Patent Application
20190345229
The invention relates to methods promoting the agglutination of Salmonella bacteria in the gut of subjects, thereby providing immune exclusion and limiting bacterial entry, therefore reducing mucosal and systemic infection.
Protection of mucosal surfaces against colonization and possible entry and invasion by microbes is provided by a combination of constitutive, non-specific substances (mucus, lysozyme and defensins), and also by specific immune mechanisms including secretory Igs (SIgs) at the humoral level (Chairatana and Nolan (2016) Crit. Rev. Biochem. Mol. Biol. 113:1-12; Mantis et al. (2011) Mucosal Immunol. 4:603-611). In vivo, experimental and clinical resistance to infection can be correlated with specific secretory IgA (SIgA) antibodies (Abs) serving as an immunological barrier at mucosal surfaces (Corthésy (2013) Autoimmun. Rev. 12:661-665; Strugnell and Wijburg (2011) Nat. Rev. Microbiol. 8:656-667). It is thought that aggregation, immobilization and neutralization of pathogens at mucosal surfaces is facilitated by the multivalency of SIgA (Mathias et al. (2013) Infect. Immun. 81:3027-3034; Levinson et al. (2015) Infect. Immun. 83:1674-1683). SIgM serving as a surrogate of SIgA in IgA-deficient individuals appears to act via a similar protective mechanism (Brandtzaeg (2009) Scand. J. Immunol. 70:505-515).
The lack of effective vaccines against several infectious agents, and the emergence of new pathogens and problems associated with antibiotic resistance, has led to a renewed interest in passive immunization. Because mucosal surfaces are the major portal of entry for microbes and because their specific neutralization is largely mediated by secretory immunoglobulins, the concept of using exogenously delivered Abs as prophylactic and therapeutic tools has arisen. In emergency situations that cannot wait for a vaccine to produce its effect, direct intervention with Ab molecules to the affected site represents the basis for topical immunotherapy (Virdi et al (2016) Cell Mol. Life Sci. 73:535-545).
We have previously established that polyreactive, serum-derived polymeric IgA, IgM and a mixture of the two isotypes (IgA/M) can be assembled into secretory Abs upon combination with recombinant secretory component (SC) (Longet et al. (2013) J. Biol. Chem. 288:4085-4094). In support of their use for local passive immunization, the molecules display high in vitro stability upon exposure to intestinal washes rich in proteases (Crottet and Corthesy (1998) J. Immunol. 161:5445-5453).
We have now surprisingly shown that immune complexes of large size form between such preparations comprising secretory Igs and the enteropathogen Salmonella. Following oral administration of such immunoglobulins, large aggregates keep existing in the intestinal lumen, thus limiting entry of the pathogen into the intestinal target tissue, namely Peyer's patches (PPs) and mesenteric lymph nodes (MLNs). Systemic dissemination assessed in the spleen is diminished as well, resulting in quenching of local inflammatory responses. Such effects were obtained as a function of the dose and molecular form of the Ab administered, with SCIgA/M performing the best. Surprisingly, the exogenously delivered Ab molecules exhibit the same functional features as locally secreted endogenous Abs in the harsh gut environment.
One aspect of the present invention is a method for promoting agglutination of Salmonella bacteria in the gut of a subject, comprising administering a composition comprising secretory IgA (SCIgA) and/or secretory IgM (SCIgM) to the gut of the subject, wherein the composition is not milk or derived from milk. Another aspect of the invention is the use of a composition comprising SCIgA and/or SCIgM for promoting agglutination of Salmonella bacteria in the gut of a subject, wherein the composition is not milk or derived from milk. Yet another aspect of the invention is a composition comprising SCIgA and/or SCIgM for use in the treatment or prevention of Salmonella infection in a subject, wherein the composition is administered to the gut of the subject and promotes agglutination of Salmonella bacteria, wherein the composition is not milk or derived from milk.
Preferably, the composition is administered orally to the subject.
Preferably, the agglutination of the bacteria leads to their immune exclusion.
Preferably, the entry of the bacteria into Peyer's patches (PPs) and their diffusion into mesenteric lymph nodes (MLNs) are inhibited. More preferably, the composition reduces mucosal infection. More preferably, the composition reduces local inflammation. Even more preferably, the composition reduces the systemic dissemination of the Salmonella bacteria.
Preferably the Salmonella bacteria are Salmonella enterica subsps, e.g. Salmonella typhi.
The composition preferably comprises IgA and/or IgM with binding specificity for Salmonella bacteria, preferably for Salmonella enterica subsps, even more preferably for Salmonella typhi.
Preferably the IgA and/or IgM is prepared from plasma, preferably from human plasma. More preferably, the IgA and/or IgM is combined in vitro with secretory component (SC). More preferably the SC is human secretory component. Even more preferably the SC is recombinant SC, expressed in a mammalian cell line.
