Pre-Existing Anti-Vector Immunity to Adenovirus-Inspired VLP Vaccines Shows an Adjuvant-Dependent Antagonism

Abstract

Background/Objectives: The use of virus-like particles (VLPs) in vaccinology has expanded significantly in recent years. VLPs have the advantage of being non-infectious while effectively stimulating B cell responses through the repetitive presentation of epitope motifs on their surface. Since VLPs are often derived from human-infecting viruses, preexisting immunity may influence the immune response they elicit, warranting further investigation. Methods: We have developed a 60-mer VLP derived from human adenovirus type 3, a common pathogen. We investigated the impact of pre-existing adenovirus immunity on the immunization outcome against the linear S14P5 epitope of SARS-CoV-2, which was engineered into the particle (Ad-VLP-S14P5). To this end, antibody responses to S14P5 were evaluated following immunization with Ad-VLP-S14P5 in either naive or vector-primed mice. Results: Mice with pre-existing anti-vector immunity exhibited significantly greater anti-S14P5 antibody responses compared to vector-naive animals, demonstrating a beneficial impact of prior anti-adenovirus responses. However, the addition of an oil-in-water adjuvant for the immunizations abolished this positive impact, even leading to a deleterious effect of the pre-existing anti-vector immunity. Conclusions: The data suggest that the immune status against immunizing VLPs must be taken into consideration when designing immunization protocols. Importantly, the effects of prior immunity may vary depending on the nature of the protocol, including factors such as adjuvant use.

Keywords: SARS-CoV-2; VLP; adenovirus; adjuvant; pre-existing immunity.

Figure 1

S14P5 insertion in Ad-VLP. (A) The S14P5 epitope is conserved amongst different SARS-CoV-2 variants. S14P5 (in green) is located just beneath the RBD domain (in red). * conserved residues. (B) The epitope is inserted in an exposed loop of the Ad-VLP, giving rise to Ad-VLP-S14P5. (C) Negative staining electron microscopy of the purified empty Ad-VLP and Ad-VLP-S14P5 (Bar 50 nm). (D) Screening by ELISA of a cohort COVID-19 infected patients serums on Ad-VLP (blue) and Ad-VLP-S14P5 (Red). Results are represented by the area under the curve (AUC).

Figure 2

Preimmunization of mice with empty Ad-VLP and immunization schedule. (A) Four groups of mice (n = 6) received the same dose of Ad-VLP-S14P5 vaccine (blue vials) at D0 and D14 without (groups I and II) or with adjuvant (groups III and IV, vials labeled with ‘A-star’). Mice from groups II and IV were preimmunized with empty Ad-VLP at D-14. Blood was collected before the first (D-1), at the second (D14) injection, and at the end of the experiment (D28). (B) Pre-existing immunity against the vector was checked by direct ELISA against empty Ad-VLP at D-1 for all groups.

Figure 3

Evaluation of anti-S14P5 and anti-vector response of mice immunized with Ad-VLP-S14P5 without adjuvant. (A) ELISA was performed on biotinylated S14P5 peptide with serial dilutions of serums from groups I and II collected at D14 and D28. Statistical analysis was performed by calculating the area under the curve (AUC) of mice from each group using unpaired T test (one-tailed) D14 * p = 0.0406, and D28 * p = 0.0156. (B) Similar experiment on empty Ad-VLP to determine the corresponding anti-vector response.

Figure 4

Evaluation of S14P5 and anti-vector response of mice immunized with adjuvanted Ad-VLP-S14P5. (A) ELISA was performed on biotinylated S14P5 peptide with serial dilution of serums from groups III and IV collected at D14 and D28. Statistical analysis was performed by calculating the area under the curve (AUC) of mice from each group using unpaired t test (one-tailed); ns: not significant. (B) Similar experiment on empty Ad-VLP to determine the corresponding anti-vector response.

Figure 5

Recognition of recombinant trimeric spike by the four groups. (A) ELISA was performed on soluble trimeric recombinant spike with serial dilution of serums from the four groups collected at D28. (B) Correlation between the result obtained on the biotinylated S14P5 and the recombinant spike. Statistical analysis was performed by calculating the area under the curve (AUC) of mice from each group on S14P5 at D28 (left panel) and on recombinant spike at D28 (right panel) using one-way ANOVA followed by Tukey’s multiple comparisons test ** p < 0.01; ns: not significant.