key: cord-1044129-9qzo18v3
authors: Wang, Yunfei; Wang, Lichun; Cao, Han; Liu, Cunbao
title: SARS‐CoV‐2 S1 is superior to the RBD as a COVID‐19 subunit vaccine antigen
date: 2020-07-21
journal: J Med Virol
DOI: 10.1002/jmv.26320
sha: fe38bf4b1634ae92335ae8169cb300c5665ea67f
doc_id: 1044129
cord_uid: 9qzo18v3

Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has developed into a global pandemic within a matter of months. While subunit vaccines are one of the prominent options for combating coronavirus disease 2019 (COVID‐19), the immunogenicity of spike protein‐based antigens remains unknown. When immunized in mice, the S1 domain induced much higher IgG and IgA antibody levels than the RBD and more efficiently neutralized SARS‐CoV‐2 when adjuvanted with alum. It is inferred that a large proportion of these neutralization epitopes are located in the S1 domain but outside the RBD and that some of these are spatial epitopes. This finding indicates that expression systems with posttranslational modification abilities are important to maintain the natural configurations of recombinant spike protein antigens and are critical for effective COVID‐19 vaccines. Further, adjuvants prone to a Th1 response should be considered for S1‐based subunit COVID‐19 vaccines to reduce the potential risk of antibody‐dependent enhancement (ADE) of infection. This article is protected by copyright. All rights reserved.

Biology, Chinese Academy of Medical Sciences & Peking Union Medical College (IMB, CAMS). Animals were randomly divided into 5 groups with 6 mice in each group (N=6). Antigens were diluted to 10 μg/mouse/dose in 25 μL of PBS and mixed with the same volume of alum adjuvant (ThermoFisher Scientific) prior to immunization. Thus, 50 μL of immunogens were administered intramuscularly into the thigh muscle three times at 2 week intervals. 2 weeks after the final immunization, mice were anesthetized with ketamine, and blood was collected via cardiac puncture. After clotting at 4 °C overnight, serum was collected by centrifugation at 3 000 rpm for 10 min and pooled by group.

All experiments were performed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals of the Animal Ethics Committee of the IMB, CAMS (permit number: SCXK (dian) K2017-0002).

Ninety-six-well plates were coated with 2 μg/ml HEK293K cell-expressed recombinant SARS-CoV-2 S1 or RBD proteins overnight at 4 °C. Plates were washed one time with wash buffer (PBS containing 0.05% (v/v) polysorbate 20) and then blocked with 5% (w/v) skim milk dissolved in wash buffer for 1 hour at 37 °C. Plates were then washed 4 times and incubated with serially diluted mouse sera for 1 hour at 37 °C. Next, plates were washed 5 times and incubated with goat anti-mouse IgG/IgA/IgG1/IgG2a HRP-conjugated secondary antibodies This article is protected by copyright. All rights reserved.

Article (ThermoFisher Scientific) for 1 hour at 37 °C. Following 5 additional washes, 3,3',5,5'-tetramethylbenzidine (TMB, BD Bioscience) substrate was added. The plate was incubated at room temperature in the dark for 10 min, and reactions were stopped by the addition of 2 M sulfuric acid. Absorbance (450 nm) was detected using a microplate reader (Bio-Tek Instruments, Inc). Antibody titers were defined by end-point dilution with a cut-off signal of OD450=0.1. Sera samples that did not produce an OD>0.1 at 1:500 were determined as 0.

The IgG1-to-IgG2a titer ratio was calculated to evaluate Th1-Th2 balance 28,29 . For neutralization, mouse sera were diluted with DMEM in a two-fold series.

Then, 50 μL of SARS-CoV-2 diluted with DMEM to 3.3 lg CCID 50 was added to 50 μL of diluted serum, incubated at 37 °C for 1 hour, and then added to 100 μL of

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Article CO 2 for 4 days, the neutralization titer was reported as the serum dilution at which SARS-CoV-2 infection was inhibited by 50%.

All SARS-CoV-2 manipulations were carried out in a biosafety level 3 (BSL-3) laboratory at IMB, CAMS.

Data are shown as the mean and standard deviation. GraphPad Prism 7.0 (San Diego, CA, USA) was used for statistical analyses.

Here, we fused S1 and RBD to the carboxyl terminus of the norovirus shell domain, which has been reported to present recombinant expressed proteins on the surface of virus-like particles to enhance the immunity of recombinant proteins 23, 24 . While both RBD and S1 were expressed well with the norovirus shell domain (S-RBD and S-S1, respectively), as certified by the corresponding band (~52 kDa for S-RBD and ~108 kDa for S-S1) by SDS-PAGE ( Figure 1A ) and western blot ( Figure 1B ), both were expressed as inclusion bodies. Following sonication, washing and dialysis, while S-RBD showed quite high purity (lane S-RBD in Figure 1A ), S-S1 showed only approximately 60% purity by SDS-PAGE (lane S-S1 in Figure 1A ). Transmission electron microscopy showed that after dialysis, only a small portion of the S-RBD and S-S1 fusion proteins formed similar but not identical virus-like particles with diameters of approximately 30-60 nm (showed by arrows in Figure 1C & 1D) , while the majority of these recombinant proteins formed irregular aggregates ( Figure 1C & 1D) . This article is protected by copyright. All rights reserved.