Preferably at least 10% of the protein in the composition is SCIgA, more preferably at least 15%, 18%, 20%, or 25%, even more preferably at least 30%, 40% or 50% of the protein in the composition is SCIgA. Preferably at least 10% of the protein in the composition is SCIgM, more preferably at least 15%, 18%, 20% or 25%, even more preferably at least 30%, 40% or 50% of the protein in the composition is SCIgM.
Preferably at least 10% of the protein in the composition is SCIgA and at least 10% of the protein in the composition is SCIgM, more preferably at least 15% is SCIgA and at least 15% is SCIgM, even more preferably at least 20% is SCIgA and at least 20% is SCIgM.
Preferably, the IgA and/or IgM may be enriched for Abs with specificity for Salmonella.
The IgA and/or IgM may alternatively be monoclonal or a mixture of two or more monoclonal Abs, with specificity for Salmonella bacteria, preferably with specificity for Salmonella enterica subsps. In this case, the IgA or IgM is co-expressed with J chain in a cell line, preferably in a mammalian cell line. SC may be co-expressed in the same cell line, or expressed separately and combined with the J chain-comprising dimeric/polymeric IgA/IgM in vitro.
Salmonella enterica species comprise as many as 2500 serovars; they comprise 8 subspecies. Around 2000 serotypes induce nontyphoidal disease, including food poisoning. Typhoid fever is caused by Salmonella serotypes which are adapted to humans or higher primates. While presently self-contained in developed countries, typhoid fever has a devastating effect in India, in some African countries and generally countries with a poor health care system, lack of clean drinking water and poor hygienic conditions. The risk of death is about 10-30% in infected patients without treatment. Based on WHO fact sheet No. 139, the onset of disease symptoms occurs 6-72 hours after ingestion of Salmonella, and illness lasts for up to 7 days. Patients are usually treated with antibiotics, but formerly used antibiotics such as streptomycin, ampicillin and chloramphenicol are no longer used as first line therapy since the emergence of multi-drug resistant typhoid. Antibiotics like ciprofloxacin, ceftriaxone or azithromycin are used, but in general, resistance to antibiotics is an increasing problem, and alternative approaches will be required for the treatment of Salmonella infections, in particular of multi-drug resistant Salmonella infections, such as multi-drug resistant typhoid.
The invention provides a method to promote agglutination of Salmonella bacteria in the gut of subjects, by administering a composition comprising SCIgA and/or SCIgM to the gut of the subject, e.g. by oral administration, wherein the composition is not milk or derived from milk.
Surprisingly this leads to the formation of large aggregates of the immunoglobulin and the bacteria, which leads to immune exclusion of the bacteria. Preferably the remaining free Salmonella bacteria are reduced by the composition by at least 10%, more preferably by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, even more preferably by at least 60%, 70%, 75%, most preferably by at least 80%.
The entry of the Salmonella bacteria into PPs and the diffusion into MLNs is inhibited by the composition. Preferably the number of colony-forming units (cfu's), i.e. the bacterial count, per mg PP is reduced by administration of the composition by at least 2 fold, more preferably by at least 2.5 fold, 3 fold, even more preferably by at least 4 fold.
The composition also reduces mucosal infection. Dissemination of Salmonella is inhibited after administration, preferably oral administration, of the immunoglobulin composition. Bacterial counts in mucosal tissues are preferably reduced by at least 2 fold, more preferably by at least 3 fold, 4 fold, or even 5 fold, even more preferably by at least 6 fold, 7 fold, 8 fold, most preferably by at least 9 fold, when observed 48 hours after administration of the immunoglobulin composition. Bacterial counts in mucosal tissues (PPs and MLNs) and/or the spleen is preferably reduced by at least 2 fold, more preferably by at least 3 fold, 4 fold, or even 5 fold, even more preferably by at least 6 fold, 7 fold, 8 fold, most preferably by at least 9 fold, when observed 6 days after administration of the immunoglobulin composition.
Another aspect of the invention is the prophylactic effect of administration, preferably oral administration, of the immunoglobulin composition. Preferably prophylactic administration of the immunoglobulin composition, e.g. 8 hours or 24 hours prior to the infection, leads to a reduction of bacterial counts in PPs, or MLNs, or spleen, or liver, by at least 2 fold, more preferably by at least 3 fold, 4 fold, or even 5 fold, even more preferably by at least 6 fold, 7 fold, 8 fold, most preferably by at least 9 fold, when observed 6 days post infection, as compared to without administration of the immunoglobulin composition.
Preferably, administration, preferably oral administration, of the immunoglobulin composition prior to infection (i.e. prophylactic administration, e.g. 8 or 24 hours prior to the infection) reduces weight loss caused by the infection by at least 20%, more preferably by at least 30%, 40%, or 50%, even more preferably by at least 60%, most preferably by at least 70%.
