The complement system is an innate immune mechanism largely conserved across mammalian species and acts against infectious agents. Components of the complement system work in an orchestrated manner to subdue pathogens. This is achieved in myriad ways, including the deposition on the target pathogen, which leads to membrane disruption or opsonization, and the recruitment of immune cells to the area of activity through complement mediators. One such mediator is the complement subunit C5a, which is produced via proteolysis of the parent C5 molecule. Human C5a is 74 amino acids in length and acts in a chemotactic manner to recruit and activate immune cells bearing the C5a receptor (1). The native C5a molecule potently induces recruitment of phagocytes, including macrophages and monocytes, and less so inflammatory granulocytes, such as neutrophils and mast cells (2). Due to its ability to recruit, activate, and induce degranulation in the latter, full-length C5a is appropriately known for its inflammatory effects.

The C5aR1/CD88 interactive peptide, EP67, is a small decapeptide derived from the C terminus of human C5a with the sequence YSFKDMP[MeL]aR where “MeL” is N-methyl leucine and “a” is D-alanine. It was designed to act as a non-inflammatory immunostimulant and immune adjuvant by selectively recruiting and activating phagocytic antigen-presenting cells whilst reducing inflammatory effects that can occur with the recruitment of granulocytic cells (3–5). EP67 has demonstrated effectiveness in murine models of viral infection, such as protection against cytomegalovirus (CMV) infection via adjuvant activity (2). The antiviral activity may be partially due to EP67’s ability to promote T_H1 immune activation via the C5aR1/CD88 receptor pathway (5). Additionally, EP67 has demonstrated efficacy as additional in bacterial and viral infections (6–8). Therefore, we posited that EP67 could work in a therapeutic manner against a potent, symptomatic coronavirus infection.

In 2020, the worldwide spread of SARS-CoV-2, a betacoronavirus, led to a pandemic with high morbidity and mortality. With the relative paucity of broad-spectrum antiviral agents, vaccine approaches were primarily employed to reduce spread and attenuate the more severe effects resulting from viral infection. Several animal models exist for modeling coronavirus infections; however, access barriers such as cost, availability, and housing requirements (e.g. BSL-3 facilities) can render these impractical for many laboratories (9). The betacoronavirus, Murine Hepatitis Virus (MHV), naturally infects mice and various disease pathologies of human SARS-CoV and SARS-CoV-2 infections can be modeled in vivo with varied animal and MHV strains (10–12). Specifically, C57BL/6 mice inoculated intranasally with MHV strain A59 (MHV-A59) have shown acute pneumonia and lung pathology due to viral spread in the lungs and leukocyte infiltration beginning as early as 2 days post-infection (2 dpi) (12). Using this model, we administered a single dose of EP67 intranasally after the onset of symptoms (3 days post-infection; 3 dpi) to evaluate the ability of EP67 treatment to alleviate severe health decline in infected mice. Mice receiving EP67 recovered quickly compared to untreated infected mice who significantly declined in health. This suggests that EP67 could serve as an effective antiviral therapeutic against the betacoronavirus MHV. Additionally, we show that EP67 increased long-term plasma IgG nucleocapsid antibodies when coadministered with inactivated SARS-CoV-2, with a similar increase in spike protein IgG. Together, these findings further support the antiviral efficacy of EP67 with potentially broader applications to additional viral diseases.

To establish the capacity of EP67 to serve as a humoral immune adjuvant to SARS-CoV-2, mice were immunized with 2.7 x 10³ PFU of γ-irradiated (inactivated) SARS-CoV-2 Vero cell lysate coadministered with either 100 µg EP67 or PBS (vehicle control group). The irradiated viral lysate was leveraged in place of infectious live virus; however, this approach limits the utilization of the T_H1 cell-mediated adaptive immune response, a known component of EP67 immune modulation (5). Nevertheless, following two immunizations spaced 24 days apart, mice receiving EP67-adjuvanted γ-irradiated SARS-CoV-2 lysate demonstrated significantly more viral nucleocapsid-specific IgG plasma antibodies compared to control mice receiving PBS and γ-irradiated SARS-CoV-2 lysate at Day 42 (p = 0.030). This suggests an apparent long-term, persistence in protection resulting from the immunizations at Days 0 and 24 ( Figure 1 ).

