Optimizing immunogenicity and product presentation of a SARS-CoV-2 subunit vaccine composition: effects of delivery route, heterologous regimens with self-amplifying RNA vaccines, and lyophilization

Abstract

Introduction: Dozens of vaccines have been approved or authorized internationally in response to the ongoing SARS-CoV-2 pandemic, covering a range of modalities and routes of delivery. For example, mucosal delivery of vaccines via the intranasal (i.n.) route has been shown to improve protective mucosal responses in comparison to intramuscular (i.m.) delivery. As we gain knowledge of the limitations of existing vaccines, it is of interest to understand if changes in product presentation or combinations of multiple vaccine modalities can further improve immunological outcomes.

Methods: We investigated a commercial-stage SARS-CoV-2 receptor binding domain (RBD) antigen adjuvanted with a clinical-stage TLR-7/8 agonist (3M-052) formulated on aluminum oxyhydroxide (Alum). In a murine immunogenicity model, we compared i.n. and i.m. dosing of the RBD-3M-052-Alum vaccine. We measured the magnitude of antibody responses in serum and lungs, the antibody-secreting cell populations in bone marrow, and antigen-specific cytokine-secreting splenocyte populations. Similarly, we compared different heterologous and homologous prime-boost regimens using the RBD-3M-052-Alum vaccine and a clinical-stage self-amplifying RNA (saRNA) vaccine formulated on a nanostructured lipid carrier (NLC) using the i.m. route alone. Finally, we developed a lyophilized presentation of the RBD-3M-052-Alum vaccine and compared it to the liquid presentation and a heterologous regimen including a previously characterized lyophilized form of the saRNA-NLC vaccine.

Results and discussion: We demonstrate that i.n. dosing of the RBD-3M-052-Alum vaccine increased IgA titers in the lung by more than 1.5 logs, but induced serum IgG titers 0.8 logs lower, in comparison to i.m. dosing of the same vaccine. We also show that the homologous prime-boost RBD-3M-052-Alum regimen led to the highest serum IgG and bronchial IgA titers, whereas the homologous saRNA-NLC regimen led to the highest splenocyte interferon-γ response. We found that priming with the saRNA-NLC vaccine and boosting with the RBD-3M-052-Alum vaccine led to the most desirable immune outcome of all regimens tested. Finally, we show that the lyophilized RBD-3M-052-Alum vaccine retained its immunological characteristics. Our results demonstrate that the route of delivery and the use of heterologous regimens each separately impacts the resulting immune profile, and confirm that multi-product vaccine regimens can be developed with stabilized presentations in mind.

Keywords: RNA vaccine; adjuvant formulation; heterologous vaccine; intranasal vaccine; lyophilized vaccine; receptor binding domain; vaccine development.

Figure 1

Generalized animal study diagram. For all presented in vivo studies, animals were vaccinated on Days 0 and 21, then euthanized on Day 42. At the termination of the study, peripheral blood, bronchial lavage (BAL), splenocytes, and bone marrow-resident cells were harvested for the indicated assays (64).

Figure 2

Route of delivery and adjuvant formulation affect antibody isotype class switching. (A) Serum titer of total anti-RBD IgG, (B) BAL titer of anti-RBD IgA, and (C) log₁₀ transform of serum ratio of exponentiated anti-RBD IgG2a/IgG1 titers. Data collected from n = 8 (4M:4F) animals on Day 42 after being vaccinated twice intramuscularly (i.m.) or intranasally (i.n.) on Days 0 and 21 with RBD in combination with the 3M-052-Alum adjuvant, or without an adjuvant in the case of the unadjuvanted RBD control. The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed for all animals simultaneously using frozen serum and BAL samples. Horizontal bars represent the mean ± SD of log-normalized data. Statistical significance was determined via two-way ANOVA followed by Holm-Sidak’s correction for multiple comparisons, fixing the family-wide error rate to 0.05. Comparisons were made for the same adjuvant between i.m. and i.n. delivery, and between the adjuvanted and unadjuvanted RBD via each route of delivery (4 comparisons total).

Figure 3

Route of delivery has minimal effect on bone marrow and splenocyte cellular responses to the RBD vaccine. (A) Bone marrow-derived anti-full-length-wt-Spike IgG antibody-secreting cells (ASC) ELISpot. T cell ELISpot measurement of splenocytes secreting (B) IFN-γ or (C) IL-5 upon stimulation with a SARS-CoV-2 peptide pool. Data collected from n = 8 (4M:4F) animals on Day 42 after being vaccinated twice intramuscularly (i.m.) or intranasally (i.n.) on Days 0 and 21 with RBD in combination with 3M-052-Alum (or without an adjuvant in the case of the unadjuvanted RBD control). The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed at the time of tissue harvest. Horizontal bars represent the mean ± SD of the log-transformed data. Statistical significance was determined via two-way ANOVA followed by Holm-Sidak’s correction for multiple comparisons fixing the family-wide error rate to 0.05. Comparisons were made for the same adjuvant between i.m. and i.n. delivery, and between the adjuvanted and unadjuvanted RBD via each route of delivery (4 comparisons total).

