key: cord-1007314-zxnyi98u
authors: Nanishi, Etsuro; Borriello, Francesco; O’Meara, Timothy R.; McGrath, Marisa E.; Saito, Yoshine; Haupt, Robert E.; Seo, Hyuk-Soo; van Haren, Simon D.; Brook, Byron; Chen, Jing; Diray-Arce, Joann; Doss-Gollin, Simon; Leon, Maria De; Chew, Katherine; Menon, Manisha; Song, Kijun; Xu, Andrew Z.; Caradonna, Timothy M.; Feldman, Jared; Hauser, Blake M.; Schmidt, Aaron G.; Sherman, Amy C.; Baden, Lindsey R.; Ernst, Robert K.; Dillen, Carly; Weston, Stuart M.; Johnson, Robert M.; Hammond, Holly L.; Mayer, Romana; Burke, Allen; Bottazzi, Maria E.; Hotez, Peter J.; Strych, Ulrich; Chang, Aiquan; Yu, Jingyou; Barouch, Dan H.; Dhe-Paganon, Sirano; Zanoni, Ivan; Ozonoff, Al; Frieman, Matthew B.; Levy, Ofer; Dowling, David J.
title: Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity
date: 2021-05-21
journal: bioRxiv
DOI: 10.1101/2021.05.20.444848
sha: d04caf87f507096a9080756e900ecc378f2a1695
doc_id: 1007314
cord_uid: zxnyi98u

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic especially for low- and middle-income countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide (AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD neutralizing Ab titers in both age groups (∼80-fold over AH), and protected aged mice from the SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and synergistically enhanced cytokine and chemokine production in human young adult and elderly mononuclear cells. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. One Sentence Summary Alum and CpG enhance SARS-CoV-2 RBD protective immunity, variant neutralization in aged mice and Th1-polarizing cytokine production by human elder leukocytes.

In order to assess the translational relevance of an adjuvant formulation it is key to confirm its 240 ability to activate human immune cells. To this end, we stimulated human peripheral blood 241 mononuclear cells (PBMCs) isolated from young adults (18-40 years old) and elder adults (≥65 242 years old) with CpG, AH, and the admixed AH:CpG formulation and measured cytokine and 243 chemokine production. Whereas AH induced limited or no cytokine production, both CpG alone 244 and AH:CpG activated young adult and elderly PBMCs in a concentration-dependent manner 245 (Fig 7A-D, Fig S2) . PBMCs of both age groups treated with AH:CpG produced significantly 246 higher levels of various proinflammatory cytokines and chemokines than those treated with CpG 247 alone (Fig 7A-D) . Of note, CpG and AH synergistically induced IL-6, IL-10, TNF, CCL3, and 248 GM-CSF production in both young adult and elderly PBMCs, as defined mathematically (D 249 value, see Methods) (Fig 7C, D, Fig S2) . 250 strategy to address this urgent need. Here, we performed a comprehensive head-to-head 257 comparison of multiple adjuvants in age-specific in vivo and ex vivo animal models, along with 258 age-specific human in vitro screening, to determine the appropriate adjuvant for a SARS-CoV-2 259 RBD vaccine in the young and the aged, focusing on the innate and humoral immune response 260 reported to align best with known correlates of protection (50, 51). We found that the AH:CpG 261 adjuvant formulation enhances anti-RBD neutralizing Ab titers and type 1 immunity (i.e. IgG2a 262 switching, Th1 polarization) in both age groups. Aged mice immunized with AH:CpG are 263 protected from live SARS-CoV-2 challenge. Of note, RBD adjuvanted with AH:CpG elicited 264 levels of neutralizing Abs comparable to the clinical-grade BNT162b2 Spike mRNA vaccine. 265

The translational relevance of our findings is also highlighted by the synergistic activation of 266 human PBMCs from older individuals upon stimulation with AH:CpG. Overall, our results 267 expand upon recent preclinical and clinical studies on the enhanced immunogenicity of Spike 268 formulated with AH:CpG by showing that a vaccine composed of RBD and AH:CpG can also 269 induce a robust anti-SARS-CoV-2 immune response across different age groups. Since an RBD 270 antigen is amenable to high-yield manufacturability (52-54), our study also supports the 271 development of RBD formulated with AH:CpG as an affordable and accessible vaccine. 272 273 Among various AH:PRR agonist formulations, AH:CpG elicited the highest immune responses 274 in both young and aged mice. We observed that vaccine immune responses were generally lower 275 in aged mice than in young adult mice, even in the group receiving RBD formulated with 276 AH:CpG. While the lower levels of anti-RBD Abs observed in aged mice are likely sufficient for 277 protection, we found that an additional booster dose in the aged overcame the observed age-278 dependent reductions in vaccine response and protected aged mice from SARS-CoV-2 challenge. 279

We employed AH, which has been used for >90 years with a firmly established record of safety 280 and efficacy (32) and AS01B, which recently demonstrated excellent adjuvant effects among 281 elderly humans (33, 34), as "benchmarking" adjuvants to compare the exploratory adjuvanted 282 formulations with more established adjuvants. In this context, we demonstrated that the AH:CpG 283 adjuvanted vaccine was superior to a vaccine adjuvanted only with AH and was non-inferior to 284 AS01B. In the context of the aged mice prime-boost setting, AH:CpG-adjuvanted SARS-CoV-2 285 RBD significantly outperformed AS01B with respect to functional anti-RBD inhibition 286 (Geometric mean (GM) with SD, 57±2% vs. 14±3%) and neutralizing Abs titers (2344 ± 7 vs. 287 elderly PBMCs in vitro. These data indicate that formulating RBD with AH:CpG represents a 367 promising approach to develop a practical (e.g., not requiring low temperature storage), scalable, 368 effective, and affordable vaccine that may be effective across multiple age groups and could 369 potentially incorporate multiple RBD proteins to achieve cross-strain protection. 370 vitro models. Sample size and age criteria was chosen empirically based on results of previous 376 studies. Mouse experiments aimed to include in total 10 mice per group and were combined from 377 two individual experiments. Mice were randomly assigned to different treatment groups. In order 378 to assess the translational relevance and potential mechanism of an adjuvant formulation, we 379 designed human in vitro study with peripheral blood collected from healthy young adults, aged 380 18-40 y (n = 6), and older participants, aged Human PBMC isolation. PBMCs were isolated based previously described protocols (77). 545