To assess the ability of EP67 to serve as an effective treatment following the onset of illness from the related betacoronavirus Murine Hepatitis Virus (MHV), groups of 7-week-old male C57BL/6J mice were evaluated following infection. The C57BL/6 mouse strain has been previously used for infection with MHV strain A59 (MHV-A59), with disease progression correlated with observable illness and tissue pathology (12). Mouse body mass has been used extensively as a broad indicator of morbidity, and MHV infection in mice is known to significantly impact mouse health and weight loss, with higher doses proving lethal (12). In contrast, viral nucleic acid levels may persist in the tissues of animals with resolved infection for several days (16, 17). In the current study, each mouse received 1.1 x 10⁶ PFU MHV-A59, or 17Cl-1 uninfected host cell lysate, delivered intranasally on day 0. By day 3 post-infection (3 dpi), infected mice demonstrated a disease state with changes in behavior associated with viral infection, in agreement with previously published observations (12). To evaluate EP67 for therapeutic treatment efficacy following initial signs of infection, 100 µg of EP67 or PBS vehicle control was delivered intranasally on day 3 post-infection, and mice were monitored daily thereafter. Infected control mice receiving PBS declined in body mass, notably from day 6 post-infection onward, while EP67-treated mice maintained their body mass from the day of treatment throughout the end of the study and rapidly increased in health ( Figure 2 ). Symptomatically, EP67-treated mice returned to health by day 3 post-treatment (corresponding to day 6 post-infection) and resembled the uninfected EP67-treated control mice. Statistical significance in the body mass percentage loss, relative to initiation of treatment on day 3 post-infection (3 dpi), was observed for infected EP67-treated versus infected PBS control mice 6 days post-treatment (9 dpi; Figure 2B ) and 7 days post-treatment (10 dpi; Figure 2C ).

Similar to SARS-CoV-2 infection in humans, intranasal infection of C57BL/6 mice by MHV strains (e.g. MHV-A59) is known to induce pathology by substantial recruitment of granulocytic cells to the lungs (12, 18, 19). The mean lung mass of EP67-treated infected mice ( Figure 3 ) showed a slight increase at two days post-treatment (5 dpi) and a modest decrease compared to the infected untreated PBS control mice at seven days post-treatment (10 dpi). As expected, uninfected mice mock-infected with 17Cl-1 cell lysate alone at day 0 (0 dpi), followed by EP67 administration at 3 dpi, maintained consistent lung masses at two and seven days post-treatment, supporting the hypothesis that EP67 treatment did not aggravate the disease state further, but instead reduced disease severity within 24 hours based on behavior and measured weights. This suggests that in the context of this study, EP67, in contrast to its parent protein human C5a, does not appear to exacerbate host-mediated innate immune cascades (i.e. granulocyte effector functions) (6). Altogether, this evidence supports that EP67 treatment results in a significant decrease in morbidity following intranasal treatment at 3 days post-infection.

The ability of EP67 to rapidly cause restored health following administration in symptomatic models of viral infection supports the view of this peptide as a potent and selective immunostimulant. It is theorized that EP67 invokes recruitment and activation of antigen-presenting cells (APCs) and enhances their antigen processing time and/or display levels, thereby driving the adaptive immune response a more rapid opportunity to develop B and T cell-mediated defenses against viral infection and propagation. Such bridging of the innate and adaptive immune responses is believed to underlie the observed rapid return to health. Previous studies with EP67 and its compositional predecessor EP54 support this hypothesis: murine dendritic cells internalized the peptide, upregulated selected activation-associated cytokines, and increased MHC expression compared to control (4). Similarly, murine splenic cells demonstrated marked increases in activation-related cytokines, and promoted enhanced cellular and humoral immune responses against the ovalbumin (OVA) antigen (5). In line with these previous findings, we show that EP67 is a significant (p < 0.0001) inducer of TNF-alpha (TNF-α) secretion from another APC: mouse RAW 264.7 macrophages ( Figure 4 ). As seen in Figure 4 , EP67 resulted in nearly the same magnitude as its parental protein, human C5a. The anti-viral capabilities of TNF-α are well-established, and patients who asymptomatically clear SARS-CoV-2 have been found with slightly higher expression compared to symptomatic patients (20). While high levels of TNF-α may contribute to deleterious inflammatory responses in some diseases, it often does so only in the context of other co-expressed inflammatory cytokines: such is the case with SARS-CoV-2, where TNF-α alone did not contribute to cell damage (21). Taken together, the EP67 molecule used here demonstrates an ability to directly activate murine antigen-presenting cells, and provide effective therapy for symptomatically infected mice, supporting its role as a selective immunostimulatory bio-mimetic of the parent C5a molecule.

The SARS-CoV-2 global pandemic reiterated the urgent need for broad-spectrum antiviral agents, ideally with a capacity to treat individuals already infected. This brief study showed that the immunostimulant decapeptide, EP67, derived from human complement protein C5a, used in a single dose reduced disease severity of infection from mouse-native MHV, a betacoronavirus. EP67’s ability to return treated mice to a healthy state emphasizes its potential for safe application in the case of viral infections known to otherwise promote tissue destruction through elevated inflammatory responses. While used as a prophylactic adjuvant or as a treatment for symptomatic disease in this study, a greater understanding of EP67’s ideal therapeutic window will likely be an important element in future study designs (8). Finally, it would be valuable to advance the understanding on the molecular mechanisms responsible for this non-inflammatory immunostimulatory activity of EP67 and how it may bridge the innate and adaptive immune responses.