Protective antibodies elicited by SARS-CoV-2 spike protein vaccination are boosted in the lung after challenge in nonhuman primates

Abstract

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike protein trimers (preS dTM) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHPs). Binding and functional neutralization assays and systems serology revealed that the vaccinated NHP developed AS03-dependent multifunctional humoral responses that targeted distinct domains of the spike protein and bound to a variety of Fc receptors mediating immune cell effector functions in vitro. The neutralizing 50% inhibitory concentration titers for pseudovirus and live SARS-CoV-2 were higher than titers for a panel of human convalescent serum samples. NHPs were challenged intranasally and intratracheally with a high dose (3 × 10⁶ plaque forming units) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days after challenge, vaccinated NHPs showed rapid control of viral replication in both the upper and lower airways. Vaccinated NHPs also had increased spike protein-specific immunoglobulin G (IgG) antibody responses in the lung as early as 2 days after challenge. Moreover, passive transfer of vaccine-induced IgG to hamsters mediated protection from subsequent SARS-CoV-2 challenge. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine were sufficient to mediate protection against SARS-CoV-2 in NHPs and that rapid anamnestic antibody responses in the lung may be a key mechanism for protection.

Fig. 1.. Vaccine design and study outline.

(A) Schematic of the SARS-CoV-2 spike protein [adapted from (24)], preS dTM, with stabilizing mutations at the S1/S2 furin cleavage site and the heptad repeat region (amino acid sequences indicated by quotation marks); the transmembrane domain was replaced with a T4 trimerization domain. SS, signal sequence. SD, subdomain. FP, fusion peptide. HR, heptad repeat. CH, central helix. CD, connector domain. HR2, heptad repeat 2. TM, transmembrane domain. CT, cytoplasmic tail. (B) Schematic of NHP immunogenicity and challenge study. Immunizations were given at study weeks 0 and 3, intranasal and intratracheal SARS-CoV-2 challenge was performed at study week 6, blood draws are approximated by red droplets, and PCR and necropsy for histopathology approximated by arrows. IM, intramuscular.

Fig. 2.. Vaccination with AS03-adjuvanted preS dTM induces spike protein–specific antibodies.

Rhesus macaques were immunized with 4 or 12 μg of preS dTM adjuvanted with AS03 adjuvant at weeks 0 and 3. (A) End point binding titers after prevaccination (week 0) or after prime (week 2) and boost (week 5) were measured by ELISA (left). Immunization time points are indicated with gray arrows. End point titers from human convalescent sera (HCS) panels were measured as a comparison (n = 42, NIH; n = 18, OWS) (right). (B) Binding titers to the S1 domain, NTD, or RBD at week 5 were measured by Meso Scale Discovery (MSD) ELISA. AUC, area under the curve. (C) Avidity index at weeks 2 and 5. (D) Plasma inhibition of ACE2 binding to spike; week 5 vaccine dose response at 1:40 dilution (left graph) or over multiple dilutions (right graph) are shown. Dotted lines indicate upper limits of quantitation (ULOQ) and lower limits of quantitation (LLOQ). (E and F) Pseudovirus (E) or live virus (F) neutralization was measured over time after vaccination in NHPs or using HCS; 50% inhibitory concentration (IC₅₀) values are plotted. Symbols represent individual animals, box plots indicate the median and interquartile range, and whiskers indicate minimum and maximum data points. Geometric mean values for binding (end point) and neutralization (ID₅₀) titers are indicated in tables below each graph (GMT, geometric mean titer ). Asterisks indicate significance compared to the PBS control group as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. T cell responses are mixed after vaccination with AS03-adjuvanted preS dTM.