Local inflammation is also reduced. Preferably the frequency of neutrophils observed in PPs is reduced by administration, preferably oral administration, of the immunoglobulin composition by at least 10%, 20%, more preferably by at least 25%, 30%, 40%, even more preferably by at least 50%, most preferably by at least 60%. Preferably the frequency of macrophages observed in PPs is reduced by administration, preferably oral administration, of the immunoglobulin composition by at least 10%, 20%, more preferably by at least 25%, 30%, 40%, even more preferably by at least 50%, most preferably by at least 60%. Preferably the secretion of pro-inflammatory cytokines, e.g. TNF-alpha or IL-6, is reduced by at least 2 fold, more preferably by at least 2.5 fold, even more preferably by at least 3 fold 6 days after administration, preferably oral administration of the immunoglobulin composition.
Another aspect of the invention is the therapeutic effect of the immunoglobulin composition. Preferably, therapeutic administration of the immunoglobulin composition, e.g. one hour after the infection, leads to a reduction of bacterial counts in PPs, or MLNs, or spleen, or liver, by at least 2 fold, more preferably by at least 3 fold, 4 fold, or even 5 fold, even more preferably by at least 6 fold, 7 fold, 8 fold, most preferably by at least 9 fold or even at least 10 fold, when observed 6 days post infection, as compared to without administration of the immunoglobulin composition.
Preferably, therapeutic administration, preferably oral administration, of the immunoglobulin composition (e.g. 1 hour or 8 hours after the infection) reduces weight loss caused by the infection by at least 20%, more preferably by at least 30%, 40%, or 50%, even more preferably by at least 60%, most preferably by at least 70%.
Preferably the IgA and/or IgM is prepared from plasma, as described in detail, for example, in WO 2013/132053.
The immunoglobulin composition preferably used in the present invention can be prepared as described in detail in WO 2013/132052. Preferably, plasma derived preparations comprising IgA and/or IgM are combined in vitro with SC, without requiring prior purification of the dimeric/polymeric J chain containing IgA/IgM. Such material is referred to as secretory-like IgA or secretory-like IgM, or abbreviated as SCIgA or SCIgM. However, this material behaves very similarly to in vivo produced secretory IgA (usually abbreviated SIgA) and in vivo produced secretory IgM (usually abbreviated SIgM).
The term “secretory component” as used herein refers to a protein that specifically binds to J chain-containing immunoglobulin, and is related to or derivable from or identical to an extracellular portion of the polymeric immunoglobulin receptor (pIgR), preferably a mammalian pIgR, more preferably a primate pIgR, most preferably a human pIgR. Preferably, the secretory component confers increased stability to the J chain containing immunoglobulin. SC comprised in the composition may be recombinant SC, preferably SC produced in a mammalian cell line. SC in its traditional, narrow meaning (referred to as “natural SC” herein) is the extracellular portion of the pIgR, which usually gets associated during secretion with dimeric or polymeric IgA or pentameric IgM comprising a J chain. J chain-containing IgA/IgM binds to the polymeric immunoglobulin receptor at the basolateral surface of epithelial cells and is taken up into the cell by endocytosis. This receptor complex then transits through the cellular compartments before being transported to the luminal surface of the epithelial cells. The transcytosed IgA/IgM-pIgR complex is then released through proteolysis, and part of the pIgR, referred to as the natural SC, stays associated with the J chain-containing IgA/IgM, releasing SIgA/SIgM. However, there is evidence that reverse transcytosis of SIgA, i.e. from the luminal surface to the basolateral surface, can also take place.
The human pIgR is cloned and sequenced, its sequence is available as SwissProt entry P01833, and shown in Seq ID NO: 1. Human pIgR is a glycoprotein with 764 amino acid residues, containing a signal peptide (residues 1 to 18), an extracellular part (residues 19 to 638), a transmembrane region (residues 639 to 661), and a cytoplasmic region (residues 662 to 764). Residues 19 to 603 are thought to associate with J chain-containing IgA as described above, and this part of this glycoprotein is usually referred to as the SC (referred to as “natural SC” herein).
SC used in the immunoglobulin composition used in the present invention can comprise any extracellular pIgR sequence that is capable of associating with J chain-containing IgA. For example, SC may comprise extracellular domains of pIgR from mammalian sources, e.g. from primates, cattle, horses, cats, dogs, rabbits, guinea pigs, rats or mice, or variants thereof. Functional hybrids of the extracellular domains from several mammalian species or variants thereof are also contemplated for use in the invention, e.g. prepared by fusing the immunoglobulin-like domains from different species into a SC-like protein. A functional SC may also be formed by fusing a selection of immunoglobulin-like domains normally present, e.g. rabbit SC is functional being composed of only domains 1, 4 and 5. Preferably, however, the human SC or functional variants thereof is used.
