Targeted Amino Acid Substitution Overcomes Scale-Up Challenges with the Human C5a-Derived Decapeptide Immunostimulant EP67 Abdulraman M

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Targeted Amino Acid Substitution Overcomes Scale-Up Challenges with the Human C5a-Derived Decapeptide Immunostimulant EP67 Abdulraman M. Alshammari, D. David Smith, Jake Parriott, Jason P. Stewart, Stephen M. Curran, Russell J. McCulloh, Peter A. Barry, Smita S. Iyer, Nicholas Palermo, Joy A. Phillips, Yuxiang Dong, Donald R. Ronning, Jonathan L. Vennerstrom, Sam D. Sanderson, and Joseph A. Vetro*

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ABSTRACT: EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8)sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67. KEYWORDS: host-directed therapy, host-derived immunostimulant, complement peptide-derived immunostimulant (CPDI), mucosal adjuvant, human C5a desArg, EP54 Downloaded via UNIV OF NEBRASKA MEDICAL CTR on May 8, 2020 at 12:58:42 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

mmunization has eliminated or greatly reduced the partially effective (e.g., influenza virus, Bordetella pertussis) for incidence of over 30 infectious diseases caused by bacterial at least 35 bacterial and viral pathogens that are suitable targets I − and viral pathogens including CRS, diptheria, Haemophilus for vaccine development.2,4 6 Furthermore, only a single influenzae, hepatitis A, hepatitis B (acute), influenza, measles, partially effective licensed vaccine is available for protozoan mumps, pertussis, pneumococcus (invasive), polio (paralytic), pathogens (Plasmodium falciparum) and no licensed vaccines 7,8 rotavirus, rubella, smallpox, tetanus, and varicella.1,2 There is are available for fungal pathogens. also clinical evidence that vaccine administration within a A key requirement for licensed vaccines is the ability to fi critical period of time after pathogen exposure can minimize or safely generate long-lived, pathogen-speci c adaptive immune prevent infectious diseases caused by the hepatitis A virus, hepatitis B virus, measles virus, mumps virus, varicella zoster Received: January 6, 2020 virus, rabies virus, and smallpox virus by increasing the rate and Published: April 1, 2020 magnitude of protective innate and/or adaptive immune responses.3 Despite this success, licensed vaccines are unavailable (e.g., Staphylococcus aureus, Mycobacterium tuber- culosis, Listeria monocytogenes, HIV, CMV, and RSV) or only

© 2020 American Chemical Society https://dx.doi.org/10.1021/acsinfecdis.0c00005 1169 ACS Infect. Dis. 2020, 6, 1169−1181 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article responses that reduce, control, and clear pathogens from the As such, numerous immunostimulant molecules are being site of infection (i.e., protective adaptive immune responses) developed to safely activate mononuclear phagocytes through above threshold levels that correlate with elimination or well-defined signaling pathways.23 significant decrease in the incidence of infectious disease in a Given their role in the potentiation of innate and adaptive − given population.9 12 The majority of licensed vaccines are immune responses against bacterial, fungal, protozoan, and proposed to prevent infectious disease primarily by generating viral infections,27,28 the majority of immunostimulant mole- one or more subsets of long-lived memory B-cells that cules are currently derived from pathogen-associated molecular maintain or quickly establish sufficient levels of neutralizing pattern molecules (PAMPs) that activate mononuclear antibodies against pathogens with surface antigens that do not phagocytes through cell-associated pattern recognition recep- − change over time.13 15 Many pathogens without licensed tors (PRRs).29,30 Two PAMP-derived PRR agonists that vaccines, however, have hypervariable surface antigens, multi- stimulate PRRs from the toll-like receptor (TLR) family, ple strains, and/or short extracellular phases that additionally CpG 1018 (TLR9), and Monophosphoryl Lipid A (TLR4) are require the generation of long-lived protective CD4+ and/or also the first immunostimulant molecules to be included alone − CD8+ memory T-cells.16 19 (CpG 1018) or in combination with other adjuvants Vaccines composed of live-attenuated bacterial, fungal, (Monophosphoryl Lipid A) in licensed vaccines.31 The large protozoan, or viral pathogens that safely mimic natural number of PRRs and differences in adaptive immune responses infection are most likely to generate adequate levels of long- generated by individual PRRs, however, may complicate lived protective memory B-cells and T-cells against the wild- identifying individual or combinations of PRR agonists that type pathogen by sufficiently activating macrophages and simultaneously increase the generation of long-lived B-cells, dendritic cells, prolonging antigen presentation by antigen- CD4+, and CD8+ T-cells.32 presenting cells (APC) and inducing the most appropriate In contrast to PAMPs, the complement system is a less protective adaptive immune responses.18 Live-attenuated exploited but equally important host defense against bacterial, vaccines, however, are (i) limited to pathogens that increase fungal, protozoan, and viral infections.30,33,34 Activation of the protection after natural infection and can be grown in culture, complement system by foreign microbes at the site of infection (ii) are difficult to establish for most bacterial pathogens, (iii) through classical, alternative, or lectin pathways initiates a take a long time to develop, (iv) are rarely safe and stable, (v) proteolytic cascade of C proteins that converges on the may not cross-protect against other pathogenic strains, (vi) are production of three immunostimulatory complement peptides, not suitable for pregnant women or immunocompromised C3a, C4a, and C5a (originally described as anaphylatoxins)35 patients, and (vii) may revert to wild-type virulence (e.g., from inactive C3, C4, and C5, respectively, which are − poliovirus).18,20 22 Nonreplicating vaccines composed of killed immediately metabolized into less potent desArg forms lacking (bacteria) or inactivated (virus) pathogens (killed-inactivated C-terminal Arg, and initiates the concurrent formation of vaccines) can potentially overcome the safety issues of live- membrane attack complexes (MAC).36 These products then attenuated vaccines but not the remaining limitations.18 Killed- “complement” host immunity by increasing local and systemic inactivated vaccines are also less immunogenic than live- inflammation, opsonization of microbes, direct lysis of bacteria, attenuated vaccines, provide a shorter duration of protection, clearance of immune complexes and apoptotic cells from and do not generate appreciable levels of CD8+ T-cells. tissues, and the generation of short-lived and long-lived Although precipitating killed-inactivated vaccines into micro- memory B-cells and T-cells.36,37 meter-sized particulates by adsorption to preformed aluminum The most potent complement peptide, C5a, is a 74-residue hydroxide or aluminum phosphate particulate gels (alum glycopeptide that exhibits a number of ideal immunostimula- adjuvant) increases antibody titers, it does not increase the tory activities, including the receptor-mediated activation of generation of long-lived CD8+ T-cells and may polarize toward monocytes and macrophages through C5a receptor 1 (C5aR1/ Th2 adaptive immune responses that are potentially less CD88)38 and the generation of long-lived adaptive immune effective against bacterial, fungal, viral, and protozoan responses.39,40 C5a, however, is also a principal mediator of pathogens.23 local and systemic inflammation through its ability to activate The limitations of live-attenuated and killed-inactivated and recruit neutrophils, induce spasmogenesis, increase vaccines and the desire for well-defined vaccines with more vascular permeability, and stimulate the secretion of secondary predictable activity have led to the development of vaccines inflammatory mediators from a wide range of cell types.41 composed of one or more immunogens from the pathogen that Furthermore, many diseases caused by chronic inflammation are targeted by protective antibody and T-cell responses after have been linked to C5a and neutrophils,42,43 and C5a- infection (subunit and recombinant vaccines).8,20,24 Recombi- activated neutrophils directly damage endothelial and meso- nant vaccines also allow incorporating protein immunogens thelial tissues.44,45 As such, C5aR1 agonists that selectively engineered to generate more potent and broadly protective activate mononuclear phagocytes over neutrophils are likely to memory B-cells and T-cells.25 Subunit and recombinant be safer than indiscriminate C5aR1 agonists.46 vaccines, however, require the inclusion of immunostimulant To begin developing complement peptide-derived immu- molecules to generate long-lived adaptive immune responses nostimulants (CPDI), we first identified the last 10 amino acid because they lack immunostimulatory molecules from the wild- residues from the C-terminus activation region of human C5a ffi 65 66 67 68 69 70 71 72 type pathogen to su ciently activate macrophages, dendritic (C5a65−74:Ile-Ser -His -Lys -Asp -Met -Gln -Leu - cells, and recruited inflammatory monocytes (mononuclear Gly73-Arg74) as the minimal pharmacophore required for phagocytes) at the vaccine administration site.18 There is also a stimulating guinea pig ileal contraction (surrogate for growing interest in using immunostimulant molecules to activation of mammalian macrophages) but 5-orders of increase the rate and magnitude of protective innate immune magnitude less potent than native human C5a (EC50 155 responses after pathogen exposure in combination with μMvs8.7nM).47 Screening for amino acid residue 26 conventional antimicrobial drugs (adjunct immunotherapy). substitutions within C5a65−74 that selectively increase the

