Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and synergistic activation of human elder type 1 immunity

TL;DR: In this paper, the aluminum hydroxide (AH) and CpG adjuvant formulation (AH:CpG) was used to enhance the anti-RBD immunogenicity of the SARS-CoV-2 vaccine.

Abstract: 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.

Summary

INTRODUCTION

• The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a serious threat to humanity.
• To this end, alternative platforms such as inactivated and protein subunit SARS-CoV-2 vaccines have entered different stages of clinical development and in some cases have already been deployed at the population level (11) (12) (13) (14) (15) (16) (17) .
• Adjuvant formulations of aluminum salts and PRR agonists enhance vaccine immune responses compared to aluminum salts or PRR agonists alone (32) .

AH:CpG-formulated RBD vaccine is immunogenic in aged mice

• To assess the vaccine response in the context of aging, the immunogenicity of RBD vaccines adjuvanted with AH:PRR agonists was further studied in aged mice (14-month-old) .
• Similar to young mice, the AH:CpG formulation also elicited the highest humoral immune response after prime-boost immunization in aged mice (Fig 2A-F ).
• Of note, the vaccine adjuvanted with AH:CpG produced significantly higher hACE2/RBD inhibition and neutralizing titers compared to the vaccine adjuvanted with AS01B, which is known as a potent adjuvant in the human elderly population (33, 34) (.

AH:CpG-formulated RBD vaccine protects aged mice from lethal viral challenge

• Abs are key to protecting from SARS-CoV-2 infection.
• Since RBD formulated with AH:CpG elicited high titers of neutralizing Abs, the authors assessed the protection of immunized mice in a challenge model.
• To this end, the authors employed the mouse-adapted SARS-CoV-2 MA10 virus strain (35) .

AH:CpG-formulated RBD and Spike mRNA vaccines elicit comparable levels of neutralizing antibodies against wild type SARS-CoV-2 and variants

• Recently, it has been reported that SARS-CoV-2 mRNA vaccines are more immunogenic than RBD adjuvanted with oil-in-water emulsions (36) .
• Along with CpG-2395, the authors also tested CpG-1018, which is included in the Heplisav-B vaccine and has also been tested in combination with Spike/RBD and AH in SARS-CoV-2 studies including human vaccine trials (12, 16, 37) .
• In accordance with previously published data, the mRNA vaccine was highly immunogenic, while RBD formulated with AddaS03 failed to induce (38) (39) (40) (41) .
• A recent report showed that the mRNA BNT162b2 vaccine maintained its effectiveness against severe COVID-19 with the B.1.351 variant at greater than 90% (42) .
• As expected, the authors observed reduced titers against the variants, especially against the B.1.351 (Fig.

Innate signaling potentiated by AH:CpG formulation is well preserved in aged mice

• Lymph nodes (LNs) are critical sites for the interaction between innate and adaptive immune systems and orchestrate the development of vaccine immune responses (43, 44) .
• To gain further insights into the mechanism of action of the AH:CpG formulation, the authors collected draining LNs (dLNs) 24 hours post injection of AH:CpG or either adjuvant alone.
• CpG induced comparable dLN expansion in both age groups (Fig, also known as CpG and AH.

AH:CpG synergistically enhances proinflammatory cytokines from human elderly PBMCs

• To this end, the authors stimulated human peripheral blood mononuclear cells isolated from young adults was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (which this version posted May 21, 2021.

DISCUSSION

• The risk of COVID-19-related morbidity and mortality increases with age (47, 48) .
• Of note, RBD adjuvanted with AH:CpG elicited levels of neutralizing Abs comparable to the clinical-grade BNT162b2 Spike mRNA vaccine.
• Was not certified by peer review) is the author/funder.

MATERIALS AND METHODS

• The aim of this study was to assess optimal combinations of RBD antigen and adjuvants in pre-clinical models that take age-dependent vaccine immune responses and COVID-19 susceptibility into account.
• To this end, the authors used age-specific mouse in vivo and human in vitro models.
• Affinity tags were cleaved off from eluted protein samples by HRV 3C protease, and tag removed proteins were further purified by size-exclusion chromatography using a Superose 6 10/300 column for full length Spike and a Superdex 75 10/300 Increase column for RBD domain in a PBS buffer (pH 7.4).
• Each PRR agonist was formulated with and without aluminum hydroxide.
• Blood samples were collected 2 weeks post-immunization.

