Immune responses during COVID-19 breakthrough cases in vaccinated children and adolescents

Abstract

Background: Vaccine effectiveness against SARS-CoV-2 infection has been somewhat limited due to the widespread dissemination of the Omicron variant, its subvariants, and the immune response dynamics of the naturally infected with the virus.

Methods: Twelve subjects between 3-17 years old (yo), vaccinated with two doses of CoronaVac^®, were followed and diagnosed as breakthrough cases starting 14 days after receiving the second dose. Total IgGs against different SARS-CoV-2 proteins and the neutralizing capacity of these antibodies after infection were measured in plasma. The activation of CD4⁺ and CD8⁺ T cells was evaluated in peripheral blood mononuclear cells stimulated with peptides derived from the proteins from the wild-type (WT) virus and Omicron subvariants by flow cytometry, as well as different cytokines secretion by a Multiplex assay.

Results: 2 to 8 weeks post-infection, compared to 4 weeks after 2^nd dose of vaccine, there was a 146.5-fold increase in neutralizing antibody titers against Omicron and a 38.7-fold increase against WT SARS-CoV-2. Subjects showed an increase in total IgG levels against the S1, N, M, and NSP8 proteins of the WT virus. Activated CD4⁺ T cells showed a significant increase in response to the BA.2 subvariant (p<0.001). Finally, the secretion of IL-2 and IFN-γ cytokines showed a discreet decrease trend after infection in some subjects.

Conclusion: SARS-CoV-2 infection in the pediatric population vaccinated with an inactivated SARS-CoV-2 vaccine produced an increase in neutralizing antibodies against Omicron and increased specific IgG antibodies for different SARS-CoV-2 proteins. CD4⁺ T cell activation was also increased, suggesting a conserved cellular response against the Omicron subvariants, whereas Th1-type cytokine secretion tended to decrease.

Clinical trial registration: clinicaltrials.gov #NCT04992260.

Keywords: CoronaVac®; SARS-CoV-2; breakthrough cases; inactivated SARS-CoV-2 vaccine; omicron variant; pediatric; phase 3 clinical trial.

Figure 1

Immunogenicity branch cohort of study volunteers and their breakthrough cases of children and adolescents vaccinated with CoronaVac^®. (A) A cohort of 188 volunteers receiving the first dose of CoronaVac^® was enrolled. Of the 188 volunteers, 137 received the second dose in a 0-28 vaccination schedule. 12 volunteers diagnosed with COVID-19 after the second dose + 4 weeks were analyzed as the study progress. (B) Blood samples were obtained at the time of the first vaccine dose on the pre-immune or day 0, at the time of the 2^nd dose + 4 weeks and 2 to 8 weeks after obtaining the positive diagnosis by qPCR for COVID-19.

Figure 2

Titers of neutralizing antibodies against SARS-CoV-2 in plasma of breakthrough cases of children and adolescents. Titers of neutralizing antibodies against SARS-CoV-2 in twelve breakthrough cases. (A, B) Neutralizing experiment titers evaluated by a surrogate viral neutralization test (sVNT) expressed as geometric mean titer (GMTs) for WT virus and Omicron variant, respectively. (C, D) Neutralizing experiment titers evaluated by pseudotyped based viral neutralization assay (pVNT) with ID80 expressed as GMTs for WT virus and Omicron variant, respectively. Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2^nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively. The values on each column indicate the geometric mean. The values below the significance line indicate the fold-change between the corresponding geometric means titers (Red: decrease, Blue: increase). Transformed data, represented as reciprocal dilution in logarithm base 2 on linear scale. Data was analyzed using a mixed effects model, ***p<0.001; *p<0.05, not significant (ns).

Figure 3

Specific IgG levels against different SARS-CoV-2 proteins in plasma of breakthrough cases of children and adolescents. Titers of specific IgG levels against different SARS-CoV-2 in twelve breakthrough cases. (A) Antibody titer against S1 protein. (B) Antibody titer against N protein. (C) Antibody titer against M protein. (D) Antibody titer against E protein. (E) Antibody titer against NSP8 protein. An in-house indirect ELISA assay was used to assess plasma IgG specific antibody titers against these proteins. Data were transformed, plotted as reciprocal dilution in log base 2 on a linear scale. Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2^nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively, by before-after graph. Black values below the significance line indicate geometric mean titers (GMT), blue values above the significance line indicate an increase in GMT of the two time points compared, and red values above the significance line indicate a decrease in GMT of the two time points compared. The data was analyzed using a mixed effects model, ***p<0.001; **p<0.01; *p<0.05, not significant (ns).

Figure 4

Immune response through activation of T cell populations using MPs of SARS-CoV-2 of breakthrough cases of children and adolescents. Cellular response in breakthrough pediatric cases was assessed in PBMCs. (A–D) Percentage (%) AIM⁺ CD4⁺ T for MPs R, S of WT SARS-CoV-2 and BA.1-MUT and BA.2-MUT subvariant Omicron MPs, respectively. (E–H). Percentage (%) AIM⁺ CD8⁺ T for MPs R, S of WT SARS-CoV-2 and BA.1-MUT and BA.2-MUT subvariant Omicron MPs, respectively. The percentage of AIM⁺ CD4⁺T (CD137⁺OX40⁺) and AIM⁺ CD8⁺ T (CD137⁺CD69⁺) was determined by flow cytometry. Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2^nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively. The data was analyzed using a mixed effects model, ***p<0.001; **p<0.01; *p<0.05. The absence of * indicates that there is no statistical significance.

Figure 5

IFN-γ and IL-2 secretion by PBMCs stimulated using MPs of SARS-CoV-2 of breakthrough cases of children and adolescents vaccinated with CoronaVac^®. IFN-γ and IL-2 secretion was quantified in supernatants of PBMCs of twelve breakthrough cases, upon stimulation with megapools of peptides derived from SARS-CoV-2 proteins by Multiplex assay. (A–D) IFN-γ secretion (pg/ml) by PBMCs stimulated with MP-R and MP-S of WT SARS-CoV-2, and MP-BA.1 and BA.2 of protein S of the Omicron variant of SARS-CoV-2, respectively. (E–H) IL-2 secretion (pg/ml). Circles, triangles and squares correspond to subjects 3-5 yo, 6-11 yo and 12-17 yo, respectively. On the other hand, the black, blue and red colors represent the samples evaluated at pre-immune, 2^nd dose + 4 weeks and 2 to 8 weeks post-infection, respectively, by before-after graph. The values below the significance line indicate the times of change between the corresponding media (Red: decrease, Blue: increase). The data was analyzed using a mixed effects model, *p<0.05, not significant (ns).