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a Table 1. Relevant Sequences and Molecular Masses of C5a, C5a desArg, EP54, EP67, and EP67 Analogues

peptide Iupac-Iub designation molecular mass (g/mol) amino acid sequence ±  65 66 67 68 69 70 71 72 73 74 C5a C5a1−74 10400 ( 1,000) ile Ser His Lys Asp Met Gln Leu Gly Arg ±  65 66 67 68 69 70 71 72 73 C5a desArg C5a1−73 10250 ( 1,000) ile Ser His Lys Asp Met Gln Leu Gly 65 67 69 71 73 01 02 03 04 05 06 07 08 09 10 Ep54 [Tyr Phe Pro Pro D-Ala ]C5a65−74 1209.6 Tyr Ser Phe Lys Pro Met Pro Leu (D-Ala) Arg 65 67 71 72 73 01 02 03 04 05 06 07 08 09 10 Ep67 [Tyr Phe Pro Me-Leu D-Ala ]C5a65−74 1241.6 Tyr Ser Phe Lys Asp Met Pro (Me-Leu) (D-Ala) Arg 7 65 67 71 72 73 01 02 03 04 05 06 07 08 09 10 [Cha ]Ep67 [Tyr Phe Cha Me-Leu D-Ala ]C5a65−74 1297.7 Tyr Ser Phe Lys Asp Met Cha (Me-Leu) (D-Ala) Arg 8 65 67 71 72 73 01 02 03 04 05 06 07 08 09 10 [Leu ]Ep67 [Tyr Phe Pro Leu D-Ala ]C5a65−74 1227.6 Tyr Ser Phe Lys Asp Met Pro Leu (D-Ala) Arg 7 8 65 67 71 72 73 01 02 03 04 05 06 07 08 09 10 [Cha Leu ]Ep67 [Tyr Phe Cha Leu D-Ala ]C5a65−74 1283.7 Tyr Ser Phe Lys Asp Met Cha Leu (D-Ala) Arg aCha = cyclohexylalanine; Me-Leu = N-methyl-leucine. Molecular masses of C5a and C5a desArg vary due to diﬀerences in glycosylation levels. Substitutions to amino acid residues in EP67 are highlighted in bold and underlined. C5a was not used in the current study because it is quickly converted to C5a desArg in vivo.

Figure 1. De novo peptide structure prediction and comparison of C5a65−74, EP67, and EP67 analogue conformations. (A) Major cluster view and α (B) RMSD traces of C5a65−74, EP67, and EP67 analogues from an -helical structure were generated by PEP-FOLD. Analogues were generated in YASARA and refined for 500 ps using the built-in md_refine macro. Each refined structure was then used in a 50 ns molecular dynamics (MD) simulation. All molecular dynamics simulations and postanalysis used Desmond, as bundled with the Schrödinger software suite. Peptides were placed in a cubic box with periodic boundaries. No dimension of the box was allowed closer than 12 Å to allow the peptides room to unfold. The box was filled with TIP4P water and neutralized by adding appropriate Na+ or Cl− ions. Salt concentration in the box was set to 0.05 M NaCl. All simulations first used Schrödinger’s built-in relaxation protocol before the main MD run. The main 50 ns MD run was an NPT ensemble with temperature at 298 K and pressure at 1 atm. Nose−́Hoover chain and Martyna−Tobias−Klein were the thermostat and barostat methods, respectively. The average structure of the major cluster of each trajectory was then extracted for comparison in YASARA.

ﬁ 65 67 69 71 73 contraction of human umbilical arteries (surrogate for identi ed EP54 ([Tyr Phe Pro Pro D-Ala ]C5a65−74) activation of human macrophages)48 over the secretion of β- (Table 1)asaﬁrst-generation, C5a-derived decapeptide glucuronidase from human polymorphonuclear leukocytes agonist of C5aR1 that selectively increases the potency of μ (PMN) (surrogate for activation of human neutrophils), we C5a65−74 775-fold in human umbilical arteries (EC50 155 M

