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WO2025038171A2 - A bacterial vesicle-based pneumococcal vaccine against influenza-mediated secondary streptococcus pneumoniae pulmonary infection - Google Patents

A bacterial vesicle-based pneumococcal vaccine against influenza-mediated secondary streptococcus pneumoniae pulmonary infection Download PDF

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WO2025038171A2
WO2025038171A2 PCT/US2024/034124 US2024034124W WO2025038171A2 WO 2025038171 A2 WO2025038171 A2 WO 2025038171A2 US 2024034124 W US2024034124 W US 2024034124W WO 2025038171 A2 WO2025038171 A2 WO 2025038171A2
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pspa
omv
spn
infection
mice
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WO2025038171A3 (en
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Wei Sun
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Albany Medical College
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to vaccines and, more particularly, to a vaccine against Streptococcus pneumoniae infections using Yersina outer membrane vesicles.
  • Spn S. pneumoniae
  • Spn S. pneumoniae
  • An initial influenza virus infection results in increased inflammation, tissue damage, and immune dysregulation in the lungs, which exacerbates pneumonia caused by subsequent Spn superinfection.
  • secondary Spn infection is a known fatal complication of influenza, which was documented as early as the 1918 influenza pandemic and in subsequent influenza pandemics, like the 2009 H1N1 pandemic.
  • pneumococcal surface protein A is an immunogenic protein expressed on the surface of all strains of Spn and plays critical roles in pneumococcal pathogenesis and virulence.
  • PspA was found to be highly immunogenic and cross-protective in mouse and human studies, even though PspA displayed high variability within three protein families that consist of six clades.
  • Yptb Yersinia pseudotuberculosis
  • OMV outer membrane vesicle
  • S. pneumoniae (Spn) is a common pathogen causing a secondary bacterial infection following influenza which leads to severe morbidity and mortality during seasonal and pandemic influenza. Therefore, there is an urgent need to develop bacterial vaccines that prevent severe post-influenza bacterial pneumonia. Accordingly, there is a need in the art for an approach that can protect against to deliver heterologous antigens to achieve protection against S. pneumoniae and corresponding respiratory bacterial infections.
  • the present invention provides an approach that can deliver heterologous antigens to achieve protection against Spn and other respiratory bacterial infections.
  • the present invention employs an improved Yptb OMV platform to deliver the highly immunogenic a-helical region of PspA (designated as OMV-PspA).
  • OMV-PspA highly immunogenic a-helical region of PspA
  • the protective efficacy of the platform was evaluated against an H1N1 CA04 infection-mediated secondary Spn infection.
  • the results demonstrated that intramuscular prime-boost immunization with OMV- PspA induced potent humoral and cellular immune responses in systemic and mucosal compartments and afforded significant protection against influenza-mediated secondary Spn infection.
  • FIG. l is a series of graphs and images showing the construction and analysis of OMVs containing PspA.
  • A Construction of pSMV92 plasmid, in which the codon- optimized a-helical region of pspA (3-285 aa) was cloned into pYA4515 plasmid.
  • B The analysis of PspA antigen by immunoblotting.
  • the PspA synthesis in the YptbS46 harboring pYA4515, an empty Asd+ plasmid, YptbS46 harboring pSMV92, containing a-helical region of pspA (Left panel);
  • M 10 to 250 kDa protein ladder (ThermoFisher Scientific).
  • C Transmission electron microscope (TEM) image (Left panel) of OMV-PspA and Dynamic Light Scattering (DLS) (Left panel) of OMV-PspA.
  • D Comparison of embryonic alkaline phosphate (SEAP) activities in HEK-blue cells with or without murine toll-like receptor 4 (mTLR4) (Invitrogen).
  • SEAP embryonic alkaline phosphate
  • mTLR4 murine toll-like receptor 4
  • OMVs from YptbS44 strain and rPspA were used as controls. Data are shown as the mean ⁇ SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; **** P ⁇ 0.0001.
  • FIG. 2 is a series of graphs of the immune responses induced by i.m. OMV immunization.
  • A Immunization schema for mouse study.
  • B Weight change rates of mice after immunization.
  • C Total serum IgG titers to PspA in Swiss Webster mice on 21 and 35 days post-vaccination (DPV).
  • E Anti-PspA IgG titers in the BALFs of mice immunized with OMV-PspA, OMV-NA, rPspA, or PBS.
  • F Lung PspA-specific T-cell responses in immunized mice. On 45 DPV, total CD4 + and CD8 + T-cell subsets and their specific cytokine (IFN-y, TNF-a, IL- 17A) were stimulated by rPspA antigen.
  • IFN-y total CD4 + and CD8 + T-cell subsets and their specific cytokine
  • FIG. 3 is a series of graphs showing that immunized mice were challenged with a sublethal dose of an influenza virus.
  • animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain.
  • A The schema for this study.
  • B Weight change rates of immunized mice after CA04 infection.
  • Virus titers in the BALF (C) and lung (D) of mice at 2, 4, and 8 days post viral infection (DPVI) were quantified using plaque assay described in Materials and Methods.
  • E Lung histopathological analysis of representative mice from each group on 4 DPVI. The lungs were microscopically examined and imaged using Nanozoomer 2.0 RS Hamamatsu slide scanner (scale bar, 100 nm). Each symbol was obtained from an individual mouse, and data were represented as the mean ⁇ SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; * ⁇ 0.05; ** ⁇ 0.01; *** P ⁇ 0.001; **** P ⁇ 0.0001.
  • FIG. 4 is a series of graphs of the short-term and long-term protective efficacy of OMV-PspA immunization against influenza-mediated secondary Spn infection.
  • animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain and monitored for 9 days.
  • F Weight change rates of immunized mice after CA04 infection. On 196 PDV, animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain.
  • G Long-term protection against secondary Spn infection. On 9 DPVI (205 DPV), animals were intranasally challenged with 1.5 x 10 4 CFU of Spn strain D39.
  • FIG. 5 is a series of graphs of the roles of antibody and T cells in protection against influenza-mediated secondary Spn infection.
  • A Comparative analysis of opsonophagocytic killing assay against Spn strains D39 and A66.1 using antisera collected from immunized mice on 35 DPV.
  • mice Serum transfer.
  • mice Twenty-four hours postinjection, the recipient mice were intranasally challenged with 50 pfu of H1N1 (CA04) and then challenged with 1.5x 10 4 cfu of Spn strain D39 on 9 DPVI.
  • C The schema of T-cell depletion and cytokine neutralization.
  • mice On 8 days post CA04 infection, antibody -treated mice were then intranasally challenged with 1.5* 10 4 cfu of Spn strain D39. The mortality and morbidity of animals were all monitored for 15 days. Statistical significance was analyzed by the log-rank (mantel Cox) test for survival analysis. Data were analyzed and presented as the mean ⁇ SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P ⁇ 0.05 **, P ⁇ 0.0 , ****, P ⁇ 0.000P
  • FIG. 6 is a series of a series of graphs of the patterns of alveolar macrophages (AMs) in the BALF and lung of immunized mice with or without infection.
  • A Gating strategy for AMs and neutrophils.
  • B Representative flow plot showing the percentage of AMs without infection.
  • FIG. 7 is a series of graphs of the patterns of neutrophils in the BALF and lung of immunized mice with or without infection and cytokine profiles in the BALF of mice subjected to co-infection.
  • A Representative flow plot showing the percentage of neutrophils without infection.
  • FIG. 8 is the nucleic acid sequence of PspA antigen and lipid A analysis of OMV-PspA (SEQ ID NO: 1).
  • A Codon optimized a-helical region of pspA encoding aa residues 3 to 285 (849 bp; 283 aa) of the mature PspA from Spn strain D39.
  • B Mass spectrum analysis of lipid A species in OMV-PspA.
  • FIG. 9 is a series of graphs of antibody titers in the BALF, T-cell gating, and spleen T-cell responses.
  • A Gating Strategy for T-cell responses for both post vaccination and infection.
  • B Representative flow plots for CD4-specific cytokines (IFN-y, TNF-a, and IL- 17 A) in the immunized mice post vaccination.
  • C Representative flow plots for CD8- specifc cytokines (IFN-y, TNF-a, and IL-17A) in the immunized mice post vaccination.
  • D Spleen PspA-specific T-cell responses in immunized mice. The detailed procedures were described in Materials and Methods.
  • FIG. 10 is a series of graphs of an opsonophagocytic killing assay, lung T-cell responses in immunized mice secondarily infected with Spn, and the role of IFN-y in protection against influenza-mediated secondary Spn infection.
  • A Comparative analysis of opsonophagocytic killing assay against clinical Spn isolates (ST552, ST554, ST556, ST558, ST560) using sera from different immunized mice.
  • B Lung T-cell responses in immunized mice secondarily infected with Spn.
  • D Weight change rates of immunized mice after CA04 infection. On 36 DPV, animals were challenged intranasally with 15 pfu of H1N1 (CA04) strain.
