ETEC is a non-invasive pathogen that mediates small intestine adherence through bacterial 53 surface structures known as colonization factors (CFs) . Once bound to the small intestine, the 54 bacteria produce toxins causing a net flow of water from enterocytes, leading to watery diarrhea. 55 Development of an effective vaccine against ETEC bacterial attachment and colonization has 56 long been considered as a promising approach against ETEC diarrhea. CFA/I is one of the most 57 prevalent CFs and is composed of CfaE, the tip minor adhesin subunit, and a homopolymeric 58 structural subunit, CfaB. In human clinical trials, oral administration of anti-CfaE bovine IgG 59 provided protection against virulent ETEC challenge in over 60% of the test group, suggesting 60 that an adhesin-based vaccine could be effective to elicit endogenous production of protective 61 antibodies (4). Recent studies in our laboratories demonstrated that when administered orally to 62 mice and non-human primates, anti-CfaE human monoclonal antibodies inhibited bacterial 63 colonization in the small intestine and protected animals from diarrhea associated with ETEC 64 infection (5).
ETEC strains are antigenically diverse with over 25 types of CFs having been identified (6-9) . 67 CFA/I represent the archetype of class 5 fimbriae, the largest class of human specific CFs that 68 causes a majority of moderate to severe ETEC diarrheal cases (7, 9, 10) . Class 5 fimbriae 69 includes three sub-classes, 5a (CFA/I, CS4, CS14), 5b (CS1, CS17, CS19, PCF071), and 5c 70 (CS2). Other non-class 5 fimbriae have also been implicated in endemic and traveler diarrheal 71 diseases including helical CS5, fibrillary CS3, CS21 and non-fimbrial CS6 adhesins (11). As 72 such, development of a broadly protective vaccine against all major pathogenic ETEC strains 73 remains one of the most important challenges in vaccine design (12, 13) . In theory, a cross-74 protective vaccine could be generated with a combination of intact CFs (14) . However, such 75 strategies could lead to unexpected instability or safety concerns and high costs for The discovery of camelid heavy-chain variable domains (VHHs or nanobodies) has produced a 80 new application for antibody-derived biologics (15). Nanobodies consist of a highly stable and 81 soluble single antigen-binding variable region (15 kDa), since they are derived from heavy chain 82 only antibodies with no associated light chains (16). Unlike conventional antibodies, nanobodies 83 can access distinct antigenic sites (e.g., enzyme active sites, recessed regions of viral 84 glycoproteins) due to smaller paratope diameters and a longer complementarity-determining 85 region 3 (CDR3) to form finger-like extensions (15, 17 101 To generate nanobodies with broad cross-reactivity against ETEC adhesins, two male llama were 102 subcutaneously immunized with N-terminal fragments of eight class 5 ETEC adhesins ( Figure   103 1A). Serum response to adhesins was measured by ELISA and PBMC were isolated from llama 104 to generate phage-displayed libraries of size around 10 8 as previously described (26). Libraries 105 were initially used for panning on immobilized colonization factor antigens CS1 and CS2, 106 because of their relatively low sequence homology. Output from those pannings were used for a 107 subsequent panning round on CfaE. Periplasmic extracts were prepared and tested for binding to 108 all 8 adhesin antigens in ELISA. Cross-reactive clones were sent for sequence determination and 109 further characterization. From these selections, 26 candidate nanobodies were selected. 128 To test the breadth of cross-reactivity, selected llama-and yeast-derived nanobodies were 129 sequenced, cloned in pET-26b vector, expressed in E.coli, and examined by ELISA for binding 130 to eight class 5 adhesins. Two yeast-derived clones (2R215 and 2R23) and two llama-derived 131 clones (1D7 and 1H4) were selected based on their broad reactivity against multiple adhesins 132 (Figure 2A-D) . The four lead nanobodies were then tested for inhibition of mannose resistant 133 hemagglutination (MRHA) against an ETEC strain expressing CFA/I (H10407), the most 134 prevalent class 5 adhesin. All nanobodies showed maximal inhibitory concentration (IC100) in the 135 micromolar concentration range (1.9 to 3.7µM) ( Figure 2E ). We next examined if these 136 nanobodies had activity against ETEC strains expressing 7 other class 5 adhesins in MRHA 137 assay. Consistent with ELISA binding data, the four nanobodies were cross-protective against all 138 eight strains with an IC100 activity ranging from 0.4125 to 13.2 µM ( Table 1) .We next examined 139 the MRHA activity of nanobodies against ETEC strains expressing other pathogenic fimbrial and 140 non-fimbrial adhesins including helical CS5, fibrillary CS3, and non-fimbrial CS6 adhesins, as 141 well as another common diarrhea causing adhesin CS21. Surprisingly, four nanobodies showed 142 broad protection against all tested strains with an IC100 ranging from 0.2063 to 6.6 µM ( Table 1) .