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Figure 2. Experimental design for determining immunostimulant activity in primary human mononuclear phagocytes and neutrophils. Monocytes (MC) and neutrophils (NP) were isolated from the whole blood of healthy human adult male donors using Miltenyi magnetic anti-CD14 MicroBeads and MACSxpress Whole Blood Neutrophil Isolation Kits, respectively. Isolated NP were plated for 2 h before treatment, whereas isolated CD14+/CD14+CD16+ MC were plated for 2 h before immunostimulant treatment or differentiated into M0-monocyte-derived macrophages (M0-MDM) (human GM-CSF 6 d) or immature monocyte-derived dendritic cells (MDDC) (human GM-CSF/IL-4 for 7 d) before treatment. For potency studies, cells were incubated with increasing concentrations of the indicated immunostimulant for 24 h, and concentrations of IL-6 and TNF-α [MC, M0-MDM, MDDC] (Figure 3) or myeloperoxidase (MPO) [NP] (Figure 5) released into cell culture media was determined by ELISA. ∼ to 200 nM) but only 64-fold in human PMN (EC50 >300 an amphipathic polymer solubilizer (APol) combined with μM to 4.7 μM) (UA/PMN selectivity = 34).49 Importantly, CpG and Montanide increases the magnitude and quality of B- systemic immunization with EP54 directly conjugated to the cell and T-cell adaptive immune responses generated by a C-terminal of peptide epitopes increases epitope-specific protective APol-solubilized membrane protein and subsequent ̈ antibodies and CD8+ T-cells in mice (Ab, CD8+ T-cells) and protection of naive mice against primary respiratory challenge rabbits (Ab), indicating that EP54 is an immunostimulant with Chlamydia trachomatis.59 In addition to acting as a molecule that increases the generation of B-cell and T-cell systemic and mucosal adjuvant, respiratory administration of adaptive immune responses.39,50 EP67 alone within a 24 h window before or after respiratory ̈ Pro amino acid residue substitutions at positions 5 and 7 challenge also increases protection of naive mice against fl 60 (positions 69 and 71 in C5a65−74)(Table 1) likely contribute primary mucosal infection with in uenza. to the immunostimulatory potency and mononuclear phag- Although EP67 is a promising candidate for the develop- ocyte selectivity of EP54 by extending the peptide backbone ment of novel vaccines and adjunct immunotherapies, the but eliminate potential side-chain contributions by the inefficient coupling efficiency between Pro7 and Me-Leu8 5 7 49,51,52 substituted Asp and Gln residues. Given that adding (positions 71 and 72 in C5a65−74)(Table 1) and challenging methyl groups to backbone amide nitrogen atoms extends the purification by HPLC greatly increase scale-up costs for clinical peptide backbone without removing contributions of adjacent use. Pro7 and Me-Leu8, however, likely induce structural side-chains, we next screened N-methylated backbone changes to the peptide backbone of EP67 that increase potency analogues of EP54 and identified a second-generation C5a- and selectivity over first-generation EP54.53 As such, we derived decapeptide agonist of C5aR1, EP67 wanted to determine if we could overcome the scale-up 65 67 71 72 73 7 8 ([Tyr Phe Pro Me-Leu D-Ala ]C5a65−74)(Table 1), as a challenges of EP67 by replacing Pro and/or Me-Leu with more potent and mononuclear phagocyte-selective immuno- large-scale amenable amino acid residues predicted to induce stimulant molecule that increases the potency of C5a65−74 similar structural changes and, consequently, preserve the μ 1107-fold in human umbilical arteries (EC50 155 M to 140 activity of EP67. ∼ μ nM) but only 1.6-fold in human PMN (EC50 >300 Mto The NMR structure of C5a alone or as a part of inactive C5 190 μM) (UA/PMN selectivity = 2951).53 is a four-helix core stabilized by three disulfide bridges35 that Systemic immunization with EP67 directly conjugated to consists of a receptor recognition region and a C-terminus whole protein,38 peptide epitopes,50 or attenuated patho- activation region that contains the minimal pharmacophore, 54,55 35 gens generates Th1- and Th17-biased adaptive immune C5a65−74. Crystallographic and NMR studies show that the responses in young and old mice.56 Respiratory immunization C-terminus activation region has an α-helical structure when with EP67 directly conjugated to peptide epitopes alone57,58 or part of C5 but an extended, flexible structure after C5 is indirectly conjugated to encapsulated protein vaccines on the cleaved to form C5a.35,61 Recent Cryo-EM studies further surface of biodegradable nanoparticles11 also increases the show that the receptor recognition region retains its α-helical generation of long-lived mucosal and systemic memory CD8+ structure when part of C5, whereas the C-terminus activation T-cells and subsequent protection against primary respiratory region likely changes conformation following conversion to challenge in mice. Furthermore, the combination of respira- C5a because it is not held in place by the disulfide bridges.62 tory/systemic immunization with EP67 directly conjugated to These data suggest that the more potent form of C5a65−74,in

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Figure 3. Dose−response curves of IL-6 and TNF-α secretion from primary human monocytes, M0-monocyte-derived macrophages, and immature monocyte-derived dendritic cells after treatment with human C5a desArg, EP54, EP67, or EP67 analogues for 24 h. Human monocytes (MC), unpolarized M0-monocyte-derived macrophages (M0-MDM), and immature monocyte-derived dendritic cells (MDDC) were prepared from the whole blood of healthy human adult male donors (Figure 2). Cells (1 × 106) were then treated with increasing concentrations of human C5a desArg pooled from the blood of multiple donors (black circles), first-generation EP54 (black squares), 2nd-generation EP67 (black triangles), [Cha7]EP67 (red open circles), [Leu8]EP67 (green open squares), or [Cha7, Leu8]EP67 (blue open triangles). After 24 h, average concentrations (±SD) (n = 2 replicates from 3 blood donors) of IL-6 or TNF-α secreted into the cell culture media of human monocytes (A and D), M0-MDM (B and E), or MDDC (C and F) were determined by ELISA and fit with a four-parameter dose−response curve where Y = min + (xHillslope) * (max − HillSlope HillSlope min)/(x +EC50 ). contrast to the helical structure observed in C5, is an extended, Primary Human Mononuclear Phagocytes. To determine flexible structure that more effectively engages the activation if replacing Pro7 and/or Me-Leu8 with large-scale amenable site of C5aR1. amino acid residues predicted to induce similar structural The major cluster view and RMSD trace of C5a65−74 alone changes affects the potency and efficacy of EP67 in human α versus an -helical structure from de novo structure prediction mononuclear phagocytes, we replaced Pro7 with Cha ([Cha7]- showed long periods of low RMSD to the major cluster, 8 8 7 α EP67), Me-Leu with native Leu ([Leu ]EP67), or Pro and suggesting that C5a65−74 maintains an -helical backbone Me-Leu8 with Cha and native Leu, respectively ([Cha7Leu8]- conformation in solution (Figure 1). In contrast to C5a65−74, 7 8 EP67) (Table 1); determined purity (Table S4); and although [Cha Leu ]EP67 is masked in the major cluster view fi (Figure 1A), RMSD traces suggest that EP67 has a more con rmed the molecular mass (Table S5) and amino acid fl residue composition (Table S6) of each purified peptide. We exible structure in solution that is maintained by replacing + + + Pro7 with the bulky trans-amino acid residue cyclohexylalanine then prepared human CD14 /CD14 CD16 monocytes (MC) fl (Cha) and/or replacing Me-Leu7 with Leu (Figure 1B). Thus, (>97% purity by ow cytometry, not shown), unpolarized we hypothesized that replacing Pro7 and/or Me-Leu8 with Cha human monocyte-derived macrophages (M0-MDM), and and/or Leu amino acid residues, respectively, will not adversely immature human monocyte-derived dendritic cells (MDDC) affect the activity of EP67. To test this hypothesis, we from the whole blood of healthy human adult male donors compared EP67 potency, efficacy, and selective activation of (Figure 2) and compared the dose-dependent secretion of IL- human mononuclear phagocytes (monocytes, unpolarized 1β, IL-6, and TNF-α after treating with pooled human C5a (M0)-monocyte-derived macrophages, immature monocyte- desArg (parent molecule), EP54, EP67, or EP67 analogues for derived dendritic cells) over human neutrophils with Cha7/ β α 8 24 h. IL-1 , IL-6, and TNF- are characteristic surrogate Leu analogues of EP67. markers for the activation of mononuclear phagocytes because they are secreted by activated tissue-resident macrophages, ■ RESULTS AND DISCUSSION dendritic cells, and recruited inflammatory monocytes during Replacing Pro7 with Cha and/or Me-Leu8 with Leu the early stages of infection to drive acute inflammation in Selectively Affects the Potency and Efficacy of EP67 in support of innate and adaptive immune responses.33,63

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Figure 4. Comparison of potencies and efficacies for IL-6 and TNF-α secretion from primary human mononuclear phagocytes after treatment with ffi ± human C5a desArg, EP54, EP67, or EP67 analogues for 24 h. Average (A) EC50 (potencies) and (B) EMAX (e cacies) 95% CI for IL-6 and TNF-α were calculated (Table S1) from dose−response curves of IL-6 and TNF-α secretion from human monocytes (MC), M0-monocyte-derived macrophages (M0-MDM), and monocyte-derived dendritic cells (MDDC) after treatment for 24 h (Figure 3). 0.05 level of statistical difference vs aEP67 (black triangles) for the indicated cytokine and cell type.

