Patent Grant
11185583
Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 114,302 Byte ASCII (Text) file named “seq_listing.txt,” created on Feb. 11, 2015.
The majority of human pathogens enter via mucosal sites. Thus, there is a significant need for novel vaccine technologies that can generate robust mucosal immunity. However, adjuvants are currently required to accomplish this. Yet, adjuvants applied to mucosal tissue increase the risk of unwanted inflammatory effects, toxicity, and may even increase susceptibility to infection.
U.S. Publication No. 20120258092 to Dahiyat relates to optimized CD20 antibodies having Fc variants, methods for their generation, and method for their application, such as methods of enhancing macrophage activation, particularly for therapeutic purposes.
U.S. Patent Application Publication No. 2009/0280181 to Slager relates to particles with nucleic acid complexes, medical devices including the same and related methods.
U.S. Pat. No. 7,459,531 to Moore relates to human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins.
U.S. Pat. No. 6,248,332 to Gosselin relates to methods of stimulating in a subject an immune response to an Ag to which the immune response is targeted. This method includes the step of administering to the subject a binding agent, which binds a surface receptor of an APC, and an Ag to which the immune response is targeted.
U.S. Pat. No. 6,258,358 to Gosselin relates to methods of stimulating in a subject an immune response to an Ag to which the immune response is targeted. Also disclosed are molecular complexes including the binding agent coupled to an Ag.
U.S. Pat. No. 7,316,812 to Keler relates to cells transformed to express on their surface a component, which binds to an Fc receptor of an effector cell are disclosed. Also disclosed are expression vectors used to transform the cells. Once transformed, the cells bind to effector cells via the Fc receptor of the effector cell to stimulate an effector cell mediated immune response.
U.S. Pat. No. 7,378,504 relates to isolated monoclonal Abs, such as human Abs that bind to CD64 with high affinity. Nucleic acid molecules encoding the Abs of the invention, expression vectors, host cells and methods for expressing the Abs of the invention are also disclosed.
U.S. Publication No. 20040109874 to Fuller relates to methods for generating an immune response at a mucosal surface.
However, none of the aforementioned documents appear to disclose or suggest using a fusion protein with binding domains FcγRI+FcRn as an adjuvant-free muscosal vaccine.
This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the present invention.
In one aspect, the vaccine platform will allow non-invasive, single dose, and highly efficient vaccine delivery to the nasal mucosa, thereby stimulating robust mucosal immunity in the absence of traditional adjuvant. This vaccine platform will have wide application not only against common pathogens such as Streptococcus pneumonia and influenza, but also emerging, re-emerging, and biodefense pathogens.
One embodiment involves the development of a unique multi-functional non-adjuvanted mucosal vaccine platform that will maximize mucosal and systemic immune responses (cellular and humoral), eliminate the need for adjuvant, eliminate the requirement for a cold chain, enable safe intranasal delivery of vaccines, as well as provide a vaccine platform that can be employed to develop vaccines against a wide range of pathogens and in particular, superior protection against pathogens that infect via the mucosal route. To accomplish these goals, this vaccine will comprise a DNA and/or recombinant fusion protein (FP) vaccine. In the case of the DNA form, in some embodiments, the DNA will be administered intranasally as a single dose and induce expression of an Fc receptor (FcR)-targeted FP. Alternative approaches will include: an FcR-targeted DNA prime-FP boost vaccine, a combined FcR-targeted DNA plus FP vaccine, or an FcR-targeted FP prime-FP boost vaccine. In some embodiments, the vaccine will be administered intranasally as either a liquid mist or powder lyophilized or precipitated onto carrier particles. When administered as a DNA vaccine, in some embodiments, the plasmid encoding the FP will transfect mucosal epithelial cells along the nasal mucosa within the upper respiratory tract, resulting in local expression of FcR-targeted FP containing the antigen (Ag). In some embodiments, the FP itself will comprise three functional components/elements (be multi-functional in nature) (
This strategy can be employed for any pathogen and is therefore a vaccine platform technology. The vaccine platform will provide a particular advantage over existing vaccines in protection against pathogens that are transmitted via the respiratory route (i.e. influenza, RSV, tuberculosis, Streptococus pneumoiae, etc.). In addition, since intranasal immunization results in the induction of mucosal responses in other compartments (i.e vagina, gut) and in the blood, the technology could also be used to develop vaccines against sexually transmitted pathogens (i.e. HIV, Chlamydia, etc.), orally transmitted pathogens (i.e. Hepatitis A, Helicobacter pylori) and blood-borne pathogens (i.e. vector borne pathogens, such as dengue and pathogens transmitted via needle sticks, such as HBV). As indicated above, the vaccine is designed to maximize Ag transport to the NALT and subsequently Ag presentation/T cell activation within the NALT, resulting in an efficient mechanism to induce both local mucosal as well as systemic humoral and cellular immunity. This technology will solve several challenges that currently cannot be met by existing technologies: 1) Most vaccines are administered systemically (injected with a needle) and induce immune responses primarily in the periphery providing a defense against a respiratory pathogen only after it has disseminated from the periphery into the mucosa. In contrast, the proposed FP vaccine will induce superior protection against pathogens transmitted via a mucosal route by inducing immune responses that block or abort transmission at the site of mucosal exposure. 2) Existing licensed or experimental vaccines require either a live attenuated virus or adjuvant to stimulate protective levels of immunity, but at the expense of inducing inflammatory responses that reduce their tolerability and safety, especially when administered intranasally. In contrast, in one embodiment, the FP vaccine does not require an adjuvant, so it can be safely administered intranasally and induce mucosal immunity with minimal risk. 3) Most DNA vaccines are poorly immunogenic due, in part, to lack of inherent immunostimulatory properties and the low amount of Ag produced. Current efforts to overcome the poor immunogenicity of DNA vaccines include delivery of multiple doses, co-administration of the DNA vaccine with an adjuvant, and/or boosting DNA vaccine primed responses with a different vaccine modality. In contrast, in one embodiment, the FP vaccine in a DNA form will overcome these issues by directly targeting the small amounts of Ag-containing FP produced by the DNA vaccine to the NALT and APCs within the NALT. In this way, the expressed Ag is more efficiently processed and presented to the immune systemic leading to more robust immunogenicity without the need for high Ag doses or a second vaccine modality. In addition, targeting Ag to the APCs results in activation of these cells without the need for an adjuvant. 4) Using the FP vaccine in either DNA or protein form and administered as either DNA or protein alone or in combination (DNA+protein, DNA/protein) will provide advantages over similar vaccine concepts administered as non-targeted recombinant protein, killed, or live attenuated vaccines. Specifically, DNA vaccines are stable at room temperature so they can be distributed without a cold chain. In addition, DNA vaccines can be rapidly scaled up with relatively minimal cost providing a more cost effective approach for wide-spread vaccination. Due to the lack of requirement for adjuvant, the protein form of the FP vaccine should provide similar advantages, Furthermore, in the event of an emerging pandemic, a FP vaccine will be able to achieve more rapid intervention than other vaccine modalities because production of the vaccine will require only the pathogen's sequence from a clinical isolate and once identified, FP vaccines can be propagated in a very short (FP as DNA) or shorter (FP as protein) period of time using well-established molecular and recombinant techniques, without the need for extensive or complex purification steps. Since both the DNA and protein forms of the FP vaccines are non-replicating, they are also safer and can be more rapidly manufactured and distributed without the need for the more extensive safety tests currently required for killed or live attenuated vaccines. In addition, the FP vaccine should be more immunogenic with fewer doses, reducing the need for repeat immunizations, further increasing patient compliance and safety profile of the vaccine.