  • FIG. 1 a platform for providing OMV-PspA vaccination that induces both memory adaptive and innate immune responses and provides significant protection against influenza-mediated secondary Spn infection in a mouse model.
  • the data strongly suggest that an OMV vaccine delivering Spn antigens can be a promising vaccine candidate for counteracting co-infection.
  • OMV-PspA immunization elicits potent humoral and cellular immune responses (FIG. 2C-F and FIG. 9), both of which are required for optimal protection against influenza-mediated secondary Spn infection (FIG. 5).
  • IgG and IgA areotypes in both systemic and lung compartments
  • intramuscular immunization generally induces robust IgG titers in systemic compartments but barely elicits mucosal responses such as secretory IgA (SIgA) in the lung.
  • SIgA secretory IgA
  • Both the systemic and mucosal PspA vaccinations induced antigen-specific IgG and provided high levels of protection.
  • Vaccine-induced specific secretory IgA (SIgA) is important for preventing Spn colonization in the nasal cavity, but their role in protection against systemic pneumococcus infection has not been well defined.
  • Intramuscular OMV-PspA immunization induced robust PspA-specific IgG titers in both sera and BALF (FIG. 2C-E) but not SIgA titers in the BALF (data not shown) and offered significant protection against secondary Spn respiratory infection (FIG. 4).
  • passive immunization can provide 80% protection against secondary Spn infection (FIG.5B) while both CD4 + and CD8 + T-cell depletion in OMV-PspA immunized mice loses 80% protection (FIG. 5D).
  • PspA-specific serum IgG and T-cell responses have a synergistic role and are sufficient in protection against a pulmonary/invasive Spn infection.
  • PAMPs pathogen-associated molecular patterns
  • OMV-PspA or OMV-NA vaccination impeded animal weight loss, reduced virus titers in the respiratory tract, and lightened lung damage post-CA04 (H1N1) challenge (FIG. 3).
  • H1N1 lung damage post-CA04
  • HIN1 unspecific antimicrobial response to CA04
  • OMV-PspA immunized mice had significantly high AMs and high levels of protective cytokines, but low neutrophils and harmful cytokines/chemokines in the respiratory tract in comparison to OMV-NA, rPpspA, or PBS immunized mice during secondary Spn infection (FIG. 6 and 7), suggesting that activated or trained AMs may be involved in pulmonary immune regulation.
  • a study has shown that T cells can reciprocally interact with AMs at the mucosal site and this helps in memory development. Therefore, to achieve optimal protection against influenza-mediated secondary bacterial infection, all arms of the innate and adaptive immune system may be required.
  • Cytokine profiling in the BALF of mice post Spn infection showed that OMV- PspA immunization induced significantly increased IFN-y, IL-17A, TNF-a, GM-CSF, and IFN-a, while significantly decreasing IL-12, IL-ip, IL-la, IL-6, G-CSF, KC, MIP, and MCP in comparison to OMV-NA, rPspA, or PBS immunization (FIG. 7E).
  • a significant increase of IFN-y, IL-17A, and TNF-a at 2 DPSI in OMV-PspA immunized mice was associated with protection against infection, which was confirmed by the neutralization of these cytokines (FIG.
  • IFN-y, IL-17A, and TNF-a coordinate innate and adaptive immune responses to counteract the secondary Spn infection.
  • Cytokines such as IFN-y, IL- 17A, and TNF-a, secreted by T-helper (Th) 1 and Thl7 cells play an important role in Spn clearance.
  • IFN-y has been shown to have a detrimental role in non-vaccinated mice subjected to influenza-mediated secondary Spn infection. Mechanically, influenza infection activates IFN-y receptor (IFN-yR) signaling and abrogates the function of AMs which are required to counteract pneumococcal infection.
  • IFN-yR IFN-y receptor
  • IFN-y is important to maximize protection in OMV-PspA immunized mice against coinfection (FIG. 5E, and 7E).
  • the discrepancy may be due to the dysregulated IFN-y production in non-vaccinated mice versus the tightly regulated IFN-y production in OMV- PspA-immunized mice after influenza virus infection.
  • the OMV-PspA immunization may imprint AMs via IFN-y signaling to improve their function for bacterial clearance.
  • IFN-a in the BALF of OMV-PspA immunized mice at 2 DPSI (FIG. 7E).
  • Type I interferons can protect against pneumococcal invasive disease by inhibiting bacterial transmigration across the lungs.
  • the defined source of these cytokines and corresponding mechanisms still needs to be further deciphered.
  • the plasmids pSMV92 and pYA4515 were introduced individually into a recombinant Yptb mutant, YptbS46 (Table 1), carrying among other things an IpxE insertion and a pmrF-J deletion, to determine the synthesis of PspA in bacteria and their OMVs.
  • High amounts of PspA antigen were synthesized in the YptbS46(pSMV92) strain and were enclosed in its OMVs, but not in the YptbS46(pYA4515) strain and its OMVs (FIG. IB).
  • OMVs from YptbS46(pSMV92) and YptbS46(pYA4514) were designated as OMV-PspA and OMV-NA, respectively.
  • the morphology and size of OMVs were examined by transmission electron microscopy (TEM) and dynamic light scattering (DLS).
  • OMVs showed a circular morphology with a bilayer structure and were in the range of 20 to 200 nm (FIG.1C).
  • Mass spectrum analysis showed that exclusive monophosphoryl lipid A species were contained in the OMV-PspA (FIG. 8B).
  • Plasmids pYA3620 Asd + , pBR ori. P-Lactamase signal sequence-based periplasmic bland C- terminal secretion plasmid pYA4515 The 2 nd and 3 rd codons of N terminus of Bia sequence was mutated into AAA AAA (Lys Lys) in pYA3620 pSMV92 Codon-optimized a-helical region of pspA encoding aa residues 3 to 285 was cloned into the EcoRI and / ///dill sites of pYA4515
  • mice immunized with either OMV-PspA or OMV-NA experienced significant weight loss 4 days post-vaccination (DPV), but animals gained weight rapidly (FIG. 2B) without any obvious disease symptoms.
  • Antibody isotyping showed that both anti-PspA IgG2a/IgGl and IgG2b/IgGl ratios in the OMV-PspA-immunized mice were almost equal to one at both 21 and 35 DPV and were significantly higher than the ratios (mean value ⁇ 0.8) in the rPspA- immunized mice (FIG. 2D).
  • bronchoalveolar lavage fluid (BALF) antibody analysis showed that anti-PspA IgG titers (mean 2.9 logic) were only detected in the OMV-PspA-immunized mice on 35 DPV (FIG. 2E). However, no secreted IgA titers were detected in any groups (data not shown). The results indicated that OMV-PspA vaccination is capable of inducing mucosal IgG responses.
  • BALF bronchoalveolar lavage fluid
  • BALF virus titers reached a peak in PBS (mean 5.2 logic pfu/ml) and rPspA (mean 3.7 logic pfu/ml) immunized mice, while slightly decreased in both OMV immunized mice.
  • mice were challenged with 50 pfu of H1N1 CA04, and weight loss was monitored for 9 days.
  • the PBS or rPspA immunized mice had -12% weight loss, whereas the OMV-PspA or OMV-NA immunized mice had -5% weight loss at 9 DPVI (FIG. 4F).
  • mice On 9 DP VI (205 days post initial immunization), groups of mice were subjected to intranasal challenge with 1.5> ⁇ 10 4 cfu of Spn strain D39.
  • Both antibodies and T cells are important for protection against influenza-mediated secondary Spn infection. Both antibody and antigen-specific T-cell responses induced after pneumococcal colonization are protective against subsequent Spn infections in humans and mice. Our results also revealed that OMV-PspA immunization induced potent PspA-specific antibody and T-cell responses (FIG. 2C-F and FIG. 9). Thus, we sought to determine the roles of antibodies and T cells in protection against influenza- mediated secondary Spn infection. The opsonophagocytic killing (OPK) assay can be used to predict the correlation of serum antigen-specific antibodies with vaccine efficacy.
  • OPK opsonophagocytic killing
  • the OPK assay showed that the OPK activity in sera from OMV-NA immunized or PBS mice exhibited a basal level.
  • Sera from OMV-PspA-immunized mice showed significantly higher OPK activity to laboratory-adapted Spn strains D39 and A66.1 and clinical isolates ST552, ST554, and ST556 than sera from rPspA, OMV-NA, or PBS immunized mice, but no OPK activity to clinical isolates ST558 and ST560 (FIG. 5A).
  • the OPK activity in sera from rPspA-immunized mice was moderate to Spn strains D39 and A66.1 but still substantially higher than those from OMV-NA-immunized or PBS mice.
  • OMV- PspA and OMV-NA immunization significantly increased the number of AMs in both the lungs and BALF in comparison to rPspA or PBS immunization.