Table 2. Summary of EP67 Analogue Eﬀects on EP67 Potency and Eﬃcacy in Primary Human Mononuclear Phagocytes and a Neutrophils

potencyb efficacyc potencyb efficacyc MC M0-MDM MDDC MC M0-MDM MDDC NP NP peptide IL-6 TNF-α IL-6 TNF-α IL-6 TNF-α IL-6 TNF-α IL-6 TNF-α IL-6 TNF-α MPO MPO [Cha7]EP67 NC NC +29% NC NC NC NC +9.5% −9.5% −16% −5.2% −16.6% NC −20% [Leu8]EP67 NC NC NC NC NC NC −9.3% NC NC −23% +41% +45% NC −26% [Cha7Leu8]EP67 NC NC +31% NC NC +39% NC NC −21% −21% −16% −10% NC NC aValues calculated from Table S1 and Table S2. Cha = cyclohexylalanine, MC = human monocytes, M0-MDM = unpolarized human monocyte- derived macrophages, MDDC = human monocyte-derived dendritic cells, NP = human neutrophils, and NC = no change. bIncreased (+) or − − Δ Δ Δ − * c decreased ( ) % potency = % EC50 or +% EC50 vs EP67 EC50, where % EC50 = (EP67 analogue EC50/EP67 EC50) 1) 100%. Increased − ffi Δ − Δ Δ − * (+) or decreased ( )%e cacy = +% EMAX or % EMAX vs EP67 EMAX, where % EMAX = (EP67 analogue EMAX/EP67 EMAX) 1) 100%.

Sigmoidal dose−response curves were observed for IL-1β EP54 (Figure 4A, black squares) ranged from 22.5- to 123.3- secretion from MC, M0-MDM, and MDDC over the fold greater than C5a desArg, depending on the secreted concentration range of human C5a desArg (Figure S1), cytokine and mononuclear phagocyte (Table S1). Further- β whereas IL-1 was not detected after treatment with up to 1 more, the EC50 of EP67 (Figure 4A, black triangles) was mM EP54, EP67, or EP67 analogues (not shown). In contrast similar to the EC50 of EP54 (Figure 4A, black squares), with to IL-1β, sigmoidal dose−response curves were observed for the exception of TNF-α secretion from MC (35% less) (121 ± α IL-6 and TNF- secretion from MC, M0-MDM, and MDDC [−0.2,+0.3] [95% CI] vs 186 [−0.2,+0.3] nM) and IL-6 over the concentration ranges of C5a desArg (Figure 3, black secretion from M0-MDM (47% less) (167 ± [−0.3,+0.3] circles), EP54 (Figure 3, black squares), EP67 (black [95% CI] vs 317 [−0.9,+1] nM) (Table S1). Similar triangles), and EP67 analogues (Figure 3, colored open magnitudes of potencies for recombinant human C5a (EC symbols). This suggests the current C5a-derived immunosti- 50 5 nM), EP67 (EC50 140 nM), and EP54 (EC50 220 nM) were mulants, in contrast to the C5a/C5a desArg parent molecule, reported for the contraction of human umbilical arteries do not stimulate the secretion of IL-1β from human 53 (surrogate for activation of human macrophages). mononuclear phagocytes. Given that C5aR1 is a G protein- The E of EP67 (Figure 4B, black triangles), with the coupled receptor (GPCR), one possibility is that EP67 and MAX exception of a similar E for TNF-α from M0-MDM, ranged EP67 analogues act as “biased agonists” of C5aR1 in MAX mononuclear phagocytes that stabilize unique active C5aR1 from 6 to 27% lower than C5a desArg (Figure 4B, black conformations, which alter the extent of Gα and β-arrestin circles), whereas the EMAX of EP54 (Figure 4B, black squares) coupling, the rate of GTP binding by Gα, the turnover of G ranged from 24 to 47% lower than C5a desArg, depending on protein, the activation of downstream signaling targets, and the the secreted cytokine and mononuclear phagocyte (Table S1). regulation of GPCR compared to C5a/C5a desArg.64 This Thus, EP67 and EP54 are both partial agonists for activating possibility is supported by the recent discovery of a human mononuclear phagocytes compared to pooled human hexapeptide (C5apep) with high affinity for C5aR1 that exhibits C5a desArg. biased agonism of neutrophil migration relative to C5a through The EMAX of EP67 (Figure 4B, black triangles) was 23.6 to decreased β-arrestin coupling and C5aR1-receptor traffick- 34% greater than the EMAX of EP54 (Figure 4B, black squares), ing.65 also depending on the secreted cytokine and mononuclear The EC50 of EP67 in MC, M0-MDM, and MDDC (Figure phagocyte. These results indicate that the potency of EP67 is 4A, black triangles) ranged from 14.6- to 89.5-fold greater than similar or greater than EP54, depending on the secreted ffi C5a desArg (Figure 4A, black circles), whereas the EC50 for cytokine and mononuclear phagocyte, whereas the e cacy of

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Figure 5. Comparison of potencies and efficacies for myeloperoxidase secretion from primary human neutrophils after treatment with human C5a desArg, EP54, EP67, or EP67 analogues for 24 h. (A) Human neutrophils (NP) were prepared from the whole blood of healthy human adult male donors (Figure 2). NP were then treated with increasing concentrations of human C5a desArg pooled from the blood of multiple donors (black circles), EP54 (black squares), EP67 (black triangles), [Cha7]EP67 (open red circles), [Leu8]EP67 (open green squares), or [Cha7, Leu8]EP67 (open blue triangles). After 24 h, average concentrations (±SD) (n = 2 replicates from 3 blood donors) of myeloperoxidase (MPO) secreted into cell culture media were determined by ELISA and fit with a four-parameter dose−response curve where Y = min + (xHillslope) * (max − min)/ HillSlope HillSlope ffi ± (x +EC50 ). Average (B) potencies (log EC50) and (C) e cacies (EMAX) 95% CI for MPO secretion (Table S2) were calculated from MPO dose−response curves. a0.05 level of statistical difference vs EP67.