T cell responses in rhesus macaques immunized with 4 or 12 μg of preS dTM adjuvanted with AS03 adjuvant at weeks 0 and 3. Cells taken before immunization (prevax) or at week (Wk) 5 were stimulated with an S1 peptide pool covering the spike protein and then assessed by intracellular cytokine staining. (A) Percent of memory CD4 T cells expressing any T_H1 cytokine (IL-2, TNF, or IFN-γ; left) or any T_H2 cytokine (IL-4 or IL-13; right). (B) Percent of memory CD4 T cells expressing the indicated cytokine. (C) Percent of CD4 T cells expressing the T_FH markers IL-21 (left) or CD40L (right) in all memory CD4 T cells (top) or the T_FH subset (bottom). (D) Proportion of memory CD4 T cells expressing any T_H1 (IL-2, TNF, or IFN-γ), T_H2 (IL-4 or IL-13), or T_FH (IL-21 or CD40L) markers by Boolean gating; week 5 responses from both vaccine dose groups are averaged. Pie arcs indicate the proportion of cells expressing any T_H1 and T_H2 cytokines in the same cell (brown arc); T_H1 cytokines only (gray arc), or T_H2 cytokines only (pink arc). (E) Percent of memory CD8 T cells expressing any T_H1 cytokine (IL-2, TNF, or IFN-γ). Symbols represent individual animals, box plots indicate the median and interquartile range, and whiskers indicate minimum and maximum data points. Asterisks indicate significance compared to the PBS control group unless otherwise indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.. Vaccination with AS03-adjuvanted preS dTM protected NHP from SARS-CoV-2 challenge.

Rhesus macaques immunized with 4 or 12 μg of AS03-adjuvanted preS dTM were challenged with 3 × 10⁶ PFU of SARS-CoV-2 by the intranasal and intratracheal routes. SARS-CoV-2 subgenomic RNA (sgRNA) in BAL (A) and nasal swabs (B) was measured at 2, 4, and 7 days after challenge. Symbols represent individual animals; bars indicate group geometric means. Red dotted lines indicate lower limit of quantitation. (C) Histopathological analysis at 7 days after challenge. Representative images from lung sections from two animals per group analyzed by hematoxylin and eosin (H&E) staining for inflammation (top; ×4 magnification) or IHC staining for viral antigen (middle; ×4 magnification; bottom; ×10 magnification). Scale bars, 200 and 100 μm for ×4 and 10× magnifications, respectively. Red arrowheads indicate foci of viral antigen. (D to F) Quantification of histopathology for four animals at days 7 to 8 after challenge. Scores indicating inflammation from H&E staining (D), viral antigen (E), or eosinophil infiltration (F). Symbols represent individual animals, box plots indicate the median and interquartile range, and whiskers indicate minimum and maximum data points. Asterisks indicate significance compared to the PBS control group as follows: *P < 0.05, **P < 0.01, and ****P < 0.0001.

Fig. 5.. Anamnestic antibody responses are initiated in the lung after SARS-CoV-2 challenge.

BAL supernatant was collected before challenge (week 5) and on days 2, 4, 7, and 14 after SARS-CoV-2 challenge. (A and B) S-2P IgG (A) and IgA (B) binding titers in BAL samples were calculated. (C and D) S-2P IgG binding titers in nasal washes (C) and plasma (D) taken before and after challenge were measured. Symbols represent individual animals, box plots indicate the median and interquartile range, and whiskers indicate minimum and maximum data points. Asterisks indicate significance compared to the PBS control group as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. Passively transferred IgG from vaccinated NHP protected hamsters from SARS-CoV-2 challenge.

Total IgG was isolated from pooled week 6 sera from rhesus macaques immunized with 3 μg of AS03-adjuvanted preS dTM. Ten or 2 mg of total IgG was transferred to hamsters; 10 mg of IgG from before (pre-) vaccination or PBS was transferred as a negative control for protection; mAb Ly-CoV555 (10 mg/kg) was transferred as a positive control. Animals were then challenged 1 day later with SARS-CoV-2; body weight was recorded daily, and oral swabs were taken for PCR on days 2, 4, and 7. (A) Passive transfer study timeline. (B) Daily change in body weight after challenge. Lines depict group mean body weight change from day 0; error bars represent SEM. (C) The correlation between serum S-2P binding titers and percent weight loss on day 6 after SARS-CoV-2 challenge is shown. Curve depicts a four-parameter logistic fit of the data. Symbols represent individual animals; whiskers indicate SEM. Asterisks indicate significance compared to the PBS control group at each time point: *P < 0.05, **P < 0.01, and ***P < 0.001.