To elucidate the interaction between anti-ETEC nanobody and adhesin at the molecular level, we Based on docking results, individual CfaE residues within or adjacent to the putative receptor 153 binding pocket formed by three arginine residues (R67, R181 and R182) were selected for 154 mutagenesis to alanines and tested by ELISA for nanobody binding (Figure 3E-H) . ELISA 155 results showed that mutating Y65, R67, E161, D184, R181, R182, and Y183 to alanine affected 156 the binding of nanobodies to CfaE suggesting that these residues are involved in the binding interactions. This is in line with findings by Baker who demonstrated R181 of CfaE as crucial for 158 ETEC binding in a human in vitro organ culture (30). In addition to the residues near the putative 159 receptor domain, we identified several other highly conserved residues such as S65, L64, Y58, 160 L110, Y156 and C143 that are required for interaction between nanobodies and adhesin.
Interestingly, sequence alignment revealed that these residues are highly conserved among at 162 least eight class 5 adhesins (31), which is consistent with our observation that these nanobody are 163 broadly cross protective against multiple ETEC strains.
The lead nanobodies were further evaluated in a mouse model of ETEC colonization as 167 described previously (29) ( Figure 4A ). Groups of five DBA/2 mice were given a mixture of 168 bacteria and anti-CfaE nanobodies by oral gavage. All nanobodies were tested at 100 mg/kg, 169 except for 2R23, which was tested at 50mg/kg due to the low production yield. Twenty four (Figure 7C, D) . Furthermore, the 1D7 228 VHH-IgA1 fusionbody retained activity when the pre-treatment time was extended to 2 hours. A 229 1.7 log reduction in total CFUs was observed in 1D7 VHH-IgA1 treated animals as compared to 230 the group treated with irrelevant control (P<0.0001) (Figure 7E) . We also compared the activity 
The initial screening was performed against CS1and CS2, followed by a second round of 510 screening against the CfaE antigen. Phages were eluted by unspecific and competitive elutions.
Eluted phage were used to infect exponentially growing E. coli TG1 that were plated on Luria 512 broth (LB) agar plates containing 2% (w/v) glucose and 100 µg/ml ampicillin. In addition, eluted 513 phages were serially diluted, infected in TG1 and 5 µl spots of these infections were plated to 514 allow estimating how many phages were eluted. Periplasmic extracts were prepared according to 515 standard protocols involving overnight production in 2YT medium and induction with IPTG.
Extracts were tested for binding to all 8 Cfa antigens in ELISA. Based on HinfI digestion pattern, 517 amino acid sequences and cross-reactive binding to Cfa antigens, unique candidate genes were 518 selected for further characterization. 
The growth conditions are the same as H10407. Nanobodies were multimerized to dimeric or trimeric forms with (G4S) linkers. The dimeric 568 forms were generated using 6X (G4S) linker to connect two monomeric VHHs in tandem N 569 terminus to C terminus orientation. Trimers were generated using two 3X (G4S) linkers between 570 monomeric nanobody units. The multimers were cloned into pET-26b vector adding a C-571 terminal 6xHis tag. Nanobodies were purified from the periplasmic fraction by Ni-NTA 572 chromatography (Gold Biotechnology) and dialyzed against PBS to remove imidazole.
To generate VHH-IgA fusionbodies, monomeric nanbody sequences were cloned into a pcDNA solution until an OD600 of 1 was reached for the assay. Other ETEC strains except #400599 599 (CS14) were cultured in ETEC medium containing 1% Casamino Acids (Sigma) and 0.15%