Therefore the SC used in the immunoglobulin composition used in the invention preferably comprises residues 19 to 603 of SEQ ID NO: 1 or functional variants thereof. Functional variants may include deletions, insertions, and/or substitutions, preferably substitutions are conservative substitutions, e.g. a basic amino acid residue is substituted for another basic amino acid, a hydrophobic amino acid is substituted for another hydrophobic amino acid, etc. The variant SC is at least 50% identical in sequence to residues 19 to 603 of SEQ ID NO: 1, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, more preferably at least 85% or even 90%, even more preferably at least 92%, 94%, 95%, 97%, 98%, or even 99% identical to residues 19 to 603 of SEQ ID NO: 1. Preferably, the SC comprises the extracellular portion of the pIgR, more preferably the extracellular portion of the human pIgR, most preferably the SC comprises or even consists of residues 19 to 603 of SEQ ID NO: 1.
The skilled person is well aware how to produce the SC by recombinant techniques. An example of expression of human SC in CHO cells has been described by Phalipon et al (Phalipon et al., (2002) Immunity 17:107-115), but the invention is not limited to SC produced by this system. For example, the desired cDNA sequence can be produced synthetically or cloned via RT-PCR, using RNA isolated from cells or tissue expressing pIgR as template. The cDNA can then be inserted into a mammalian expression vector such as pcDNA3—many alternative expression vectors are available. The recombinant expression vector will then be introduced into a suitable host cell line, such as CHO, COS-1 or COS-7, HEK293, or BHK. Other cell lines are available and can also be used. Methods for introducing such vectors into a cell line include lipofection, electroporation and other techniques well known to the skilled person. Usually cells harboring the expression vector and expressing the protein of interest are then selected and cloned. Viral expression systems can also be used, for example, vaccinia virus can be used to express proteins at high levels in mammalian cells, baculovirus expression systems can be used to express proteins at high levels in insect cells. Yeast or bacterial expression systems can also be envisaged, and such expression systems are known to the skilled person. Likewise, plant expression systems can also be envisaged, and such systems are known to the skilled person.
SC or variant thereof used in the composition of the invention may also comprise a tag, such as a hexa-Histidine tag, which can aid in the purification of the resulting protein. If such a tag is attached via a cleavable linker, the tag may be cleaved off prior to use in the invention. Similarly, the SC may be produced as a fusion protein. Again, a cleavable linker may be used so that the fusion partner may be cleaved off the SC prior to use in the invention.
The skilled person can then purify the expressed protein with standard methods. Recombinant SC may be purified to high purity with a suitable method, for example size-exclusion and/or ion exchange chromatography. Preferably the final preparation of recombinant SC will be essentially free of contaminants, particularly host cell proteins. However, SC can also specifically associate with J chain containing immunoglobulin in unpurified form, thus purification prior to association with the J chain-containing immunoglobulin is not essential.
The SC may also be obtained from a natural source, preferably from milk, saliva or mucus. Preferably the SC is of human origin, but SC from other species can also be used in the invention.
The molar ratio between SC and J chain containing IgA dimers/polymers or IgM pentamers in the composition of step (a) ranges between 1:10 and 10:1, preferably between 1:5 and 5:1, more preferably between 1:2 and 2:1.
The molar ratio between SC and J chain within the composition of step (a) is between 1:10 and 10:1, preferably between 1:5 and 5:1, more preferably between 1:2 and 2:1.
The amount of SC used in step (b) may be at least 1 part (by weight) of SC to 50 parts (by weight) of protein in the composition of step (a), preferably at least 1 part to 40, 30, 20, 15, 10, most preferably at least 1 part of SC to 5 parts of protein in the composition of step (a).
Alternatively the IgA and/or IgM may be monoclonal anti-Salmonella Abs. In the composition used in the invention, a single monoclonal Ab or a mixture of one or more monoclonal Abs may be used. It may be a mixture comprising monoclonal Abs specific for different Salmonella species. The monoclonal IgA and/or IgM is then co-expressed with J chain, in a cell, preferably a mammalian cell. Following expression, the resulting J chain containing IgA and/or IgM is then combined in vitro with SC as described for plasma-derived IgA and/or IgM above. However, SCIgA and/or SCIgM can also be produced by co-expressing IgA or IgM, J chain and SC in the same cell (see, for example, U.S. Pat. No. 6,300,104 B1).
Administration is preferably orally.
Determination of the appropriate doses of the immunoglobulin composition for treating or preventing Salmonella infections is well within the capabilities of those skilled in the art. For example, a dose of 0.1 g to 10 g, preferably a dose of 0.2 g to 5 g, more preferably a dose of 0.5 g to 2 g, even more preferably a dose of 1 g is administered. Preferably the dose is administered four times per day, more preferably three times per day, even more preferably twice per day, most preferably once per day, as long as symptoms of the infection are present, preferably a dose of 1 g once per day could be used.
