Heterologous booster with a novel formulation containing glycosylated trimeric S protein is effective against Omicron

Abstract

In this study, we evaluated the efficacy of a heterologous three-dose vaccination schedule against the Omicron BA.1 SARS-CoV-2 variant infection using a mouse intranasal challenge model. The vaccination schedules tested in this study consisted of a primary series of 2 doses covered by two commercial vaccines: an mRNA-based vaccine (mRNA1273) or a non-replicative vector-based vaccine (AZD1222/ChAdOx1, hereafter referred to as AZD1222). These were followed by a heterologous booster dose using one of the two vaccine candidates previously designed by us: one containing the glycosylated and trimeric spike protein (S) from the ancestral virus (SW-Vac 2µg), and the other from the Delta variant of SARS-CoV-2 (SD-Vac 2µg), both formulated with Alhydrogel as an adjuvant. For comparison purposes, homologous three-dose schedules of the commercial vaccines were used. The mRNA-based vaccine, whether used in heterologous or homologous schedules, demonstrated the best performance, significantly increasing both humoral and cellular immune responses. In contrast, for the schedules that included the AZD1222 vaccine as the primary series, the heterologous schemes showed superior immunological outcomes compared to the homologous 3-dose AZD1222 regimen. For these schemes no differences were observed in the immune response obtained when SW-Vac 2µg or SD-Vac 2µg were used as a booster dose. Neutralizing antibody levels against Omicron BA.1 were low, especially for the schedules using AZD1222. However, a robust Th1 profile, known to be crucial for protection, was observed, particularly for the heterologous schemes that included AZD1222. All the tested schedules were capable of inducing populations of CD4 T effector, memory, and follicular helper T lymphocytes. It is important to highlight that all the evaluated schedules demonstrated a satisfactory safety profile and induced multiple immunological markers of protection. Although the levels of these markers were different among the tested schedules, they appear to complement each other in conferring protection against intranasal challenge with Omicron BA.1 in K18-hACE2 mice. In summary, the results highlight the potential of using the S protein (either ancestral Wuhan or Delta variant)-based vaccine formulation as heterologous boosters in the management of COVID-19, particularly for certain commercial vaccines currently in use.

Keywords: COVID-19; Omicron; SARS- CoV-2; heterologous booster; spike-based vaccine.

Figure 1

Specific ancestral Spike (SW) IgG and IgG isotypes levels induced by homologous and heterologous vaccination schedule. (A) K18-hACE2 mice (n =7/group) were immunized on Days 0 and 14 with commercial vaccines mRNA1273 or AZD1222 plus a booster with formulations containing purified spike protein from ancestral (SW) or Delta (SD) SARS-CoV-2 variant on Day 60 delivered intramuscularly. (B) SW specific IgG levels induced by 2 dose primary series. Serum collected on Days 14 and 45 after the second dose was analyzed by ELISA. (C) SW specific IgG, (D) IgG1 and (E) IgG2c levels induced by 3-dose homologous and heterologous schemes. Sera from immunized and non-immunized were collected 14 Days after the 3^rd dose and analyzed by ELISA. Serum antibody levels are expressed as EC50 ± SD for each group. For statistical analysis, antibody levels were analyzed by a one-way ANOVA with Bonferroni’s multiple comparisons test. The * symbol indicate significant differences among immunized: ****p<0.0001, *** p<0.001, *p<0.05. The # symbol indicate significant differences between non-immunized and immunized animals: #### p <0,0001.

Figure 2

Neutralizing antibodies induced by homologous and heterologous vaccination schedule. Mice were immunized as shown in Figure 1A. Serum was collected on Day 14 after the 2^nd and 3^rd dose. Neutralizing titers were measured by 50% inhibition for the pseudotyped virus (CoV2pp-GFP) expressing W (A, B) or Omicron (C, D) spike protein. Data from mRNA1273 vaccinated mice are presented in panels (A) y (C) and those from AZD1222 in panels (B, D). Absolute inhibitory concentration was calculated as the corresponding point between the 0% and 100% assay controls. Fifty % inhibition was defined by the controls for all the samples on the same plate. For statistical analysis, values were analyzed by a one-way ANOVA with Bonferroni`s multiple comparisons test. The * symbol indicate significant differences among immunized: ****p<0.0001, ***p<0.001, **p<0,01, *p<0.05. The # symbol indicate significant differences between non-immunized and immunized animals: ####p<0.0001,##p<0,01, #p<0,05.