Adjuvants and immunization. The

• Briefly, high-binding flat-bottom 96-well plates (Corning, NY) were coated with 50 ng/well RBD or 25 ng/well Spike and incubated overnight at 4 °C.
• Plates were washed three times and incubated for 1 hour at RT with HRP-conjugated anti-mouse IgG, IgG1, IgG2a, or IgG2c (Southern Biotech).
• The copyright holder for this preprint (which this version posted May 21, 2021.
• Each serum sample was diluted 1:160, pre-incubated with 3 ng of RBD-Fc in 1% BSA PBS for 1 hour at RT, and then transferred to the hACE2-coated plate.
• The optical density was read at 450 nm with SpectraMax iD3 microplate reader (Molecular Devices).

SARS-CoV-2 neutralization titer determination.

• All serum samples were heat-inactivated at 56°C for 30 min to remove complement and allowed to equilibrate to RT prior to processing for neutralization titer.
• Samples were diluted in duplicate to an initial dilution of 1:5 or 1:10 followed by 1:2 serial dilutions (vaccinated sample), resulting in a 12-dilution series with each was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (which this version posted May 21, 2021.
• BioRxiv preprint well containing 100 µL. Dilution plates were then transported into the BSL-3 laboratory and 100 µL of diluted SARS-CoV-2 (WA-1, courtesy of Dr. Natalie Thornburg/CDC) inoculum was added to each well to result in a multiplicity of infection (MOI) of 0.01 upon transfer to titering plates, also known as /10.1101/2021.05.20.444848 doi.

Pseudovirus neutralization assay.

• The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to as described previously (75, 76) .
• To determine the neutralization was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (which this version posted May 21, 2021.
• Three-fold serial dilutions of heat inactivated serum or plasma samples were prepared and mixed with 50 µL of pseudovirus.

Splenocyte restimulation assay.

• Immunized mice were sacrificed 2 weeks after the final immunization, and spleens were collected.
• To isolate splenocytes, spleens were mashed through a 70 µm cell strainer, and the resulting cell suspensions were washed with PBS and incubated with 2 mL of ACK lysis buffer for 2 minutes at RT to lyse erythrocytes.
• Splenocytes were washed again with PBS and plated in flat-bottom 96-well plates (2 x 10 6 cells per well).
• Then, SARS-CoV-2 Spike peptides (PepTivator SARS-CoV-2 Prot_S, Miltenyi Biotec) were added at a final concentration of 0.6 nmol/ml in the presence of 1 μg/mL anti-CD28 antibody (total cell culture volume, 200 µL per well).
• After 24 (for IL-2 and IL-4) and 96 (for IFNγ) hours, supernatants were harvested, and cytokine levels were measured by ELISA according to the manufacturer's protocol.

SARS-CoV-2 mouse challenge study. Mice were anesthetized by intraperitoneal injection 50

• Mice were then intranasally inoculated with 1 x 10 3 PFU of mouse-adapted SARS-CoV-2 (MA10, was not certified by peer review) is the author/funder.
• The copyright holder for this preprint (which this version posted May 21, 2021.
• SARS-CoV-2 lung titers were quantified by homogenizing harvested lungs in PBS (Quality Biological Inc.) using 1.0 mm glass beads (Sigma Aldrich) and a Beadruptor (Omni International Inc.).
• A pathologist was blinded to information identifying the treatment groups and fields were examined by light microscopy and analyzed.
• The severity of interstitial inflammation was evaluated and converted to a score of 0-4 with 0 being no inflammation and 4 being most severe.