This will be the first instance of a mucosal vaccine employing FP as either a DNA or protein vaccine form, which targets both FcRn and FcγRI. More generally, to our knowledge, expression/use of FP vaccine designed to both target Ag to the NALT via the FcRn transeptithelial transport pathway, and subsequently to APCs within the NALT via FcγRI, has not been tested or published. Furthermore, DNA vaccination is not an obvious choice for this approach because, in the absence of FcR targeting, DNA vaccines are poorly immunogenic and require high doses to induce an immune response that cannot be achieved via intranasal delivery. In fact, most DNA vaccines are administered via the intramuscular route and designed to express and present the Ag primarily in the muscle cells not nasal cells in vivo. Therefore, a DNA vaccine, which produces a FP that targets the Ag to APC in a mucosal compartment, is not obvious and is novel. Furthermore, the expression of the FP by a DNA vaccine, which targets enhanced delivery of the Ag (via FcRn-targeted FP) to the NALT, and subsequently APCs within the NALT will likely overcome poor immunogenicity of DNA vaccines, thus providing a novel method to overcome current limits of DNA vaccines. Based on current state of art, people would also not combine the three FP components (Anti-FcγRI-Ag-anti-FcRn) into a single FP. However, we have thus far demonstrated we can generate an FP that maintains FcRn and FcγRI targeting functions. Furthermore, an FP DNA vaccine will provide two concurrent mechanisms for Ag presentation—first, in the cells transfected and adjacent interdigitating DCs, and second, in the APCs within the NALT targeted by the expressed Ag-containing FP. This dual Ag presentation will not only increase immunogenicity, but should also expose a broader range of antigenic epitopes than a traditional recombinant protein or DNA vaccine, resulting in a broader specificity in the immune response that will be more effective for protection. DNA vaccine transfection of nasal epithelium in vivo will also provide an advantage over nasal delivery of killed or live attenuated vaccines because presentation in these vaccine forms is very transient, whereas a DNA vaccine can express Ag in the transfected cells for days or weeks, thus providing longer term exposure in vivo to the vaccine Ag and targeting of the Ag to APCs within the NALT. This effect should translate into more durable immunity when compared to mucosal administration of non-targeted protein-based vaccines. Taken together, the vaccine strategy will result in a unique sequence of events (
In some embodiments the mucosal vaccine platform can be broadly employed for any pathogen, especially those transmitted via the respiratory route. Gene delivery in the case of CF or drug delivery are also contemplated.
Some embodiments inhere advantages, for example: 1) Improve potency of DNA vaccines; 2) Eliminates the cold chain; 3) Non-invasive administration; 4) Eliminates adjuvant/potential toxicity; 5) Reduced dose, administrations, and thus cost and potential for adverse effects; 6) No adjuvant and no live replicating vectors so increased safety profile (minimal risk for inflammation, no risk of reversion); 7) Can generate both humoral and cellular immunity; 8) Can generate both mucosal and peripheral immunity; 9) Mucosal immunity induced by the vaccine is expected to confer enhanced protection against mucosally transmitted pathogens when compared to existing vaccines that induce responses only in the periphery; 10) Universal vaccine platform that will be applicable to bacterial and viral as well as mucosal and non-mucosal pathogens; 11) Likely increased compliance due to elimination of booster immunizations and injection with needle.
It is to be understood that both the foregoing summary and the following descriptions are exemplary, and thus do not restrict the scope of the embodiments.
Reference will now be made in detail to several embodiments which, together with the drawings and the examples herein, serve to better describe the invention in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Current Protocols in Molecular Biology (Ausubel et. al., eds. John Wiley & Sons, N.Y. and supplements thereto), Current Protocols in Immunology (Coligan et al., eds., John Wiley St Sons, N.Y. and supplements thereto), Current Protocols in Pharmacology (Enna et al., eds. John Wiley & Sons, N.Y. and supplements thereto) and Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilicins, 2Vt edition (2005), for example.
Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).
For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.” As used herein, the term “about” means at most plus or minus 10% of the numerical value of the number with which it is being used.
In some embodiments, a broadly applicable vaccine platform is provided.
In some embodiments, the need for an adjuvant is eliminated. In some embodiments, an adjuvant is not present.
In some embodiments, vaccine safety is improved.
In some embodiments, the manufacture and approval of vaccines is improved or streamlined, or the costs thereof are reduced.
In some embodiments, an adjuvant-independent mucosal vaccine platform is provided, which includes a single recombinant molecule comprising one or more multiple interdependent components, or any combination of: 1) an antigen or antigen-binding component; 2) a component that targets the vaccine antigen to human FcRn; 3) a component that targets the vaccine antigen to human FcγRI; 4) a component that facilitates purification of the vaccine when in protein form. Linker sequences designed to minimize steric interference between adjacent components can connect the individual components. In some embodiments, the components act sequentially and in concert in order to maximize vaccine potency and thereby eliminate the requirement for adjuvant. In some embodiments, the antigen or antigen-binding component provides the immunogen that stimulates protective immunity. In some embodiments, the human FcRn-binding component functions to initially deliver the antigen from the nasal tract to the nasal-associated lymphoid tissue (NALT) via FcRn-containing nasal epithelial cells. In some embodiments, the human FcγRI-binding component will then direct the antigen to human FcγRI-expressing antigen presenting cells within the NALT. In some embodiments, both of the latter events are employed to sufficiently increase vaccine potency to a level that permits the elimination adjuvant. In some embodiments, the human FcRn-binding component will first maximize antigen delivery to the NALT, while the human FcγRI-binding component will subsequently maximize immune stimulation by antigen presenting cells within the NALT. In some embodiments, as a result of this sequence of events, the recombinant vaccine will generate immune protection equal or superior to that of vaccines containing adjuvant.
In some embodiments, the invention provides an immunogenic fusion protein for use as a mucosal vaccine comprising:
In some embodiments, the invention provides an immunogenic fusion protein for use as a cancer vaccine comprising:
In some embodiments, the invention provides an immunogenic fusion protein for use as a mucosal vaccine comprising:
In some embodiments, the invention provides nucleic acids and vectors encoding the immunogenic fusion proteins.
In some embodiments, the vectors can be useful as DNA vaccines. In one embodiment, the invention provides a vector comprising the nucleotide sequence of SEQ ID NO:17 (pUW160s). An antigen or gene of interest can be inserted into SEQ ID NO:17 immediately after nt position 2304. In one embodiment, the invention provides a vector comprising the nucleotide sequence of SEQ ID NO:18 (pJV7563). An antigen or gene of interest can be inserted into SEQ ID NO:18 immediately after nt position 1993.
In some embodiments, the invention provides pharmaceutical compositions comprising the immunogenic fusion proteins.
In some embodiments, the invention provides pharmaceutical compositions comprising the nucleic acids or vectors.
In some embodiments, the invention provides a method of inducing an immune response, comprising administering to a subject in need thereof an immunologically-effective amount of an immunogenic fusion protein of the invention.
In some embodiments, the invention provides a method of inducing an immune response, comprising administering to a subject in need thereof an immunologically-effective amount of a nucleic acid encoding an immunogenic fusion protein of the invention.
In some embodiments, the invention provides a method of inducing an immune response, comprising administering to a subject in need thereof an immunologically-effective amount of nucleic acid encoding any one of the following, or a combination thereof;
In some embodiments, the invention provides a method of inducing an immune response, comprising administering to a subject in need thereof an immunologically-effective amount of a fusion protein of any one of the following, or a combination thereof;
In some embodiments, the one or more FcγR1 binding domains is an anti-FcγR1 antibody or a fragment thereof. In some embodiments, the anti-FcγR1 antibody fragment comprises SEQ ID NO:24. In some embodiments, the FcγR1 is human.
In some embodiments, the one or more FcRn binding domains is selected from the group consisting of an anti-FcRn antibody or a fragment thereof and a mammalian serum albumin protein or a fragment thereof. In some embodiments, the FcRn binding domain is human serum albumin or a fragment thereof. In some embodiments, the FcRn binding domain is a fragment of human serum albumin comprising domain III. In some embodiments, the FcRn binding domain is a variant of domain III of human serum albumin having at least 90% amino acid identity. In some embodiments, the fragment of human serum albumin comprising domain III comprises SEQ ID NO: 25. In some embodiments, the FcRn is human.
