Beta variant COVID-19 protein booster vaccine elicits durable cross-neutralization against SARS-CoV-2 variants in non-human primates

All animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and were approved by the Institutional Animal Care and Use Committee (IACUC) from the University of Louisiana at Lafayette New Iberia Research Center (IACUC number 2020-8733-013).


For primary immunization, the mRNA vaccines were SARS-CoV-2 prefusion spike constructs 2 P, GSAS, 2 P/GSAS, 2 P/GSAS/ALAYT, and 6 P/GSAS described in ref. 28, the subunit vaccines were CoV2 preS dTM-AS03 vaccines, where the antigens were produced using the phase-I/-II manufacturing process, 1.3- and 2.6-µg doses, or using an intermediate manufacturing process, 2.4 µg dose.

For the booster, the CoV2 preS dTM derived from the ancestral strain (D614) and the Beta variant were produced using an optimized purification process to ensure a minimum of 90% purity.

The antigens were formulated in monovalent or bivalent formulations with AS03 adjuvant. The CoV2 preS dTM was produced from a Sanofi proprietary cell culture technology based on the insect cell—baculovirus system, referred to as the Baculovirus Expression Vector System (BEVS). The CoV2 preS dTM (ancestral D614) sequence was designed based on the Wuhan YP_009724390.1 strain S sequence, modified with 2 prolines in the S2 region, deletion of the transmembrane region, and addition of the T4 foldon trimerization domain. The CoV2 preS dTM (Beta) was designed based on the Beta (B.1.351) sequence (GISAID Accession EPI_ISL_1048524) and contains the same modifications.

AS03 is a proprietary adjuvant system composed of α-tocopherol, squalene, and polysorbate-80 in an oil-in-water emulsion manufactured by GSK. Vaccine doses were formulated by diluting the appropriate dose of preS dTM with PBS–tween to 250 µL, then mixing with 250 µL of AS03, followed by inversion five times for a final volume of 500 µL. Each dose of AS03 contains 11.86 mg of α-tocopherol, 10.69 mg of squalene, and 4.86 mg of polysorbate-80 (Tween 80) in PBS.

Animals and study design

Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and were conducted with approved animal protocols from the Institutional Animal Care and Use Committee (IACUC) at the research facilities. NHP studies were conducted at the University of Louisiana at Lafayette New Iberia Research Center.

Two cohorts of vaccinated NHPs received a booster immunization after randomizing each group within a cohort based on their baseline characteristics (Fig. 1).

In the mRNA-primed cohort, six adult male and six adult female Mauritius cynomolgus macaques (Macaca fascicularis) aged 4–10 years, selected based on their responses to the primary vaccination, were randomly allocated to three groups of four animals according to their baseline characteristics.

In the subunit-primed cohort, 15 adult male Indian rhesus macaques (Macaca mulatta) aged 4–7 years were randomly allocated to three groups of five animals. In the priming phase, animals received two immunizations of either Sanofi’s mRNA COVID (ancestral D614) experimental candidate vaccines or CoV2 preS dTM-AS03 (ancestral D614) vaccine through the intramuscular route in the deltoid at day 0 and day 21. Seven months after the primary immunization, both cohorts were immunized with CoV2 preS dTM (ancestral)–AS03, CoV2 preS dTM (Beta)–AS03, and a bivalent CoV2 preS dTM (ancestral + Beta)–AS03. All groups received a total dose of 5 µg of CoV2 preS dTM antigen. All immunologic analyses were performed blinded on serum collected at 7, 14, 21, 28, 56, 84 days, and 6 months post-boost injection for D614G and Beta seroneutralizations; on D14, 56, 84, and 6 months for Delta; on D14 and 6 months for Omicron (BA.1), Omicron BA.4/5, and SARS-CoV1. Animal studies were conducted in compliance with all relevant local, state, and federal regulations, and were approved by the New Iberia Research Center.

Pseudovirus-based virus neutralization assays

Serum samples were diluted 1:4 or 1:20 in media (FluoroBrite™ phenol-red-free DMEM + 10% FBS +  10 mM HEPES + 1% PS + 1% GlutaMAX™) and heat-inactivated at 56 °C for 30 min. Further, a twofold, 11-point, dilution series of the heat-inactivated serum were performed in media. Diluted serum samples were mixed with reporter-virus particle (RVP)-GFP (Integral Molecular) listed in Table 1 diluted to contain ~300 infectious particles per well and incubated for 1 h at 37 °C. Ninety-six well plates of ~50% confluent 293T-hsACE2 clonal cells (Integral Molecular, Cat# C-HA102) in 75 µL volume were inoculated with 50 µL of the serum + virus mixtures and incubated at 37 °C for 72 h. At the end of the 72-h incubation, plates were scanned on a high-content imager and individual GFP-expressing cells were counted. The neutralizing antibody titer was reported as the reciprocal of the dilution that reduced the number of virus plaques in the test by 50%.