Briefly, heparinized whole blood was centrifuged at 500 g for 10 min, then the upper layer of 546 platelet-rich plasma was removed. Plasma was centrifuged at 3000 g for 10 min, and platelet-547 poor plasma (PPP) was collected and stored on ice. The remaining blood was reconstituted to its 548 original volume with heparinized DPBS and layered on Ficoll-Paque gradients (Cytiva) in 549

Accuspin tubes (Sigma-Aldrich). PBMCs were collected after centrifugation and washed twice 550 with PBS. were analyzed by one-or two-way ANOVAs followed by post-hoc Tukey's test or Dunnett's test 570 for multiple comparisons. Non-normally distributed data were log-transformed. In the animal 571 experiences, time to event were analyzed using Kaplan-Meier estimates and compared across 572 groups using the Log-rank test. For human in vitro PBMC assay, unpaired Mann-Whitney tests 573 were applied at each concentration. We conducted gene expression analyses with R 4.0.4 using 574 packages 'ggplot2', 'dplyr', and 'MASS' for the transcript abundance determination of gene arrays in each group. We log-transformed data before performing principal component analysis 576 (PCA) and unsupervised hierarchical clustering using R packages 'prcomp' and 'pheatmap' 577 respectively. We analyzed the differential gene expression using generalized linear models 578 (GLMs) with treatment and age as fixed effects. We then enriched the differentially expressed 579 genes using the blood transcriptional module method based on an existing protocol (Li et 

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

SARS-CoV-2 mRNA vaccine design 617 enabled by prototype pathogen preparedness

Safety, 680 tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy 681 adults aged 18-59 years: a randomised, double-blind, placebo-controlled, phase 1/2 682 clinical trial

CoV-2 vaccines strategies: a comprehensive review of phase 3 candidates

Biological E's novel Covid-19 vaccine of SARS-CoV-2 for protection against

Covid-19 disease

Emerging concepts in the science of vaccine 773 adjuvants

The continued advance of vaccine adjuvants -775 'we can work it out'

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific

CpG Oligonucleotides as Vaccine

TLR7/8 adjuvant overcomes newborn 932 hyporesponsiveness to pneumococcal conjugate vaccine at birth

A 954 SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of

ACE2-spike protein-protein interaction

Cervini for their 980 support of the Precision Vaccines Program. We thank Dr. Barney S. Graham (NIH Vaccine hACE2/RBD inhibition rate, and (F) anti-Spike IgG were assessed. N=10 per group. Data were 1041 combined from two individual experiments. (G-J) Serum samples were collected on Day 210, 1042 and (G) Anti-RBD IgG

Blue and red colored asterisks respectively indicate 1046 comparisons to RBD and AH adjuvanted RBD groups. Box-and-whisker plots represent the 1047 minimum, first quartile, median, third quartile, and maximum value. LLD, lower limit of 53 /mL)

IL-4) and 96 (for IFNγ) hours, supernatants were harvested and cytokine levels were 1061 measured by ELISA. N=4-5 per group

ANOVAs followed by post-hoc Tukey's test for multiple comparisons. *P <0.05, **P <0

Blue and red colored asterisks respectively indicate comparisons 1064 to RBD and AH adjuvanted RBD groups. Box-and-whisker plots represent the minimum

AH:CpG-adjuvanted vaccine protects aged mice from SARS-CoV-2 challenge

14-month-old BALB/c mice were immunized as in Figure 2. On Day 70 (6 weeks post 2 nd 1082 boost), mice were challenged IN with 10 3 PFU of mouse-adapted SARS-CoV-2 (MA10)

Data represent mean and 1084 SEM with body weights shown for surviving mice at each time-point (one mouse in RBD group 1085 died at 4 days post infection). Data were analyzed by one-way ANOVA followed by Dunnett's 1086 Test for comparisons between PBS group. (B) Viral titer in lung homogenates at 4-days post 1087 1095 Figure 5. AH:CpG-adjuvanted RBD vaccines and an authorized spike mRNA vaccine

Aged, 14-month-old BALB/c mice were immunized IM on Days 0 and 14 with monomeric

Anti-RBD 1100 binding ELISA, (B) anti-Spike binding ELISA, (C) hACE2/RBD inhibition rate, and (D) SARS-B.1.351 variants were assessed. N=5 per group. The numbers indicate GMT. Each symbol 1105 represents an animal. *P <0.05, **P <0.01, ***P <0.001, **** P <0.0001. Blue colored asterisks 1106 indicate comparisons to PBS group. Box-and-whisker plots represent the minimum, first quartile, 1107 median, third quartile, and maximum value. LLD, lower limit of detection. 1108 containing treatments

Generalized linear model comparing treatment and age with each gene was performed. The top 4 1122 significant genes (Ddx58, Ifit2, Isg15, Stat1) were selected and plotted with their relative 1123 expression values by age and treatment

Wallis test to compare mean differences across groups and Wilcoxon test to compare between 1125 ages. (E) Enrichment analysis of differentially expressed genes using the blood transcriptional 1126 modules

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