EP67 for IL-6 and TNF-α secretion is greater than EP54 in In summary (Table 2), (i) [Cha7]EP67 does not affect human mononuclear phagocytes. potency but selectively increases EP67 efficacy in MC, [Cha7]EP67 (Figure 4A, red open circles) did not affect the selectively increases potency but decreases EP67 efficacy in α ff EC50 of EP67 (Figure 4A, black triangles) for IL-6 and TNF- M0-MDM, and does not a ect potency but decreases EP67 α ffi 8 ff from MC, decreased EC50 for IL-6 (29%) but not TNF- from e cacy in MDDC; (ii) [Leu ]EP67 does not a ect potency in ff α M0-MDM, and did not a ect EC50 for IL-6 and TNF- from MC, M0-MDM, or MDDC but selectively decreases EP67 MDDC. [Leu8]EP67 (Figure 4A, green open squares) did not efficacy in MC and M0-MDM and greatly increases efficacy in ff α 7 8 ff a ect the EC50 of EP67 for IL-6 and TNF- from MC, M0- MDDC; and (iii) [Cha Leu ]EP67 does not a ect EP67 MDM, or MDDC. [Cha7Leu8]EP67 (Figure 4A, blue open potency or efficacy in MC, selectively increases potency but ff α ffi triangles) did not a ect the EC50 of EP67 for IL-6 and TNF- decreases EP67 e cacy in M0-MDM, and selectively increases ff ffi from MC, decreased EC50 for IL-6 (31%) without a ecting potency but decreases EP67 e cacy in MDDC. Thus, α ff 7 8 ff EC50 for TNF- from M0-MDM, and did not a ect EC50 for replacing Pro with Cha and/or Me-Leu with Leu a ects α ffi IL-6 but decreased EC50 for TNF- (39%) from MDDC. the potency and e cacy of EP67 depending on the secreted Thus, [Cha7]EP67 selectively increases EP67 potency in M0- cytokine and human mononuclear phagocyte. These observa- MDM without affecting potency in MC or MDDC; [Leu8]- tions, like the inability to stimulate the secretion of IL-1β at up EP67 does not affect EP67 potency in MC, M0-MDM, or to 1 mM, are consistent with the possibility that EP67 and MDDC; and [Cha7Leu8]EP67 selectively increases EP67 EP67 analogues act as biased agonists of C5aR1 in potency in M0-MDM and MDDC without affecting potency mononuclear phagocytes compared to C5a desArg. in MC (Table 2). Replacing Pro7 with Cha and/or Me-Leu8 with Leu [Cha7]EP67 (Figure 4B, red open circles) did not affect the Does Not Affect Potency but Selectively Decreases the ffi EMAX of EP67 (Figure 4B, black triangles) for IL-6 but E cacy of EP67 in Primary Human Neutrophils. To α 7 8 increased EMAX for TNF- (9.5%) from MC and decreased determine if replacing Pro with Cha and/or Me-Leu with Leu α ff ffi EMAX for IL-6 and TNF- from M0-MDM (9.5%, 16%) and a ects the potency and e cacy of EP67 in human neutrophils MDDC (5.2%, 16.6%). [Leu8]EP67 (Figure 4B, green open (NP), we isolated human neutrophils (NP) from the whole squares) decreased the EMAX of EP67 for IL-6 (9.3%) without blood of healthy human adult male donors (>94% purity by ff α ff fl a ecting EMAX for TNF- from MC, did not a ect EMAX for IL- ow cytometry, not shown) (Figure 2) and then compared α 6 but decreased EMAX for TNF- (23%) from M0-MDM, and, dose-dependent secretion of myeloperoxidase (MPO) from α surprisingly, greatly increased EMAX for IL-6 and TNF- from NP after treating with pooled human C5a desArg, EP54, EP67, MDDC (41%, 45%) to levels similar (IL-6) or greater (TNF- or EP67 analogues for 24 h (Figure 5). Sigmoidal dose− α) than C5a desArg (Figure 4B, black circles). [Cha7Leu8]- response curves were observed for MPO secretion from NP ff EP67 (Figure 4B, blue open triangles) did not a ect the EMAX over the concentration ranges of C5a desArg, EP54, EP67, and α of EP67 for IL-6 and TNF- from MC but decreased EMAX for EP67 analogues (Figure 5A). α IL-6 and TNF- from M0-MDM (21%, 21%) and MDDC The EC50 of EP67 for MPO secretion from NP (Figure 5B, (16%, 10%), respectively. Thus, [Cha7]EP67 selectively black triangle) was 22857-fold greater than C5a desArg (Figure increases EP67 efficacy in MC but decreases efficacy in M0- 5C, black circle) (160 [−17,+20] μMvs7± 1 nM [95% CI]), 8 MDM and MDDC, and [Leu ]EP67 selectively decreases whereas the EC50 of EP54 (Figure 5B, black square) was 3000- EP67 efficacy in MC and M0-MDM but significantly increases fold greater (21 [−3,+4] μMvs7± 1 nM [95% CI]) (Table ffi 7 8 ff e cacy in MDDC, whereas [Cha Leu ]EP67 does not a ect S2). The EMAX of EP67 for MPO secretion from NP (Figure EP67 efficacy in MC but decreases efficacy in M0-MDM and 5C, black triangle) was also 62.4% less than C5a desArg MDDC. (Figure 5C, black circle) (3.5 ± 0.1 [95% CI] vs 9.3 ± 0.3 ng/

1175 https://dx.doi.org/10.1021/acsinfecdis.0c00005 ACS Infect. Dis. 2020, 6, 1169−1181 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article

