WO2019217665A1 - Administration de peptide récepteur d'acquisition de fer pour la vaccination contre pseudomonas aeruginosa - Google Patents

Administration de peptide récepteur d'acquisition de fer pour la vaccination contre pseudomonas aeruginosa Download PDF

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WO2019217665A1
WO2019217665A1 PCT/US2019/031499 US2019031499W WO2019217665A1 WO 2019217665 A1 WO2019217665 A1 WO 2019217665A1 US 2019031499 W US2019031499 W US 2019031499W WO 2019217665 A1 WO2019217665 A1 WO 2019217665A1
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vaccine composition
fpva
peptide
aeruginosa
group
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PCT/US2019/031499
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WO2019217665A4 (fr
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Mariette BARBIER
Fredrick Heath DAMRON
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West Virginia University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • Various embodiments disclosed herein relate generally to various iron-acquisition receptor peptides that may be administered to vaccinate against infections caused by Pseudomonas aeruginosa.
  • Pseudomonas aeruginosa is a major bacterial pathogen causing a wide range of infections ranging from acute superficial infections (burn wounds, keratitis and dermatitis), to chronic respiratory infections in immunocompromised patients (Cystic Fibrosis (CF), Bronchiectasis, Chronic Obstructive Pulmonary Disease). It is one of the main causative agents of pneumonia in patients under assisted mechanical ventilation and is responsible for life threatening infections such as endocarditis and septicemia. This bacterium is intrinsically resistant to a large number of antibiotics and the medical community is currently running out of therapeutic options to treat the infections it causes. In patients with CF for example, these infections can often not be eradicated. There is no preventative treatment such as vaccines for these diseases.
  • Vaccines targeting LPS have also been pursued by scientists. Hepta- and octavalent vaccines
  • Vaccines targeting secreted products have also been pursued.
  • P. aeruginosa secretes proteins that act as virulence factors during infection.
  • Various studies have attempted to design vaccines using exotoxin A, alkaline protease, and elastase as vaccine antigens. Most of these components did not show efficacy alone and moderate efficacy when combined. Efficacy studies are very limited and have not been followed up.
  • Vaccines targeting alginate exopolysaccharide have also been studied.
  • P. aeruginosa secretes an exopolysaccharide coating that protects the bacterium against environmental stresses and the immune system.
  • Vaccination with exopolysaccharide produces opsonic (help the immune system clear the bacteria) and non-opsonic (do not help the immune system) antibodies in various murine and rat models of vaccination.
  • vaccination with the exopolysaccharide only triggered production of opsonic antibodies in 2 out of 23 subjects. Another trial showed production of antibodies in 30% of the subjects. However, no long-term benefits from this vaccination were observed.
  • aeruginosa pili have shown moderate protection while vaccines targeting PcrV have shown to decrease lung injury and virulence in burn wound models, although only with strains producing low amounts of toxins. Overall, these vaccines are the most promising but none have been brought to market.
  • a vaccine composition including an outer membrane protein of P. aeruginosa , wherein the outer membrane protein is an iron acquisition receptor protein.
  • iron acquisition receptor protein includes a peptide selected from a group that includes SEQ ID NOS: 1-4.
  • peptide includes a modification at the C- or N-terminus.
  • a vaccine composition further including additional adjuvants, such as curdlan and carrier proteins, such as keyhole limpet hemocyanin (KLH), recombinant tetanus toxoid (rTTHc) and recombinant diphtheria toxoid (CRM 197 ).
  • additional adjuvants such as curdlan and carrier proteins, such as keyhole limpet hemocyanin (KLH), recombinant tetanus toxoid (rTTHc) and recombinant diphtheria toxoid (CRM 197 ).
  • KLH keyhole limpet hemocyanin
  • rTTHc recombinant tetanus toxoid
  • CCM 197 recombinant diphtheria toxoid
  • compositions wherein the composition is administered via a mucosal delivery method or via injection.
