The ability to induce innate immunity safely has long been a goal for treating viral, bacterial, and fungal infections. Increasing resistance to pulmonary infection following non-specific immune stimulation has been recognized for over thirty years 
. Such a strategy requires development of therapeutic entities able to induce various elements of innate immunity with little/no adverse inflammatory side effects.
The anaphylatoxin C5a is pharmacologically active byproduct of the complement cascade. C5a acts to bring inflammatory cells to the site of tissue injury/infection and to subsequently activate their effector responses. These functions are dependant upon binding of C5a to the cell-surface receptor CD88 (reviewed in 
. Like native C5a, EP67 function is critically dependent upon CD88 ligation 
. However, although it is able to activate antigen presenting cells, EP67 neither binds to nor signals through the CD88 surface receptor on inflammatory PMNs 
. Therefore, insuffulation of EP67 induces the acute phase proteins TNF and IL-6 in the absence of accompanying neutrophil stimulation 
The current study shows that delivery of EP67 to the airways induces a potent anti-viral response characterized by rapid cytokine induction and sequential influx of innate effector cell populations. A single insufflated dose of EP67 was sufficient to initiate a cascade of molecular and cellular events covering a period of days. The resultant innate immune response provided 100% protection following infection with a lethal dose of influenza. This EP67-induced cascade of events displayed the essential characteristics and kinetics expected for a protective innate response to infection. The cytokines and chemokines detected in the airways within two hours of EP67 treatment (TNF, IL-6, GM-CSF, and KC/CXCL1) are all important mediators of direct antiviral protection. TNF is by itself a robust inhibitor of influenza A replication 
. During an active infection, TNF also enhances the recruitment of leukocytes to the site of infection and activates innate immune responses 
. IL-6 exhibits pleiotropic effects, including the activation of NK cells and macrophages and stimulation of T cell differentiation during influenza infection 
. GM-CSF enhances survival, proliferation, maturation, and differentiation of myeloid cells. GM-CSF also blocks mortality from lethal influenza infection and is critical to alveolar growth and repair 
. The chemokine KC/CXCL1 plays a critical role in preventing bacterial pneumonia after influenza infection 
In addition to direct anti-viral and protective functions, the cytokines and chemokines induced by EP67 are all associated with chemotaxis of innate immune effector cell populations. A single administration of EP67 induces the recruitment of neutrophils within two hours. Neutrophils are one of the first inflammatory cell type to appear in large numbers during acute infection 
. Neutrophils may contribute to innate immune protection during the early stages of influenza infection, although their requirement is debated 
. Although neutrophils exhibit chemotaxis directly to native C5a, EP67 is devoid of C5a-like engagement of CD88-bearing PMNs 
. The cytokines and chemokines detected two hours after EP67 treatment are all associated with neutrophil chemotaxis 
, and both TNF and GM-CSF act to increase neutrophil survival times in tissue from hours to days 
. The peak neutrophil response (both total number and percentage of BAL) occurred twenty-four hours after EP67 treatment. Stimulated neutrophils produce a large number of proinflammatory mediators, including TNF and IL-1β 
. If EP67 were directly activating the neutrophil population, it would be anticipated that the local concentration of these cytokines would greatly increase at or near the peak of the neutrophil response. However, the local concentration of TNF and IL-1ß show no correlation to the kinetics of neutrophil influx and disappearance. This argues against neutrophils as the cellular source of the airway cytokines, underscoring an important therapeutic attribute of EP67; i.e., its ability to recruit neutrophils for the initial assault on an infection, but no capacity to activate them directly once recruited. In the absence of a wide-spread infection, this avoids induction of an unnecessary or overly-aggressive inflammatory outcome.