Alternatively a dose of 1 to 100 mg/kg bodyweight could be administered, preferably 1 to 50 mg/kg, even more preferably 5 to 20 mg/kg, most preferably 10 to 15 mg/kg.
Preferably the immunoglobulin composition is administered during the acute phase of the disease, for example for as long as the patient shows disease symptoms such as fever, diarrhea, abdominal pain, headaches, and/or as long as bacteria can be found in blood and/or stool.
Another aspect of the invention is the use of the immunoglobulin composition as described above as treatment in addition to standard antibiotic treatment. For this aspect of the invention the immunoglobulin is administered as described above to assist the antibiotics in eliminating the Salmonella bacteria from the patient. For example, the immunoglobulin composition is administered using the same dosage as outlined above during the acute phase of the infection while the patient is receiving antibiotic therapy. Preferably, the immunoglobulin composition is administered during the first two days, more preferably during the first three days, even more preferably during the first four days, most preferably during the first five days of infection, in addition to standard antibiotic therapy.
The invention also provides a method to prevent Salmonella infection by administration of the immunoglobulin composition as described above. For example, when travelling into a country with poor hygienic conditions, a dose of 0.1 g to 10 g, preferably a dose of 0.2 g to 5 g, more preferably a dose of 0.5 g to 2 g, even more preferably a dose of 1 g is administered. Preferably the dose is administered twice per day, more preferably once per day, preferably a dose of 1 g once per day could be used. However, the dose can be adjusted, depending on the risk of exposure to Salmonella and the likely bacterial load to be encountered. Another aspect of the invention is the use of the immunoglobulin composition as described above for the prevention of Salmonella infection. Yet a further aspect of the invention is the immunoglobulin composition as described above for the use of prevention of Salmonella infection. The doses that can be used prophylactically are as outlined above.
Preferably the immunoglobulin composition is formulated as a liquid, for example a syrup. For example, it may be a liquid comprising 1 to 20% immunoglobulin, preferably 2 to 20% immunoglobulin, more preferably 2 to 15% immunoglobulin, even more preferably 5 to 10% immunoglobulin. Most preferably, it comprises 5% or 10% immunoglobulin. The liquid preferably also comprises a stabilizer, such as an amino acid (e.g. proline, glycine, histidine, arginine), a saccharide, or a sugar alcohol, or a protein such as albumin; other stabilizers that can be used are well known to the skilled person. The liquid may additionally comprise agents that enhance the taste, such as flavouring. The skilled person will be well aware of how to select suitable excipients and taste-enhancing agents.
Alternatively, the immunoglobulin composition may be administered in form of a capsule, preferably an acid-resistant capsule that releases its content in the gut of the subject. The skilled person will be well aware of how to prepare such dosage forms.
Alternatively, the immunoglobulin composition may be administered in form of a gel or a powder.
In fact, any form that can be taken orally can be envisaged.
The following examples are intended to exemplify, but not limit the invention.
Materials and Methods
Preparation of Human Plasma-Derived and Mouse Monoclonal Abs Human plasma-derived IgG preparations (IgPro10, Privigen) were prepared as reported (Cramer et al. Vox Sang 2009; 96:219-25). Preparations containing IgA and IgM were obtained from an ion-exchange chromatographic side fraction used in the large-scale manufacture of IgG from human plasma. The elution fraction containing IgA and IgM was concentrated and re-buffered to 50 g/l protein in PBS by tangential-flow filtration (TFF; Pellicon XL Biomax 30, Merck Millipore). The resulting IgA/M solution, which contained IgA and IgM in a 2:1 mass ratio, was further processed to: (i) SCIgA/M, by combining in vitro IgA/M with recombinant human SC (Phalipon et al., Immunity 2002; 17:107-15); (ii) polymeric IgA (pIgA) at >50% (by mass), through removal of IgM from IgA/M by affinity chromatography specific for human IgM (CaptureSelect® IgM, BAC B.V., Naarden, Netherlands); (iii) IgM, through removal of IgA from IgA/M by affinity chromatography specific for human IgA (CaptureSelect® IgA). The secretory counterpart of pIgA and IgM was prepared by combination with recombinant human SC (Phalipon et al., Immunity 2002; 17:107-15). Mouse monoclonal secretory IgASal4 (SIgASal4) specific for S. enterica Typhimurium surface carbohydrates was prepared as described previously (Michetti P et al Infect Immun 1992; 60:1786-92). Ab preparations were labeled with Cyanin 5 (Cy5) fluorescent dye (Innova Biosciences Ltd) according to the manufacturer's protocol.