  • Various embodiments further recite a method of preventing an infection, by administering a vaccine composition including an outer membrane protein of P. aeruginosa, wherein the outer membrane protein is an iron acquisition receptor protein.
  • Various embodiments further recite a method of preventing an infection wherein the vaccine composition generates antibodies that induce iron starvation in low iron conditions.
  • Various embodiments further recite a method of preventing an infection wherein the vaccine composition triggers the production of opsonic antibodies.
  • FIG. 1 illustrates a representation of the FpvA peptides of the disclosure
  • FIG. 2A illustrates a schematic diagram of the vaccination timeline and experimental design in a CD1 mouse model
  • FIG. 2B illustrates ELISA for the detection of IgG and IgM antbPAOl antibodies in WCV vaccinated mice over time
  • FIG. 2C illustrates ELISA for the detection of IgG and IgM anti-unconjugated FpvA peptide antibodies in FpvA-vaccinated mice
  • FIG. 2D illustrates ELISA for the detection of IgG and IgM anti-unconjugated FpvA peptide antibodies in FpvA-KLH vaccinated mice
  • FIG. 3A illustrates IgG and IgM antibody titers of NYC, FpvA peptides only, and FpvA- KLH vaccinated and challenged mice groups against unconjugated FpvA peptides, with each circle representing data from one mouse of NYC, WCV, FpvA and FpvA-KLH vaccine groups at 16 hours post-challenge;
  • FIG. 3B illustrates a Western blot analysis of pooled NVC and FpvA-KLH sera against purified FpvA protein expressed in E. coli using the vector pHERD20T ⁇ 3 ⁇ 4Al-His6.
  • FIG. 3C illustrates IgG and IgM antibody titers of NVC, FpvA peptides only and FpvA- KLH vaccinated and challenged mice groups against heat-killed whole-cell Pseudomonas aeruginosa PAOl, with each circle representing data from one mouse of NVC, WCV, FpvA and FpvA-KLH vaccine groups at 16 hours post-challenge;
  • FIG. 3D illustrates IgG response of pooled sera from NVC and FpvA-KLH vaccinated challenged mice against P. aeruginosa cystic fibrosis isolates and lab strains, with each circle representing different P. aeruginosa strains, and triangles showing the PAOl strain used for mice infection;
  • FIGS. 4A-4B illustrate, respectively, the bacterial burden of NVC, WCV and FpvA-KLH vaccinated mice in the nasal wash and lung;
  • FIG. 4C illustrates the lung weight of each vaccinated mouse measured 16 hours after challenge, with each circle representing data from one mouse;
  • FIG. 4D illustrates H&E staining of the lungs from the immunized and adjuvant only control mice 16 hours post challenge with lxlO 8 P. aeruginosa P AOl;
  • FIG. 5A illustrates elicited IgG subtypes: IgGl, IgG2b, IgG2a; of FpvA-KLH vaccinated sera against FpvA peptides at 16 hours post-challenge with each circle representing data from one mouse;
  • FIG. 5B illustrates the percentage of RORyT+ CD4+ (Thl7) T cell population in the spleen
  • FIG. 5C illustrates an ELISpot assay for identifying an IFN-g specific response of intranasal
  • FIG. 5D illustrates an ELISpot assay for identifying an IL-17 specific response of intranasal curdlan only, FpvA-KLH vaccination with curdlan and FpvA-KLH vaccination with alum in the presence or absence of heat-killed P. aeruginosa at day 34 post-vaccination;
  • FIG. 6A illustrates a schematic representation of the gating strategy used for differentiating CD4 + T central memory (TCM) (CD4+CD44+CD62L+), T effector memory-like (LEM) (CD4+CD44+CD62L-) and Tissue-resident memory T cells (TRM) (CD4+CD44+CD62L- CD103+CD69+) from naive T cells (TN) (CD44-CD62L+);
  • TCM T central memory
  • LEM T effector memory-like
  • TRM Tissue-resident memory T cells
  • FIG. 6B illustrates the percentage of naive T cell population in CD4 + ;
  • FIG. 6C illustrates the percentage of central memory T cells and effector memory-like T cells in CD4 + T cell population
  • FIG. 6D illustrates the percentage of tissue-resident memory cells in CD4 + T cell population
  • FIG. 7 illustrates the promotion of opsonophagocytosis by anti-FpvA antibodies
  • FIG. 8A illustrates FpvA vaccination using the carrier proteins rTTHc and CRM197 and production of antibodies specific for the peptides
  • FIG. 8B illustrates production of FpvA peptide-specific antibodies in blood across time.