In some embodiments, the infectious disease organism is a bacterial or viral pathogen. In some embodiments, the pathogen is selected from the group consisting of Streptococcus pneumonia, Neisseria meningitidis, Haemophilus influenza, Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., and Group B streptococci, Bacillus anthracis adenoviruses; Bordetella pertussus; Botulism; Bovine rhinotracheitis; Brucella spp.; Branhamella catarrhalis; canine hepatitis; canine distemper; Chlamydiae; Cholera; coccidiomycosis; cowpox; tularemia; filoviruses; arenaviruses; bunyaviruses; cytomegalovirus; cytomegalovirus; Dengue fever; dengue toxoplasmosis; Diphtheria; encephalitis; Enterotoxigenic Escherichia coli; Epstein Barr virus; equine encephalitis; equine infectious anemia; equine influenza; equine pneumonia; equine rhinovirus; feline leukemia; flavivirus; Burkholderia mallei; Globulin; Haemophilus influenza type b; Haemophilus influenzae; Haemophilus pertussis; Helicobacter pylori; Hemophilus spp.; hepatitis; hepatitis A; hepatitis B; hepatitis C; herpes viruses; HIV; HIV-1 viruses; HIV-2 viruses; HTLV; Influenza; Japanese encephalitis; Klebsiellae spp. Legionella pneumophila; leishmania; leprosy; lyme disease; malaria immunogen; measles; meningitis; meningococcal; Meningococcal Polysaccharide Group A, Meningococcal Polysaccharide Group C; mumps; Mumps Virus; mycobacteria; Mycobacterium tuberculosis; Neisseria spp; Neisseria gonorrhoeae; ovine blue tongue; ovine encephalitis; papilloma; SARS and associated coronaviruses; parainfluenza; paramyxovirus; paramyxoviruses; Pertussis; Plague; Coxiella burnetti; Pneumococcus spp.; Pneumocystis carinii; Pneumonia; Poliovirus; Proteus species; Pseudomonas aeruginosa; rabies; respiratory syncytial virus; rotavirus; Rubella; Salmonellae; schistosomiasis; Shigellae; simian immunodeficiency virus; Smallpox; Staphylococcus aureus; Staphylococcus spp.; Streptococcus pyogenes; Streptococcus spp.; swine influenza; tetanus; Treponema pallidum; Typhoid; Vaccinia; varicella-zoster virus; and Vibrio cholera and combinations thereof.
The antigen from the pathogen is not limiting. In some embodiments, the antigen is selected from the group consisting of: PspA (Streptococcus pneumonia), gp120 (HIV), hemagglutinin (influenza) and neuraminidase (influenza). In some embodiments, the antigen is PspA. The nucleotide sequence of PspA is provided in SEQ ID NO:16.
The one or more antigen binding components that bind antigen from one or more infectious disease organisms is not limiting. In some embodiments, the one or more antigen binding components comprises C reactive protein (CRP) (HGNC: 2367) or a fragment thereof, C3 (HGNC: 1318) or a fragment thereof, myelin basic protein (MBP) (HGNC: 6925) or a fragment thereof, CD6 (HGNC: 1691) or a fragment thereof, CD163 (HGNC: 1631) or a fragment thereof, and combinations thereof.
The one or more antigens from one or more cancers is not limiting. In some embodiments, the one or more antigens from one or more cancers is selected from the group consisting of wherein the antigen is selected from the group consisting of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B e antigen (HBeAg), N53, NS4a, NS5a and NS5b, HPV E6/E7, EBV LMP, HBV, HCV, mutated k-ras, p53, bcr-abl, HER-2, hTERT, ganglioside GD3, NY-ESO-1, MAGE/BAGE/GAGE, Hu, Yo, GAD, MART-1/melan-A, gp-100, tyrosinase, PSA and combinations thereof.
The cancer to be treated is not limiting and can include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical cancer, AIDS-related cancers, AIDS-related lymphoma, anal cancer, astrocytoma (including, for example, cerebellar and cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor (including, for example, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, visual pathway and hypothalamic glioma), cerebral astrocytoma/malignant glioma, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor (including, for example, gastrointestinal), carcinoma of unknown primary site, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-Cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's Family of tumors, extrahepatic bile duct cancer, eye cancer (including, for example, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (including, for example, extracranial, extragonadal, ovarian), gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, squamous cell head and neck cancer, hepatocellular cancer, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma (including, for example, endocrine pancreas), Kaposi's sarcoma, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (including, for example, non-small cell), lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma!plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oral cavity cancer, osteosarcoma, oropharyngeal cancer, ovarian cancer (including, for example, ovarian epithelial cancer, germ cell tumor), ovarian low malignant potential tumor, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (including, for example, nonmelanoma or melanoma), small intestine cancer, supratentorial primitive neuroectodermal tumors, T-Cell lymphoma, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (including, for example, gestational), unusual cancers of childhood and adulthood, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, viral induced cancers (including, for example, HPV induced cancer), vulvar cancer, Waldenström's macroglobulinemia, and Wilms' Tumor.
In some embodiments, the immunogenic fusion protein enhances transepithelial transport of the fusion protein to the nasal-associated lymphoid tissue (NALT) and enhances FcγR1 crosslinking by the fusion protein on antigen presenting cells (APC) within the NALT.
In some embodiments, the fusion protein comprises two or more FcγR1 binding domains. In some embodiments, the fusion protein comprises three FcγR1 binding domains.
In some embodiments, the immunogenic fusion protein comprises bivalent Anti-FcγRI-PspA-HuSA. In some embodiments, the bivalent Anti-FcγRI-PspA-HuSA comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the bivalent Anti-FcγRI-PspA-HuSA comprises the nucleotide sequence of SEQ ID NO:23.
In some embodiments, the immunogenic fusion protein comprises trivalent Anti-FcγRI-PspA-HuSA. In some embodiments, the trivalent Anti-FcgRI-PspA-HuSA comprises SEQ ID NO:13. In some embodiments, the trivalent Anti-FcγRI-PspA-HuSA comprises the nucleotide sequence of SEQ ID NO:20.
In some embodiments, the immunogenic fusion protein comprises a cleavable protein sequence and/or affinity tag to aid in purification. In some embodiments, the affinity tag comprises at least 6 histidine residues. In some embodiments, the immunogenic fusion protein comprises a secretion signal to facilitate secretion of the protein through plasma membrane. In some embodiments, the secretion signal is a lysozyme secretion signal.
In some embodiments, wherein upon administration to a subject, the fusion protein generates protection against both mucosal and non-mucosal pathogens. In some embodiments, the fusion protein generates protection at mucosal and non-mucosal sites.
In some embodiments, the vaccine platform includes a recombinant molecule containing single or multiple antigens from a single infectious disease organism.
In some embodiments, the vaccine platform includes a recombinant molecule containing single or multiple antigens from multiple infectious disease organisms.
In some embodiments, the one or more antigen binding components includes a component that binds to live attenuated infectious disease organisms.
In some embodiments, the one or more antigen binding components includes a component that binds to inactivated infectious disease organisms.
In some embodiments, the vaccine platform includes an antigen from cancerous or tumor cells.
In some embodiments, the vaccine platform includes a single or multiple human FcRn-binding domains.
In some embodiments, the vaccine platform includes a single or multiple human FcγRI-binding domains.
In some embodiments, the vaccine platform includes a cleavable protein purification component.
In some embodiments, the vaccine platform includes an additional immune modulatory component.
In some embodiments, the additional immune modulatory component is or includes a TLR agonist, Complement component, or cytokine analogue.
In some embodiments, the vaccine platform includes a DNA or RNA vaccine administered intradermally or intranasally.
In some embodiments, the vaccine platform includes a DNA or RNA vaccine, which lacks the FcRn-binding component when administered intradermally.
In some embodiments, the vaccine platform includes a immunogenic fusion protein which lacks the FcRn-binding component.
In some embodiments, trivalent Anti-FcγRI-PspA can be utilized in the invention, which lacks a FcRn-binding component. In some embodiments, the amino acid sequence of trivalent Anti-FcγRI-PspA is SEQ ID NO:11 and the nucleotide sequence is SEQ ID NO:19.
In some embodiments, bivalent Anti-FcγRI-PspA can be utilized in the invention, which lacks a FcRn-binding component. In some embodiments, the amino acid sequence of bivalent Anti-FcγRI-PspA is SEQ ID NO:12 and the nucleotide sequence is SEQ ID NO:21.
In some embodiments, the vaccine platform includes a DNA or RNA vaccine, which induces secretion of the recombinant protein vaccine into the nasal tract, when administered intranasally.
In some embodiments, the vaccine platform includes a protein vaccine administered intradermally or intranasally.
In some embodiments, the vaccine platform includes a protein vaccine, which lacks the FcRn-binding component, when administered intradermally. In some embodiments, Non-FcγRI targeted PspA-HuSA can be useful. In some embodiments, the amino acid sequence of PspA-HuSA is SEQ ID NO:14 and the nucleotide sequence is SEQ ID NO:22.
In some embodiments, the vaccine platform is administered as a DNA vaccine intradermally and subsequently a protein vaccine intranasally.
In some embodiments, the vaccine platform is administered as a protein vaccine intradermally and subsequently a DNA vaccine intranasally.
In some embodiments, the vaccine platform generates protection against both mucosal and non-mucosal pathogens.
In some embodiments, the vaccine platform generates protection at mucosal and non-mucosal sites.
In some embodiments, the vaccine platform includes a single vaccine containing a population of recombinant vaccine molecules, each containing a different infectious disease antigen or a set of infectious disease antigens.
