Possible inhibition of GM-CSF production by SARS-CoV-2 spike-based vaccines

A SARS-like coronavirus 2 (SARS-CoV-2) has caused a pandemic Coronavirus Disease 2019 (COVID-19) that killed more than 3.3 million people worldwide. Like the SARS-CoV, SARS-CoV-2 also employs a receptor-binding motif (RBM) of its spike protein to bind a host receptor, the angiotensin-converting enzyme 2 (ACE2), to gain entry. Currently, several mRNA or adenoviral vaccines encoding for the spike protein of SARS-CoV-2 are being used to boost antibodies capable of inhibiting spike-ACE2 interaction and viral entry. However, recent evidence has also suggested an anti-inflammatory effect of spike-reactive antibodies, suggesting that some SARS-CoV-2 spike-based vaccines may elicit protective antibodies capable of inhibiting GM-CSF production and COVID-19 progression.


Background
The recent emergence and rapid spread of SARS-like coronavirus 2, SARS-CoV-2, has caused a pandemic COVID-19 that is catastrophically harming human health (Qiang et al. 2021). As of 12 May 2021, approximately 159 million people have been infected, leading to more than 3,300,000 deaths worldwide (https:// www. who. int/ emerg encies/ disea ses/ novel-coron avirus-2019). Like the β-coronavirus that caused the 2003 outbreak of the severe acute respiratory syndrome (SARS-CoV), SARS-CoV-2 virus also employs its envelope spike (S) glycoproteins to bind a host cell surface receptor, the angiotensin-converting enzyme 2 (ACE2), to gain host cell membrane fusion and viral entry (Hoffmann et al. 2020).

SARS-CoV-2 Spike-reactive antibodies impair viral entry
To boost adaptive antibody responses against SARS-CoV-2 infections, several mRNA and adenoviral vaccines encoding a surface fragment of a SARS-CoV-2, the spike (S) protein, are currently being employed worldwide to fight against COVID-19. It is hoped that antibodies raised against SARS-CoV-2 S protein may inhibit viral interaction with host ACE2 receptor, thereby preventing viral entry (Amanat and Krammer 2020). Indeed, neutralizing antibodies targeting the receptor-binding domain (RBD) or the receptor-binding motif (RBM) of SARS-CoV-2 S protein were found in the blood of COVID-19 patients (Zost et al. 2020;Pinto et al. 2020), and some of which could indeed impair viral entry (Zost et al. 2020;Pinto et al. 2020).