  • the rPspA immunization slightly increased the number of AMs in the lungs and BALF but without significant difference (FIG. 6C).
  • the co-infection further increased the number of AMs in OMV-PspA immunized mice but retained comparable AM number in the lungs and BALF of other groups of mice (FIG. 6D and E).
  • the neutrophil analysis showed that the number of neutrophils in the lungs and BALF of all groups of mice was comparable at pre-infection (FIG.
  • cytokine/chemokine in the BALF of mice at 2 DPSI were measured, which also reflected lung inflammation and damage during infection.
  • the results showed that the levels of cytokines/chemokines such as IFN-y, TNF-a, IL-17A, GM-CSF, and IFN-a which appeared to be associated with protection were significantly higher in the BALF of OMV-PspA immunized mice than those in PBS, OMV-NA, or rPspA immunized mice (FIG. 7E).
  • cytokines/chemokines such as IL-ip, IL-la, IL-6, IL-12, keratinocyte-derived chemokine (KC), granulocyte stimulating factor (GCSF), and macrophage inflammatory protein 1 (MIP) were substantially higher in PBS, OMV-NA, or rPspA immunized mice than those in OMV-PspA immunized mice (FIG. 7E).
  • OMV-PspA immunization effectively increased activated AMs, restrained massive neutrophil infiltration, and orchestrated the production of cytokines/chemokines in the immunized mice after the secondary Spn infection, facilitating clearing of bacteria and limiting lung damage.
  • the pSMV92 plasmid was verified by PCR using PspA-opt-1 (5’ ccatgggctctccggtagccag 3’)(SEQ ID NO: 2) / PspA-opt-2 (5’aagcttttatgctttcttaagg 3’) primers (SEQ ID NO: 3).
  • OMV isolation and analysis OMVs were isolated from YptbS46 harboring pYA4515 or pSMV92 following our previous description with slight modification. Briefly, strains were grown at 28°C in 1 L LB broth for 24 hr. The bacterial cultures were kept on ice for 1 hr. Then, the bacterial cells were pelleted by centrifugation at 10,000 x g and 4°C for 20 min. The culture supernatant was filtered using a 0.45-pm pore membrane (Millipore) to remove any residual bacteria and cell debris and concentrated with a 100-kDa filter using a Vivaflow 200 system (Sartorius).
  • Millipore 0.45-pm pore membrane
  • OMVs were harvested by ultracentrifugation (120,000 x g) for 2 h at 4°C.
  • the vesicle pellet was resuspended in 150 pL of 0.1 x sterilized PBS (pH 7.4), centrifuged at 5000 rpm for 5 min, filtered through a 0.22-pm pore membrane (Millipore), and stored at 4°C for subsequent experiments. Protein and lipid contents were analyzed as described previously.
  • the size and morphology of the OMVs were characterized using DynaProTM Dynamic Light Scattering (DLS) (Wyatt Technology, Santa Barbara, CA, USA) and negative staining EM using a Tecnai F20 electron microscope (FEI, Hillsboro, OR, USA).
  • DLS DynaProTM Dynamic Light Scattering
  • FEI Tecnai F20 electron microscope
  • TLR4 cell line The OMV stimulation via TLR4 pathway was analyzed on HEK-BlueTM hTLR4 cells (Invivogen, CA, USA) as described previously. The cells were maintained in complete DMEM (supplemented with 10% FBS and 100 pg/mL penicillin, lOOpg/mL streptomycin, and lOOpg/mL Normocin) media in presence of 5% CO2 at 37°C. 5* 10 4 cells were seeded on a 96 well plate and stimulated with 4 pg/mL concentration of OMV (20pL volume) for 8 hr. PBS and rPspA (3pg/mL) were used as positive and negative control. HEK-blue NULL cells were used as experimental controls. Relative NF-KB activity was measured by embryonic alkaline phosphate (SEAP) activity as described in manufacturer’s protocol.
  • SEAP embryonic alkaline phosphate
  • Lipid A isolation and analysis by mass spectrometry The lipid A isolation from OMV-PspA and analysis was the same as described previously.
  • ELISA enzyme-linked immunosorbent assay
  • Opsonophagocytic killing assay The opsonophagocytic killing assay was performed as described previously. Briefly, HL-60 cells (ATCC, CCL-240) were differentiated into granulocyte-like cells in the Iscove's Modified Dulbecco's Medium (IMDM) (ATCC) containing 100 mM N’, N-dimethylformamide (Sigma) for 5 days.
  • IMDM Iscove's Modified Dulbecco's Medium
  • Sera samples from immunized mice containing opsonic antibodies were heat-inactivated (56°C, 30 min) and serially diluted with opsonization buffer (mixture of 80 mL of sterile water, 10 mL of 10 x Hank’s balanced solution, 10 mL of 1% gelatin, and 5.3 mL of fetal bovine serum).
  • opsonization buffer mixture of 80 mL of sterile water, 10 mL of 10 x Hank’s balanced solution, 10 mL of 1% gelatin, and 5.3 mL of fetal bovine serum.
  • Each well in a 96-well plate contained 40 pL of 4 * 10 5 HL60 cells, 10 3 CFUs of different S.

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Abstract

A Yersinia pseudotuberculosis strain (designated as YptbS46) was tailored with an Asd+ plasmid pSMV92 that can synthesize high amounts of the Spn pneumococcal surface protein A (PspA) antigen and monophosphoryl lipid A as an adjuvant. The recombinant strain produced outer membrane vesicles (OMVs) enclosing a high amount of PspA protein (designated as OMV-PspA). A prime-boost intramuscular immunization with 30 pg of OMV- PspA induced both memory adaptive and innate immune responses in co-infected mice, reduced the viral and bacterial burden, and provided complete protection against secondary Spn infection. Also, the OMV-PspA immunization afforded significant cross-protection against the secondary Spn A66.1 infection and long-term protection against the secondary Spn D39 challenge. An OMV vaccine delivering Spn antigens can thus be a new pneumococcal vaccine candidate.

Description

TITLE
A BACTERIAL VESICLE-BASED PNEUMOCOCCAL VACCINE AGAINST INFLUENZA-MEDIATED SECONDARY STREPTOCOCCUS PNEUMONIAE PULMONARY INFECTION
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with government support under Grant Nos. R01 AI162670 and R56 AI146434 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
BACKGROUND
1. FIELD
[0002] The present disclosure relates to vaccines and, more particularly, to a vaccine against Streptococcus pneumoniae infections using Yersina outer membrane vesicles.
2. DESCRIPTION OF THE RELATED ART
[0003] S. pneumoniae (Spn) is a common human nasopharyngeal microbiota, but it is also a leading pathogen that causes many non-invasive and invasive diseases with significant morbidity and mortality, especially in children under five years of age, the elderly, and immunocompromised individuals. An initial influenza virus infection results in increased inflammation, tissue damage, and immune dysregulation in the lungs, which exacerbates pneumonia caused by subsequent Spn superinfection. During seasonal and pandemic influenza, secondary Spn infection is a known fatal complication of influenza, which was documented as early as the 1918 influenza pandemic and in subsequent influenza pandemics, like the 2009 H1N1 pandemic.
[0004] Currently, two types of capsular polysaccharide-based vaccines covering certain Spn serotypes have been approved by FDA. Administration of these vaccines was found to reduce the overall rates of invasive disease and pneumococcal pneumonia in elderly adults by 50-75% but did not afford protection against community-acquired pneumonia. Moreover, new higher-valent PCV15 and PCV20 vaccines expand protection to pneumococcal diseases caused by a few prominent nonvaccine serotypes responsible for Invasive pneumococcal diseases (IPD). However, the introduction of capsular serotype-based pneumococcus vaccines was found to lead to the occurrence of serotype replacement. Over one hundred pneumococcal capsular serotypes have been identified, while polysaccharide- based vaccines only target a small subset of clinical strains. To address the current dilemma, it is imperative to develop new types of serotype-independent pneumococcal vaccines by seeking relatively conserved surface proteins which are common to most or all pneumococcal strains as vaccine formulations.
[0005] Among several leading candidate antigens (Ags), pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Spn and plays critical roles in pneumococcal pathogenesis and virulence. PspA was found to be highly immunogenic and cross-protective in mouse and human studies, even though PspA displayed high variability within three protein families that consist of six clades. To enhance the efficacy of protein vaccines and promote mucosal immune responses, there is emergent interest in the application of bacterial vesicles or other nanoparticles delivering candidate pneumococcal proteins as vaccines. For example, a self-adjuv anting Yersinia pseudotuberculosis (Yptb) outer membrane vesicle (OMV) platform has been developed to provide protection against Yersinia pestis.
[0006] S. pneumoniae (Spn) is a common pathogen causing a secondary bacterial infection following influenza which leads to severe morbidity and mortality during seasonal and pandemic influenza. Therefore, there is an urgent need to develop bacterial vaccines that prevent severe post-influenza bacterial pneumonia. Accordingly, there is a need in the art for an approach that can protect against to deliver heterologous antigens to achieve protection against S. pneumoniae and corresponding respiratory bacterial infections.