Table 1 Description of the reporter-virus particles (RVPs) used in the pseudovirus-neutralization assay

Enzyme-linked immunosorbent assay (ELISA)

Nunc microwell plates were coated with SARS-CoV S-GCN4 protein (GeneArt, expressed in Expi 293 cell line) at 0.5 µg/mL in PBS at 4 °C overnight. Plates were washed three times with PBS–Tween 0.1% before blocking with 1% BSA in PBS–Tween 0.1% for 1 h. Samples were heat-inactivated at 56 °C for 30 min and plated at a 1:450 initial dilution followed by threefold, seven-point serial dilutions in blocking buffer. Plates were washed three times after 1-h incubation at room temperature before adding 50 µL of 1:8000 Goat anti-human IgG (Jackson Immuno Research, CAT# 109-036-098) to each well. Plates were incubated at room temperature for 1 h and washed thrice. Plates were developed using Pierce 1-Step Ultra TMB-ELISA Substrate Solution for 6 min and stopped by TMB STOP solution. Plates were read at 450 nm in a SpectraMax® plate reader, and the data analyzed using Softmax® Pro 6.5.1 GxP software and the proprietary software, Sanofi Universal Exporter 2.1. Antibody titers were reported as the highest dilution that is equal to 0.2-OD cutoff.

Enzyme-linked immunospot (ELISpot)

Memory B cells were analyzed using Human IgG Single-color B cell ELISpot kit (CTL, CAT# NC1911372). Cryo-preserved PBMCs were quickly thawed in a 37 °C water bath. A Fetal Calf Serum (FCS) /DNAse I (200 unit/mL) mixture was slowly added to PBMCs, before being transferred to a complete cell culture medium (CM) (RPMI 1640 with 10% FCS and antibiotic cocktail). After centrifugation and resuspension into 6 mL of CM, PBMCs were transferred into 6-well plates and incubated at 37 °C with 5% of CO2 for 1 h. Then, B-Poly-STM was added at 1:1000 dilution for cell stimulation, for up to 4 days at 37 °C with 5% of CO2.

Pre-stimulated PBMCs were harvested and centrifuged at 433 × g for 5 min at room temperature (RT). After washing, PBMCs were counted using Guava® easyCyte cell counter and the cells were adjusted to the desired concentration with CM.

Ninety-six well-plates with PVDF membrane were permeabilized with 15 μl of 70% ethanol for a maximum of 1 min, then washed three times with sterile phosphate-buffered saline (PBS) 1X before being coated with 80 µL of human Ig capture antibody (Ab) diluted at 1:50 or with SARS-CoV2 S-GCN4 protein, Wuhan, (GeneArt, expressed in Expi 293 cell line) at 4 µg/mL, or with PBS. The plates were incubated overnight at 4 °C and then washed three times with sterile PBS and blocked with CM for 1 h at RT. CM was then removed and PBMCs were added at 3 × 105 cells in the S-GCN4 protein and PBS-coated wells, and at 5 × 103 cells in the Ig capture Ab-coated wells, under 100 μL/well. Each condition was tested in duplicate, and plates were incubated for 18 h at 37 °C with 5% CO2.

To reveal antibody-secreting cells, plates were first washed twice with PBS, then twice with 0.05% Tween PBS, and then 80 µL of anti-human IgG detection solution was added. After 2 h of incubation at RT, plates were washed three times with 0.05% Tween PBS, and 80 µL of a tertiary solution was added. Plates were incubated for 1 h at RT, then washed twice with 0.05% Tween PBS and twice with distilled water. Eighty microliters of a blue developer solution was added and incubated at RT for 15–20 min. The reaction was stopped by rinsing the plate membrane with water and decanting it three times. The plates were air-dried, then scanned and read using Cytation 7 analyzer. The number of spots with the PBS only (background) was subtracted from the number of S-specific or total IgG spots. The results are expressed as S-specific IgG-secreting memory B cells/million PBMCs and as % of S-specific IgG-secreting memory B cells among all circulating IgG-secreting memory B cells.

Statistical analyses

For both mRNA-primed and CoV2 preS dTM-AS03-primed cohorts, at the time of the assignment, the characteristics at baseline (sex, age, and weight) were balanced to have comparable groups. The pseudovirus-neutralizing titers were also taken into account as well as the previous vaccine groups. ELISA titers and neutralizing titers were log10-transformed prior to statistical analysis.

Due to the differences in species and gender repartition between both cohorts, no direct comparison was performed on immune responses between the two cohorts.

The time-effects post-booster (nAb decay) were modeled by-product for a given PsV variant using a linear regression model or quadratic model with plateau when applicable. If the decay was linear for all the products, an ANCOVA (Analysis of Covariance) model was used, the product was considered as a categorical factor and the time a continuous and repeated factor. When the interaction between group and time was statistically non-significant (<10%), meaning that all products have the same behavior according to the time, a common slope model was used. When the interaction was statistically significant, the slopes were calculated for each product separately. The models were considered acceptable if the studentized residual distribution was considered normal.

Analyzes were performed using SEG SAS v9.4®.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.


Leave a Reply

Your email address will not be published. Required fields are marked *