Bacterial Strain and Culture Conditions
Bacteria used were the virulent strain SL1344 of S. enterica serovar Typhimurium (abbreviated St; Hoiseth S K, Stocker B A. Nature 1981; 291:238-9). Bacteria from frozen stock were grown on Luria-Bertani (LB) agar plates containing 90 μg/ml streptomycin (Sigma-Aldrich) for 24 hours at 37° C. Colonies were amplified in 3 ml of LB liquid broth supplemented with 90 μg/ml streptomycin for 16 hours at 37° C., with shaking. 25-fold dilution of the overnight culture was pursued for 1.5 hour at 37° C. to yield bacteria in the exponential phase. Bacterial density was determined with the knowledge that 1 OD600nm corresponds to 9.5×108 cfu/ml. In specific experiments, the GFP-expressing St strain obtained in the laboratory was prepared under the same conditions, except that 50 μg/ml ampicillin was added for culture.
ELISA
Assessment of the interaction between St and human plasma-derived Abs was carried out by ELISA as described in Longet et al. (J Biol Chem 2014; 289:21617-26) with minor modifications: Serial dilutions of human plasma-derived monomeric IgA (mIgA), polymeric IgA (pIgA), SIgA, IgM, SIgM, IgA/M, SCIgA/M or IgG (starting at 0.5 μM) or SC (starting at 6.25 μM) were added to St coated in PBS. Bound proteins were detected by incubation with biotinylated goat anti-human kappa chain (Pierce, 1/1000 dilution) or rabbit anti-human SC (Rindisbacher et al. J Biol Chem 1995; 270:14220-8; 1/500 dilution), followed by Extravidin-HRP (Sigma, 1/4000 dilution) or goat anti-rabbit-HRP (Sigma, 1/4000 dilution).
Preparation of Immune Complexes For administration to ligated intestinal loop, 2×106 St or St-GFP were mixed with 100 μg of SIgASal4 or human plasma-derived pIgA, SIgA, IgM, SIgM, IgA/M, SCIgA/M or IgG, or with 20 μg of SC in 100 μl PBS. For oral infection, 2×107 bacteria were mixed with 200 μg, 1 mg or 5 mg of human plasma-derived IgAM or SCIgA/M in 150 μl PBS. The mixtures were incubated for 1 hour at room temperature on a rotating wheel, and subsequently used as such.
Observation by Laser Scanning Confocal Microscopy of Immune Complexes
Immune complexes formed by St-GFP and plasma-derived Abs were laid onto glass slides (Thermo Scientific), mounted with anti-fading Vectashield reagent (Vector Laboratories), and visualized immediately using a Leica SP5 confocal microscope equipped with a 63×objective. Images were processed with Imaris 8 software. To evaluate agglutination efficiency, the number of free St-GFP, the number of bacteria aggregates and their estimated size were determined on 10 different fields, in 5 independent experiments.
Mice
Four week-old female Balb/c mice were obtained from Charles River Laboratories (L'Arbresle, France) and used at the age of 7-8 weeks. They were housed in the animal facility of the Lausanne University State Hospital under standard conditions. All experiments were approved by the State Veterinary Office.
Ligated Intestinal Loops
Intestinal ligated loops were performed according to the procedure of Rey et al. (Rey et al. J Immunol 2004; 172:3026-33) with minor modifications: 100 μl of a solution containing 2×106 St in a free form or as immune complexes were delivered into the lumen of an intestinal ligated loop containing a PP. Mice were sacrificed 1.5 hour later, the PP was removed from the intestinal tissue, and incubated 30 min in DMEM containing 2% FCS and 100 μg/ml gentamycin to kill extracellular bacteria. A PP upstream of the ligated loop was collected to serve as noninfected control. The bacterial load was determined as described (Pigny et al. J Infect Dis 2016; 214:438-46).
Preparation of Tissue Sections and Observation by Laser Scanning Confocal Microscopy
Intestinal segments containing a PP were immediately fixed in 500 μl of PBS-4% paraformaldehyde (Fluka) for 2 hours at 4° C., and further processed as described (Kadaoui K A, Corthésy B. J Immunol 2007; 179:7751-7), with minor modifications: Bacteria were detected upon incubation in PBS-5% FCS-0.1% saponin with biotinylated IgASal4 (1/50), followed by Cy5-labeled streptavidin (1/500; Amersham Biosciences). After washing, cell nuclei were stained with DAPI. Laser scanning confocal microscopy images were obtained using a Leica SP5 in multi-track mode. Raw images were analyzed and processed with Imaris 8 software. All the images presented in the paper are representative of at least 4 sections were obtained from 3D reconstructions generated from the same sections.
Ab Degradation Assay
In vitro: intestinal washes were prepared form BALB/c mice (4-6 weeks old) as described (Crottet and Corthesy (1998) J. Immunol. 161:5445-5453). 300 ng of SCIgA/M, IgA/M and IgG were mixed with 2.5 μl of intestinal washes in a final volume of 20 μl of PBS and incubated for the indicated times at 37° C. Addition of 2.5 μl of Complete® protease inhibitor (Roche Applied Science) stopped the reaction and the degradation products were stored at −20° C. Detection of the reduced form of the heavy chain of each Ab mixture was performed by immunoblotting using goat anti-human alpha chain (1/1000; Cappel), followed by HRP-labeled rabbit anti-goat Ab (1/4000; Sigma-Aldrich) or biotinylated anti-human mu chain (1/1000; KPL) or biotinylated anti-human gamma chain (1/1000; Sigma-Aldrich) sera, followed by Extravidin-HRP (1/4000; Sigma-Aldrich).
