key: cord-0685586-ql30rv83
authors: Hou, Yu-xuan; Peng, Cheng; Han, Zheng-gang; Zhou, Peng; Chen, Ji-guo; Shi, Zheng-li
title: Immunogenicity of the spike glycoprotein of Bat SARS-like coronavirus
date: 2010-02-12
journal: Virol Sin
DOI: 10.1007/s12250-010-3096-2
sha: 82c629c5878c63023651e3d44f00885525ecdae8
doc_id: 685586
cord_uid: ql30rv83

A group of SARS-like coronaviruses (SL-CoV) have been identified in horseshoe bats. Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity, SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64% amino acid identity, suggesting there are fundamental differences between these two groups of coronaviruses. To gain insight into the basis of this difference, we established a recombinant adenovirus system expressing the S protein from SL-CoV (rAd-Rp3-S) to investigate its immune characterization. Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein. Moreover, a strong cellular immune response demonstrated by elevated IFN-γ and IL-6 levels was also observed in these mice. However, the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein, and did not neutralize HIV pseudotyped with SARS-CoV S protein. These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV, which may cause the immunological differences between human SARS-CoV and bat SL-CoV. Furthermore, the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.

genome organization and high sequence identities in gene products (92%-100%) with the main exception of the N-terminus of the spike glycoprotein (S protein) which shares only 64 % amino acid sequence identity, implying that the bat SL-CoV S protein has different properties from that of SARS-CoV [20] . The S protein of SARS-CoV is responsible for binding the cellular receptor, angiotensin-converting enzyme 2 (ACE2) [19] , and mediates viral entry [6, 16, 28] . Additionally, the S protein induces protective humoral and cellular responses in the host [4, 5, 10, 11, 13-15, 21, 32, 34, 37] . For the bat SL-CoV S protein, by using the HIV-pseudotyped SARS-CoV and SL-CoV S protein, we have demonstrated that SL-CoV could not use ACE2 as its receptor.

However, the replacement of the SARS-CoV receptor binding domain (RBD) to SL-CoV S protein could convert the SL-CoV pseudovirus to be susceptible to the human ACE2 expressing cells [26] . Furthermore, our previous study showed that viral-like particles containing the SL-CoV S protein have a stronger ability to stimulate dendritric cells (DCs) to cytokine induction than those containing the SARS-CoV S protein. And SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than the SARS-CoV S DNA in mice [1] .

These results showed that the SL-CoV S protein has properties that are distinct from SARS-CoV S protein.

Thus it is of great importance to elucidate its immunogenicity.

In this study, we constructed a recombinant adenovirus expressing the full-length codon-optimized S gene of bat SL-CoV Rp3 isolate (rAd-Rp3-S), and investigated its ability to induce humoral and cellular immune responses in mice. The ELISA data showed that the rAd-Rp3-S immunized mice generated strong humoral immune responses against the HIV-pseudotyped SL-CoV S protein. However, the induced antibody had weaker cross-reaction with the HIV-pseudotyped SARS-CoV S protein; neither neutralized the pseudovirus. ELISPOT assay found that the secretion levels of IFN-γ and IL-6 in the rAd-Rp3-S immunized mice were higher than that of the negative control, demonstrating that the cellular immune responses were also elicited in the rAd-Rp3-S immunized mice. Our study demonstrated that the S protein from bat SL-CoV can elicit effective immune responses. It also suggested that rAd-Rp3-S can be a potential vaccine candidate against the group of SL-CoVs in the future.

The AdEasy adenoviral vector system (Stratagene), comprising the shuttle vector pAdTrack-CMV and the backbone vector pAdEasy-1, was used to construct a recombinant adenovirus carrying the full length codon-optimized S gene of bat SL-CoV Rp3 isolate (GenBank accession NO.DQ071615) [26] , named rAd-Rp3-S. Briefly, the full-length codon-optimized S gene of bat SL-CoV Rp3 isolate was amplified from the plasmid pcDNA3.1-Rp3-S with primers 5'-GTA GTCGACACCATGGACGCCATGAAGAGGGG-3', was blocked for 2 h with 3% skimmed milk in TBS buffer (20 mmol/L Tris base, 137 mmol/L NaCl, pH 7.6). Following, the blocked membrane was probed with F26G8, mouse IgG mono-antibody against the SARS-CoV S protein that was kindly provided by Dr.

Berry [3] and bound with goat anti-mouse AP-conjugated antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). The results were finally revealed by using chemical substrate NBT and BCIP coloration.

The six-week-old female BALB/c mice (n = 5 per group) were intraperitoneally immunized every 2 weeks with rAd-Rp3-S (1×10 7 pfu per dose) or with the negative controls (rAd-GFP or PBS). Mice were immunized three times for each condition before bleeding.

Sera from the immunized mice of different groups were collected on day 0, 14, 28 and 42 d. S protein specific antibodies (IgGs) were titered by ELISA.