Full text

1
Title: Alum:CpG adjuvant enables SARS-CoV-2 RBD-induced protection in aged mice and 1
synergistic activation of human elder type 1 immunity 2
3
One Sentence Summary: Alum and CpG enhance SARS-CoV-2 RBD protective immunity, 4
variant neutralization in aged mice and Th1-polarizing cytokine production by human elder 5
leukocytes. 6
7
Authors: Etsuro Nanishi
1, 2†
, Francesco Borriello
1, 2, 3†
, Timothy R. O’Meara
1‡
, Marisa E. 8
McGrath
4‡
, Yoshine Saito
1
, Robert E. Haupt
4
, Hyuk-Soo Seo
5, 6
, Simon D. van Haren
1, 2
, Byron 9
Brook
1, 2
, Jing Chen
7
, Joann Diray-Arce
1, 2
, Simon Doss-Gollin
1
, Maria De Leon
1
, Katherine 10
Chew
1
, Manisha Menon
1
, Kijun Song
5
, Andrew Z. Xu
5
, Timothy M. Caradonna
8
, Jared 11
Feldman
8
, Blake M. Hauser
8
, Aaron G. Schmidt
8, 9
, Amy C. Sherman
1, 10
, Lindsey R. Baden
10
, 12
Robert K. Ernst
11
, Carly Dillen
4
, Stuart M. Weston
4
, Robert M. Johnson
4
, Holly L. Hammond
4
, 13
Romana Mayer
12
, Allen Burke
12
,
Maria E. Bottazzi
13, 14
, Peter J. Hotez
13, 14
, Ulrich Strych
13, 15
, 14
Aiquan Chang
16
, Jingyou Yu
16
, Dan H. Barouch
16
, Sirano Dhe-Paganon
5, 6
, Ivan Zanoni
2, 3
, Al 15
Ozonoff
1, 2
,
Matthew B. Frieman
4§
, Ofer Levy
1, 2, 17§
, David J. Dowling
1, 2§
* 16
17
Affiliations: 18
1
Precision Vaccines Program, Division of Infectious Diseases, Boston Children’s Hospital, 19
Boston, MA, USA. 20
2
Department of Pediatrics, Harvard Medical School, Boston, MA, USA. 21
3
Division of Immunology, Boston Children’s Hospital, Boston, MA, USA. 22
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.444848doi: bioRxiv preprint

2
4
Department of Microbiology and Immunology, University of Maryland School of Medicine, 23
Baltimore, MD, USA. 24
5
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA. 25
6
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 26
Boston, MA, USA. 27
7
Research Computing Group, Boston Children’s Hospital, Boston, MA, USA. 28
8
Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA. 29
9
Department of Microbiology, Harvard Medical School, Boston, MA, USA. 30
10
Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA. 31
11
Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, 32
MD, USA. 33
12
Department of Pathology, University of Maryland Medical Center, Baltimore, MD, USA. 34
13
Texas Children's Hospital Center for Vaccine Development, Baylor College of Medicine, 35
Houston, TX, USA.
36
14
National School of Tropical Medicine and Departments of Pediatrics and Molecular Virology 37
& Microbiology, Baylor College of Medicine, Houston, TX, USA. 38
15
National School of Tropical Medicine and Department of Pediatrics, Baylor College of 39
Medicine, Houston, TX, USA. 40
16
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard 41
Medical School, Boston, MA, USA. 42
17
Broad Institute of MIT & Harvard, Cambridge, MA, USA.
43
44
†
These authors contributed equally to this manuscript. 45
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.444848doi: bioRxiv preprint

3
‡
These authors contributed equally to this manuscript. 46
§
Co-senior authors. 47
48
*Corresponding author: David J. Dowling, Precision Vaccines Program, Division of 49
Infectious Diseases, Boston Children’s Hospital; Harvard Medical School, Rm 842, Boston, MA 50
02115, USA. Tel: +1 617-919-6890. e-mail: david.dowling@childrens.harvard.edu 51
52
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.444848doi: bioRxiv preprint

4
ABSTRACT 53
Global deployment of vaccines that can provide protection across several age groups is still 54
urgently needed to end the COVID-19 pandemic especially for low- and middle-income 55
countries. While vaccines against SARS-CoV-2 based on mRNA and adenoviral-vector 56
technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 57
vaccines are needed to meet global demand. In this context, protein subunit vaccines formulated 58
with appropriate adjuvants represent a promising approach to address this urgent need. Receptor-59
binding domain (RBD) is a key target of neutralizing antibodies (Abs) but is poorly 60
immunogenic. We therefore compared pattern recognition receptor (PRR) agonists, including 61
those activating STING, TLR3, TLR4 and TLR9, alone or formulated with aluminum hydroxide 62
(AH), and benchmarked them to AS01B and AS03-like emulsion-based adjuvants for their 63
potential to enhance RBD immunogenicity in young and aged mice. We found that the AH and 64
CpG adjuvant formulation (AH:CpG) demonstrated the highest enhancement of anti-RBD 65
neutralizing Ab titers in both age groups (~80-fold over AH), and protected aged mice from the 66
SARS-CoV-2 challenge. Notably, AH:CpG-adjuvanted RBD vaccine elicited neutralizing Abs 67
against both wild-type SARS-CoV-2 and B.1.351 variant at serum concentrations comparable to 68
those induced by the authorized mRNA BNT162b2 vaccine. AH:CpG induced similar cytokine 69
and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and 70
aged mice and synergistically enhanced cytokine and chemokine production in human young 71
adult and elderly mononuclear cells. These data support further development of AH:CpG-72
adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups. 73
74
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.444848doi: bioRxiv preprint