In some embodiments, the vaccine platform includes a single vaccine containing a population of recombinant molecules, each containing a different tumor antigen or a set of tumor antigens.
In some embodiments, the vaccine platform is humanized.
In some embodiments, the vaccine platform is modified to eliminate autoreactive and/or antigenic sequences unrelated to the infectious disease or tumor antigen(s).
In some embodiments, a fusion protein comprising Streptococcus pneumonia antigen PspA is provided. In some embodiments, a divalent anti-hFcγRI-PspA-HSA (human serum albumin fragment) FP is provided. In some embodiments, a trivalent anti-hFcγRI-PspA-HSA FP is provided.
In some embodiments, the divalent and trivalent anti-hFcγRI-PspA-HSA FP is functional in vivo based on FcR binding (hFcγRI, FcRn), enhanced FP internalization via hFcγRI, enhanced transepithelial transport of Ag via FcRn, and enhanced hFcγRI-mediated presentation of PspA to PspA-specific T cells.
In some embodiments, PspA, anti-hFcγRI-PspA, and anti-hFcγRI-PspA-HSA containing DNA vaccine vectors are provided.
In some embodiments, a formulation (bi- vs. trivalent FP; FcRn vs. non-FcRn-binding; DNA, FP, or DNA prime-FP boost), route (i.n., i.d., or i.m.), and dose, is provided based on Streptococcus pneumonia immunogenicity and protection.
In some embodiments, the Streptococcus pneumonia vaccine platform affords broad protection against 2 or more strains of Streptococcus.
In some embodiments, immunogenicity and protection is comparable or superior to the vaccines such as Prevnar®13.
In some embodiments, the flu HA FP induces broad protection against homologous and drifted strains of flu.
In some embodiments, immunogenicity and protection is comparable or superior to vaccines such as Fluzone.
The present invention further provides pharmaceutical compositions comprising the fusion proteins and nucleic acids in combination with a pharmaceutically acceptable excipient.
In one embodiment, pharmaceutically acceptable means a material that is compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose, and is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or exipients include, without limitation, any of the standard pharmaceutical carriers or excipients such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions, and the like.
The fusion proteins and nucleic acids described herein can be prepared and/or formulated without undue experimentation for administration to a mammal, including humans, as appropriate for the particular application. The pharmaceutical compositions may be manufactured without undue experimentation in a manner that is itself known, e.g., by means of conventional mixing, dissolving, dragee-making, levitating, emulsifying, encapsulating, entrapping, spray-drying, or lyophilizing processes, or any combination thereof.
Suitable routes of administration may include, for example, oral, lingual, sublingual, rectal, transmucosal, nasal, buccal, intrabuccal, intravaginal, or intestinal administration; intravesicular; intraurethral; topical administration; transdermal administration; administration by inhalation; parenteral delivery, non-parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, and optionally in a depot or sustained release formulation. Furthermore, one may administer the compound in a targeted drug delivery system, for example in a liposome. Combinations of administrative routes are possible.
Proper dosages of the fusion proteins can be determined without undue experimentation using standard dose-response protocols. In one embodiment, the dosage of the compound, salt thereof, or a combination thereof, or pharmaceutical composition, may vary from about 0.001 μg/kg to about 1000 mg/kg. This includes all values and subranges therebetween, including 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1 μg/kg, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mg/kg, and any combination thereof.
In one embodiment, the fusion proteins and/or nucleic acids are administered mucosally.
The fusion proteins and nucleic acids can also be prepared for nasal administration. As used herein, nasal administration includes administering the compound to the mucous membranes of the nasal passage or nasal cavity of the subject. Pharmaceutical compositions for nasal administration of the compound include therapeutically effective amounts of the compound prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the compound may also take place using a nasal tampon or nasal sponge.
The compositions may also suitably include one or more preservatives, anti-oxidants, or the like.
Some examples of techniques for the formulation and administration of the fusion proteins and nucleic acids may be found in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins Publishing Co., 20 addition, incorporated herein by reference.
In one embodiment, the pharmaceutical compositions contain the fusion protein or nucleic acid in an effective amount to achieve their intended purpose. In one embodiment, an effective amount means an amount sufficient to prevent or treat the disease. In one embodiment, to treat means to reduce the development of, inhibit the progression of, or ameliorate the symptoms of a disease in the subject being treated. In one embodiment, to prevent means to administer prophylactically, e.g., in the case wherein in the opinion of the attending physician the subject's background, heredity, environment, occupational history, or the like, give rise to an expectation or increased probability that that subject is at risk of having the disease, even though at the time of diagnosis or administration that subject either does not yet have the disease or is asymptomatic of the disease.
While the invention has been described with reference to certain particular examples and embodiments herein, those skilled in the art will appreciate that various examples and embodiments can be combined for the purpose of complying with all relevant patent laws (e.g., methods described in specific examples can be used to describe particular aspects of the invention and its operation even though such are not explicitly set forth in reference thereto).
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect applies to other aspects as well and vice versa. Each embodiment described herein is understood to be embodiments that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any device, method, or composition, and vice versa. Furthermore, systems, compositions, and kits of the invention can be used to achieve methods of the invention.
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
A highly potent mucosal vaccine platform, which does not require adjuvant, can be generated utilizing a human FcγRI (hFcγRI)-specific-antigen (Ag) fusion protein (FP), which: 1) Incorporates a human FcRn (hFcRn)-binding sequence to increase transepithelial transport of FP to the nasal-associated lymphoid tissue (NALT). 2) Has increased valency in its hFcγRI-targeting component to further enhance Ag internalization, Ag processing, dendritic cell (DC) maturation, and Ag presentation in the NALT. 3) Combines an i.n. hFcR-targeted DNA vaccine-prime with an i.n. FP-boost to maximize and sustain vaccine potency.
The mucosa is the first line of defense against many pulmonary bacterial and viral pathogens and their antigenic variants. Also, mucosal immunity, induced by natural infection or vaccination, can afford better protection than peripheral immune responses, including improved cross-protection against antigenic variants1-4. Without wishing to be bound by theory, it is believed that this is likely due, in part, to a crucial early opportunity to prevent a disseminated infection by eliminating/blocking the infection at its most vulnerable stage, while the infection is still limited. However, most licensed vaccines induce responses primarily in the periphery, producing a suboptimal immune response at the initial mucosal site of exposure. In addition, the most commonly used vaccine platforms—live attenuated and adjuvanted protein vaccines—often cause adverse inflammatory effects when administered via a mucosal route. Unadjuvanted protein vaccines are safer, but usually poorly immunogenic. Thus, we propose the development of a novel and highly potent mucosal FP vaccine platform, which does not require the use of adjuvant. The FP will: 1) Target Ag to hFcRn to increase Ag transepithelial transport to the NALT5. 2) Increase hFcγRI crosslinking on APCs within the NALT to enhance Ag presentation and T cell activation6-10. 3) Combine an i.n. administered hFcR-targeted DNA vaccine-prime with an i.n. FP-boost. A strategy that has been shown to enhance the magnitude, quality, and longevity of protein vaccines11. Data, which support the successful development of the proposed adjuvant-independent mucosal vaccine platform include the use of a hFcγRI transgenic mouse model in which pneumococcal surface protein A (PspA) targeted to hFcγRI i.n. generates enhanced mucosal immunity and protection against a mucosal challenge with Streptococcus pneumoniae (Sp), without the use of adjuvant7. We have also shown that a mAb-bound inactivated Francisella tularensis (mAb-iFt) complex administered i.n. enhances protection against mucosal Ft challenge. This protection is FcγR-dependent and requires FcRn8. Subsequent studies have also demonstrated that FcRn mediates enhanced transport of mAb-iFt versus iFt alone from the nasal passage to the NALT9. Thus, a strong potential for the successful development of the proposed adjuvant-free mucosal vaccine platform has been demonstrated using two distinct FcR-targeted vaccine strategies and two distinct mucosal infectious disease models in which enhanced protection has been observed without the use of traditional adjuvant. A novel/unique FP will now be generated/developed to maximize adjuvant-free FP potency, which will be crucial to its successful application, as well as altering long established perceptions regarding the requirement for adjuvant. Specifically, we will incorporate molecular modifications to our bivalent prototype FP to further improve FP targeting and immunogenicity: 1) A sequence, which targets FPs to hFcRn12,13, will be added to increase FP delivery to the NALT. 2) The divalent anti-hFcγRI-PspA FP will be converted to a trivalent hFcγRI-targeted FP to further enhance Ag internalization, Ag processing, DC maturation, and subsequent Ag presentation9,10,14,15 within the NALT. 3) An i.n. administered hFcR-targeted DNA vaccine-prime will be combined with an i.n. FP-boost to further improve vaccine potency and durability16-20.