BRIEF SUMMARY
[0007] The present invention provides an approach that can deliver heterologous antigens to achieve protection against Spn and other respiratory bacterial infections. The present invention employs an improved Yptb OMV platform to deliver the highly immunogenic a-helical region of PspA (designated as OMV-PspA). The protective efficacy of the platform was evaluated against an H1N1 CA04 infection-mediated secondary Spn infection. The results demonstrated that intramuscular prime-boost immunization with OMV- PspA induced potent humoral and cellular immune responses in systemic and mucosal compartments and afforded significant protection against influenza-mediated secondary Spn infection.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) [0008] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:
[0009] FIG. l is a series of graphs and images showing the construction and analysis of OMVs containing PspA. (A) Construction of pSMV92 plasmid, in which the codon- optimized a-helical region of pspA (3-285 aa) was cloned into pYA4515 plasmid. (B) The analysis of PspA antigen by immunoblotting. The PspA synthesis in the YptbS46 harboring pYA4515, an empty Asd+ plasmid, YptbS46 harboring pSMV92, containing a-helical region of pspA (Left panel); The PspA antigen in 8 pg of OMVs isolated from YptbS46(pSMV92) (designated as OMV-PspA), YptbS46(pYA4515) (designated as OMV-NA), 2 pg of purified recombinant PspA were used as a positive control (Right panel). M, 10 to 250 kDa protein ladder (ThermoFisher Scientific). (C) Transmission electron microscope (TEM) image (Left panel) of OMV-PspA and Dynamic Light Scattering (DLS) (Left panel) of OMV-PspA. (D) Comparison of embryonic alkaline phosphate (SEAP) activities in HEK-blue cells with or without murine toll-like receptor 4 (mTLR4) (Invitrogen). HEK-blue mTLR4 cells were cocultured with 40 pg/mL of OMV-PspA for 8 hours. OMVs from YptbS44 strain and rPspA were used as controls. Data are shown as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; **** P<0.0001.
[0010] FIG. 2 is a series of graphs of the immune responses induced by i.m. OMV immunization. Swiss Webster mice (n=10/group, equal males and females, 7-weeks old) were immunized i.m. with 30 pg of OMV-PspA, OMV-NA, 3 pg of rPspA/ Alhydrogel or Alhydrogel alone in 50pL of PBS and boosted on day 22 after priming. (A) Immunization schema for mouse study. (B) Weight change rates of mice after immunization. (C) Total serum IgG titers to PspA in Swiss Webster mice on 21 and 35 days post-vaccination (DPV). (D) The ratio of IgG2a/IgGl and IgG2b/IgGl for antibodies specific to PspA antigen on 21 and 35 DPV. (E) Anti-PspA IgG titers in the BALFs of mice immunized with OMV-PspA, OMV-NA, rPspA, or PBS. (F) Lung PspA-specific T-cell responses in immunized mice. On 45 DPV, total CD4+ and CD8+ T-cell subsets and their specific cytokine (IFN-y, TNF-a, IL- 17A) were stimulated by rPspA antigen. Each symbol was obtained from an individual mouse, and data were represented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; * P<0.05,' ** P<0.0P, *** P<0.00P, **** P<0.0001.
[0011] FIG. 3 is a series of graphs showing that immunized mice were challenged with a sublethal dose of an influenza virus. Swiss Webster mice (n=10/group, equal males and females, 7-weeks old) were i.m. immunized with OMV-PspA, OMV-NA, rPspA, or PBS as described above. On 36 PDV, animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain. (A) The schema for this study. (B) Weight change rates of immunized mice after CA04 infection. Virus titers in the BALF (C) and lung (D) of mice at 2, 4, and 8 days post viral infection (DPVI) were quantified using plaque assay described in Materials and Methods. (E) Lung histopathological analysis of representative mice from each group on 4 DPVI. The lungs were microscopically examined and imaged using Nanozoomer 2.0 RS Hamamatsu slide scanner (scale bar, 100 nm). Each symbol was obtained from an individual mouse, and data were represented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; * <0.05; ** <0.01; *** P<0.001; **** P<0.0001.
[0012] FIG. 4 is a series of graphs of the short-term and long-term protective efficacy of OMV-PspA immunization against influenza-mediated secondary Spn infection. Swiss Webster mice (n=10/group, equal numbers of males and females, 7-weeks old) were immunized intramuscularly with OMV-PspA, OMV-NA, rPspA/Alhydrogel, or PBS/Alhydrogel alone (negative control) and then boosted on day 22 after the priming immunization as described above. On 36 PDV, animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain and monitored for 9 days. (A) On 9 DPVI, animals were intranasally challenged with 1.5 x 104 CFU of Spn strain D39. (B) The bacterial burden was evaluated in the lungs, blood, and spleen. The experiments were performed twice, and the data were combined for analysis. (C) Lung histopathological analysis of representative mice from each group on 2 days post Spn infection (DPSI). The lungs were microscopically examined and imaged using Nanozoomer 2.0 RS Hamamatsu slide scanner (scale bar, 100 nm). (D) On 9 DPVI, animals were intranasally challenged with 1 * 103 CFU of Spn strain A66.1. (E) The schema used for long-term study. Groups of Swiss Webster mice (n=5-10 females, 7-weeks old) were i.m. immunized with OMV-PspA, OMV-NA, rPspA, or PBS. Bloods were collected at 60, 90, 120, 150 and 180 DPV for measuring serum anti-PspA IgG titers. (F) Weight change rates of immunized mice after CA04 infection. On 196 PDV, animals were challenged intranasally with 50 pfu of H1N1 (CA04) strain. (G) Long-term protection against secondary Spn infection. On 9 DPVI (205 DPV), animals were intranasally challenged with 1.5 x 104 CFU of Spn strain D39. The mortality and morbidity of animals were all monitored for 15 days. Statistical significance was analyzed by the log-rank (mantel Cox) test for survival analysis. Data were analyzed and presented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05 **, P< 0.0P, ****, P< 0.000L [0013] FIG. 5 is a series of graphs of the roles of antibody and T cells in protection against influenza-mediated secondary Spn infection. (A) Comparative analysis of opsonophagocytic killing assay against Spn strains D39 and A66.1 using antisera collected from immunized mice on 35 DPV. Data obtained from two experiments were pooled, analyzed, and presented as the mean ± SD. (B) Serum transfer. (Left) a schema for serum transfer and (Right) survival of naive Swiss Webster mice (n=5/group, females, 7-weeks old) which were received corresponding sera and subjected to the co-infection. Naive mice were i.p. injected with 100 pl of serum collected from PBS-, rPspA-, 0MV-NA-, and OMV-PspA- immunized mice on 35 DPV, respectively. Twenty-four hours postinjection, the recipient mice were intranasally challenged with 50 pfu of H1N1 (CA04) and then challenged with 1.5x 104 cfu of Spn strain D39 on 9 DPVI. (C) The schema of T-cell depletion and cytokine neutralization. OMV-PspA immunized Swiss Webster mice (n=5/group, females) were intranasally challenged with 50 pfu of H1N1 (CA04). On 8 DPVI, mice were i.p. administrated either with anti-CD4, anti-CD8, or anti-CD4 plus anti-CD8 mAbs (200pg/each mouse in 200pL) at a two-day interval for the depletion of CD4+ and/or CD8+ T cells, or anti- TNF-a, anti-IFN-y, and/or anti-IL-17A (200pg/each mouse in 200pL) at a two-day interval for the neutralization of these cytokines. Mice were injected with the isotype control mAbs as controls. (D) Protection against secondary Spn infection in T-cell depleted mice. (E) Protection against secondary Spn infection in cytokine-neutralized mice. On 8 days post CA04 infection, antibody -treated mice were then intranasally challenged with 1.5* 104 cfu of Spn strain D39. The mortality and morbidity of animals were all monitored for 15 days. Statistical significance was analyzed by the log-rank (mantel Cox) test for survival analysis. Data were analyzed and presented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05 **, P< 0.0 , ****, P< 0.000P
[0014] FIG. 6 is a series of a series of graphs of the patterns of alveolar macrophages (AMs) in the BALF and lung of immunized mice with or without infection. Swiss Webster mice (n=5 females) were immunized with OMV-PspA, OMV-NA, rPspA, or PBS as described above. Single cells were collected from the lung and BALF samples of these mice on 35 DPV before the CA04 challenge and on 2 DPSI and stained with fluorochrome-labeled molecular markers for characterizing AMs using flow cytometry. (A) Gating strategy for AMs and neutrophils. (B) Representative flow plot showing the percentage of AMs without infection. (C) Quantitative analysis of the number of AMs in the lung (per lung) and BALF (per mL) without infection. (D) Representative flow plot showing the percentage of AMs at 2 DPSI. (C) Quantitative analysis of the number of AMs in the lung (per lung) and BALF (per mL) at 2 DPSI. Data were analyzed and presented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05 **, P< 0.0P, ****, P< 0.000L
[0015] FIG. 7 is a series of graphs of the patterns of neutrophils in the BALF and lung of immunized mice with or without infection and cytokine profiles in the BALF of mice subjected to co-infection. Swiss Webster mice (n=5 females) were immunized with OMV- PspA, OMV-NA, rPspA, or PBS as described above. Single cells were collected from the lung and BALF samples of these mice on 35 DPV before the CA04 challenge and on 2 days post Spn infection and stained with fluorochrome-labeled molecular markers for characterizing neutrophils using flow cytometry. (A) Representative flow plot showing the percentage of neutrophils without infection. (B) Quantitative analysis of the number of neutrophils in the lung (per lung) and BALF (per mL) without infection. (C) Representative flow plot showing the percentage of neutrophils at 2 DPSI. (D) Quantitative analysis of the number of neutrophils in the lung (per lung) and BALF (per mL) at 2 DPSI. (E) Cytokine profiles in the BALF of mice secondarily infected Spn. Cytokines/chemokines in the BALF samples were measured using the Bio-Plex Pro Mouse Cytokine 23-plex Assay kit (Bio-rad). Data were analyzed and presented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05.: **, P< 0.0 , ****, P< 0.0001.