  • identical reference numerals have been used to designate elements having substantially the same or similar structure or substantially the same or similar function.
  • This disclosure relates to a peptide-based vaccine composition that specifically targets the major iron acquisition systems for P. aeruginosa.
  • Iron acquisition proteins are expressed during both acute and chronic infection with P. aeruginosa and are important to the survival and virulence of the bacteria. Preventing iron acquisition results in iron starvation and decreasing bacterial virulence.
  • prevention of P. aeruginosa infection involves utilizing the outer membrane protein receptor FpvA to bind the siderophore pyoverdine. This siderophore delivers iron into the cell.
  • Embodiments of the vaccine composition of the disclosure may take advantage of the following:
  • FpvA is an outer-membrane protein of P. aeruginosa ⁇
  • previous vaccine studies have shown that outer-membrane proteins are the most viable candidates for vaccine design for P. aeruginosa, ⁇ 2.
  • FpvA is expressed during infection: unlike previous vaccine approaches based on the use of the most abundant proteins which might not be relevant during infection, FpvA is expressed during infection with P. aeruginosa, ⁇
  • FpvA is important for survival and pathogenesis of the bacterium: P. aeruginosa bacteria in which the gene fpVL encoding for the protein FpvA has been removed have strongly decreased virulence during infection;
  • Peptide-based vaccines are safe: they do not contain adverse-effect inducing LPS;
  • peptides of the embodiments are specifically formulated to have very low cross-species reactivity to reduce the risks of immune disease or reaction against commensal bacteria as a result from vaccination.
  • the vaccine composition based on the outer membrane protein receptor FpvA is a peptide consisting essentially of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO:4.
  • the peptides may include a modification at the C- and/or N-terminus.
  • the C-terminus is modified to include a cysteine residue.
  • the vaccine composition may include an FpvA peptide formulated with an effective amount of an adjuvant.
  • Suitable adjuvants include, but are not limited to, high molecular weight polymers of glucose, alum and the like.
  • the vaccine composition may include an FpvA peptide formulated with an effective adjuvant amount of a high molecular weight polymer of glucose, such as b-glucan, dextran and the like.
  • Preferred b-glucans include curdlan.
  • the vaccine composition of the invention includes an FpvA peptide and an effective amount of a carrier protein.
  • Preferred carrier proteins include keyhole limpet hemocyanin (KLH), recombinant tetanus toxoid (rTTHc) and recombinant diphtheria toxoid (CRM 197 ).
  • prevention of P. aeruginosa infection involves utilizing a vaccine composition further including additional outer membrane proteins including other siderophore receptors, such as FpvB and FptA; heme receptors such as PhuR; hemophore receptors such as HasR; and iron transport proteins, such as FeoB and pseudopaline system proteins.
  • additional outer membrane proteins including other siderophore receptors, such as FpvB and FptA; heme receptors such as PhuR; hemophore receptors such as HasR; and iron transport proteins, such as FeoB and pseudopaline system proteins.
  • the vaccine composition may be formulated for intranasal administration.
  • the vaccine composition may be administered using alternative routes of administration including, without limitation, parenteral administration methods, such as subcutaneous (SC) injection, transdermal, intramuscular (IM) injection, intradermal (ID) injection, as well as non-parenteral, e.g., oral, intravaginal, pulmonary, ophthalmic and or rectal administration.
  • parenteral administration methods such as subcutaneous (SC) injection, transdermal, intramuscular (IM) injection, intradermal (ID) injection, as well as non-parenteral, e.g., oral, intravaginal, pulmonary, ophthalmic and or rectal administration.
