Humoral Immune Response of Heterologous ChAdOx1 nCoV-19 and mRNA-1273 Prime-Boost Vaccination against SARS-CoV-2 Variants in Korea

Safe and effective vaccination is considered one of the most important preventive measures to contain a pandemic [1]. Vaccination is currently the most effective and safest method for the prevention of coronavirus disease 2019 (COVID-19) [2]. In a short period of time and with intense research, several highly effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed [3]. Five SARS-CoV-2 vaccines have been approved in Korea through a fast-track system, and these are based on three technologies. Chimpanzee adenoviral (ChAdOx1 nCoV-19/AZD1222, Vaxzevria; AstraZeneca, Cambridge, UK) [4] or replication-incompetent human adenoviral (Ad26.COV2; Janssen, Beerse, Belgium) vaccines [5] are vector vaccines that can transduce the genetic information of the SARS-CoV-2 S protein into host cells. In case of the messenger ribonucleic acid (mRNA) vaccines BNT162b2 (Pfizer/BioNTech, New York, NY, USA) and mRNA-1273 (Moderna, Cambridge, MA, USA) [6], the genetic information for the S protein has been optimized, and the mRNA is packaged in liposomes. A recombinant nanoparticle vaccine from Novavax (NVX-2373, Gaithersburg, MD, USA) was constructed with the full-length spike glycoprotein from the prototype strain and Matrix-M adjuvant [7]. Additionally, the SKYCovione vaccine by SK Bioscience (SKYCovione, Seoul, Korea) received domestic approval by the Ministry of Food and Drug Safety, Korea, on June 29, 2022 [8].

The Korean government released the plan for vaccination schedule, and the vaccination started in February until December 2021. The government sequentially provided vaccines such as those manufactured by AstraZeneca, Janssen, Pfizer, and Moderna according to the vaccination plan based on age, clinical results of the vaccine, licensing, and expert advice. For the 2nd dose, the same vaccine was used, unless in cases when the demand of the vaccine exceeded its supply. Cross vaccination was conducted after deliberation by the Special Immunization Committee for some people who had received the AstraZeneca vaccine as the 1st dose. Prior to Korea, several countries, including Canada and Spain, had already approved this dose mix, primarily because of the concerns about rare and potentially lethal blood clots associated with ChAdOx1 nCoV-19/AZD1222 [9]. Each COVID-19 vaccine was approved according to the efficacy and safety data confirmed in large-scale clinical trials; however, the immunogenicity results after cross-vaccination differed country- and study group-wise [10, 11].

During domestic cross-vaccination, some people were vaccinated with the Moderna vaccine (mRNA-1273) instead of the Pfizer vaccine (BNT162b2) as the 2nd dose due to a misinoculation during domestic cross-vaccination. To obtain basic domestic data on the immunogenicity of the cross-vaccination with mRNA-1273, the humoral immune response was analyzed in voluntary applicants receiving ChAdOx1 nCoV-19 as the 1st dose and mRNA-1273 as the 2nd dose. Serum samples were obtained from 28 individuals after 2 weeks (the 2-week group) and from 11 individuals after 3 months (the 3-month group) of cross-vaccination. Immunogenicity was analyzed by evaluating the titers of the S protein-binding antibody and neutralizing antibody against the major variants of SARS-CoV-2.

Antigen-specific humoral immune response was analyzed using the Elecsys AntiSARS-CoV-2 S assay (Roche Diagnostics, Mannheim, Germany) - a commercial electrochemiluminescence immunoassay (ECLIA) that detects antibodies (including IgG) against the SARS-CoV-2 S protein on the Cobas e module (Roche Diagnostics). For enzyme-linked immunosorbent assay (ELISA), 96-well plates were coated with 50 ng/well of SARS-CoV-2 S1+S2 ECD, S1, S2, and RBD proteins (Sino Biological, Beijing, China) and incubated overnight at 4°C. The wells were blocked using 1% bovine serum albumin in phosphate-buffered saline (PBS) for 2 h at 37°C. After washing with PBS containing 0.02% Tween-20 (0.02% PBST), 2-fold serial dilutions of sera (initial dilution 1: 100) from vaccinated human individuals were added to the wells, and the plates were incubated at 37°C for 1 h. After washing thrice with 0.02% PBST, HRP-conjugated anti-mouse IgG was added, and the plates were incubated at 37°C for 1 h. The plates were washed five times and incubated with tetramethyl benzidine substrate for 10 min at 25°C. After adding the stop solution, the absorbance was measured at 450 nm on a SpectraMax i3x microplate reader.

The plaque reduction neutralization test was performed to measure the 50% neutralizing dilution (ND50) titers against the wild-type (BetaCoV/Korea/KCDC03/2020, NCCP43326) [12], alpha (NCCP), beta (NCCP), gamma (NCCP), delta (hCoV-19/Korea/KDCA119861/2021, NCCP43390), and omicron (B.1.1.529) variants in serum samples. Vero E6 cells (ATCC; 2.5 × 10⁵ cells/well) were seeded in 12-well plates and incubated overnight at 37°C in a 5% CO₂ incubator. The 60 PFU of virus was mixed with an equal volume of 2-fold serially diluted heat-inactivated sera and incubated at 37°C for 1 h. The mixture was infected cell. After 1 h, the monolayer was overlaid with 0.75% agar in 4% fetal bovine serum 2X modified Eagle medium and incubated at 37°C in a 5% CO₂ incubator for 2 - 3 days. The cells were fixed with formaldehyde and stained using crystal violet. The Spearman–Kärber method was used to calculate ND50 titer values. The tests were independently performed twice on each sample. Statistical analysis was conducted Student’s t-test, One-way ANOVA test, and the graphs were plotted using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Continuous variables were analyzed using the Student’s t-test or one-way analysis of variance with Tukey’s post-test. All tests of significance were two-tailed. P <0.05 was considered significant.