5
INTRODUCTION 75
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory 76
syndrome coronavirus 2 (SARS-CoV-2) resulted in a serious threat to humanity. Rapid 77
deployment of safe and effective vaccines is proving key to reducing morbidity and mortality of 78
COVID-19, especially in high-risk populations such as the older adults (1). Novel vaccine 79
technologies including mRNA and adenoviral vector vaccines have dramatically accelerated the 80
process of vaccine development, shown high efficacy in preclinical and clinical studies, and 81
therefore been granted Emergency Use Authorization by the Food and Drug Administration (2-9). 82
Unfortunately, worldwide access to these vaccines may be limited by the need for ultra-cold 83
storage (mRNA vaccines), cost, and concerns regarding global scalability especially in the third 84
world (1). This situation not only represents a major ethical problem but may also promote the 85
emergence of vaccine-resistant SARS-CoV-2 strains due to high infection rates in unvaccinated 86
regions (10). Thus, ongoing efforts are needed to investigate additional affordable, easily 87
scalable, and effective vaccine approaches against SARS-CoV-2 to improve global access. To 88
this end, alternative platforms such as inactivated and protein subunit SARS-CoV-2 vaccines 89
have entered different stages of clinical development and in some cases have already been 90
deployed at the population level (11-17). These approaches may play an essential role in the 91
global fight against COVID-19 since they utilize well-established technologies, do not require 92
low temperature storage, and have proven safety and effectiveness in various age groups 93
including young children and the elderly. 94
95
With the exception of inactivated viruses, most SARS-CoV-2 vaccine candidates aim to target 96
the SARS-CoV-2 Spike glycoprotein, as it is required for binding to the human receptor 97
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 21, 2021. ; https://doi.org/10.1101/2021.05.20.444848doi: bioRxiv preprint

Frequently asked questions

Q1. What was the RNA used to determine the RNA content of dLNs?

(B–E) RNA isolated from dLNs was subjected to a quantitative real-1116 time PCR array comprised of 157 genes related to cytokines, chemokines, and type 1 IFN 1117 responses. 

Q2. What is the significance of the AH:CpG synergistically?

7. AH:CpG synergistically enhances proinflammatory cytokine production from 1130 human adult and elderly PBMCs 1131 Human PBMCs collected from young adult (A, C) and elderly individuals (B, D) were cultured 1132 in vitro for 24 h with CpG alone (4, 10, 20, 40, and 100 μg/mL), aluminum hydroxide (AH) 1133 alone (8, 20, 40, 80, and 200 μg/mL), or a combination of both. 

Q3. What is the role of CpG in the immune response of hepatitis B?

G. Sen, Q. Chen, C. M. Snapper, Immunization of aged mice with a pneumococcal 906 conjugate vaccine combined with an unmethylated CpG-containing oligodeoxynucleotide 907 restores defective immunoglobulin G antipolysaccharide responses and specific CD4+-T-908 cell priming to young adult levels. 

Q4. What was the ad hoc analysis of the sars-cov-2?

(A–F) Serum samples were 1039 collected on Day 28, and (A) Anti-RBD IgG, (B) IgG1, (C) IgG2a, (D) IgG2a/IgG1 ratio, (E) 1040 hACE2/RBD inhibition rate, and (F) anti-Spike IgG were assessed. 

Q5. What was the significant gene in the blood?

(E) Enrichment analysis of differentially expressed genes using the blood transcriptional 1126 modules (Li et al., 2013- PMC: 24336226) was performed from the significant gene results after 1127 the generalized linear model by treatment. 

Q6. What was the level of synergy calculated?

Level of 1139 synergy was calculated using an adapted Loewe definition of additivity (D <1: synergy, D=1: 1140 additivity, D >1: antagonism).