Aim 1: Produce and verify in vitro, the functional capacity of bivalent, trivalent, and hFcRn-targeted anti-hFcγRI-PspA FPs. The bivalent, trivalent, and hFcRn-targeted anti-hFcγRI-PspA FPs have been produced. FP functions tested will include: hFcRn binding and transepithelial transport of FPs, FP binding to hFcγRI, FP internalization by APCs, and the ability of FPs to induce DC maturation and FP-enhanced Ag presentation/T cell activation.
Aim 2: Identify the optimal (most protective) FP configuration utilizing a hFcγRI/hFcRn-expressing mouse model. Bivalent versus trivalent hFcRI-targeted FPs (plus or minus the hFcRn binding component) will be administered i.n. at varying FP doses. Protection against Sp challenge and PspA-specific T and B cell responses will be measured. The optimal FP will be identified based primarily on superior protection provided.
Aim 3: Maximize FP vaccine platform potency and protective longevity utilizing an i.n. administered hFcR-targeted DNA vaccine-prime plus i.n. FP-boost regimen. The ability of FP, hFcR-targeted DNA, and hFcR-targeted DNA vaccine-prime plus FP-boost regimens, each administered i.n., to further enhance protection and extend immune memory, without the use of adjuvant, will be tested. The optimal Sp vaccine regimen will then be compared to licensed Sp vaccine, followed by cross-protection studies to validate the cross-protective potential of this vaccine platform, in particular as it applies to Sp vaccination.
Current approaches to vaccine development are as diverse as the infectious organisms they are designed to protect against. However, most current protein-based vaccine strategies require two primary components: identification of a protective antigen (Ag) and the use of an appropriate adjuvant. Adjuvants can be divided into two primary categories: immune modulators and delivery systems21. Immune modulators include: bacterial products [lipopolysaccharides, peptidolglycans, lipoproteins, DNA (CpGs), and enterotoxins], plant products (saponins and glycosylceramides), and cell products (heat shock proteins and cytokines). Mechanisms of action include: dendritic cell (DC)/macrophage activation, up-regulation of costimulatory molecules (required for efficient T cell activation), and induction of cytokines involved in immune regulation. However, most immune modulators have broad specificity and activity, increasing potential toxic side effects and raising safety concerns. Vaccine delivery systems are more limited in number. Delivery systems function primarily by slowing Ag release at the site of injection and/or enhancing Ag uptake by Ag presenting cells (APCs). The only delivery system currently approved for human use in the U.S. is mineral salt, specifically Alum, which induces a potent antibody (Ab) (humoral, Th2-type) response, but is ineffective at boosting cellular (Th1-type or CD8 T cell) responses. Another delivery system consists of emulsions (Ag mixed with oil and water) such as MF59. However, similar to mineral salt (Alum), MF59 stimulates a potent humoral (Th2-type) immune response, but fails to stimulate cellular immune responses. A third delivery system consists of particulate Ag, such as the incorporation of Ag into lipid-containing vesicles or nanoparticles. Particulate Ag can stimulate both humoral and cellular immune responses. However, manufacture and maintenance of particle consistency can be difficult and expensive, potentially resulting in a vaccine product, which 3rd world countries cannot afford. In addition, the adjuvants discussed above are generally used in parenteral immunizations, which often do not produce strong mucosal immunity22. Yet, the majority of pathogens enter via mucosal routes. In contrast, mucosal immunization can provide potent protection at both mucosal and non-mucosal sites5. Furthermore, as specifically pointed out in a number of reviews, including in Nature Medicine and Nature Reviews, this applies to human vaccination, as well as that of mice23-25. Thus, there is a need for more effective and safe mucosal vaccination strategies and/or mucosal adjuvants. While both Cholera Toxin B (CTB) and IL-12 can be effective as mucosal adjuvants in mice26-28, and possibly humans, as with many adjuvants, there is significant concern regarding their toxicity/safety. Therefore, a mucosal vaccine platform, which does not require adjuvant and can stimulate both humoral and cellular immune responses, such as the use of FcR-targeted mucosal immunogens [anti-FcR fusion proteins (FPs)]5,29, would significantly advance vaccine technology, while eliminating many of the issues associated with the use of adjuvants. As stated by Dr. Jerry McGhee, a world renown mucosal immunologist, “The development of effective strategies for mucosal vaccination would revolutionize medicine”5. In addition, targeting Ag to hFcγRI bypasses the inhibitory receptor FcγRIIB, which can limit FcγR-induced DC maturation and Ab production14,15. Accordingly, we propose the development of a highly innovative adjuvant-independent (recombinant) mucosal vaccine platform, which sequentially targets FP Ag to: 1) hFcRn for enhanced transepithelial transport of Ag to the nasal-associated lymphoid tissue (NALT), and 2) hFcγRI on APCs/DCs for enhanced Ag processing/presentation and T cell/B cell activation within the NALT (
The current paradigm for vaccine development involves the identification of a protective Ag and the requirement for a safe and effective adjuvant. Given the myriad of problems adjuvants face, including safety concerns and FDA approval, and the paucity of researchers challenging this paradigm by developing adjuvant-free vaccine strategies, the proposed adjuvant-free FP platform is a highly innovative approach to mucosal vaccine development. Furthermore, the creation of a unique/novel dual (hFcγRI/hFcRn)-targeted multi-functional FP, which mediates sequential targeting of FP Ag to hFcRn i.n. for enhanced transepithelial transport of FP Ag to the NALT and subsequently FP Ag to hFcγRI on APCs for enhanced T and B cell activation within the NALT (
Regarding the potential for developing an adjuvant-free mucosal vaccine, published studies and preliminary data validate the potential for success of the proposed FP-based mucosal vaccine platform. Specifically, partial protection against the highly virulent F. tularensis (Ft) SchuS4 organism is achieved when immunizing i.n. with adjuvant-free inactivated Ft (iFt) targeted to FcR via mAb-iFt complex8. Furthermore, these studies demonstrate important roles for FcγR and FcRn in this protection. Specifically, mice immunized i.n. with mAb-iFt are not protected in the absence of FcγR or FcRn8. Also, subsequent mechanistic studies indicate that FcRn plays a critical role in enhancing the transport of iFt to the NALT, when immunizing i.n. with mAb-iFt9. Importantly, the enhanced transport of iFt to the NALT is eliminated in the absence of FcRn. In addition, subsequent studies by other laboratories have demonstrated a similar function for FcRn, when utilizing i.n. administered recombinant Fc-Ag immunogens plus adjuvant30,31. In 2012, one of the present inventors also published studies utilizing a hFcγRI transgenic (Tg) mouse model and a prototype bivalent [mono (hFcγRI)-specific] anti-hFcγRI-PspA FP. These studies demonstrated that administering this prototype anti-hFcγRI-PspA FP i.n. to hFcγRI Tg mice in the absence of adjuvant enhances protection against subsequent S. pneumoniae (Sp) challenge (
The FP DNA constructs to be used in these studies, which have been generated, are depicted in
Development of vaccine platform technology such as that proposed, which eliminates the requirement for adjuvant, will fundamentally alter the paradigm by which vaccines are generated and administered. In addition, as stated by Dr. McGhee5 “The development of effective strategies for mucosal vaccination would revolutionize medicine, allowing protection from the many viral and bacterial pathogens that enter the body via the mucosa . . . ”5,30,31. Specifically, the proposed FP platform will be the first recombinant unadjuvanted mucosal vaccine platform to take full advantage of this “Mucosal Gateway” for vaccination (
Aim 1: Produce and verify in vitro, the functional capacity of bivalent, trivalent, and FcRn-targeted anti-hFcγRI-PspA FPs.