Figure 4

Vaccine regimen influences antibody titer and isotype class switching. (A) Serum titer of total anti-RBD IgG, (B) serum titer of total anti-full-length intact wt Spike IgG, (C) BAL titer of anti-RBD IgA, and (D) log₁₀ transform of serum ratio of exponentiated anti-RBD IgG2a/IgG1 titers. Data were collected from n = 8 (4M:4F) animals on Day 42 after being vaccinated intramuscularly (i.m.) on Days 0 and 21 with the indicated vaccines in the indicated order. Arrow symbols demarcate heterologous prime-boost vaccinations (Prime → Boost). The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed for all animals simultaneously using frozen serum and BAL samples. Horizontal bars represent the mean ± SD of log-normalized data. Statistical significance was determined via one-way ANOVA followed by Holm-Sidak’s correction for multiple comparisons, fixing the family-wide error rate to 0.05.

Figure 5

Vaccination regimen impacts cellular response phenotype. (A) Bone marrow-derived anti-full-length-wt-Spike IgG antibody-secreting cells (ASC) ELISpot. T cell ELISpot measurement of splenocytes secreting (B) IFN-γ or (C) IL-5 upon stimulation with a SARS-CoV-2 peptide pool. Data were collected from n = 8 (4M:4F) animals on Day 42 after being vaccinated intramuscularly (i.m.) on Days 0 and 21 with the indicated vaccines in the indicated order. Arrow symbols demarcate heterologous prime-boost vaccinations (Prime → Boost). The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed at the time of tissue harvest. Horizontal bars represent the mean ± SD of the log-transformed data. Statistical significance was determined via one-way ANOVA followed by Holm-Sidak’s correction for multiple comparisons fixing the family-wide error rate to 0.05.

Figure 6

Pseudovirus neutralization is not significantly altered by heterologous regimens. Data were collected from n = 3-6 animals on Day 42 after being vaccinated intramuscularly (i.m.) on Days 0 and 21 with the indicated vaccines in the indicated order. Arrow symbols demarcate heterologous prime-boost vaccinations (Prime → Boost). The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed for all animals simultaneously using frozen serum samples. Day 42 serum samples were tested in a pseudovirus neutralization assay against a Wuhan (wt) pseudovirus. Horizontal bars represent the mean ± SD of log-normalized data. Statistical significance was determined via one-way ANOVA, followed by a Holm-Sidak’s correction for multiple comparisons, fixing the family-wide error rate to 0.05.

Figure 7

Identification of the most desirable dosing regimen. (A) Heatmap of individual desirability index scores (d_ij ) for the j ^th group and i ^th response. Columns are ordered left to right by decreasing weight (see Supplementary Table S1 ). Scores are normalized within each response variable. D = Day, BM = bone marrow, BAL = bronchoalveolar lavage. (B) Weighted, aggregate desirability scores (D_j ) per group (j), ordered left to right from highest to lowest score. The saRNA-NLC prime-RBD-3M-052-Alum boost and the RBD-3M-052-Alum prime-boost regimens were the most desirable based on the weights outlined in Supplementary Table S1 . Desirability index results were calculated using formulas described in the Supplementary Methods . Arrow symbols demarcate heterologous prime-boost vaccinations (Prime → Boost).

Figure 8

The lyophilization process does not significantly impact the humoral immune response. (A) Serum titer of total anti-RBD IgG, (B) BAL titer of anti-RBD IgA, and (C) ratio of exponentiated serum anti-RBD IgG2a/IgG1 titers. Data were collected from n = 8 (4M:4F) animals on Day 42 after being vaccinated twice intramuscularly (i.m.) on Days 0 and 21 with the indicated vaccines in the indicated order. Arrow symbols demarcate heterologous prime-boost vaccinations (Prime → Boost). The study was divided in half and vaccinations/harvests were staggered 1 week apart to reduce operator burden. Assays presented here were performed for all animals simultaneously using frozen serum and BAL samples. Horizontal bars represent the mean ± SD of log-normalized data. Statistical significance was determined via two-way ANOVA followed by Holm-Sidak’s correction for multiple comparisons, fixing the family-wide error rate to 0.05. The study was performed simultaneously with Figure 4 and Figure 5 ; liquid material data are presented in both figures.