Molecular Medicine
Although expressing recombinant proteins in E. coli is relatively easy and cost-effective, cysteine-rich proteins (such as the full-length spike protein of SARS-CoV-2) can be difficult to produce in prokaryotes partly because the reducing environment of the bacterial cytoplasm favors the formation of incorrect disulfide bond and production of insoluble protein aggregates (i.e., inclusion bodies) that are often difficult to refold correctly after denaturation. Indeed, even a small fragment of the SARS-CoV-2 spike protein (such as the RBM and RBD) formed insoluble inclusion bodies in E. coli, and a high concentration of denaturant (8 M urea) had to be employed to solubilize these inclusion bodies before subsequent chromatography purification and refolding (Qiang et al. 2021). To prevent excessive oxidation and cross-linking of the nine and two cysteines in RBD and RBM, respectively, these recombinant proteins were renatured and refolded in a buffer containing a reducing agent [Tris (2-carboxyethyl) phosphine (TCEP)]. Even so, recombinant RBD still exhibited an extremely low affinity to human ACE2 with an estimated K D of 161,000 nM, possibly because the cysteine-rich RBD was not refolded into a "correct" conformation suitable for RBM-ACE2 interaction, as the high probability of "incorrect" disulfide cross-linking was factorially proportional to the number of cysteine residues (Qiang et al., 2021).
The intricate mechanism by which RBM-reactive antibodies selectively blocked the RBM-induced GM-CSF induction without affecting the release of other cytokines/chemokines remains an exciting subject of future investigation. Because human or murine GM-CSF does not contain any segment that even remotely resembles the epitope sequence (NDALYEYLRQ) of the TN/RBM-reactive antibodies, these antibodies are not expected to bind human or murine GM-CSF or to interfere with its immune-detection by Cytokine Antibody Arrays. Furthermore, our immunoblotting analysis revealed that these TN/RBM-reactive antibodies did not cross-react with any proteins in the whole-cell lysates of murine macrophages or human peripheral blood mononuclear cells (huPBMCs), confirming a lack of cross-reactivity of these TN/RBM-reactive mAbs to any other endogenous proteins of innate immune cells (Qiang et al. 2021). We speculate that SARS-CoV-2 spike protein activates innate immune cells through multiple signaling molecules that might include ACE2 and other yet-to-be identified pathogen pattern recognition receptors (PRRs). It is thus possible that some RBM-reactive antibodies might selectively prevent its interaction with a receptor involved in the GM-CSF induction, but did not interfere with its engagement with other pattern recognition receptors responsible for the induction of other cytokines or chemokines. Furthermore, we did not generate full-length spike protein in E. coli to confirm its innate immune stimulatory properties, because the fulllength spike protein might similarly fail to fold correctly after urea denaturation. Therefore, future studies are needed to confirm the innate immune stimulatory properties of the full-length spike protein of SARS-COV-2 using recombinant proteins expressed in other eukaryotic cells. Nevertheless, our surprising findings fully supported the emerging notion that GM-CSF might be a key biomarker for SARS-CoV-2-induced cytokine storm in a subset of COVID-19 patients with more severe pneumonia often escalating to respiratory failure and death (Hue et al. 2020;Gibellini et al. 2020;Blot et al. 2020;Thwaites et al. 2021;Zhao et al. 2021).
In response to severe SARS-CoV-2 infections, some hosts mount hyperactive inflammatory responses characterized by the excessive infiltration and activation of myeloid cells and consequent production of various cytokines and chemokines -the "cytokine storm" (Qiang et al. 2021). GM-CSF might be a key mediator of SARS-CoV-2-induced cytokine storm in a subset of COVID-19 patients with severe outcomes (Hamilton, 2020;Hue et al. 2020;Gibellini et al. 2020;Blot et al. 2020;Thwaites et al. 2021;Zhao et al. 2021). On the one hand, GM-CSF can promote myelopoiesis as well as recruitment of innate immune cells (e.g., monocytes, macrophages and dendritic cells) to SARS-CoV-2 infection sites (Hamilton, 2020;Qiang et al. 2021). On the other hand, GM-CSF can also polarize innate immune cells into pro-inflammatory phenotypes, promoting the production of proinflammatory cytokines (e.g., TNF, IL-1β and IL-6) and chemokines (e.g., MCP-1) (Hamilton, 2020).
Currently, a number of clinical trials are being planned to assess the efficacy of mAbs against GM-CSF (Clinical Trial Registry #: NCT04341116, NCT04351243, NCT04351152, NCT04376684) (Mehta et al. 2020;Lang et al. 2020;Hamilton et al. 2016) or GM-CSF receptor (Clinical Trial Registry #: NCT04399980, NCT04463004, and NCT04492514) . For instance, intravenous infusion of an anti-GM-CSF mAb (Lenzilumab, 600 mg, thrice) significantly reduced blood levels of IL-1α and IL-6 in 11 out of 12 patients with severe COVID-19 (Temesgen et al. 2020). Similarly, a neutralizing antibody against human GM-CSF receptor (mavrilimumab) significantly improved clinical outcome in 13 patients with severe COVID-19 pneumonia . In light of the emerging notion that GM-CSF might be a key biomarker of SARS-CoV-2-induced cytokine storm in a subset of COVID-19 patients with more severe outcome (Hue et al. 2020;Gibellini et al. 2020;Blot et al. 2020;Thwaites et al. 2021;Zhao et al. 2021), future GM-CSFtargeting clinical trials should be conducted in a subset of COVID-19 patients with particularly elevated blood GM-CSF levels.

Conclusions
It is thus possible that SARS-CoV-2 spike protein-based vaccines may elicit protective antibodies capable of preventing SARS-CoV-2-elicited GM-CSF production and hyperactive "cytokine storm". In light of the on-going efforts to develop effective SARS-CoV-2 vaccines, it may be important to assess the innate immune-modulating properties of all mRNA or adenoviral vaccine candidates and respective antibodies in experimental and clinical settings.