Published studies by one of the inventors demonstrate that a prototype bivalent anti-hFcγRI-PspA FP administered i.n. to hFcγRI Tg mice, enhances protection against subsequent i.n. challenge with Sp in the absence of adjuvant7. Others, when targeting Ag to FcRn i.n., but with adjuvant, have obtained similar results30,31. Evidence suggests that the above FcRn-mediated enhancement is due, in part, to increased transport of Ag from the nasal passage to the underlying NALT5,31. Thus, to further optimize the potency of our current prototype divalent anti-hFcγRI-PspA FP, a sequence from human serum albumin (HSA-Domain III), which binds FcRn and mediates FcRn-dependent transepithelial transport of Ags13, has been added to the divalent anti-hFcγRI-PspA FP. We have also converted the bivalent FP to a trivalent FP to more extensively crosslink hFcγRI on APCs, thereby enhancing Ag internalization, DC maturation9,10,14,15, Ag presentation/T cell activation, and ultimately vaccine potency. In regard to the use of a bivalent versus trivalent FP, it has been clearly demonstrated that anti-hFcγRI-targeted Ags are significantly more potent immunogens in trivalent vs. divalent form6,10. Furthermore, maximizing FP potency will be crucial to establishing a new paradigm in which the use of this adjuvant-free mucosal vaccine platform is a viable and an acceptable alternative to adjuvant.
Objectives: 1) Generate DNA constructs and produce FPs; 2) Verify FP function(s) in vitro.
Aim 1.1: Generation of DNA constructs and production of FPs.
FP DNA constructs depicted in
Aim 1.2: Verification of FP function in vitro.
FP binding to hFcγRI and hFcRn: Binding of hFcγRI-specific FPs to hFcγRI and HSA-containing FPs to hFcRn will be measured by ELISA (
Transepithelial transport of HSA-containing (FcRn-targeted) FPs: Modification of a standard in vitro IgG transport assay33 will be used to assess HSA-containing FPs versus FPs lacking HSA to interact with FcRn from human epithelial cells and subsequently transit an epithelial layer. Briefly, T84 cells expressing human FcRn will be grown on transwell filter inserts to form a monolayer exhibiting transepithelial electrical resistance (300 ohms/cm2) as measured via a tissue-resistance meter equipped with planar electrodes. In addition to electrical resistance, confocal microscopy, immunohistochemistry, and bulk protein transport, will also be used to verify monolayer integrity32,34. Monolayers will be equilibrated in Hanks balanced salt solution. FPs will be applied to the apical compartment, and incubated with DMEM medium plus or minus competitor for FcRn (Free HSA) at varying concentrations for 1-4 hours at 37° C. Time point samples taken from the basolateral compartment will then be assayed for FPs via Western blot or ELISA. IgG will also be used as a positive control for FcRn-mediated transepithelial transport. Once, transepithelial transport has been verified using T84 cells, similar assays will be conducted using tracheal epithelial cells from WT, hFcRn Tg, hFcγRI Tg, and hFcγRI/hFcRn Tg mice.
Induction of DC maturation by FPs: Bivalent and trivalent FPs will be incubated with BMDCs and DC maturation measured by monitoring maturation markers (MHC Class II, CD40, CD80, CD86, and CD205) by flow cytometry, as previously described9.
FP-mediated PspA presentation to PspA-specific T cells: This assay will also be conducted as previously described7. Briefly, the PspA-specific T cell hybridoma (B6D2) (1×105 cells/well) will be co-cultured with hFcγRI/FcRn-expressing, non-hFcRn-expressing, and/or non-hFcγRI-expressing BMDCs (2×105 cells/well) with titrating amounts of PspA or PspA-containing FPs. Equivalent PspA concentrations will be used as the basis for equilibrating FP concentrations and thereby comparing presentation of the various FPs, with concentrations ranging from 0 to 10 μg/ml of PspA. Cells will then be incubated for 30 hours at 37° C. in 5% CO2, supernatants will be collected, and IL-2 production will be measured via Luminex assay. In addition to BMDCs from non-hFcγRI/FcRn-expressing mice, wells lacking BMDCs or FP Ag will also serve as negative controls, as well as wells containing soluble F(ab′)2 anti-hFcRn and/or anti-hFcγRI blocking mAbs, to further confirm hFcγRI and/or FcRn involvement.
Potential outcomes and alternative approaches: Bivalent and trivalent FPs, as well as anti-FcγRI-PspA-HSA FP have been produced and bind both hFcRn and/or hFcγRI (
Aim 2: Optimize FP platform immunogenicity and protective efficacy utilizing the FPs generated and tested in Aim 1 and a hFcγRI/hFcRn-expressing mouse model.
It will be critical to first identify the FP generated in Aim 1, which is most protective against Sp challenge. Thus, we will compare the protective capacity of bivalent vs. trivalent FPs (plus and minus FcRn-binding HSA) using a hFcγRI/FcRn-expressing mouse model. Once the optimal (most protective) FP formulation is identified, the optimal dose will be determined, and humoral and cellular immune responses to FP vaccination will also be examined. While mucosal and parenteral immunization routes have proven successful in the case of Sp vaccines, mucosal routes are generally believed to be superior for mucosal pathogens in mouse and humans23-25. Thus, we will focus on the i.n. immunization route.
Objectives: 1) Utilizing a hFcγRI/FcRn-expressing mouse model, identify the FP formulation providing optimal protection against Sp challenge; 2) Using the above optimal FP formulation, identify the optimal FP dose that provides maximal protection in this challenge model; 3) Examine humoral and cellular immune responses to the FP vaccine.
Aim 2.1: Determine which FP provides optimal protection against Sp challenge utilizing a hFcγRI/FcRn-expressing mouse Sp protection model.
We currently maintain a colony of hFcγRI Tg and non-Tg littermate mice by breeding one male C57BL/6 hFcγRI Tg+/− mouse with two female C57BL/6 WT mice per breeding colony. The hFcγRI Tg mice are then identified by PCR. To generate hFcγRI/FcRn-expressing mice, we will similarly breed a male hFcγRI Tg+/− mouse with two C57BL/6 hFcRn (+/+) mice (Jackson Laboratories). Mice expressing both hFcγRI and hFcRn will again be identified by PCR, as is done to verify the presence of hFcγRI. Mice will be divided into groups of 8 mice/group, 8-12 weeks of age. Human FcγRI/FcRn-expressing mice will be immunized i.n. with 20 μl of PBS, PspA, PspA-HSA, bivalent anti-hFcγRI-PspA, bivalent anti-hFcγRI-PspA-HSA, trivalent anti-hFcγRI-PspA, or trivalent anti-hFcγRI-PspA-HSA on day 0 and boosted on day 21. In each case, the amount of PspA administered will be equivalent (10 μg/mouse). Immunized mice will then be challenged i.n. two weeks post-boost with 1×106 CFU Sp and monitored 21 days for survival. Infectious challenge of animals will be performed with strain A66.17, a mouse virulent capsule type 3 pneumococcus (family 1, clades 1,2), serologically similar to the family 1, clade 2, PspA of strain Rx137 utilized to construct the PspA-containing FPs. Culturing and counting the inoculums subsequent to challenge will be done to verify the exact CFU administered. Importantly, the above immunization regimen and challenge dose produces approximately 50% protection in hFcγRI Tg mice when immunizing with bivalent anti-hFcγRI-PspA7. The latter dosing/result will thus allow detection of differences in survival (positive and negative) between immunized groups. Should 100% protection be achieved with multiple FPs, the challenge dose will be increased to better distinguish between degrees of protective efficacy between FPs. To further delineate the protective efficacy of the FPs following immunization and challenge, bacterial burden will also be measured at various time intervals post-challenge in lung, liver, and spleen, as previously described by Dr. Gosselin7. The remaining tissue homogenate will be spun at 14,000×g for 20 min, and the clarified supernatant will be stored at −20° C. for future cytokine analysis.
Aim 2.2: Using the optimal FP formulation from Aim 2.1, determine the optimal FP dose that provides optimal protection in this challenge model.
Challenge experiments will be repeated utilizing the optimal FP formulation identified above and doses of PspA within FPs ranging from 3-30 μg/mouse. The challenge dose will be the highest dose used in Aim 2.1 for which protection was observed in Aim 2.1. Both survival and bacterial burden will then be measured. Once the optimal dose has been determined, immunizations will be carried out in WT, hFcRn Tg, hFcγRI Tg, versus hFcγRI/hFcRn-expressing mice to verify hFcRn and hFcγRI roles in the protection observed.
Aim 2.3: Examine the humoral and cellular immune responses to the FP vaccination.
Ab production: Sp-specific Ab production will be measured by ELISA as previously described by Dr. Gosselin7. Wells will be coated with live Sp or PspA. BSA-coated wells will be used as specificity controls for Sp and PspA. PspA and Sp-specific IgM, IgG isotypes, and IgA will be measured and responses compared between that of bronchioalveolar lavage (BAL) and serum from the same animals. We will also test for Abs cross-reactive with mouse and human tissues (potentially autoreactive Abs). In the case of human tissues, this will be done using paraffin embedded tissues from heart, lung, liver, kidney, and lymph node obtained commercially from anonymous donors. Tissues will be stained immunohistochemically as previously described by Dr. Gosselin38 using serum from FP-immunized mice. Isotype controls and Abs with known tissue specificity will be used as negative and positive controls, respectively. The ability of sera from FP-immunized mice to block FP binding to hFcγRI or FcRn will also be examined.