In vivo: 2 μg unlabeled Abs were injected in a final volume of 20 μl of PBS in a mouse ligated intestinal loop. After 6 hours, mice were sacrificed, the loop's tissue and luminal content were collected and the tissue was further cut into small pieces. The samples were incubated in 50 mM HCl and Complete® protease inhibitor (Roche Applied Science) for 16 hours at 4° C. under gentle agitation, then centrifuged for 10 minutes at 2,350×g. The clear supernatant was collected and stored at −20° C. prior to use. SDS-PAGE and immunoblot analyses were performed as for the in vitro assay.
Oral Infection of Mice and Bacterial Load Determination
Mice were orally infected with 2×107 St alone or complexed with plasma-derived Abs using a round tip stainless steel needle. Infected BALB/c were sacrificed 6 days post-infection and bacterial loads in PPs, MLNs and spleen were determined as described (Pigny et al. J Infect Dis 2016; 214:438-46).
Oral Infection of Mice and Passive Administration of Ab
Mice were orally infected with 2×107 (prophylactic and therapeutic settings) or 2×106 (survival in therapeutic setting) St using a round tip stainless steel needle (Pigny et al. (2016) J. Infect. Dis. 214:438-446). Mice infected with the lowest dose exhibited the expected reduced bacterial load in all tissues tested, and died at the latest on day 11 as compared to days 6-7 with a 2×107 dose. In the prophylactic setting, 10 mg of polyreactive SCIgA/M, IgA/M or IgG were orally administered 24 hours and 8 hours prior to bacteria infection. In the therapeutic setting, 10 mg of SCIgA/M were orally administered 1 hour or 8 hours post-infection. Bacterial loads in PPs, MLNs, spleen and liver were assessed as described (Pigny et al. (2016) J. Infect. Dis. 214:438-446). Weight of mice and disease score (Acred et al. (1994) Laboratory Animals 28:13-18) based on fur ruffling, activity, posture, eye/nose discharge and aspect of feces were recorded on a daily basis.
Assessment of Inflammation
Six days post-infection, mice were sacrificed, 4-6 PPs were collected per mouse and processed to cell suspensions (Mikulic et al. Cell Mol Immunol 2016). Cells (1.5×106) were labeled with anti-CD16/32 mAbs, followed by anti-CD45, -CD3, -CD19, -CD11b, -CD11c, -Ly-6C, -Ly-6G, -F4/80 and DAPI. Frequency of neutrophils (DAPI−CD45+CD3−CD19−autofluorescence−CD11b+CD11c−Ly-6C−F4/80−Ly-6G+) and macrophages (DAPI−CD45+CD3−CD19−autofluorescence+CD11b+CD11c−Ly-6C−F4/80+Ly-6G−) were recorded with a Gallios flow cytometer (Beckman Coulter). Alternatively, 1×106 cells were seeded in wells of round bottom 96-well culture plates and incubated for 24 hours at 37° C.; LPS (100 ng/ml) was added in control wells. TNF-α and IL-6 secreted in the culture supernatants were quantified by ELISA (ELISA Max kits, BioLegend).
Histological Analysis of Intestinal Tissues
At sacrifice, 1-cm sections of duodenum and jejunum were collected, fixed in paraformaldehyde and cut into 5 parts before embedding in paraffin. 7-μm sections were then prepared and stained with hematoxylin and eosin to assess tissue integrity. At least 10 tissue sections per animal were observed with an Axio Imager Z1 microscope (Zeiss).
Statistical Analysis
Statistical analysis was performed using Prism software (GraphPad Software, Inc., La Jolla, Calif.). Bars represent median of each experimental group. The unpaired, non-parametric Mann-Whitney test was used to compare two experimental groups. The unpaired, non-parametric Kruskal-Wallis test, corrected with Dunn's test for multiple comparisons, was applied to compare experimental groups. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
We first tested the ability of different molecular forms of plasma-derived IgA and IgM, or IgG, or recombinant SC to interact with St by ELISA. All Abs preparations displayed a dose-dependent recognition of the bacteria, yet at different levels (
The agglutination capacity was quantitated by counting free (i.e. not contained in aggregates) St-GFP as compared to immune complexes prepared at different Ab/SC to bacteria ratios (5, 1, 0.2 or 0.04 μg Ab and 1 or 0.2 μg SC per 2×104 bacteria). About 200 free St-GFP were counted by observation field in the absence of Abs (
These data demonstrate that the interaction of (S)IgA and (S)IgM with St translates into the formation of bacterial aggregates, an essential prerequisite for the prevention of pathogen entry into mucosal tissues.
