ****p<0.0001. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. and CD8 + (AIM + [CD69 + , CD137 + ]) T cells was determined by flow cytometry, upon All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint stimulation for 24h with MP-S+R (A-C) and MP-CD8A+B (D-E) in samples obtained at pre-immune, two weeks after the 2 nd dose, four weeks after the 2 nd dose, twenty weeks the 2 nd dose, and four weeks after the 3 rd dose. Results were obtained from a total of 33 volunteers (A-D), 14 were of them were adults between 18-59 years old (B-E), and 19 of them were ≥ 60 years old (C-F). Data shown represent means + 95%CI. Data from flow cytometry was normalized against DMSO and analyzed separately by One-way ANOVA with mixed effect analysis. *P<0.05; **p<0.005; ****p<0.0001. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint Table   Table 1 GMT: Geometric mean titer; GMU: Geometric mean units.

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The copyright holder for this this version posted November 17, 2021. ; https://doi.org/10.1101/2021.11.16.21266350 doi: medRxiv preprint

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