Within one day of EP67 treatment, both NK cells and exMacs are present in the airways. Three days after EP67, mDCs can be detected as a large percentage of the airway cells. The involvement of these cell types in the clearance of various airway infections is well established (reviewed in 
). NK cells lyse virally-infected cells, acting to contain viral infection during maturation of the acquired immune response. NK cells also play a critical role in activation of the CTL response to influenza 
. Macrophages phagocytize pathogens, and loss of the exMac population is associated with highly increased pulmonary influenza titers and a decrease in the subsequent T cell response 
. The pulmonary DCs initiate the adaptive immune response by presenting antigen to naïve T lymphocytes 
. EP67 also enhanced expression of MHC II and costimulatory surface markers on the APC populations (i.e., AM, exMac, and mDC) in the airways in a dose dependant fashion. An increased antigen-presenting capacity secondary to this observed maturation of APC populations is consistent with our previous work showing that that EP67 (as well as the earlier sister analogue EP54) are effective adjuvants in both young and aged mice 
In the normal lung, the AM and the bronchial epithelium (BE) are continually exposed to the outside world during respiration. Both cell types can rapidly release cytokines in response to inflammation/infection. Furthermore, both AM and BE express the C5aR CD88 and are able to secrete cytokines in response to both natural C5a and EP67 or its earlier sister analogs (
and Sanderson, unpublished data). It is thus quite likely that the AM and the bronchial epithelium both contribute to the immediate cytokine response following EP67 insufflation. The cellular source of those cytokines that peak two days after EP67 treatment (IL-1ß, IL-12p70, and MCP-1, as well as the second peak of IL-6) is less clear. By this late time point, it is unlikely that any EP67 remains in the airways. This second wave of mediators is most likely derived from continual activation of the initiating responder cells and/or to maturation of different innate effector cell populations in the airways. For example, mDCs can release multiple chemokines over several days 
, with the later phases biased toward recruitment of monocytes and T-lymphocytes. This would be consistent with the presence of IL-12p70, considered a “Th1-type” cytokine, as well as MCP-1, a monocyte chemoattractant. The second peak of IL-6 deserves mention. IL-6 acts as both a pro-inflammatory and an anti-inflammatory cytokine. The latter function is critical to the resolution of the innate immune response (reviewed in 
). The mechanisms involved include antagonism of the TNF response and induction of neutrophil apoptosis. The rapid loss of neutrophils three days after EP67 treatment corresponds both with the limits of apoptosis protection induced by TNF and GM-CSF as well as the second local increase in the IL-6 concentration.
The EP67-induced release of TNF and IL-6 followed by an influx of innate immune effector cells resembles the innate immune response to influenza infection (reviewed in 
. However, the innate immune response to influenza infection does not normally commence for a matter of days due to antagonism of the antiviral type 1 IFN pathway by the NS-1, PB1-F2, and PB2 viral proteins 
. Suppression acts at the level of the infected cell, delaying initiation of the innate immune response during the early stages of viral replication and spread. Because of this both mice and humans experience unconstrained viral replication during the earliest time points following infection. In the absence of treatment, the endogenous anti-viral immune response is generally initiated two days after infection, with effector cells appearing in the lungs a day later 
. Not until initiation of the innate immune response do viral titers begin to diminish. Early treatment with EP67 induced a robust response even in the presence of an established infection, initiating an anti-viral response up to two days earlier than in the untreated animals. This led to a significant decrease in the pulmonary viral burden, reflected in protection from weight loss and death.
Although early treatment was clearly protective, it was unsurprising that a single dose of EP67 delivered two or three days after infection shows no clear therapeutic benefit. By this time post-infection, the endogenous innate response has been initiated. In the case of uncomplicated (non-lethal) infection, there may be no therapeutic benefit to increasing the already-initiated innate response. This limited therapeutic window is similar to that seen with neuraminidase inhibitors, which are also most effective during early stages of rapid viral replication 
and less so once symptoms are present, particularly for children 
. Unfortunately, human exposure to an infectious agent is generally unrecognized until symptoms appear; but there are times when the risk of exposure is greatly increased. The most obvious such cases are health care professionals as well as coworkers and family members of patients. In such cases, post-exposure prophylaxis with EP67 should be within the window of window of therapeutic efficacy. Furthermore, the experiments herein used only a single dose of EP67 given at early stages of infection. It remains to be determined how multiple doses of EP67 impact the course of disease. These studies will be particularly important in the context of lethal infection or in the presence of underlying immunodeficiency.