Figure 3

Homologus and heterologous immunization induces Ag-specific Th1 (IFN-γ)/Th17 (IL-17)/Th2 (IL-5). (A) K18-hACE2 mice (n =7/group) were immunized on Days 0 and 14 with commercial vaccines mRNA1273 or AZD1222 plus a booster with formulations containing purified spike protein from ancestral (SW) or Delta (SD) SARS-CoV-2 variant on Day 60 delivered intramuscularly. Mice were challenged 14 days after the 3^rd dose with SARS-CoV-2 Omicron BA.1 variant, 5 days after challenge mice were sacrificed and spleens were harvested. Levels of secreted IFN-γ (B), IL-17 (C) and IL-5 (D) following splenocytes stimulation with medium or recombinant spike protein from W o D variants were determined by ELISA. Bars are means ± SEM of pg/ml. The * symbol indicate significant differences among immunized: ***p<0.001, **p<0.01, *p<0.05. The # symbol indicate significant differences between non-immunized and immunized animals: ####p<0.0001, ###p<0.001, ##p<0.01, #p<0.05.

Figure 4

T CD4+ populations induced after Omicron BA.1 challenge in mice vaccinated with homologous or heterologous schemes. Five days post challenge immunized and non-immunized mice were sacrificed and spleen cells were collected. For FACS analysis the spleen cells were incubated with CD16/CD32 Fc g RII to block IgG Fc receptors. Cells were incubated with LIVE/DEAD Violet (Invitrogen), followed by surface staining with fluorochrome-conjugated anti-mouse Abs for various markers. (A) CD4+CD44+CD62L-, (CD4+ Teff), (B) CD4+CD44+CD62L+ (CD4+ Tcm) and (C) CD4+CXCR5+CD127Low/high(CD4+ Tfr/Tfh). Flow cytometry analysis was performed on an BD FACS Aria Fusion. The results were analyzed using Flow Josoftware (TreeStar). The represented data correspond to the mean of absolut cells counts ± SEM. Two-way ANOVA followed by Bonferroni post-test were used for statistical analysis. ***p<0.001; **p < 0.01, *p < 0.05.

Figure 5

Protection assay against Omicron BA.1 SARS-CoV-2 infection in the intranasal challenge mouse model. (A) K18-hACE2 mice (n =7/group) were immunized on Days 0 and 14 with commercial vaccines mRNA1273 or AZD1222 plus a booster with formulations containing purified spike protein from ancestral (SW) or Delta (SD) SARS-CoV-2 variant on Day 60 delivered intramuscularly. Mice were challenged 14 days after the 3^rd dose with SARS-CoV-2 Omicron BA.1 variant, 5 days after challenge mice were sacrificed and nasal washes and lungs were collected. (B, E) Changes in body weight were expressed as the percentage of body weight in comparison to day 0 (mean ± SEM.). Post-challenge weights were zoomed in for each treatment in the right part of the graph. (C, F) nasal and (D, G) lungs SARS-CoV-2 viral load after Omicron BA.1 challenge in animals vaccinated with schedules that contain mRNA-1273 (C, D) or AZD1222 (F, G). Results are expressed as Log number Rdrp copies/ml nasal wash or as Log number Rdrp copies/mg lungs. One-way or Two-way ANOVA followed by Bonferroni post-test, were used for statistical analysis. ****p < 0.0001, ***p<0.001, **p < 0.01, *p<0.05.

Figure 6

Pathology score in lungs and brains for non-immunized and immunized animals. K18-hACE2 mice (n = 7/group) were immunized on Days 0 and 14 with commercial vaccines mRNA1273 or AZD1222, along with a booster containing purified spike protein from ancestral (SW) or Delta (SD) variants of SARS-CoV-2 on Day 60, administered intramuscularly. Fourteen days after the third dose, mice were challenged with the Omicron BA.1 variant of SARS-CoV-2. Five days after the challenge, mice were euthanized, and their lungs and brains were collected. An experienced pathologist performed blinded assessments of the lesions detected in the lungs (A) and brains (B) using a scale ranging from 0 to 3 (where 0 indicates no lesion, 1 mild, 2 moderate, and 3 severe). The data obtained are expressed as the percentage of animals that exhibited each degree of lesion.