  • the P. aeruginosa infections that may be prevented using the vaccine composition of the disclosure include respiratory system infections, urinary tract infections, dermatitis, skin and soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and other systemic infections.
  • FpvA peptide To design the FpvA peptide, three dimensional structure modeling was used to predict the extracellular and membrane domains of FpvA using the software Chimera v.1.10.2. To further select areas of interest, extracellular portions of the protein were analyzed using the Kyle-Doolittle hydrophobicity values of each peptide to determine antigenicity, and with the Bepipred linear prediction algorithm to predict B cell linear epitopes. The regions with the highest scores on both analyses were then Blasted against P. aeruginosa (BLAST-P) to determine if these regions were conserved across P. aeruginosa isolates.
  • BLAST-P P. aeruginosa
  • WCV Whole-cell vaccine
  • P. aeruginosa PAOl“Vasil” The strain was grown for 16 hours at 37°C on Pseudomonas Isolation Agar (PIA)(Becton Dickinson). Swabbed bacteria were resuspended in Phosphate Buffered Saline (PBS), and heat killed at 60°C for 1 hour.
  • PBS Phosphate Buffered Saline
  • Each whole-cell vaccine dose consisted of 4-5x10 7 heat killed colony-forming units (CFU) of P. aeruginosa per dose in PBS.
  • CFU heat killed colony-forming units
  • FpvA peptide-only and FpvA-KLH acellular vaccine compositions were prepared as follows: lyophilized FpvA and FpvA-KLH peptides were re-suspended in 125 mM NaOH.
  • FpvA acellular vaccine doses consisted of a mix of 35 pg each FpvA peptide in PBS.
  • FpvA-KLH acellular vaccine doses consisted of a mix of 35 pg of each FpvA-KLH peptide in PBS. All vaccines were prepared in final volume of 20 pi and contained 100 pg of curdlan as an adjuvant.
  • a curdlan-only control was formulated as 100 pg of curdlan in PBS.
  • FpvA-KLH acellular vaccine was formulated with a mix of 35 pg of each FpvA-KLH peptide and 62.5 pg of alum in PBS.
  • mice were divided into the following groups:
  • Non-vaccinated non-challenged mice these mice were administered the adjuvant curdlan only;
  • mice were administered the adjuvant curdlan only and challenged;
  • FpvA FpvA peptides
  • FpvA-KLH peptides (FpvA-KLH): these mice were vaccinated with a cocktail of 4 FpvA peptides linked to the carrier protein KLH adjuvanted with curdlan;
  • mice from the adjuvant only group were administered with PBS (non-vaccinated non-challenged, NVNC). Blood was collected by cardiac puncture after euthanasia at day 35.
  • the lungs of each mouse were aseptically removed and weighed. Nares were flushed with 1 ml of sterile PBS to collect the nasal washes (NW). Lungs were homogenized in 1 ml of PBS using a glass Dounce homogenizer. One hundred microliters of homogenized lung samples and NW from each mouse were then serially diluted and plated on PIA plates to determine viable CFU counts. To measure cytokine response, 200 m ⁇ of lung homogenate from each mouse were pelleted by centrifugation, the supernatant was collected and stored at -80°C until cytokine analysis.
  • mice challenged with 10 7 CFU were collected at day 7 post-challenge and homogenized gently using a 100 pm pore cell strainer. The remaining lung homogenates and splenocytes were further analyzed by flow cytometry. ELISA Assays
  • the antibody titers in sera at days 7, 14 and 20 post- vaccination, and day 1 post-challenge were assayed by ELISA in Costar® 96 well high-binding microtiter plates.
  • the microtiter plates were coated with a volume of 50 m ⁇ /well of 2x10 7 CFU of heat-killed P. aeruginosa PAOl, 500 ng of individual FpvA peptides, or a mix of all peptides (125 ng of each peptide; 500 ng total) overnight at 4°C.