Figure 6. Comparison of selective activation of primary human mononuclear phagocytes vs human neutrophils after treatment with human C5a desArg, EP54, EP67, or EP67 analogues for 24 h. Average selectivities ± propagated 95% CI for stimulating the secretion of IL-6 or TNF-α from human (A) MC, (B) M0-MDM, or (C) MDDC over secretion of MPO from human neutrophils (NP) were calculated from respective EC50 values after treatment for 24 (Table S3). mL), whereas the EMAX of EP54 (Figure 5C, black square) was (Figure 6, black triangles) were 9- to 15-fold greater than EP54 39.8% less (5.6 ± 0.2 [95% CI] vs 9.3 ± 0.3 ng/mL) (Table (Figure 6, black squares) within the same cell type, again S2). Furthermore, the EC50 for EP67 was 7.6-fold greater than depending on the secreted cytokine and mononuclear EP54 (160 [−17,+20] [95% CI] vs 21 [−3,+4] μM), and the phagocyte (Table S3). Thus, EP67 selectivity for stimulating ± ± EMAX was 60% less than EP54 (3.5 0.1 [95% CI] vs 5.6 0.2 human mononuclear phagocytes over human neutrophils ng/mL) in NP (Table S2). Similar potencies for MPO versus pooled human C5a desArg is lower than the selective secretion from human PMNs (surrogate for activation of activation of human umbilical artery contraction (surrogate for human neutrophils) were reported for recombinant human activation of human macrophages) over human PMNs μ μ C5a (EC50 6 M), EP54 (EC50 4.4 M), and EP67 (EC50 190 (surrogate for activation of human neutrophils) but still μM).53 Thus, consistent with previous studies, (i) the potency much greater than EP54. A much higher selectivity for of EP67 and EP54 is well below the potency of human C5a stimulating the contraction of human umbilical artery desArg in human neutrophils, and (ii) EP67 is less potent and activation over MPO secretion from human PMNs was efficacious in human neutrophils than EP54. reported for EP67 (2951), whereas a similar, low selectivity 53 None of the EP67 analogues (Figure 5B, colored open was reported for EP54 (34). This may be due to differences ff symbols) a ected the EC50 of EP67 for MPO secretion from between EP67 activity in whole tissues versus isolated cells. NP (Figure 5B, black triangle). In contrast, [Cha7]EP67 None of the EP67 analogues (Figure 6, colored open (Figure 5C, red open circle) and [Cha7Leu8]EP67 (Figure 5C, symbols) affected the selectivities of EP67 for stimulating IL-6 α blue open triangle) decreased the EMAX of EP67 (Figure 5C, or TNF- secretion from MC (Figure 6A), M0-MDM (Figure black triangle) for MPO secretion by 20% (2.8 ± 0.1 [95% CI] 6B), or MDDC (Figure 6C) over stimulating the secretion of vs 3.5 ± 0.2 ng/mL) and 26% (2.6 ± 0.1 [95% CI] vs 3.5 ± 0.2 MPO from NP (Figure 6C, black triangles) (Table S3). Thus, 7 8 ng/mL), respectively (Table S2), whereas [Leu8]EP67 (Figure replacing Pro with Cha and/or Me-Leu with Leu does not affect the selective activation of human mononuclear 5C, green open square) had a similar EMAX compared to EP67. Thus, replacing Pro7 with Cha and/or Me-Leu8 with Leu does phagocytes over human neutrophils by EP67 versus pooled not affect EP67 potency but selectively decreases efficacy in human C5a desArg. human neutrophils (Table 2). These observations, like the differences in potency and efficacy observed in mononuclear ■ CONCLUSION phagocytes, are consistent with the possibility that EP67 and We found that replacing Pro7 with Cha and/or Me-Leu8 with EP67 analogues act as biased agonists of C5aR1 in both Leu does not significantly affect EP67 potency, efficacy, or mononuclear phagocytes and neutrophils compared to C5a selective activation of human mononuclear phagocytes over desArg. human neutrophils. Thus, replacing Pro7 and Me-Leu8 with Replacing Pro7 with Cha and/or Me-Leu8 with Leu large-scale amenable amino acid residues predicted to induce Does Not Affect Selective Activation of Primary Human similar structural changes is a suitable strategy to overcome Mononuclear Phagocytes over Human Neutrophils by scale-up challenges with EP67. EP67. To determine if replacing Pro7 with Cha and/or Me- Leu8 with Leu affects the selective activation of human ■ METHODS mononuclear phagocytes over human neutrophils, we Experimental Methods. Peptide Synthesis, Purification, compared the selective stimulation of IL-6 or TNF-α secretion and Characterizations. Using an AAPPTEC Apex 396 from human MC, M0-MDM, and MDDC over secretion of synthesizer, all peptides were assembled on preloaded Fmoc- MPO from human NP 24 h after treatment with EP54, EP67, Arg(pbf)-Wang resin using Fmoc-amino acid derivatives and or EP67 analogues versus pooled human C5a desArg (Figure N-(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-yl- 6). The selectivity of EP67 for stimulating MC, M0-MDM, and methylene]-N-methylmethanaminium hexafluorophosphate N- MDDC over NP versus C5a desArg (Figure 6, black triangles) oxide (HATU) in the presence of excess DIEA. A solution of ranged from 302 to 1413, whereas the selectivity of EP54 piperidine in DMF (1/4, v/v) was used to remove the Fmoc versus C5a desArg (Figure 6, black squares) ranged from 23 to group. Peptides were cleaved from the resin and freed of side 126, depending on the secreted cytokine and mononuclear chain protecting groups by stirring the peptide resin in a phagocyte (Table S3). Furthermore, the selectivities of EP67 mixture of TFA (87.5%), phenol (5%), water (5%), and

1176 https://dx.doi.org/10.1021/acsinfecdis.0c00005 ACS Infect. Dis. 2020, 6, 1169−1181 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article triisopropylsilane (2.5%) for 2 h at room temperature. The monocytes were plated in 24-well plates (1 × 106 cells/well) crude products were purified by preparative reversed-phase (BD Biosciences) with complete culture medium (CCM) (1 high performance liquid chromatography (RP-HPLC) on C18- mL) (RPMI 1640, 2 mM L-glutamine, 1% autologous plasma, bonded silica columns under optimized gradient elution 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1× conditions employing 0.25 N triethylammonium phosphate, vitamins, 100 U/mL penicillin G, 100 μg/mL streptomycin ff ff ° pH 2.25, (bu er A) and a mixture of bu er A and acetonitrile sulfate; Invitrogen, US) and incubated (37 C/5% CO2). After (2/3, v/v, buffer B).66 Peptides were loaded onto the same 2 h, the media was aspirated, and the adherent cells were column, previously equilibrated with a mixture of 10 mM washed 2× with sterile D-PBS (1 mL) (“PBS” without Ca2+ or hydrochloric acid and acetonitrile (95/5, v/v), and quickly Mg2+; GE Healthcare Life Sciences, SH30028.02) before (i) eluted by raising the amount of acetonitrile in the eluent to adding CCM alone (1 mL) or CCM containing increasing 60% over 30 min to produce the hydrochloride salt. concentrations of the indicated immunostimulant and Peptide purity was determined by analytical reversed-phase incubating for 24 h or (ii) adding CCM (1 mL) containing HPLC using three different columns with the same gradient the required concentrations of factors for differentiation into elution conditions: an ACE 5 C18−300 column (250 × 4.6 macrophages or dendritic cells over 6 days (described below). mm, 5 μm, 300 Å pore size, catalog number ACE-221-2546), Neutrophils were isolated from the same donors (10 mL of an ACE 5 phenyl-300 column (250 × 4.6 mm, 5 μm, 300 Å blood/donor) using a MACSxpress whole blood neutrophil pore size, catalog number ACE-225-2546), and an ACE 5 CN- isolation kit, a MACSmix tube rotator, and a MACSxpress 300 column (250 × 4.6 mm, 5 μm, 300 Å pore size, catalog separator (Miltenyi Biotec, Germany), according to the number ACE-224-2546) from MAC-MOD Analytical Inc. manufacturer’s protocol, and counted (Auto T4, Nexcelom). (Chadds Ford, PA). Solvent A was water containing 0.1% Isolated neutrophils were plated in 24-well plates (1 × 106 trifluoroacetic acid (TFA, v/v), and solvent B was a mixture of cells/well) with CCM (1 mL) and incubated (37 °C/5% acetonitrile and water (3/2, v/v) containing 0.1% TFA. CO2). After 2 h, the media was aspirated, and the adherent Peptides were eluted from the columns by increasing the cells were washed 2× with sterile D-PBS (1 mL) before adding percentage of solvent B from 5 to 100% over 50 min, and the CCM alone (1 mL) or CCM containing increasing column effluent was continuously monitored at 214 nm. concentrations of the indicated immunostimulant. Peptide masses were measured on an Orbitrap Fusion Lumos The purity of isolated monocytes and neutrophils was from ThermoScientific (Waltham MA) and with ≤0.5 ppm determined on the day of isolation by staining the cells with error (Mass Spectrometry and Proteomics Core Facility at the recombinant CD14-PE-Vio615, CD-15APC, and CD-16PE University of Nebraska Medical Center). Amino acid human antibodies (Miltenyi Biotec, Germany), before and compositions of all peptides were determined using a Hitachi after separation, and by analyzing via flow cytometry. Isolated 8800 amino acid analyzer after being subjected to vapor-phase cells (1 × 106) were resuspended in cell staining buffer (BD hydrolysis in constant boiling 6 M hydrochloric acid for 24 h Biosciences) (0.100 mL) containing the indicated antibodies (Protein Structure Core Facility at the University of Nebraska (5 μL/each antibody). The cells were gently mixed, incubated Medical Center). in the dark (2−8 °C, 10 min.), pelleted (300 RCF, RT, 10 Peptide Structure Prediction. PEP-FOLD67 was used to min), resuspended in staining buffer (1 mL), pelleted (300 ff generate the initial conformation of C5a65−74. The analogues of RCF, RT, 10 min.), and resuspended in FACS staining bu er fl C5a65−74 were generated in YASARA (www.yasara.org) and (0.5 mL) for analysis by ow cytometry. Cells were analyzed refined for 500 ps using the built-in md_refine macro. Each on a BD LSR II flow cytometer (Becton and Dickinson, La refined structure was then used in a 50 ns molecular dynamics Jolla, CA) with a BD high-throughput sampler. The flow (MD) simulation. All MD simulations and postanalysis used cytometer was compensated using single stained cells, and the Desmond68 as bundled with the Schrödinger software suite. maximum number of events were acquired and analyzed by Each peptide was placed in a cubic box with periodic FlowJo software (Tree Star, Ashland, OR, US). boundaries. No dimension of the box was allowed closer Generation of Unpolarized Human Monocyte-Derived than 12 Å to allow the peptides room to unfold. The box was Macrophages (M0-MDM) and Immature Human Monocyte- filled with TIP4P water and neutralized by adding the Derived Dendritic Cells (MDDC). Unpolarized human appropriate Na+ or Cl− ions. The salt concentration in the monocyte-derived macrophages (M0-MDM) were generated box was set to 0.05 M NaCl. All simulations first used by culturing freshly isolated monocytes in 24-well plates (1 × Schrödinger’s built-in relaxation protocol before the main MD 106 cells/well) with CCM (1 mL) containing recombinant run. The main 50 ns MD run was an NPT ensemble with the human M-CSF (rhM-CSF, Miltenyi Biotec) (50 ng/mL) for 3 temperature at 298 K and pressure at 1 atm. Nose−́Hoover days, replacing half the media (0.5 mL) from each well with chain and Martyna−Tobias−Klein were used as the thermostat fresh CCM (0.5 mL) and rhM-CSF (50 ng/mL), and and barostat methods, respectively. The average structure of incubating for another 3 days.69 Immature human monocyte- the major cluster of each trajectory was then extracted for derived dendritic cells (MDDC) were generated in the same comparison in YASARA. manner as M0-MDM but incubated with CCM containing Isolation of Human Monocytes and Neutrophils from recombinant human IL-4 (rhIL-4, Miltenyi Biotec) (50 ng/ Human Whole Blood. Fresh whole blood (1 unit = 450 mL) mL) and recombinant human GM-CSF (Miltenyi Biotec) was drawn from healthy human male donors (aged 19−40 (160 ng/mL).70 On day 7, the media was aspirated, and the years) into vacutainer bags containing EDTA (Innovative cells were washed once with warm, sterile PBS (37 °C) and + + + ° Research, US). CD14 and CD14 /CD16 monocytes were incubated (37 C/5% CO2) with CCM (1 mL) containing the isolated from whole blood (180 mL of blood/donor) using indicated immunostimulant for 24 h. magnetic StraightFrom Whole Blood CD14 MicroBeads Potency of EP67 Analogues in Human Mononuclear (Miltenyi Biotec, Germany), according to the manufacturer’s Phagocytes and Neutrophils. Mononuclear phagocytes (MC, protocol, and counted (Auto T4, Nexcelom). Isolated M0-MDM, MDDC) or NP were plated in 24-well plates (1 ×