[0016] FIG. 8 is the nucleic acid sequence of PspA antigen and lipid A analysis of OMV-PspA (SEQ ID NO: 1). (A) Codon optimized a-helical region of pspA encoding aa residues 3 to 285 (849 bp; 283 aa) of the mature PspA from Spn strain D39. (B) Mass spectrum analysis of lipid A species in OMV-PspA.
[0017] FIG. 9 is a series of graphs of antibody titers in the BALF, T-cell gating, and spleen T-cell responses. (A) Gating Strategy for T-cell responses for both post vaccination and infection. (B) Representative flow plots for CD4-specific cytokines (IFN-y, TNF-a, and IL- 17 A) in the immunized mice post vaccination. (C) Representative flow plots for CD8- specifc cytokines (IFN-y, TNF-a, and IL-17A) in the immunized mice post vaccination. (D) Spleen PspA-specific T-cell responses in immunized mice. The detailed procedures were described in Materials and Methods. Each symbol was obtained from an individual mouse, and data were represented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05 **, P< 0.01; ****, P< 0.0001.
[0018] FIG. 10 is a series of graphs of an opsonophagocytic killing assay, lung T-cell responses in immunized mice secondarily infected with Spn, and the role of IFN-y in protection against influenza-mediated secondary Spn infection. (A) Comparative analysis of opsonophagocytic killing assay against clinical Spn isolates (ST552, ST554, ST556, ST558, ST560) using sera from different immunized mice. (B) Lung T-cell responses in immunized mice secondarily infected with Spn. On 2 DPSI, total CD4+ and CD8+ T-cell subsets and their specific cytokine (IFN-y, TNF-a, IL-17A) were measured in the lungs of immunized mice. (C) The schema of immunization. C57/BL6 and IFN-y ' mice (n=5, mixed gender) were intramascularly immunized with 20 pg of OMV-PspA, OMV-NA, or 3 pg of rPspA. Mice administered PBS were used as a negative control group. (D) Weight change rates of immunized mice after CA04 infection. On 36 DPV, animals were challenged intranasally with 15 pfu of H1N1 (CA04) strain. (E) Protection against secondary Spn infection. On 9 DPVI (45 DPV), animals were intranasally challenged with 1.5 x 104 CFU of Spn strain D39. The mortality and morbidity of animals were monitored for 15 days. Statistical significance was analyzed by the log-rank (mantel Cox) test for survival analysis. Data were analyzed and presented as the mean ± SD. The statistical significance of difference among groups was analyzed by two-way ANOVA with Turkey post hoc test: ns, no significance; *, P< 0.05 **, P< 0.0P, ****, < 0.0001.
DETAILED DESCRIPTION
[0019] Referring to the figures, wherein like numerals refer to like parts throughout, there is seen in FIG. 1, a platform for providing OMV-PspA vaccination that induces both memory adaptive and innate immune responses and provides significant protection against influenza-mediated secondary Spn infection in a mouse model. The data strongly suggest that an OMV vaccine delivering Spn antigens can be a promising vaccine candidate for counteracting co-infection.
[0020] Bacterial OMVs composed of immunogenic membranes and specific antigens stimulate humoral and cellular immune responses, which are required for protection against Spn lung infection. OMV-PspA immunization according to the present invention elicits potent humoral and cellular immune responses (FIG. 2C-F and FIG. 9), both of which are required for optimal protection against influenza-mediated secondary Spn infection (FIG. 5). Intranasal immunization has been shown to induce antigen-specific IgG and IgA isotypes in both systemic and lung compartments, while intramuscular immunization generally induces robust IgG titers in systemic compartments but barely elicits mucosal responses such as secretory IgA (SIgA) in the lung. Both the systemic and mucosal PspA vaccinations induced antigen-specific IgG and provided high levels of protection. Vaccine-induced specific secretory IgA (SIgA) is important for preventing Spn colonization in the nasal cavity, but their role in protection against systemic pneumococcus infection has not been well defined. Intramuscular OMV-PspA immunization induced robust PspA-specific IgG titers in both sera and BALF (FIG. 2C-E) but not SIgA titers in the BALF (data not shown) and offered significant protection against secondary Spn respiratory infection (FIG. 4). In addition, passive immunization can provide 80% protection against secondary Spn infection (FIG.5B) while both CD4+ and CD8+ T-cell depletion in OMV-PspA immunized mice loses 80% protection (FIG. 5D). Thus, these results suggest that PspA-specific serum IgG and T-cell responses have a synergistic role and are sufficient in protection against a pulmonary/invasive Spn infection.
[0021] OMVs possess pathogen-associated molecular patterns (PAMPs) from bacteria that can strongly activate the innate immune response. OMV-PspA or OMV-NA vaccination impeded animal weight loss, reduced virus titers in the respiratory tract, and lightened lung damage post-CA04 (H1N1) challenge (FIG. 3). These results imply that the innate immune memory, i.e., trained immunity, is activated by OMV immunization, resulting in possible functional reprogramming of innate immune cells and mounting an unspecific antimicrobial response to CA04 (HIN1) infection, which appears to correlate with the increased number of AMs due to OMV immunization (FIGS. 6 B-C). However, complete protection against secondary Spn D39 infection was only achieved by OMV-PspA vaccination, whereas rPspA and OMV-NA vaccination provided low and bare protection, respectively (FIG. 4A), suggesting that PspA-specific adaptive immunity to Spn is more important for containing secondary Spn infection than trained immunity. Previous studies have shown that activated or trained AMs establish intercellular signaling pathways in the lung microenvironment to effectively clear pathogens and orchestrate acute responses to lung inflammation, injury, and repair. Here, OMV-PspA immunized mice had significantly high AMs and high levels of protective cytokines, but low neutrophils and harmful cytokines/chemokines in the respiratory tract in comparison to OMV-NA, rPpspA, or PBS immunized mice during secondary Spn infection (FIG. 6 and 7), suggesting that activated or trained AMs may be involved in pulmonary immune regulation. A study has shown that T cells can reciprocally interact with AMs at the mucosal site and this helps in memory development. Therefore, to achieve optimal protection against influenza-mediated secondary bacterial infection, all arms of the innate and adaptive immune system may be required. In support of this, the data revealed a role for antibodies, T cells, and corresponding cytokines in protection against co-infection (FIG. 2, 4, and 5). Further studies are needed to delineate the precise mechanisms by which AMs coordinate adaptive immune responses in immunized mice before and after infection to establish a robust immunity against viral-bacterial coinfection.
[0022] Cytokine profiling in the BALF of mice post Spn infection showed that OMV- PspA immunization induced significantly increased IFN-y, IL-17A, TNF-a, GM-CSF, and IFN-a, while significantly decreasing IL-12, IL-ip, IL-la, IL-6, G-CSF, KC, MIP, and MCP in comparison to OMV-NA, rPspA, or PBS immunization (FIG. 7E). A significant increase of IFN-y, IL-17A, and TNF-a at 2 DPSI in OMV-PspA immunized mice was associated with protection against infection, which was confirmed by the neutralization of these cytokines (FIG. 5E). Our data suggest that IFN-y, IL-17A, and TNF-a coordinate innate and adaptive immune responses to counteract the secondary Spn infection. Cytokines, such as IFN-y, IL- 17A, and TNF-a, secreted by T-helper (Th) 1 and Thl7 cells play an important role in Spn clearance. Interestingly, IFN-y has been shown to have a detrimental role in non-vaccinated mice subjected to influenza-mediated secondary Spn infection. Mechanically, influenza infection activates IFN-y receptor (IFN-yR) signaling and abrogates the function of AMs which are required to counteract pneumococcal infection. However, the results demonstrated that IFN-y is important to maximize protection in OMV-PspA immunized mice against coinfection (FIG. 5E, and 7E). The discrepancy may be due to the dysregulated IFN-y production in non-vaccinated mice versus the tightly regulated IFN-y production in OMV- PspA-immunized mice after influenza virus infection. In addition, the OMV-PspA immunization may imprint AMs via IFN-y signaling to improve their function for bacterial clearance. Moreover, we also observed substantially increased levels of IFN-a in the BALF of OMV-PspA immunized mice at 2 DPSI (FIG. 7E). Type I interferons can protect against pneumococcal invasive disease by inhibiting bacterial transmigration across the lungs. However, the defined source of these cytokines and corresponding mechanisms still needs to be further deciphered.