To test whether IgA/M and SCIgA/M can indeed mediate immune exclusion and prevent St entry into PPs, we administered immune complexes formed at a ratio of 1 μg Ab per 2×104 bacteria into ligated intestinal loops containing one PP. We first determined that the St-specific SIgASal4 mAb protecting mice when given as bag-back (Michetti et al. Infect Immun 1992; 60:1786-92) was able to significantly reduce the number of bacteria in PPs in this experimental context (
After having demonstrated that IgA/M and SCIgA/M Abs bound to St limit its diffusion from the lumen into PPs, it turned out highly relevant to evaluate whether biological activity was preserved after oral administration. An infective dose of 2×107 St was defined in trial experiments to ensure a reliable bacterial load in PPs and MLNs at 2 days post-oral administration, together with an associated local and systemic infection after 6 days. When combined with IgA/M and SCIgA/M at a ratio of 1 μg Ab per 2×104 bacteria, the number of St recovered from PPs and MLNs 2 days post-infection were significantly reduced as compared to the administration of the bacteria alone (
SIgA has been demonstrated to dampen inflammatory processes in the GI tract (Mikulic J. et al (2016) Cell Mol Immunol, Mantis N et al (2011) Mucosal Immunol. 4:603-611), which is considered beneficial for the host by preventing tissue destruction in pathological situations. After oral infection with St, rapid GI inflammation is triggered; relevant markers include an increase in frequencies of neutrophils and macrophages, as well as elevated production of TNF-α and IL-6 (
Once in the intestine, SIgA/M, but not IgA/M and IgG, is anchored to the epithelium through strong interaction with mucus (data not shown). The stability in the intestinal environment of the same set of Abs was examined by testing their susceptibility to degradation by mouse intestinal washes (See Materials and Methods). After 2 hours of incubation in intestinal washes, immunoblots performed in reducing conditions indicated the presence of intact alpha chain (62 kDa) and some degradation products (
Together, these data demonstrate that after administration in the intestinal environment, the SCIgA/M preparation, as compared to IgA/M and IgG, combines the most optimal anchoring and stability properties, two parameters that have been associated with protective efficacy in the gut.
To test the biological activity of SCIgA/M, as well as the importance of SC in the preparation, prophylactic oral administration of 10 mg of SCIgA/M, IgA/M and IgG (as control) was performed at 24 hours and 8 hours before intragastric inoculation with 2×107 CFU of St. Mice were kept for 6 days post-infection, and the protective efficacy of the Abs was assessed by measuring the local intestinal and systemic bacterial load. Prophylactic passive immunization with SCIgA/M turned out to be the more potent at reducing the bacterial load in PPs, MLNs, and spleen of infected mice, and displayed similar characteristics than IgA/M when the liver was examined (
St infection and its associated inflammatory response are known to damage the intestinal epithelium, leading to additional alteration of the gut function. Consistent with its capacity to limit infection, prophylactic administration of SCIgA/M, and to a lesser extent IgA/M, contributed to largely maintain the architecture of the intestinal tissue post-infection. As depicted in
The strict correlation between the residual bacterial load and a possible effect on the survival of prophylactically treated animal is important to validate protection in the physiological context timewise. To address this issue, the experimental focus was put on animals having received prophylactically the best protective candidate Ab as defined above, i.e. SCIgA/M at a dose of 10 mg given orally 24 hours and 8 hours prior to infection with 2×107 St. In contrast to control animals rapidly losing weight and reaching high disease scores (
The promising results obtained with the prophylactic administration of polyreactive SCIgA/M prompted us to assess the therapeutic protective efficacy of this Ab preparation. When 2×107 St were used to orally infect mice, administration of 10 mg SCIgA/M 1 hour later was able to significantly reduce the bacterial load recovered at day 6 post-infection from both intestinal (PPs and MLNs) and systemic (spleen and liver) tissues (
The protective efficacy of a single dose of SCIgA/M observed 6 days post-infection led us to hypothesize that therapeutic application of these polyreactive Abs may have a positive effect on the survival of mice post-infection. We therefore orally infected mice with 2×106 St, and applied 10 mg of SCIgA/M 8 h later; the weight, the disease score and the survival of animals were recorded for 20 days. In the untreated control group, mice rapidly lost weight (
Altogether, our data demonstrate that a single oral therapeutic administration of polyreactive SCIgAM at a time when St infection is already ongoing entails the reduction of the intestinal and systemic bacterial load and of the symptoms associated with infection. This results in increasing the overall survival rate of infected mice to 50%, comparing with 100% mortality in the absence of therapeutic SCIgA/M.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16205456.3 | Dec 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/083693 | 12/20/2017 | WO | 00 |
