SARS-CoV-2 vaccines elicit durable immune responses in infant rhesus macaques

Abstract

The inclusion of infants in the SARS-CoV-2 vaccine roll-out is important to prevent severe complications of pediatric SARS-CoV-2 infections and to limit transmission and could possibly be implemented via the global pediatric vaccine schedule. However, age-dependent differences in immune function require careful evaluation of novel vaccines in the pediatric population. Toward this goal, we assessed the safety and immunogenicity of two SARS-CoV-2 vaccines. Two groups of 8 infant rhesus macaques (RMs) were immunized intramuscularly at weeks 0 and 4 with stabilized prefusion SARS-CoV-2 S-2P spike (S) protein encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or the purified S protein mixed with 3M-052, a synthetic TLR7/8 agonist in a squalene emulsion (Protein+3M-052-SE). Neither vaccine induced adverse effects. Both vaccines elicited high magnitude IgG binding to RBD, N terminus domain, S1, and S2, ACE2 blocking activity, and high neutralizing antibody titers, all peaking at week 6. S-specific memory B cells were detected by week 4 and S-specific T cell responses were dominated by the production of IL-17, IFN-γ, or TNF-α. Antibody and cellular responses were stable through week 22. The immune responses for the mRNA-LNP vaccine were of a similar magnitude to those elicited by the Moderna mRNA-1273 vaccine in adults. The S-2P mRNA-LNP and Protein-3M-052-SE vaccines were well-tolerated and highly immunogenic in infant RMs, providing proof-of concept for a pediatric SARS-CoV-2 vaccine with the potential for durable immunity that might decrease the transmission of SARS-CoV-2 and mitigate the ongoing health and socioeconomic impacts of COVID-19.

Fig. 1.. Study design: Evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant RMs.

Infant RMs (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 μg of mRNA-encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 μg of S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of eight animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18, and 22, and LN biopsies were obtained at week 6.

Fig. 2.. SARS-CoV-2 vaccine–elicited binding Ab responses in infant RMs.

Plasma and saliva were collected before vaccination (week 0); at week 4 before the second dose; at 2 weeks after second dose (week 6); and at weeks 8, 14, 18, and 22 from infant RMs vaccinated with 30 μg of mRNA-encoding S-2P S protein in LNP (n = 8; red) or with 15 μg of prefusion SARS-CoV-2 S-2P S protein formulated with 3M-052 adjuvant (n = 8; blue). (A) S-2P protein–specific Ab responses were measured by ELISA. Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 S. Data are reported as log₁₀ AUC values. (B) Ab epitope specificity measured by binding antibody multiplex assay (BAMA). Plasma was diluted 1:10,000 to measure binding to different domains of the S protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding Ab responses are reported as log₁₀-transformed MFI after subtraction of background values. (C) Salivary RBD-specific IgG was measured by BAMA using serial dilutions of saliva, and responses are reported as RBD-specific IgG (nanograms)/total IgG (micrograms). Different symbols represent individual animals (Table 1). Arrows in (A) indicate times of immunizations.

Fig. 3.. SARS-CoV-2 vaccine–elicited functional Ab responses in infant RMs.

(A) The capacity of plasma Abs to mediate blocking of the RBD-ACE2 interaction was measured with an ELISA-based ACE2 blocking assay at 1:10 plasma dilution. Data are reported as ACE2 blocking (%). (B and C) Neutralization capacity was measured using S D614G-pseudotyped viruses in 293 T/ACE2 cells (B) and whole-virus (D614G) assay with Vero E6 cells (C); results are expressed as reciprocal 80% inhibitory dilution (ID₈₀). Gray dotted lines represent detection cutoff. Different symbols represent individual animals (Table 1). Longitudinal data for each animal in the mRNA-LNP (red) or Protein+3M-052-SE group (blue) are represented by separate lines. Arrows in (C) indicate times of immunizations.

Fig. 4.. Characterization of S-specific B cell responses 2 weeks after boost.

(A) CD20⁺CD27⁺ memory B cells that costained with fluorochrome-conjugated SARS-CoV-2 S protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood. Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in fig. S14. (B) In LNs, we determined total bcl-6⁺Ki-67⁺ GC B cells and CD27⁺ memory B cells as percent of total CD20⁺ B cells (left) (see fig. S15 for gating strategy) and also the percent of S-specific memory B cells (right). MNC, mononuclear cell. (C) ASC as measured by B cell ELISpot in PBMCs from mRNA-LNP or Protein+3M-052-SE vaccinees, whereas (D) is showing mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at week 6. Different symbols represent individual animals (Table 1). Solid lines represent median values.

Fig. 5.. Immunophenotype of LN T cell population 2 weeks after boost.

(A) CD4⁺ T cells positive for IL-4 or IL-21, T_FH markers CD279/PD-1 and CD185/CXCR5, or bcl6⁺ T_FH were measured and are represented as percent of total LN CD4⁺ T cells. The gating strategy for these panels is provided in fig. S15. (B) SEB-activated T_FH frequencies assessed by the AIM assay: CXCR5⁺CD185⁺ cells that coexpressed CD134 and CD137 for Protein+3M-052-SE and mRNA-LNP, respectively. (C) The frequency of CXCR5⁺CD185⁺CD134⁺CD137⁺CXCR3⁺CD196⁻ T_FH1, CXCR3⁻CCR6⁻ T_FH2, and CXCR3⁻ CD196⁺ T_FH17 cells is shown. The gating strategy for these populations is given in fig. S17. Red symbols, mRNA-LNP; blue symbols, Protein+3M-052-SE. Different symbols represent individual animals (Table 1). Solid lines define the median.

Fig. 6.. S-specific CD4⁺ T cell responses in SARS-CoV-2 immunized infant macaques.

Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α [indicated at the x axis in (D)] was performed on PBMCs at weeks 4 (A), 6 (B), 8 (C), and 14 (D) to assess T cell responses to a peptide pool encompassing the entire SARS-CoV-2 S protein (see fig. S16 for gating strategy). Responses detected in mRNA-LNP recipients are displayed in red, and cytokine responses from Protein+3M-052 and SE vaccinees are in blue. The dashed lines represent week 0 values plus 2 SDs and define the cutoff for positive cytokine responses. Different symbols represent individual animals (Table 1).