  • the plates were washed three times with PBS with 0.05% Tween 20 (PBS-T) and blocked with 2% Bovine Serum Albumin (BSA) in PBS overnight at 4°C. Blocked plates were washed three times with PBS-T. Serum samples were prepared in 2% BSA in PBS, added to the wells, and serially diluted from 1:100 to 1:25,600. After 1 hour of incubation at 37°C, plates were washed four times with PBS-T.
  • PBS-T PBS with 0.05% Tween 20
  • BSA Bovine Serum Albumin
  • Plates were incubated with anti-IgG, TgM, -IgGl, TgG2a or -IgG2b alkaline-phosphatase conjugated goat anti-mouse antibodies at 1:2,000 per well for 1 hour at 37°C. Plates were then washed five times with PBS-T and incubated with Pierce ⁇ -Nitrophenyl Phosphate (PNPP) for 30 minutes. The absorbance of the plates was read at 405 nm using SpectraMax i3. If the detected signal was above the background threshold, antibody titers were considered positive.
  • PNPP Pierce ⁇ -Nitrophenyl Phosphate
  • Purified FpvA protein was resuspended in Lemmli buffer and boiled for 5 minutes. A total of 5 pg of protein was loaded into each well and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to a previously rehydrated PYDF membrane. After the transfer, the membrane was blocked with 5% skim milk in PBS-T at 4°C overnight.
  • the membrane was incubated with pooled murine sera from the FpvA-KLH and from NYC challenged at a dilution of 1:2,000, or with anti-Fiis antibodies at 1:5,000 in 5% skim milk in PBS-T for 16 hours
  • the membrane was washed 3 times with PBS-T and incubated with anti-mouse IgG antibodies conjugated with HRP at 1:5,000 in 5% skim milk in PBS- T for one hour.
  • the membrane was washed again 3 times with PBS-T, and finally developed using SuperSignal West Femto Maximum Sensitivity Substrate. Chemiluminescence signal was detected using a Chemidoc Touch Imaging System.
  • Tissue homogenates were strained with 4 ml of PBS through a disposable 100 pm pore cell strainer. Samples were centrifuged at 1,000 x g for 5 minutes, and supernatants were discarded. Red blood cells were lysed for 2 minutes at 37°C. The remaining cells were centrifuged at 1,000 x g for 5 minutes. The pellets were re-suspended in PBS with 1% fetal bovine serum (FBS) for blocking and incubated on ice for 15 minutes. Lung cells were then stained with specific cell surface markers. Each cell suspension sample was incubated with the specific antibody cocktails for 1 hour at 4°C in the dark. Cell suspension samples were pelleted and resuspended in PBS before flow cytometry analysis.
  • FBS fetal bovine serum
  • Samples were processed using a BD LSR Fortessa flow cytometer and analyzed using Flowjo vlO.
  • Spleens from day 7 post-challenge were strained using 4 ml RPMI 1640 medium with 10% FBS and 0.5% penicillin-streptomycin (RPMI complete medium) through a disposable 100 pm pore cell strainer.
  • the splenocytes were red blood cell lysed and blocked with 1% FBS.
  • Splenocytes were stained for T cells with the specific cell surface receptor and intracellular transcription factor markers. The cells were then centrifuged at 1,000 x g for 5 minutes, and pellets were re-suspended in PBS.
  • the concentration of IFN-y, FL-Ib, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-oc and IL-17 cytokine levels m the lung were determined by quantitative sandwich immunoassays using the Meso Scale Diagnostics Y-PLEX Plus Proinflammatory Panell Mouse Kit (K15048G-1) and Mouse IL-17 Ultra-Sensitive kits (K152ATC-1).
  • the electrochemiluminescence signal was detected using MSD Multi -Array Imaging Platform. Lung supernatant from NYNC, NVC, and FpvA-KLH groups were diluted to 1:5 and 1:300 to avoid saturation of the signal.
  • ELISpot assays were performed to determine the number of antigen-specific splenocytes in vaccinated animals secreting IL-17 in response to antigen stimulation.