EP67 treatment during the early stage of infection was associated with a significant reduction in viral burden and protection from morbidity/mortality. Despite the reduced viral burden, the protective humoral response to the infection was not compromised. Although there was a possible trend towards decreased IgG1, there was no reduction in the concentrations of either IgG2b or IgG2c (the homolog of IgG2a found in C57BL/6 mice) in the groups treated with EP67 compared to the non-treated control animals. Members of the IgG2 subclass are the most potent mediators of influenza virus neutralization in vitro
and elimination in vivo
. A decrease in these critical immunoglobulin subtypes could manifest itself as impaired humoral protection. Long term protective immunity is also critically dependent upon establishment of a robust acquired immune response. Therefore, maintenance of these critical immunoglobulin subtypes following EP67 treatment indicates that induction of the early, robust innate response does not have a deleterious effect on humoral protection. Further studies to uncover the mechanism of robust humoral immunity despite the decreased viral burden, as well as the effect on CD4 and CD8 cell function following EP67 treatment, are ongoing.
The overall role of the innate immune response is to provide defense against pathogens that is both immediate and non-specific. Expanding upon the results shown herein, the response generated by EP67 in the airway should display little pathogen specificity, and thus activating the non-specific innate immune response with EP67 should prove efficacious in treating diverse viral, bacterial, and fungal infections. Beyond the promiscuity of their protection, therapeutics that operate via the innate immune response minimize mutational pressures on pathogens, since the therapeutic effect is neither directed toward nor imposed directly upon the pathogen 
These studies were designed to determine whether EP67, previously used primarily as an adjuvant, could provide direct protection in the face of respiratory infection. The results indicate that the response-selective stimulation of CD88-bearing effector cells in the airways provides immediate protection even from established infection, without compromising the subsequent acquired response to infection. More broadly, the results indicate that EP67 may function as a broad-spectrum emergency therapeutic for diverse respiratory infection. Ideally, an emergency therapeutic for infectious disease should exhibit several characteristics. It should provide protection when administered either prior to infection (prophylactic protection) or after infection has been established (therapeutic efficacy), and it should provide protection against multiple, diverse pathogens. In the current report, we show that EP67 fulfills the first two characteristics. Because protection was mediated via the innate immune response, EP67-induced protection should prove efficacious against diverse infectious agents, including non-viral pathogens as well as pathogens not confined to the respiratory tract. This is supported by our findings that EP67 induces effective innate immunity against both bacterial 
and fungal (Phillips, unpublished) infection.
The peptide sequence of EP67 (YSFKDMP(MeL)aR) was originally derived from the biologically active C-terminal sequence of human C5a (C5a65–74: ISHKDMQLGR, 40% identity). Despite the efficacy shown in the current study, EP67 bears very little sequential similarity to the same region of murine C5a (SPHKPVQLGR). That EP67 binds to and engages C5aRs on both human and murine antigen presenting cells reflects more on the similarities of the secondary binding pocket within these C5aRs that accommodate the unique topochemical features of EP67 than it does sequential similarities of the C-terminal region of the murine and human C5a ligand.
In summary, this report shows that the C5a agonist peptide EP67 provides both prophylactic and therapeutic protection against influenza infection. Protection results from the rapid induction of a robust innate immune response that includes high local concentrations of anti-viral cytokines and the influx of several populations of innate immune effector cell types. These results have profound implications for influenza therapeutic development and, ultimately, for broad-spectrum emergency therapy against unidentified respiratory pathogens.