Attempts to develop vaccines against ExPEC have previously focused on specific virulence factors (O-antigens, OMP fractions, fimbriae, toxins, and iron-acquisition systems), or whole cells, but most of them were either not safe, poorly immunogenic, or did not provide cross-protection against ExPEC strains [7C12]. In order to develop a more effective vaccine against ExPEC sepsis, we tested siderophore receptors (IutA and IroN), which are highly common among human being ExPEC isolates [13]; and common pilus (ECP) [14] that takes on a synergistic part in multiple methods of the infectious process [15C18]. and non-lethal sepsis challenges. Moreover, passive immunization against these four antigens resulted in significant reductions of bacteria in internal organs and blood of the mice, especially when the challenge strain was produced in iron-restricted media. Inclusion of antibodies to PNAG increased the efficacy of the passive immunization under conditions where the challenge bacteria were produced in LB medium but not in iron-restricted media. The information and data presented are the first step toward the development of a broadly protective vaccine against sepsis-causing strains. Keywords: vaccine, sepsis, antibodies, sepsis, antigens, challenge, (ExPEC) normally reside in the human intestine but are capable of infecting extraintestinal sites like the blood, urinary tract, and meninges, using specific virulence attributes [1, 2]. ExPEC are major causes of both community and nosocomial bacterial sepsis, with mortality ranging from 30%C50% [3C5]. Clinical failure of antibiotic therapies, mainly due to multidrug resistance, increases the cost of care and results in prolonged morbidity for patients [6]. As a result, the prevention and control of these infections is usually a pressing concern. A protective vaccine would be a useful strategy to prevent ExPEC infections. Efforts to develop vaccines against ExPEC have previously focused on specific virulence factors (O-antigens, OMP fractions, fimbriae, toxins, and iron-acquisition systems), or whole cells, but most of them were either not safe, poorly immunogenic, or did not provide cross-protection against ExPEC strains [7C12]. In order to develop a more effective vaccine against ExPEC sepsis, we tested siderophore receptors (IutA and IroN), which are highly prevalent among human ExPEC isolates [13]; and common pilus (ECP) [14] that plays a synergistic role in multiple actions of the infectious process [15C18]. Additionally chosen for passive vaccine studies were antibodies raised to a synthetic, deacetylated glycoform of the bacterial surface polysaccharide poly–(1C6)-[19]. 2. Methods 2.1. Ethics statement New-Zealand-White rabbits and female BALB/c mice were obtained from Charles River Labs (Wilmington, MA). Vaccination and contamination of animals were performed in accordance with protocols approved by the Arizona State University (ASU) Institutional Animal Care and Use Committee (IACUC) in dedicated facilities at the Biodesign Institute, ASU (Protocol number 1168R). 2.2. Antigens preparation Genes encoding the selected antigens (EcpA, EcpD, IutA, IroN) (Table S1) were PCR amplified and cloned into pET-101/D-TOPO? vectors (Invitrogen). Recombinant proteins were expressed in BL21 and purified from inclusion bodies as His-tagged protein, using ProBond Ni-NTA resin columns (Invitrogen). The expressed proteins were 78 kDa (IroN), 74 kDa (IutA), 45 kDa (EcpD), and 21 kDa (EcpA), respectively. 2.3. Production of rabbit antibodies Antisera to EcpA, EcpD, IutA, and IroN were raised by injecting subcutaneously (s.c.) rabbits with 250 g of individual recombinant antigens (rAgs) in complete Freunds adjuvant, followed by two boosts at 3 weekly intervals CD163 with 250 g of rAg in incomplete Freunds and two boosts in Montanide? ISA 71 VG adjuvant. The concentration of antigen-specific rabbit IgG was measured by indirect ELISA using a goat-derived anti-rabbit IgG standard (Southern Biotech, Birmingham, AL). Rabbit antibodies raised to 9GlcNH2-TT were prepared as previously described [19]. 2.4. Bacterial challenge strain Mice were challenged with urosepsis CFT073 [20] (Table S1) produced in either Lysogeny Broth (LB) [21] at 37C with or without 2,2-bipyridyl (100 M) with aeration until an OD600 of ~0.85 or in Dulbeccos Modified Eagle Medium (DMEM) + 0.5% Mannose + 2,2-bipyridyl (100 M), at 28C for 48h standing and the OD600 value of culture was adjusted to ~0.85. The strain was stored at ?80C in peptone-glycerol medium. According to NCBIs BLASTn, the genome of CFT073 (AE014075.1) contains the sequences for (NP_755498.1), (AAN79707.1), and locus encoding [22]. 2.5. Vaccination and challenge 2.5.1. Active immunization As shown in Physique S1, mice were s.c. injected with rAgs, either alone or in combinations [two or four antigens (high or Chlorhexidine digluconate low doses)] (Table S2), in phosphate buffered saline (PBS), emulsified in ISA 71 VG adjuvant, and boosted on day 23 (Table S2). On days 21 and 40, blood was collected by submandibular bleeding and sera were stored at ?80C. On day 42, mice were challenged intraperitoneally (i.p.) with CFT073. In the non-lethal challenge (~3.5 x 107 CFU), necropsies were performed after 24 or 48 h of challenge. In the lethal challenge (~1.8 x 108 CFU), death was recorded for 48 h after inoculation; survived mice were euthanized and necropsied. Bacterial Chlorhexidine digluconate loads were decided in blood and organs. 2.5.2. Passive immunization Mice were i.p. immunized with 200 l of either pre-immune rabbit serum (control) or antigen-specific rabbit IgG antiserum (50 g). Twenty-hour later, mice were boosted with the same Chlorhexidine digluconate serum samples. After.