  • EMD Millipore Multiscreen 96-well assay plates were pre -wetted with 15 m ⁇ of 35% ethanol for 1 minute. The plates were washed 5 times with 200 m ⁇ of sterile water and coated with anti-IL-17 (5mg/ml) and anti-IFN-g (5pg/ ml) antibodies overnight at 4°C.
  • Splenocytes were isolated from immunized and boosted CD-I mice groups as described above. The spleen of each mouse was collected at day 34 post-vaccination and homogenized using a 100 pm sterile cell strainer.
  • the splenocytes were counted using trypan blue with a Countess II FL Automated Cell Counter. The cells were diluted to 106 cells/ml using RPMI complete medium. The splenocytes were then aliquoted in duplicate and incubated with RPMI complete medium only, or RPMI complete medium containing 107 heat-killed P. aeruginosa using pre -wetted and antibody coated multiscreen 96-well assay plates. The splenocytes from non- vaccinated control mice were also incubated with RPMI complete medium containing 50 ng/ml Phorbol 12-myristate 13-acetate (PMA) as a positive stimulant control.
  • PMA Phorbol 12-myristate 13-acetate
  • IL-17 antibody- coated plates were incubated for 72 hours and IFN-g antibody-coated plates were incubated for 16 hours at 37°C with 5% CO z .
  • wells were washed and incubated with biotinylated secondary antibody IL-17 (0.25 pg/ml), IFN-g (2 pg/ml) for 2 hours at room temperature.
  • the wells were then rewashed and incubated with streptavidin-alkaline phosphatase for 1 hour at room temperature.
  • the wells were washed again and developed using a filtered substrate solution of BCIP/NBT.
  • FpvA peptides were formulated as vaccines with the adjuvant curdlan.
  • Cocktails of FpvA peptides, FpvA-KLH peptides, WCY, or curdlan alone were intranasally administered to 6-week-old female CD-I mice. Twenty-one days later, mice were boosted intranasally with the same vaccine preparations (FIG. 2A). WCY was used as a positive control while curdlan alone (non-vaccinated challenged, NYC) was used as a vehicle control. Blood was collected from the mice at days 0, 7, 14, 20, and 35. To determine the immunogenicity profile of each vaccine, ELISAs were performed on the serum samples to detect the immunoglobulins specific to FpvA peptides or P.
  • aeruginosa Mice vaccinated with the WCY, which was used as a positive control, elicited the production of significant anti-P. aeruginosa PAOl IgM and IgG antibody titers over time, as shown in FIG. 2B. It was observed that unconjugated FpvA peptides elicited low anti-FpvA peptide-specific IgM antibody titers, that did not result in isotype switching to IgG, as shown in FIG. 2C.
  • mice immunized with FpvA-KTH peptides compared to unconjugated FpvA peptides resulted in 1000-fold increase in IgG and 10-fold increase in IgM antibody responses against unconjugated FpvA peptides (FIG. 3A).
  • Immunization with FpvA-KMT peptides lead to a significant increase in IgG and IgM antibody responses against unconjugated FpvA peptides when compared to the NVC group.
  • mice While the mice were immunized with a mixture of the four FpvA peptides conjugated to KTH, anti-peptide 2 (SEQ ID NO: 2) antibodies accounted for most of the elicited IgG serum titers, and anti-peptide 4 (SEQ ID NO: 4) antibodies were detected in 3 out of 9 mice.
  • anti-peptide 2 SEQ ID NO: 2
  • anti-peptide 4 SEQ ID NO: 4
  • recombinant FpvA protein expressed in Escherichia coli (E. coli ) using the vector pHERD20T ⁇ vAl-His6, was first purified with protein identity confirmed by mass spectrometry. Western blot analysis was then performed with the purified recombinant FpvA protein and it was determined that the serum from FpvA-KLH vaccinated mice, but not curdlan- only vaccinated mice, recognized the purified recombinant FpvA protein (FIG. 3B).