Cytokine production: ELISPOT will be used following immunization to identify T cell subsets producing specific cytokines using previously published methods39. Cytokine production will also be measured after immunization, in vitro, as previously described by Dr. Gosselin7,8. Levels of selected cytokines produced in spleen cell supernatants in response to Ag (IL-2, IL-4, TNF-α, IFN-γ, and IL-5) will be measured using the multiplex Luminex assay. Cytokine mRNA generated following immunization will also be measured by RT-PCR at 1, 2, and 3 days post primary and booster immunization.
Histological analysis: It is possible that crosslinking hFcγRI could induce local toxicity or negative side effects, such as inflammation or local depletion of CD64+ cells. Therefore, histological and immunohistochemical analysis of infected tissues will be conducted to monitor and identify immune cell infiltrates and potential tissue damage/toxicity due to the immunogen/FP itself, as well as tissue damage due to infection. Mice will be euthanized at 12, 24, 48, 72, and 92 hours post-immunization or post-challenge. Post-immunization, lungs and nasal tract, including NALT, will be harvested. Post-challenge, lungs, spleen, lymph nodes (cervical, mediastinal, mesenteric, and submandibular), and nasal tract, including NALT, will be harvested. Tissues will be fixed in 2% paraformaldehyde in PBS. Tissues will then be processed for histology as previously described by one of the inventors8,40.
Potential outcomes and alternative approaches: Should there be any issues with the hFcRn component of the hFcγRI/hFcRn Tg mouse, we have the option of substituting the human FcRn-targeting sequence with the mouse equivalent, and using our hFcγRI Tg mouse model. In addition, hFcRn targeting of the anti-hFcγRI-PspA-HSA FP can be verified independently in the hFcRn Tg mice. In regard to the trivalent anti-hFcγRI-PspA, two separate studies using anti-hFcγRI mAb (22 mAb), from which our FP construct was derived, indicate trivalent interaction of this molecule with hFcγRI will produce a more effective immunogen6,10m. However, should the trivalent FP not produce protection superior to bivalent FP, we will focus on further testing/optimizing the bivalent anti-hFcγRI-HSA FP approach and subsequently combining it with an FcR-targeted DNA-prime FP-boost regimen (Aim 3). In regard to FcRn binding of the anti-hFcγRI-PspA-HSA FP, studies in WT mice and preliminary data herein (
It is also possible that crosslinking hFcγRI could induce local toxicity or negative side effects, such as inflammation or local depletion of CD64+ cells. For example, studies demonstrating hFcγRI modulation as a consequence of extensive cross-linking mediated by whole mAb H22 (humanized anti-hFcγRI), and subsequent inhibition of opsinophagocytosis has been observed, which thereby impairs normal immune function50. However, the observed inhibition in this case appeared to be dependent on the ability of whole 22 mAb to bind to hFcγRI via both Fab and Fc binding domains50. In contrast, our anti-hFcγRI-Ag FPs lack the Fc domain, with the Fab regions also binding outside the IgG-Fc binding site (
Aim 3: Optimize FP platform immunogenicity and protective efficacy/longevity utilizing a DNA-prime FP-boost strategy.
DNA vaccines are a lower cost alternative to protein vaccines, can be more easily distributed, and stimulate humoral and cellular parenteral and mucosal immune responses52,53. DNA vaccine studies utilizing PspA as a protective immunogen have also been successfully conducted in a mouse model.54 Finally, a DNA-prime Protein-boost regimen has been shown to increase vaccine potency (humoral and cellular immune responses), as well as vaccine durability.16-20. Thus, we will utilize the latter strategy to determine if we can further increase the potency and efficacy/longevity of our mucosal FP vaccine. A significant advantage of utilizing PspA as the immunogen is its potential to cross-protect against multiple Sp strains55-57. Therefore, additional challenge studies utilizing the optimized FP platform will also be conducted to determine the ability of FcR-targeted FP to cross-protect against Sp infection with several different Sp strains. While one object of the present application is establishing a highly potent adjuvant-free mucosal vaccine platform applicable to many pathogens, the latter studies will ultimately be important to also initially establishing this vaccine platform as a potentially viable approach for Sp vaccination.
Objectives: 1) Generate and test PspA and anti-hFcγRI-PspA FP-producing DNA vaccine vectors; 2) Identify the optimal human FcγRI-targeted DNA and DNA-prime FP-boost regimens; 3) Determine the breadth of protective immunity against multiple Sp challenge strains and compare the optimized FP vaccine to a licensed conjugate vaccine (Prevnar®13).
Aim 3.1: Generation and testing of PspA and anti-hFcγRI-PspA FP-producing DNA vaccine vectors.
PspA and anti-hFcγRI-PspA genes have been inserted into an optimized DNA vaccine expression cassette (
Aim 3.2: Identify optimal DNA and DNA-prime FP-boost regimens.
Immunogenicity and challenge experiments: Intradermal and i.n. DNA-prime i.n. FP-boost regimens will be compared to the optimized i.n. FP regimen identified in Aim 2. Importantly, we will initially focus on DNA immunization via the i.n. route. Subsequently we will use i.d. DNA immunization to determine if the i.d. route might be superior to the use of i n immunization either independently and/or in the DNA-prime FP-boost approach. Intradermal DNA immunization will also be used should i.n. DNA immunization prove less stimulatory than that of the i.d. route. Experimental groups will include WT, hFcRn Tg, hFcγRI Tg, and hFcγRI/FcRn-expressing mice. Controls will also include mice receiving empty vector and vector plus PspA. Vaccinations will consist of 2 doses, 4 weeks apart. Blood will be collected before and 2 and 4 weeks post-immunization to measure Sp-specific Ab. Four weeks post-final boost, mice will be sacrificed to collect BAL, splenocytes, and lung lymphocytes to measure mucosal Ab and T cell responses. Mucosal and systemic Ab, T cell responses, and protection from Sp challenge will be examined as described in Aim 2. The optimal regimen that induces a significant improvement in protection, when compared to controls and the optimal FP regimen identified in Aim 2, will then be compared to Prevnar®13. DNA immunizations will be done as described below.
Intranasal DNA vaccination: We will use an established method for i.n. immunization with plasmid DNA Our successful use of this immunization strategy to enhance PspA (Sp)-specific Ab responses with our hFcγRI-targeted FP is demonstrated in
Gene gun DNA immunizations: This procedure has also been used successfully in our laboratory. Gene gun, or particle-mediated epidermal delivery (PMED) of vaccines, is a common method used for transcutaneous injection (TCI) of DNA vaccines into the epidermal layer of the skin. It differs from IM or ID DNA injection in that it results in direct intracellular delivery of the DNA into non-professional APCs (i.e. keratinocytes) and professional APC (i.e. Langerhans cells) in the epidermis62,63. A major benefit of immunizing the skin is the induction of both systemic and mucosal responses64-72 including highly disseminated mucosal IgA responses73,74. TCI in mice and NHPs has generated CD8 CTL66,75,76 and mucosal IgA/Ab-secreting cells in the intestine77-80 female reproductive tract73,81, upper71,76 and lower respiratory tract82 and in the oral cavity71,81,83. In humans, TCI induced vaccine-specific IgA in saliva84 and the intestine85,86. The mechanism by which TCI induces mucosal responses is not entirely clear. Studies in mice suggest two possible mechanisms: 1) APCs migrate from the skin to mucosal inductive sites and activate local T cells64,66,68,70,87 and/or 2) APCs migrate to regional draining lymph nodes and induce a mucosal homing phenotype on activated lymphocytes, which then home to mucosal effector sites88,89. PMED is one of the most efficient methods for DNA vaccine delivery and has induced protective levels of systemic Ab and CD8 T cell responses against a wide variety of diseases in mice, NHP and humans78,90-92. Importantly, like other TCI methods, PMED induces mucosal responses that contribute to improved protection from mucosal challenges71,75,76. PMED delivery of plasmid expressing the FcR-targeted vaccine may thus offer a potent strategy to induce/increase mucosal immunity. Therefore, DNA immunizations will be administered into the epidermal layer of the skin using the PowderJect XR-1 particle mediated epidermal delivery (PMED) research device as previously described59,93. Optimum DNA vaccine doses in mice consist of 2 μg DNA and 1 mg gold divided into two tandem sites per mouse. Two doses (prime+boost) will be administered per animal 4 weeks apart. Controls will include PBS or empty DNA vector.