Article SARS-CoV-2 S1 induces higher IgG and IgA titers than RBD 2 weeks after the third intramuscular immunization (Figure 2A) , both S1-specific (S1-coated plate in Figure 2 ) and RBD-specific (RBD-coated plate in Figure 2 ) antibodies were analyzed.

HEK293K cell-expressed recombinant S1 (S1 immunized) and E.coli-expressed norovirus shell domain-S1 fusion protein (S-S1 immunized) induced similar S1-specific IgG titers (64 000) and similar RBD-specific titers (8 000) ( Figure 2B ). HEK293K cell-expressed recombinant RBD (RBD immunized) induced low S1-specific IgG titers (8 000) and RBD-specific IgG titers (6 000), implying low immunogenicity of the RBD alone. Unlike S1 (similar IgG titers between S1 and S-S1 may be attributed to low purity and thus low S1 content in S-S1), fusion of the RBD with the norovirus shell domain (S-RBD immunized) elevated both RBD-specific IgG titers (from 6 000 to 32 000) and S1-specific IgG titers (from 8 000 to 32 000).

As SARS-CoV-2 is a respiratory virus, mucosal immunity is important to fight infection and we therefore detected IgA titers ( Figure 2C ). While both HEK293K cell-expressed recombinant S1 (S1 immunized) and E.coli-expressed norovirus shell domain-S1 fusion protein (S-S1 immunized) induced equivalent levels of S1-specific IgA titers and IgG titers (64 000), both HEK293K cell-expressed recombinant RBD (RBD immunized) and E.coli-expressed norovirus shell domain-RBD fusion proteins (S-RBD immunized) induced half the level of S1-specific IgA titers as IgG titers (4000 vs 8000 for RBD and 16 000 vs 32 000 for S-RBD, respectively). While RBD-specific IgA titers were the lowest of all the IgG and IgA titers tested, there was a tendency for norovirus shell domain to elevate RBD immunogenicity on RBD-specific IgA titers (RBD coated, S-RBD This article is protected by copyright. All rights reserved.

Article immunized vs RBD immunized in Figure 2C ), as was observed for the S1-specific IgA titers (S1-coated, S-RBD immunized vs RBD immunized in Figure 2C ). SARS-CoV-2 S1 induced more balanced Th1-Th2 responses than the RBD Similar to S1-specific total IgG titers ( Figure 2B , S1-coated), both HEK293K cell-expressed recombinant S1 (S1 immunized) and E.coli-expressed norovirus shell domain-S1 fusion protein (S-S1 immunized) induced the highest S1-specific IgG1 ( Figure 3A , S1 coated) and IgG2a ( Figure 3B , S1 coated) titers and comparably low RBD-specific IgG1 ( Figure 3A , RBD coated) and IgG2a ( Figure   3B , RBD coated) titers. HEK293K cell-expressed recombinant RBD (RBD immunized) and E.coli-expressed norovirus shell domain-RBD fusion proteins (S-RBD immunized) induced low levels of IgG1 and IgG2a titers specific to both S1 (S1 coated in Figure 3A&B ) and the RBD (RBD coated in Figure 3A&B ).

Notably, the IgG1 titers in each group in Figure 3A are considerably higher than the IgG2a titers in Figure 3B . To enable direct comparisons between groups, we compared the IgG1/IgG2a ratios in each group induced by their own antigens ( Figure 3C ). While both HEK293K cell-expressed recombinant S1 (S1 immunized) and E.coli-expressed norovirus shell domain-S1 fusion proteins (S-S1 immunized) induced an IgG1/IgG2a ratio of 4, HEK293K-expressed recombinant RBD (RBD immunized) induced an IgG1/IgG2a ratio as high as 16. This ratio could be lowered by ligation to the E.coli-expressed norovirus shell domain (the S-RBD immunized IgG1/IgG2a ratio was 8) but was still higher than in that in the S1 and S-S1 immunized groups. Higher IgG1-to-IgG2a ratios, including the those for the S1 and S1-RBD groups, imply a Th2-biased immune response for these antigens.

IgG2a subtype antibodies, which are present in neutralizing sera 49 . This result is consistent with the observed eosinophilic infiltration following vaccination and virus exposure, a typical characteristic of Th2 immune responses with elevated IgG1/IgG2a proportions 28,29 . Unfortunately, both SARS-CoV-2 S1 and RBD showed a Th2-like immune response with high proportions of IgG1 when immunized with alum as adjuvant (Figure 3) , implying a similar immunopathological risk to those reported for other coronaviruses. Though no ADE has been reported in animal models re-exposed to SARS-CoV-2 or exposed following vaccine immunization, special attention should be paid to Th1-biased adjuvants, as reported in the development of SARS-CoV-1 and MERS-CoV vaccines 31,32 .

In conclusion, the SARS-CoV-2 S1 domain is more immunogenic than the RBD domain, inducing higher IgG and IgA antibodies and also efficient virus neutralization antibodies. We infer that a large proportion of these neutralization epitopes exist within the S1 domain but outside of the RBD and that some of these are spatial epitopes. While S1 induced a more balanced Th1/Th2 response than the RBD when adjuvanted with alum, increased levels of IgG1 antibodies still indicate a potential risk of ADE, and adjuvants prone to a Th1 response should be considered for S1 subunit-based COVID-19 vaccines.

No potential conflicts of interest.

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