1177 https://dx.doi.org/10.1021/acsinfecdis.0c00005 ACS Infect. Dis. 2020, 6, 1169−1181 ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article 106 cells/well) as described above and incubated (37 °C/5% ■ AUTHOR INFORMATION CO2) with CCM (1 mL) containing serial concentrations of Corresponding Author pooled human C5a desArg (Complement Technology Inc., Joseph A. Vetro − Center for Drug Delivery and Nanomedicine TX, USA), EP54, EP67, or EP67 analogues. After 24 h, the and Department of Pharmaceutical Sciences, College of average concentrations of IL-1β, IL-6, and TNF-α ( ± SD) (n Pharmacy, University of Nebraska Medical Center, Omaha, = 2 wells from 3 independent donors) in the media of Nebraska 68198, United States; orcid.org/0000-0003- mononuclear phagocytes or the average concentrations of 2446-9797; Email: [email protected] myeloperoxidase (MPO) ( ± SD) (n = 2 wells from 3 independent donors) in the media of NP were determined by Authors ELISA (BioLegend, US). The average maximal concentration − “ ffi ” Abdulraman M. Alshammari Department of Pharmaceutical of secreted protein (EMAX/ e cacy ) and molar concentration “ ” ± Sciences, College of Pharmacy, University of Nebraska Medical of peptide that stimulated 50% EMAX (EC50/ potency )( Center, Omaha, Nebraska 68198, United States 95% CI) was then calculated by fitting the dose−response data − − D. David Smith Department of Biomedical Sciences, Creighton with a four-parameter dose response curve where Y = min + University, Omaha, Nebraska 68178, United States; Hillslope * − HillSlope HillSlope (x ) (max min)/(x +EC50 ) (Graphpad orcid.org/0000-0002-8956-9687 Prism 8). Jake Parriott − Department of Pharmaceutical Sciences, College Selective Activation of Human Mononuclear Phagocytes of Pharmacy, University of Nebraska Medical Center, Omaha, over Neutrophils. The average selective activation of the Nebraska 68198, United States indicated mononuclear phagocyte versus the activation of NP ( − ± Jason P. Stewart Department of Pharmaceutical Sciences, propagated 95% CI) (n = 3 independent donors) by EP54, College of Pharmacy, University of Nebraska Medical Center, EP67, and EP67 analogues relative to C5a desArg “ ” −Δ Omaha, Nebraska 68198, United States ( selectivity ) was calculated as the following: antilog(( C5a Stephen M. Curran − Department of Pharmaceutical Sciences, desArg mononuclear phagocyte) − (−ΔC5a desArg NP)), Δ − College of Pharmacy, University of Nebraska Medical Center, where C5a desArg = pD2 (C5a desArg) pD2 (peptide) and − α Omaha, Nebraska 68198, United States pD2 = log(EC50(M)) for IL-6 and TNF- secretion from the Russell J. McCulloh − Department of Pediatrics, Children’s indicated mononuclear phagocyte or MPO secretion from 53 Hospital and Medical Center, Omaha, Nebraska 68114, United NP. The selectivity of human C5a desArg was set to a value States of1usingthisequationbecauseitisequipotentin Peter A. Barry − Center for Immunology and Infectious Diseases, mononuclear phagocytes and NP. Thus, the greater the Pathology and Laboratory Medicine, UC Davis School of selectivity value, the greater the selective activation of Medicine, Davis, California 95817, United States mononuclear phagocytes over neutrophils relative to C5a Smita S. Iyer − Center for Immunology and Infectious Diseases, desArg. Pathology, Microbiology & Immunology, UC Davis, School of Veterinary Medicine, California National Primate Research ■ ASSOCIATED CONTENT Center, Davis, California 95817, United States *sı Supporting Information Nicholas Palermo − Holland Computing Center, University of The Supporting Information is available free of charge at Nebraska-Lincoln, Lincoln, Nebraska 68588, United States − https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00005. Joy A. Phillips Donald P. Shiley BioScience Center, San Diego State University, San Diego, California 92182, United States The Supporting Information includes the following: Yuxiang Dong − Department of Pharmaceutical Sciences, ffi α Table S1, potencies and e cacies of IL-6 and TNF- College of Pharmacy, University of Nebraska Medical Center, secretion from primary human monocytes, M0-mono- Omaha, Nebraska 68198, United States; orcid.org/0000- cyte-derived macrophages, and monocyte-derived den- 0003-1412-3178 dritic cells after treatment with human C5a desArg, Donald R. Ronning − Department of Pharmaceutical Sciences, EP54, EP67, or EP67 analogues for 24 h; Table S2, College of Pharmacy, University of Nebraska Medical Center, ffi potencies and e cacies of myeloperoxidase secretion Omaha, Nebraska 68198, United States; orcid.org/0000- from primary human neutrophils after treatment with 0003-2583-8849 human C5a desArg, EP54, EP67, or EP67 analogues for Jonathan L. Vennerstrom − Department of Pharmaceutical 24 h; Table S3, selectivities of human C5a desArg, EP54, Sciences, College of Pharmacy, University of Nebraska Medical EP67, and EP67 analogues for the activation of primary Center, Omaha, Nebraska 68198, United States; orcid.org/ human monocytes, M0-monocyte-derived macrophages, 0000-0003-0075-2336 and monocyte-derived dendritic cells versus human Sam D. Sanderson − Department of Pharmaceutical Sciences, neutrophils; Table S4, RP-HPLC characteristics of College of Pharmacy, University of Nebraska Medical Center, EP54, EP67, and EP67 analogues; Table S5, measured Omaha, Nebraska 68198, United States molecular masses of EP54, EP67, and EP67 analogues; Table S6, amino acid residue compositions of EP54, Complete contact information is available at: EP67, and EP67 analogues; Figure S1, dose−response https://pubs.acs.org/10.1021/acsinfecdis.0c00005 curves of IL-1β secretion from primary human monocytes, unpolarized M0-monocyte-derived macro- Notes fi phages, and immature monocyte-derived dendritic cells The authors declare no competing nancial interest. after treatment with human C5a desArg for 24 h; Figure S2, RP-HPLC chromatograms of EP67 and EP67 ■ ACKNOWLEDGMENTS analogues; and Figure S3, mass chromatograms of This work was supported by NIH/NIAID 5R01AI125137 EP67 and EP67 analogues (PDF) (S.D.S., J.A.V., and J.L.V.), NIH/NIAID 1R01AI121050