The median age of the 2-week and 3-month groups was 63.4 and 63.3 years, respectively, and the male-to-female ratio was 19:9 and 5:6, respectively (Table 1). No participant reported a history of SARS-CoV-2 infection or exposure.

Table 1
Characteristics of the study participants

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The mean titer of anti-S antibodies in the 3-month group was 1,712 U/mL, exhibiting a reduction by approximately 4.6-fold compared with that in the 2-week group (7,918 U/mL; Fig. 1A). ELISA of each domain of the SARS-CoV-2 S protein revealed an overall decrease 3 months after the 2nd dose, and the IgG titer (EC50) of the RBD domain was the highest at 2 weeks and 3 months after 2nd dose (Fig. 1B). In the 3-month group, the median ND50 titers decreased approximately 6.7 times compared with those in the 2-week group (Fig. 2A). In both groups, the median ND50 titers for all variants except alpha were significantly lower than those for the wild-type (P <0.01 - 0.0001). The overall median ND50 titers for the wild-type, alpha, beta, gamma, delta, and omicron variants were 2554, 2062, 385.2, 666.9, 552.6, and 58.7, respectively, in the 2-week group and 381, 338, 81.2, 107.1, 77.4, and 17.4, respectively, in the 3-month group (Fig. 2B). Among all variants, the neutralizing antibody titer for the omicron variant was the lowest, which was approximately 43.5 and 21.9 times lower than that of the wild-type in the 2-week and 3-month groups, respectively.

Figure 1
Anti-S antibody titer and antibody titer as per S protein domains. Horizontal bars represent geometric mean with 95% confidence interval. (A) Anti-SARS-CoV-2 S protein-specific total IgG levels were measured using electrochemiluminescence immunoassay (B) Sera from individuals were serially diluted, and anti-SARS-CoV-2 S1+S2 ECD, S1, S2, and RBD protein-specific total IgG levels were measured using enzyme-linked immunosorbent assay.
^aP <0.01, ^bP <0.001, ^cP <0.1.

SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

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Figure 2
Neutralizing antibody titers against SARS-CoV-2 wild-type and other variants. The variants were wild-type (BetaCoV/Korea/KCDC03/2020), alpha (B.1.1.7), beta (B.1.351), delta (AY.69), and omicron (B.1.1.529). Horizontal bars represent geometric mean with 95% confidence interval. (A) Neutralizing antibody titers against SARS-CoV-2 wild-type after 2 weeks (n = 28) and 3 months (n = 11) of 2nd dose. (B) Neutralizing antibody titers against the wild-type and other variants 2 weeks and 3 months after 2nd dose. Comparisons between groups were analyzed using two-way analysis of variance.
^aP <0.0001.

ND50, 50% neutralization dilution; ns, not significant; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

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Immunogenicity was analyzed in a small number of subjects with ChAdOx1 nCoV-19 and mRNA-1273 cross-vaccination. This study had some limitations, such as the absence of homologous vaccination group and inflexible time point setting for the analysis. However, an indirect comparison was possible using some previous studies on the cross-vaccination of COVID-19 vaccines, where generally, higher vaccine efficacy was reported in the heterologous group than in the homologous group [13]. The titers of neutralizing antibody against the alpha (B.1.1.7), beta (B.1.351), and gamma (P.1) SARS-CoV-2 variants were 20 to >60 times higher in the ChAdOx1–BNT162b group than in the homologous ChAdOx1 nCoV-19 group [14]. In addition, according to a domestic study, heterologous ChAdOx1 nCoV-19–BNT162b2 vaccination induces stronger immunogenicity than homologous ChAdOx1 nCoV-19 vaccination does against the SARS-CoV-2 delta variant, with a tolerable reactogenicity profile [15]. Neutralizing antibody titers for the ChAdOx1 nCoV-19–mRNA-1273 cross-vaccination in our study were similar to those reported for the ChAdOx1 nCoV-19–BNT162b2 cross-vaccination [15], and the cross-vaccination exhibited higher immunogenicity than the ChAdOx1 nCoV-19 homologous vaccination. In Sweden, heterologous prime-boost vaccination with ChAdOx1 nCoV-19 as the 1st dose and BNT162b2 or mRNA-1273 as the 2nd dose was associated with 67% and 79% effectiveness against symptomatic COVID-19, respectively [16].

Our results showed that the neutralizing antibody titer was the lowest for the omicron variant in the 2-week group and decreased significantly after 3 months. In another domestic study, neutralizing antibody titers increased after 2nd dose in young and the elderly; however, those for the omicron variant were the lowest [17]. Collectively, these results suggest that co-vaccination or cross-vaccination may not provide adequate protection against infection by the omicron variant, and a variant-specific vaccine may be required for effective protection against this variant [18]. However, T-cells and humoral immunity play important roles; a study has reported that a conserved humoral immune response to the omicron variant can play a major role in protection against severe illness and death [19].

Despite the limitations of case-specific studies, our study reported antibody-neutralizing activity after a heterologous 2nd dose of mRNA-1273 vaccine that could induce a positive effect on the immune evasion of the variants. In addition, to the best of our knowledge, this is the first study to report the immunogenicity of ChAdOx1 nCoV-19 and mRNA-1273 cross-vaccination in Korea. However, the limitation of this study is that since a misinoculation subjects were recruited, factors that could affect immunogenicity such as underlying diseases were not excluded. Also, there is a possibility that the subjects may be somewhat biased, so caution is needed in interpreting the research results. Our results provide basic data for vaccination strategies in Korea and for designing and planning cross-vaccination studies if required.