[0023] Although the antigenic/genetic diversity of PspA is present among prevalent serotypes, PspA vaccines are cross-protective in animal models against multiple serotypes causing carriage and invasive diseases. Consistent with these reports, it was showed that OMV-PspA vaccination also affords significant cross-reaction in in vitro OPK-killing assays (FIG. 5A) and cross-protection in vivo survival evaluation (FIG. 4A and D). A previous study showed that a PspA fusion combining the a-helical domain of PspA from Rxl (family 1) with the proline-rich and a-helical domains of PspA from EF5668 (family 4) delivered by a recombinant attenuated Salmonella vaccine (RASV) elicited significantly greater protection than PspA/Rxl or PspA/EF5668 against multiple Spn strains. Therefore, delivering a similar PspA hybrid by the current or improved OMVs in future studies may enhance long-term protection and cross-protection against secondary Spn infection. In addition, future studies may further evaluate whether the OMV vaccine can booster efficacy of PC Vs in mice against primary and secondary Spn infection.
[0024] EXAMPLE
[0025] Delivery of pneumococcal PspA antigen using the Yptb OMV platform. Codon-optimized a-helical region of pspA encoding aa residues 3 to 285 (849 bp; 283 aa) of the mature PspA from Spn D39 strain (FIG. 8A) was cloned into an empty vector, pYA4515, to generate the pSMV92 plasmid (FIG. 1 A and Table 1). The plasmids pSMV92 and pYA4515 were introduced individually into a recombinant Yptb mutant, YptbS46 (Table 1), carrying among other things an IpxE insertion and a pmrF-J deletion, to determine the synthesis of PspA in bacteria and their OMVs. High amounts of PspA antigen were synthesized in the YptbS46(pSMV92) strain and were enclosed in its OMVs, but not in the YptbS46(pYA4515) strain and its OMVs (FIG. IB). OMVs from YptbS46(pSMV92) and YptbS46(pYA4514) were designated as OMV-PspA and OMV-NA, respectively. The morphology and size of OMVs were examined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). OMVs showed a circular morphology with a bilayer structure and were in the range of 20 to 200 nm (FIG.1C). Mass spectrum analysis showed that exclusive monophosphoryl lipid A species were contained in the OMV-PspA (FIG. 8B).
Table 1 - Strains and plasmids
Strain or Plasmid Genotype or relevant characteristics
Strains
E. coli /6212 F- - </>80 E(lacZYA-argF) endAl recAl hsdR17 deoR thi-1 glnV44 gyrA96 relAl EasdA4
I. pseudotuberculosis
YptbS44 EtolR Easd ElacI iP\vv IpxE EhmsHFRS pYV ElacZ .caflR- caflM-caflA-cafl
YptbS46 EtolR Easd ElacI. AR IpxE EhmsHFRS pYV ElacZ .caflR- caflM-caflA-cafl EpmrF-J
S. pneumoniae
S. pneumoniae D39 Wild-type virulent, encapsulated serotype 2
S. pneumoniae A66.1 Wild-type virulent, encapsulated serotype 3
S. pneumoniae ST552 Clinical isolate, Serotype 14 S. pneumoniae ST554 Clinical isolate, Serotype 23F
S. pneumoniae ST556 Clinical isolate, Serotype 19F
S. pneumoniae ST558 Clinical isolate, Serotype 6B
S. pneumoniae ST560 Clinical isolate, Serotype 9V Influenza virus H1N1 A/California/4/2009 (CA04) virus
Plasmids pYA3620 Asd+, pBR ori. P-Lactamase signal sequence-based periplasmic bland C- terminal secretion plasmid pYA4515 The 2nd and 3rd codons of N terminus of Bia sequence was mutated into AAA AAA (Lys Lys) in pYA3620 pSMV92 Codon-optimized a-helical region of pspA encoding aa residues 3 to 285 was cloned into the EcoRI and / ///dill sites of pYA4515
Figure imgf000012_0001
[0026] Furthermore, the secreted embryonic alkaline phosphatase (SEAP) activity of HEK-blue mTLR4 cells cultured with different OMVs was compared. The results showed that the TLR4 stimulatory activity of OMV-PspA was significantly less than that of OMVs isolated from YptbS44 constructed in previous work, but substantially higher than that of purified PspA protein (FIG. ID). No SEAP activity was detected in HEK-blue null cells with the same stimulation (FIG. ID). Results indicated that the monophosphoryl lipid A decorated OMVs from recombinant YptbS46 strains displayed decreased TLR4 activation.
[0027] Immune responses induced by the OMV-PspA immunization. Groups of Swiss Webster mice (n=10/group, equal males and females) were intramuscularly immunized with 30 pg of OMV-PspA, 30 pg of OMV-NA, or 3 pg of rPspA/Alhydrogel in 50 pL of PBS. An equal volume of I xphosphate-buffered saline (PBS) injection was used as a mock, and then boosted on day 22 after priming immunization (Fig 2A). In comparison to the rPspA and PBS groups, mice immunized with either OMV-PspA or OMV-NA experienced significant weight loss 4 days post-vaccination (DPV), but animals gained weight rapidly (FIG. 2B) without any obvious disease symptoms.
[0028] Antibody analysis indicated that high serum total IgG titers of anti-PspA were induced in both rPspA and OMV-PspA immunized mice at 21 DPV and were further increased after booster at 35 DPV. However, serum anti-PspA IgG titers in OMV-PspA immunized mice were substantially higher than those in rPspA immunized mice at both 21 and 35 DPV (FIG. 2C). No anti-PspA titers were detected in OMV-NA immunized or PBS- injected mice (FIG. 2C). Antibody isotyping showed that both anti-PspA IgG2a/IgGl and IgG2b/IgGl ratios in the OMV-PspA-immunized mice were almost equal to one at both 21 and 35 DPV and were significantly higher than the ratios (mean value ~0.8) in the rPspA- immunized mice (FIG. 2D). The results indicated that the OMV-PspA immunization induced balanced Thl and Th2 responses, whereas the rPspA immunization induced a Th2 -biased response. In addition, bronchoalveolar lavage fluid (BALF) antibody analysis showed that anti-PspA IgG titers (mean 2.9 logic) were only detected in the OMV-PspA-immunized mice on 35 DPV (FIG. 2E). However, no secreted IgA titers were detected in any groups (data not shown). The results indicated that OMV-PspA vaccination is capable of inducing mucosal IgG responses.
[0029] To determine antigen-specific T-cell responses, single lung cells isolated on day 45 from immunized mice were stimulated in vitro with rPspA for 48 hr, stained with indicated antibodies, and analyzed using flow cytometry as per the gating strategy mentioned (FIG. 9A, B, and C).
Table 2 - Flow cytometry antibodies
Figure imgf000013_0001
[0030]
Quantitative plots showed that the number of both PspA-specific lung CD4+ and CD8+ T cells from OMV-PspA immunized mice was significantly higher than that of rPspA, OMV- NA, or PBS immunized mice (FIG. 2F). Similar profiles were seen in the number of lung CD4+ and CD8+ T cells producing interferon-gamma (IFN-y), tumor necrosis factor (TNF-a), or interleukin 17A (IL-17A) (FIG. 2F). In addition, patterns of PspA-specific T cells in the spleen were similar to those in the lung (FIG. 9D). Our data thus indicate that OMV-PspA is highly immunogenic, and the induced antibody and T-cell responses may provide robust protection against pneumococcal pneumonia.