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(J.A.V.), the Weitz Family Foundation (S.D.S. and J.A.V.), (7) RTS (2017) S/AS01E malaria vaccine (MoSQUIRIX*) NIH/OD UG1OD024953 (R.J.M.), NIH/NIAID Children living in malaria-endemic regions: little efficacy, poorly 5R21AI134618 (P.A.B.), NIH/NIAID 5P01AI131568 documented harms. Prescrire Int. 26 (178), 5−8. (P.A.B.), NIH/NIAID 1P01AI129859-01A1 (P.A.B.), NIH/ (8) Nami, S., Mohammadi, R., Vakili, M., Khezripour, K., Mirzaei, OD K01 OD023034 (S.S.I.), NIH/NIAID R03AI138792 H., and Morovati, H. (2019) Fungal vaccines, mechanism of actions and immunology: A comprehensive review. Biomed. Pharmacother. (S.S.I.), NIH/NIAID 1R21AI34368 (S.S.I.), NIH/NIAID 109, 333−344. R01AI105084 (D.R.R.), UNMC Department of Pharmaceut- (9) Nguipdop Djomo, P., Thomas, S. L., and Fine, P. E. M. (2013) ical Sciences Startup (D.R.R.), a Ministry of Education in Correlates of vaccine-induced protection: methods and implications, Scholarship, King Saud University (Riyadh, Saudi Arabia) Vol. 1,p1−49, WHO Document Production Services, Geneva, (A.M.A.), and the Holland Computing Center of the Switzerland. University of Nebraska (N.P.), which receives support from (10) Plotkin, S. A. (2013) Complex correlates of protection after the Nebraska Research Initiative. We greatly appreciate the vaccination. Clin. Infect. Dis. 56 (10), 1458−1465. expert technical guidance of Dr. Juliana Lewis (Miltenyi (11) Tallapaka, S. B., Karuturi, B. V. K., Yeapuri, P., Curran, S. M., Biotec), Victoria Smith M.S. and Dr. Philip Hexley (UNMC Sonawane, Y. A., Phillips, J. A., David Smith, D., Sanderson, S. D., and Flow Cytometry Facility), and Dr. Laurey Steinke (UNMC Vetro, J. A. (2019) Surface conjugation of EP67 to biodegradable Protein Structure Core Facility). The UNMC Flow Cytometry nanoparticles increases the generation of long-lived mucosal and systemic memory T-cells by encapsulated protein vaccine after Research Facility and Protein Structure Core Facility is ffi respiratory immunization and subsequent T-cell-mediated protection managed through the O ce of the Vice Chancellor for against respiratory infection. Int. J. Pharm. 565, 242−257. Research and supported by state funds from the Nebraska (12) Siegrist, C.-A. (2012) in Vaccine Immunology. In Vaccines Research Initiative (NRI) and The Fred and Pamela Buffet (Plotkin, S., Orenstein, W., and Offit, P., Eds.), 6th ed., pp 14−33, Cancer Center’s National Cancer Institute Cancer Support Elsevier, Amsterdam, The Netherlands. Grant. (13) Rappuoli, R. (2007) Bridging the knowledge gaps in vaccine design. Nat. Biotechnol. 25 (12), 1361−1366. ■ DEDICATION (14) Ionescu, L., and Urschel, S. (2019) Memory B Cells and Long- lived Plasma Cells. Transplantation 103 (5), 890−898. We dedicate this manuscript to the memory of Dr. Sam (15) Thakur, A., Pedersen, L. E., and Jungersen, G. (2012) Immune Sanderson, the inventor of EP54 and EP67. His larger-than-life markers and correlates of protection for vaccine induced immune personality, enthusiasm for research, and unwavering friend- responses. Vaccine 30 (33), 4907−4920. ship will be greatly missed. (16) Amanna, I. J., and Slifka, M. K. (2011) Contributions of humoral and cellular immunity to vaccine-induced protection in ■ ABBREVIATIONS humans. Virology 411 (2), 206−215. (17) Plotkin, S. A. (2020) Updates on immunologic correlates of APC, antigen-presenting cells; C5a, complement component − 5a; C5aR1/CD88, C5a receptor 1; Cha7, cyclohexylalanine7 vaccine-induced protection. Vaccine 38 (9), 2250 2257. (18) Vetter, V., Denizer, G., Friedland, L. R., Krishnan, J., and amino acid residue in EP67; CPDI, complement peptide- Shapiro, M. 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