  • mice vaccinated with FpvA-KLH peptides resulted in a 100-fold increase in IgG and 10-fold increase in IgM antibody response compared to unconjugated FpvA peptides (FIG. 3C).
  • Anti-FpvA antibodies were also shown to promote opsonophagocytosis, as shown in FIG. 7.
  • mice were challenged on day 34 post- vaccination (FIG. 2A).
  • Challenged experiments were conducted with FpvA-KLH, WCY (positive control), and NYC (vehicle control) vaccinated mice.
  • the bacterial burden in the nares and the lung at 16 hours post-challenge were determined by plating the serial dilutions and viable bacteria counts.
  • the bacterial burden in the airways of the WCV mice were found to be significantly lower than in NVC.
  • Mice vaccinated with the FpvA-KLH peptide vaccine had significantly decreased bacterial burden in nasal cavities and the lung compared to the NVC control group, as shown in FIGS.
  • P. aeruginosa is known to cause pneumonia with acute lung injury, inflammation, and pulmonary edema during infection.
  • lung weights of mice were measured as an indicator of lung edema and histopathological analysis of lungs was performed to observe pulmonary damage and inflammation at 16 hours post-challenge.
  • the lung weight of mice vaccinated with FpvA-KLH was significantly decreased compared to the NYC after challenge. There was no significant difference in lung weight between FpvA-KLH vaccinated, naive non-challenged adjuvant only (NYNC) and WCV groups (FIG. 4C).
  • FpvA-KLH vaccination helped decrease pulmonary edema caused by P. aeruginosa infection compared to the NVC group.
  • FpvA- KLH vaccinated mice similar to the WCV vaccinated mice, had less alveolar disruption, vascular leakage, damage and recruitment of polymorphonucleated cells compared to NVC (FIG. 4D).
  • FpvA-KLH vaccination reduced bacterial burden in the lung and nares as well as lung damage and edema in an acute lung pneumonia mouse model.
  • IFN-g and IL-17 ELISpot assays were performed using splenocytes at day 34 post-vaccination.
  • splenocytes In the mice vaccinated with curdlan alone or FpvA-KLH with curdlan no difference in IFN-g secreting cells in response to stimulation with P. aeruginosa was observed (FIG. 5C).
  • splenocytes from mice vaccinated intranasally with FpvA-KLH adjuvanted with curdlan had a significantly higher number of IL-17 secreting cells than those vaccinated with curdlan alone in response to stimulation with P.
  • FpvA protein was shown to be capable of stimulating Thl7 response in mice splenocytes in vitro.
  • the FpvA-KLH vaccine was re-formulated with alum and the mice vaccinated intranasally.
  • mice intranasally vaccinated with FpvA-KLH had significant IgA titers against FpvA peptides at 16 hours post-challenge compared to non-vaccinated challenged mice in lung supernatants (FIG. 6A).
  • the T effector memory-like cells (TEM-like) CD4+CD44+CD62L-
  • TCM T central memory cells
  • FpvA peptides of SEQ ID NO: 2 were conjugated to recombinant tetanus toxoid (rTTHc) and recombinant diphtheria toxoid (CRM 197 ) . Immunogenicity was measured over time in mice and it was demonstrated that rTTHc and KGH lead to the production of equivalent levels of antibodies, while CRM 197 allowed for the production of much higher levels of antigen-specific immunoglobulins, as shown in FIGS. 8A and 8B.

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Abstract

L'invention concerne une composition de vaccin comprenant une protéine de membrane externe de P. aeruginosa, la protéine de membrane externe étant une protéine de récepteur d'acquisition de fer et une méthode de prévention d'une infection par P. aeruginosa impliquant l'administration de la composition de vaccin.
PCT/US2019/031499 2018-05-09 2019-05-09 Administration de peptide récepteur d'acquisition de fer pour la vaccination contre pseudomonas aeruginosa WO2019217665A1 (fr)

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WO2022271257A3 (fr) * 2021-04-08 2023-04-20 West Vrginia University Antigènes de protection croisée pour la vaccination

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