[0031] Evaluation of protection in OMV-immunized mice against influenza- mediated secondary Spn infection. A mouse model of influenza-mediated secondary Spn infection has successfully recapitulated clinical observations and been used for vaccine evaluation. Similar to the published mouse models of viral-bacterial co-infection, the immunized mice were challenged with 50 pfu of CA04 (H1N1) on 36 DPV and monitored animal weight loss, virus load, and lung histopathology in 8 days (FIG. 3 A). The viral infection caused significant weight loss in rPspA immunized (maximal 8% loss) or PBS (maximal 10% loss) mice (FIG. 3B). However, significant weight loss was not observed in OMV-PspA or OMV-NA immunized mice. Further, viral burden showed that BALF virus titers in PBS (mean 4.3 logic pfu/ml) and rPspA (mean 3.1 logic pfu/ml) immunized mice were substantially higher than those in OMV-PspA (mean 1.1 logic pfu/ml) and OMV-NA (mean 1.0 logic pfu/ml) immunized mice at 2 days post viral infection (DP VI) (FIG. 3C). At 4 DPVI, BALF virus titers reached a peak in PBS (mean 5.2 logic pfu/ml) and rPspA (mean 3.7 logic pfu/ml) immunized mice, while slightly decreased in both OMV immunized mice. At 8 DPVI, BALF virus titers largely dropped in all groups of mice, but virus titers in PBS (mean 2.0 logic pfu/ml) and rPspA (mean 1.4 logic pfu/ml) immunized mice remained substantially higher than those in OMV-PspA (mean 0.54 logic pfu/ml) and OMV-NA (mean 0.44 logic pfu/ml) immunized mice (FIG. 3C). Similar patterns were observed in the corresponding lungs (FIG. 3D). Histopathological analysis showed that mild immune cell infiltration and inflammation were observed in the lungs of both OMV-PspA and OMV-NA immunized mice at 4 DPVI, whereas large amounts of immune cell infiltration, edema, and congestion were observed in the lungs of the PBS or rPspA immunized mice (FIG. 3E). Results imply that OMV vaccination can provide non-specific protection against influenza viral infection.
[0032] Afterwards, the influenza virus-infected mice were then intranasally challenged with 1.5 * 104 cfu of the Spn strain D39 on 9 DPVI (i.e. 45 DPV) (FIG.3 A). Results showed that OMV-PspA, OMV-NA, or rPspA immunized mice had 100%, 20%, and 40% survival, respectively, and PBS mice succumbed to the same challenge within 3 days (FIG. 4A). On 2 days post Spn infection (DPSI), the lungs of PBS mice had the highest bacterial titers (mean value: 6.0 logic CFU/g tissue). Spn rapidly disseminated into the blood (mean value: 6.4 logic CFU/g tissue) and spleen (mean 4.2 value: logic CFU/g tissue) at significant bacterial titers (FIG. 4B). In rPspA or OMV-NA vaccinated mice, bacterial titers in the lung, blood, and spleen were moderate but remained substantially higher than those in OMV-PspA vaccinated mice. Bacteria were almost completely cleared in the organs of OMV-PspA vaccinated mice at 2 DPSI (FIG. 4B). Lung histopathology revealed huge amounts of immune cell influx and severe tissue damage in the PBS and OMV-NA immunized mice and high levels of lung damage in the rPspA immunized mice, whereas significantly low levels of lung damage in the OMV-PspA immunized mice at 2 DPSI (FIG. 4C). Furthermore, the OMV-PspA immunization afforded 90% protection against secondary Spn strain A66.1 (serotype 3) infection post influenza (FIG. 4D). This shows that OMV-PspA mediated protection is not Spn serotype-specific and that it has potential to provide wider protection against different Spn serotypes.
[0033] To evaluate the long-term protection, a prime-boost immunization regimen was conducted, and blood was collected from mice on 60, 90, 120, 150, and 180 DPV to monitor antibody titer variations (FIG. 4E up). High anti-PspA IgG titers were comparable between OMV-PspA and rPspA immunized mice at 60 and 90 DPV, which peaked at 90 DPV and retained up to 180 DPV. The antibody titers in the OMV-PspA-immunized mice started to significantly decline at 150 DPV, whereas in the rPspA-immunized mice started to decrease at 120 DPV. Beyond 120 DPV, the anti-PspA IgG titers in the rPspA-immunized mice were significantly lower than those in the OMV-PspA-immunized mice (FIG. 4E dawn). No anti-PspA titers were detected in PBS and OMV-NA mice as expected (FIG. 4E dawn). On 196 DPV, mice were challenged with 50 pfu of H1N1 CA04, and weight loss was monitored for 9 days. The PBS or rPspA immunized mice had -12% weight loss, whereas the OMV-PspA or OMV-NA immunized mice had -5% weight loss at 9 DPVI (FIG. 4F). On 9 DP VI (205 days post initial immunization), groups of mice were subjected to intranasal challenge with 1.5>< 104 cfu of Spn strain D39. The OMV-PspA immunization afforded 80% protection against the secondary Spn challenge, while the rPspA immunization only provided 20% protection, and neither the OMV-NA immunization nor PBS offer any protection against the same challenge (FIG. 4G). Therefore, results demonstrated that OMV-PspA was highly immunogenic and the OMV-PspA immunization induced long-term protection against the secondary Spn infection.
[0034] Both antibodies and T cells are important for protection against influenza-mediated secondary Spn infection. Both antibody and antigen-specific T-cell responses induced after pneumococcal colonization are protective against subsequent Spn infections in humans and mice. Our results also revealed that OMV-PspA immunization induced potent PspA-specific antibody and T-cell responses (FIG. 2C-F and FIG. 9). Thus, we sought to determine the roles of antibodies and T cells in protection against influenza- mediated secondary Spn infection. The opsonophagocytic killing (OPK) assay can be used to predict the correlation of serum antigen-specific antibodies with vaccine efficacy. Here, the OPK assay showed that the OPK activity in sera from OMV-NA immunized or PBS mice exhibited a basal level. Sera from OMV-PspA-immunized mice showed significantly higher OPK activity to laboratory-adapted Spn strains D39 and A66.1 and clinical isolates ST552, ST554, and ST556 than sera from rPspA, OMV-NA, or PBS immunized mice, but no OPK activity to clinical isolates ST558 and ST560 (FIG. 5A). The OPK activity in sera from rPspA-immunized mice was moderate to Spn strains D39 and A66.1 but still substantially higher than those from OMV-NA-immunized or PBS mice. No OPK activity was seen in sera from rPspA, OMV-NA, or PBS-immunized mice to all clinic Spn strains (FIG. 5A). To further determine whether antibodies could mediate the protection against the co-infection in vivo, immune sera collected from mice were adoptively transferred to naive Swiss Webster mice (FIG.5B). Intraperitoneal (IP) injection with 100 pl of sera from OMV-PspA and rPspA immunized mice provided 80% and 40% protection against the co-infection, respectively, whereas administration with 100 pl of sera from OMV-NA-immunized or PBS mice failed to offer any protection (FIG. 5B).
[0035] Upon the secondary Spn challenge, the number of both lung CD4+ and CD8+ T cells and cells producing IFN-y, TNF-a, or IL-17A in the OMN-PspA-immunized mice was substantially higher than that in the rPspA, OMV-NA, or PBS immunized mice (FIG. 10B). To further determine the roles of CD4+, CD8+ T cells, and corresponding cytokines in protection, CD4+ T cells, CD8+ T cells, or both were depleted by IP injection with corresponding monoclonal antibodies (mAb) (FIG. 5 C). Results revealed that OMV- PspA-immunized mice depleted of CD4+ T cells only had 40% survival, depleted of CD8+ T cells had 60% survival, and depleted of both T cells had 20% survival (FIG. 5D). In addition, individual neutralization of IFN-y or TNF-a reduced protection against co-infection to 60%, individual neutralization of IL-17A, double neutralization of TNF-a and IFN-y, or double neutralization of TNF-a and IL-17A reduced protection to 40%, and double neutralization of IFN-y and IL-17A reduced protection to 20% (FIG. 5E), while OMV-PspA-immunized mice injected with the isotype control completely survived the co-infection (FIG. 5D and E). Moreover, the OMV-PspA immunization only conferred 40% protection in IFN-y KO mice against the co-infection (FIG. IOC, D, and E). Thus, the results highlighted that antibodies, T cells, and corresponding cytokines (IL-17A, IFN-y, or TNF-a) played synergistic roles in protection against influenza-mediated secondary Spn infection.
[0036] Alterations of lung alveolar macrophages and neutrophils in immunized mice pre- and post-secondary Spn infections. Both alveolar macrophages (AMs) and neutrophils are required to effectively control pulmonary Spn superinfection. An effective coordination between AMs and neutrophils can rapidly clear bacteria from the lower airway and orchestrate lung homeostasis, whereas dysregulation of AMs and neutrophils contributes to the severity of Spn infection. The number of AMs and neutrophils in the lungs of immunized mice pre- and post-infection were therefor examined as per a gating strategy shown in FIG. 6A. Based on the flow plots (FIG. 6B) and quantitative plots (FIG. 6C), OMV- PspA and OMV-NA immunization significantly increased the number of AMs in both the lungs and BALF in comparison to rPspA or PBS immunization. In comparison to PBS, the rPspA immunization slightly increased the number of AMs in the lungs and BALF but without significant difference (FIG. 6C). Compared with pre-infection, the co-infection further increased the number of AMs in OMV-PspA immunized mice but retained comparable AM number in the lungs and BALF of other groups of mice (FIG. 6D and E). However, the neutrophil analysis showed that the number of neutrophils in the lungs and BALF of all groups of mice was comparable at pre-infection (FIG. 7A and B). Compared with pre-infection, the co-infection drastically increased the number of neutrophils in the lungs and BALF in PBS, rPspA, or OMV-NA immunized mice, but retained a comparable number in the lungs and BALF of OMV-PspA immunized mice (FIG. 7C and D).