Firstly, HIV/BJ01-S and HIV/Rp3-S, pseudotyped with SARS-CoV (BJ01 isolate, AY278488) and SL-CoV (Rp3 isolate, DQ071615) S proteins, were produced as described previously [26] . The two pseudoviruses were used antigens and coated in monitored by measurement of luciferase intensity as described previously [26] . Due to a lack of permissive cell lines for bat SARS-like CoV, the sera neutralization assay to this virus was not conducted.

To test cellular immune responses in mice, ELISPOT assays were performed to detect the secretion levels of 

The recombinant adenovirus, rAd-Rp3-S was observed by EM. The recombinant virus displayed a typical morphology characteristic of adenovirus (data not shown). The titers of rAd-Rp3-S and rAd-GFP, determined by GFP fluorencence, were up to 6.45×10 7 PFU/mL and 5.53×10 7 PFU/mL, respectively.

showed that an expected 1956bp fragment of bat SL-CoV S gene was obtained only in 293 cells infected by rAd-Rp3-S (Fig. 1A ). An expected 450 bp fragment specific for β-actin was produced in 293 cells infected by rAd-Rp3-S and rAd-GFP (Fig. 1A) . For westernblot, an expected 150-200kDa fragment that was in agreement with the size of SL-CoV S protein was observed in 293 cells infected by rAd-Rp3-S, but was not found in cells infected by rAd-GFP (Fig. 1B) . 

The mouse sera collected on day 0, 14, 28, and 42 was used to detect specific antibody responses against HIV/BJ01-S and HIV/Rp3-S. ELISA results showed that the specific antibody against HIV/BJ01-S or HIV/ Rp3-S was detected in rAd-Rp3-S immunized mouse sera on the 14 th day, the titer boosted dramatically after day 28 and was 10-fold higher than that in rAd-GFP immunized mice by day 42; no specific antibody responses were found at detectable levels in negative control groups, rAd-GFP or PBS immunized mice (Fig. 2) . Further, the antibody titer against HIV/Rp3-S was much higher than that against HIV/BJ01-S in rAd-Rp3-S immunized mice. These results indicated that the specific antibody against SL-CoV S protein and the weaker cross with SARS-CoV S protein were generated in rAd-Rp3-S immunized mice.

Due to lack of permissive cell lines for SL-CoV, HIV/BJ01-S which has a similar infectivity to wild SARS-CoV [26] , was used to detect the neutralization Percentage of neutralization activity was calculated for each antiserum as described in reference [26] , and the average values were plotted.

The splenocytes of the immunized mice that were harvested on the eighth week were used to detect the secretion levels of IFN-γ and IL-6 by ELISPOT assay. [20] . The RBD of SARS-CoV S protein contains neutralizing epitopes and can induce neutralizing antibodies [5, 10, 13, 14] . The minor mutations in the S protein, particularly the receptor binding domain (RBD), abolished viral entry into the susceptible cells and failed to induce cross-protection neutralizing antibodies [12, 35] . An important difference between the bat SL-CoV and SARS-CoV S proteins occurs in the RBD, the RBD of bat SL-CoV S protein has two deletions (5 and 12-13 aa, respectively). This important difference led to different usage of the cellular receptor between SARS-CoV and SL-CoV; the bat SL-CoV cannot use ACE2 as its receptor [26] . Our current study further demonstrated that the divergence between the SARS-CoV and SL-CoV S protein also may cause the antibody induced by SL-CoV S protein to have a weaker cross reaction with SARS-CoV S protein and such that it did not neutralize HIV/ BJ01-S. Cellular immune responses are very important for host defense against the viral infection. IFN-γ is synthesized almost exclusively by activated natural killer (NK) and T cells in response to viral infection [24] . IL-6 plays a central role in both innate and acquired immune response. In our study, high secretion level of IFN-γ (120 cells per 10 6 cells) and IL-6 (160 cells per 10 6 cells) in rAd-Rp3-S immunized mice splenocytes, indicated that both Th1 and Th2 cells were elicited in rAd-Rp3-S immunized mice. Similar responses have been observed in SARS patients and animal models, for example, the level of IFN-γ in SARS patients at the early stages of disease onset correlated with resolution of the viral infection [29] , and IL-6 was up-regulated as a consequence of being induced by SARS-CoV S protein in murine macrophages [31] .

It is clear that the recently discovered bat SL-CoVs are not the immediate progenitors of SARS-CoV which caused the outbreaks in 2002/2003 [26] . It is envisaged that the ongoing vaccine against the SARS-CoV may not protect the infection by bat SL-CoVs.

Considering the wide distribution and genetic diversity of bat SL-CoVs in China [18, 20, 25, 33] , co-infection of same bat species by different coronaviruses [18, 20, 30] and the capability of recombination of coronaviruses [2, 8] , it is likely new coronaviruses in bats that can cross species to infect humans. Moreover, high density of bat habitats and increasing contacts between bats and human will further increase the virus transmission opportunities from bats to human. So, it is highly important to be prepared for prevention and control of the emerging disease resulting from this group of viruses. The recombinant adenovirus constructed in this study provides a potential vaccine candidate against the infection by diverse bat SL-CoVs.

Virus-like particles of SARS-like coronavirus formed by membrane proteins 42

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We thank Dr. Ying Zhu of Wuhan University, China for kindly providing AdEasy adenoviral system.