[0037] Further, the levels of cytokine/chemokine in the BALF of mice at 2 DPSI were measured, which also reflected lung inflammation and damage during infection. The results showed that the levels of cytokines/chemokines such as IFN-y, TNF-a, IL-17A, GM-CSF, and IFN-a which appeared to be associated with protection were significantly higher in the BALF of OMV-PspA immunized mice than those in PBS, OMV-NA, or rPspA immunized mice (FIG. 7E). In contrast, the levels of cytokines/chemokines such as IL-ip, IL-la, IL-6, IL-12, keratinocyte-derived chemokine (KC), granulocyte stimulating factor (GCSF), and macrophage inflammatory protein 1 (MIP) were substantially higher in PBS, OMV-NA, or rPspA immunized mice than those in OMV-PspA immunized mice (FIG. 7E). The results revealed that OMV-PspA immunization effectively increased activated AMs, restrained massive neutrophil infiltration, and orchestrated the production of cytokines/chemokines in the immunized mice after the secondary Spn infection, facilitating clearing of bacteria and limiting lung damage.
[0038] Materials and Methods:
[0039] Plasmid construction. The codon-optimized a-helical region of pspA encoding aa residues 3 to 285 (849 bp; 283 aa) of the mature PspA from S. pneumoniae D39 strain (FIG. 8) was synthesized by Gene Universal Inc. (NJ, USA). The codon-optimized a- helical region of pspA was excised and cloned into EcoRI and Hindlll sites of an empty vector, pYA4515, to generate pSMV92 plasmid. The pSMV92 plasmid was verified by PCR using PspA-opt-1 (5’ ccatgggctctccggtagccag 3’)(SEQ ID NO: 2) / PspA-opt-2 (5’aagcttttatgctttcttaagg 3’) primers (SEQ ID NO: 3).
[0040] OMV isolation and analysis. OMVs were isolated from YptbS46 harboring pYA4515 or pSMV92 following our previous description with slight modification. Briefly, strains were grown at 28°C in 1 L LB broth for 24 hr. The bacterial cultures were kept on ice for 1 hr. Then, the bacterial cells were pelleted by centrifugation at 10,000 x g and 4°C for 20 min. The culture supernatant was filtered using a 0.45-pm pore membrane (Millipore) to remove any residual bacteria and cell debris and concentrated with a 100-kDa filter using a Vivaflow 200 system (Sartorius). OMVs were harvested by ultracentrifugation (120,000 x g) for 2 h at 4°C. The vesicle pellet was resuspended in 150 pL of 0.1 x sterilized PBS (pH 7.4), centrifuged at 5000 rpm for 5 min, filtered through a 0.22-pm pore membrane (Millipore), and stored at 4°C for subsequent experiments. Protein and lipid contents were analyzed as described previously. The size and morphology of the OMVs were characterized using DynaPro™ Dynamic Light Scattering (DLS) (Wyatt Technology, Santa Barbara, CA, USA) and negative staining EM using a Tecnai F20 electron microscope (FEI, Hillsboro, OR, USA). For negative staining EM, 3 pL of the samples were applied to carbon-coated copper EM grids previously glow-discharged in a PDC-3XG plasma cleaner/ sterilizer (Harrick Plasma). After waiting 1 minute, the grid was washed with dd H2O followed by a staining of 3 pL 3% uranyl acetate for 1 minute. The staining solution was blotted away using a #1 filter paper and grids were air-dried for at least one hour. EM imaging was performed using a Tecnai F20 electron microscope operated at 200 kV. Micrographs were recorded using a 4K x 4K CMOS CCD camera (TVIPS Temcam F416).
[0041] Stimulation Cytotoxicity assay on TLR4 cell line. The OMV stimulation via TLR4 pathway was analyzed on HEK-Blue™ hTLR4 cells (Invivogen, CA, USA) as described previously. The cells were maintained in complete DMEM (supplemented with 10% FBS and 100 pg/mL penicillin, lOOpg/mL streptomycin, and lOOpg/mL Normocin) media in presence of 5% CO2 at 37°C. 5* 104 cells were seeded on a 96 well plate and stimulated with 4 pg/mL concentration of OMV (20pL volume) for 8 hr. PBS and rPspA (3pg/mL) were used as positive and negative control. HEK-blue NULL cells were used as experimental controls. Relative NF-KB activity was measured by embryonic alkaline phosphate (SEAP) activity as described in manufacturer’s protocol.
[0042] Lipid A isolation and analysis by mass spectrometry. The lipid A isolation from OMV-PspA and analysis was the same as described previously.
[0043] Measurement of antibody responses. An enzyme-linked immunosorbent assay (ELISA) was used to assay the titers of antibodies against PspA in serum. Polystyrene 96-well flat-bottom microtiter plates were coated with 100 ng/well of purified rPspA. Antigens suspended in sodium carbonate bicarbonate coating buffer (pH 9.6) were applied in a 100 pL volume in each well. The coated plates were incubated overnight at 4°C. Free binding sites were blocked with a blocking buffer (phosphate-buffered saline [pH 7.4], 0.1% Tween 20, and 1% bovine serum albumin). The procedures for measuring total IgG and subtype IgG titers in the sera and bronchoalveolar lavage fluids (BALF) were described in our previous report. The highest dilution of serum resulting in an OD405 value at least 2-fold higher than that obtained with sham mouse serum was considered the antibody titer.
[0044] Opsonophagocytic killing assay. The opsonophagocytic killing assay was performed as described previously. Briefly, HL-60 cells (ATCC, CCL-240) were differentiated into granulocyte-like cells in the Iscove's Modified Dulbecco's Medium (IMDM) (ATCC) containing 100 mM N’, N-dimethylformamide (Sigma) for 5 days. Sera samples from immunized mice containing opsonic antibodies were heat-inactivated (56°C, 30 min) and serially diluted with opsonization buffer (mixture of 80 mL of sterile water, 10 mL of 10 x Hank’s balanced solution, 10 mL of 1% gelatin, and 5.3 mL of fetal bovine serum). Each well in a 96-well plate contained 40 pL of 4 * 105 HL60 cells, 103 CFUs of different S. pneumoniae strains in 10 pL of opsonophagocytic buffer, 20 pL of serum with different dilutions (1 :25, 1 :50, and 1 : 100), and 10 pL of 1% infant rabbit serum as a complement source (Sigma). Blank wells with the same system in the absence of mouse serum were used as negative controls. After 2 hr incubation, 10 pL of each sample was plated on Trypticase™ Soy Agar with 5% Sheep Blood (BD). Each sample was performed in triplicate. The opsonophagocytic killing ability was defined as a reduction in CFUs compared with the CFUs in the sera from unimmunized mice.

Claims

CLAIMS What is claimed is:
1. A vaccine platform, comprising a plurality of outer membrane vesicles expressed from a recombinant Yersinia pseudotuberculosis to include an amount of the a- helical region of pneumococcal surface protein A and an amount of monophosphoryl lipid A.
2. The platform of claim 1, wherein the a-helical region of pneumococcal surface protein A from Streptococcus pneumoniae strain D39.
3. The platform of claim 1, wherein the a-helical region of pneumococcal surface protein A comprises amino acid residues 3 to 285.
4. A non-naturally occurring organism, comprising a recombinant Y. pseudotuberculosis having a combination of a pmrF- J mutation and an IpxE insertion which exclusively produces monophosphoryl lipid A.
5. The organism of claim 4, wherein the a-helical region of pneumococcal surface protein A from S. pneumoniae strain D39.
6. The organism of claim 5, wherein the a-helical region of pneumococcal surface protein A comprises amino acid residues 3 to 285.
7. A method of immunization against S. pneumoniae infection, comprising the step of administering a pharmaceutically effective amount of outer membrane vesicles expressed from a recombinant Y. pseudotuberculosis that includes an amount of the a-helical region of pneumococcal surface protein A and an amount of monophosphoryl lipid A.
8. The method of claim 1, wherein the a-helical region of pneumococcal surface protein A from S. pneumoniae strain D39.
9. The platform of claim 1, wherein the a-helical region of pneumococcal surface protein A comprises amino acid residues 3 to 285.
PCT/US2024/034124 2023-08-15 2024-06-14 A bacterial vesicle-based pneumococcal vaccine against influenza-mediated secondary streptococcus pneumoniae pulmonary infection Pending WO2025038171A2 (en)

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