Aim 3.3: Determine the breadth of protective immunity against multiple Sp challenge strains.
While one object of the present application is establishing a potentially highly potent adjuvant-free mucosal vaccine platform applicable to many pathogens, cross-protection studies will ultimately be important in initially establishing this vaccine platform as a potentially viable approach for Sp vaccination. Utilizing the optimal immunization regimen identified above, hFcγRI/FcRn-expressing mice will be immunized with the optimal vaccine regimen versus Prevnar®13 and will then be challenged with two additional strains of Sp [D39 (Serotype 2, PspA family 1, PspA clade 2) and 3JYP2670 (Serotype 3, PspA family 2, PspA clade 4)]. Protection will then be compared to that of strain A66.1 (Serotype 3, PspA family 1, PspA clades 1,2)94. In regard to strain variability, while pairwise comparisons of PspA genes and proteins have been made, only the Rx1 strain from which the FP PspA was generated, was included37. Thus, direct gene/protein comparisons of Rx1, A66.1, D39, and 3JYP2670 are not available. However, strains from family 1, clades 1,2 (analogous to A66.1) share 65-86% identity with the Rx1-derived PspA. The case is similar for family 1, clade 2 strains (analogous to D39). Strains from family 2, clade 4 (analogous to 3JYP2670) share 54-57% identity with the Rx1 PspA. We will examine both protection and bacterial burden in the case of each challenge strain.
While we also recognize that the level of nasopharyngeal carriage is also an important correlate of vaccine efficacy against Sp infection, given one object of the present application is to development a novel and paradigm-changing vaccine platform, applicable to a range of infectious disease agents, we presently consider Sp carriage studies beyond the scope of this specific project.
Based on our preliminary studies using a DNA vaccine derived from the bivalent anti-hFcγRI-PspA FP administered i.n., in which we observed enhanced anti-Sp Ab production (
It is contemplated that continued studies will focus on moving the innovative and paradigm altering (adjuvant-free) mucosal vaccine platform toward clinical trials. These studies will also take advantage of the ability of our FP(s) to bind to FcγRI in non-human primates (NHPs) (
The present application is not limited to the use of recombinant protein/peptide epitopes, but, in the case of the FP vaccine, it is contemplated that it may be adapted to use with whole inactivated vaccines. This will be accomplished by substituting antigen with a moiety that binds inactivated organisms, such as inactivated F. tularensis, a biodefense pathogen, which the PI's laboratory has previously shown can enhance protection against F. tularensis challenge when targeted to FcR intranasally8,9.
Protection after FP immunization is eliminated in wildtype (WT-negative control) mice, which lack hFcγRI. Similar differences in immunity are also obtained when comparing PspA immunization of Tg mice with that of hFcγRI-targeted PspA FP, with the latter also producing enhanced anti-Sp Ab responses and protection. Data is provided in
Generation of DNA Constructs: These constructs were generated from the original bivalent anti-FcγRI-PspA construct within the pjG582 plasmid.
Trivalent Anti-FcγRI-PspA (Construct B):
The cloning strategy of this construct was modified due to the repetitive sequence/domain nature of the construct itself. Instead of using an “inverse PCR” approach to insert two new restriction sites for the cloning of the third FcγRI, I had to use a “modular PCR” approach. Briefly, two PCR products were generated. The first used primers:
This generated a PCR fragment that was 792 bp in length and encompassed nucleotides 1729 to 2520 in the parent construct. The second PCR used primers: FW 5′-AGAATTCATGGAAGAATCTCCCGTAGCCA-3′ (SEQ ID NO:3) and RV 5′-ATAAGCGCTGGTCGAGCCTCCCCCACCGGT-3′ (SEQ ID NO:4). This generated a PCR fragment that was 7.3 kb in length and encompassed nucleotides 2521 to 1728 (wrapping). Each PCR product was digested with AfeI and EcoRI and ligated together to form the “new” parent construct, depicted below. Restriction sites on the top were introduced in the cloning reactions. Restriction sites on the bottom were present in the parent construct.
This construct was verified by sequencing.
Construct A is shown in
The next step was to PCR the FcγRI portion using primers with flanking NheI and EcoRI sites (FW: 5′-TGCTAGCGGAGGCGGAGGTTCTAGTGA-3′ (SEQ ID NO:5) and RV: 5′-AGAATTCAGTCGAGCCTCCCCCACCGGT-3′ (SEQ ID NO:6)). This PCR product and the new parent construct (above) were digested with NheI and EcoRI and ligated together to form the construct below.
This construct was verified by sequencing with the caveat that the middle FcγRI section could not be fully sequenced (see below). However, this region was sequenced in Construct A (shown in
Trivalent Anti-FcγRI-PspA (Construct B):
Construct A was then subject to inverse PCR for introduction of two new restriction sites downstream of PspA for cloning in the HuSA. One PCR was performed using the primers: FW 5′-ATCGTACGCACCACCACCACCACCACTGA-3′ (SEQ ID NO:7) and RV 5′-ATCGTACGTGTACAACCGCCTGATCCACCCTCGAGTTCTGGGGCTGGAGTTTC T-3′ (SEQ ID NO:8). This PCR product was digested with BsiWI and ligated together to form the following construct.
Bivalent Anti-FcγRI-PspA-HuSA (Construct D):
Construct C is shown in
The HuSA sequence was amplified using the following primers: FW 5′-tgtacactagagaagtgctgtgccgct-3′ (SEQ ID NO:9) and RV 5′-cgtacgtaagcctaaggcagcttgactt-3′ (SEQ ID NO:10). This PCR product and Construct C was digested with BsiWI and BsrGI and ligated together to form the following construct. This construct was verified by sequencing.
Bivalent Anti-FcγRI-PspA-HuSA (Construct D):
Construct D is shown in
Construct B was subject to the same inverse PCR as Construct A to form the following construct:
Trivalent Anti-FcγRI-PspA-HuSA (Construct F):
Construct E is shown in
The HuSA PCR product and Construct E was digested with BsiWI and BsrGI and ligated together to form the following construct. This construct was verified by sequencing with the caveat that the middle FcγRI section could not be fully sequenced (see below). However, this region was sequenced in Construct D.
Trivalent Anti-FcγRI-PspA-HuSA (Construct F):
Construct F is shown in
To form the non-targeting PspA-HuSA construct, Construct D was digested with AgeI, the large, vector band was gel extracted and ligated to form the following construct. This construct was verified by sequencing.
Non-FcγRI-Targeted PspA-HuSA (Construct G):
Construct G is shown in
Each construct has been verified by sequencing to the extent that the technology allows. The middle FcγRI domain for Construct B and Construct F could not be verified because unique sequencing primers could not be designed to cover this region due to the repetitive nature of the construct. However, because this region was sequence verified in Construct A and D, it is believed that it is mutation free in the trivalent forms.
For unknown reasons, the AfeI site that was introduced through modular PCR caused problems with sequencing reactions and subsequent PCR. Regardless, the constructs do cut with AfeI with the correct predicted sizes, therefore, it is believed that this site is structurally sound even though it has not been proven with sequence data.
A comparison of the proposed Sp and flu FP vaccines to licensed vaccines is presented in Table I. It is contemplated that the Ag component within the FP may be replaced with a molecule, which binds inactivated microorganisms such as inactivated F. tularensis (iFt), with which one of the present inventors has demonstrated the ability to protect against the highly lethal human virulent strain Ft SchuS4 via FcR targeting (i.n administered iFt). Importantly, this would add yet another major capability to this platform, significantly expanding its application to inactivated microbial vaccines.
One object of the present application is to identify a single FcR-targeted vaccine platform that affords optimal protection to bacterial and viral mucosal challenge utilizing mouse bacterial and viral infection models, which is also broadly applicable to the majority of respiratory biodefense (Table 2), as well as non-biodefense pathogens. However, it is recognized that the FP, DNA, or DNA prime-FP boost platforms could each provide unique advantages suitable to specific pathogens. Thus, these studies could also lead to the advancement of pathogen-specific vaccine design(s) for individual and combination vaccines, dependent on the pathogen (s) of interest (bacterial or viral), route of entry (mucosal or parenteral), and protective response(s) (humoral or cellular) required. Once the optimal vaccine regimen/platform is identified, similar studies will then be conducted in NHPs. Interim's will be: 1) To determine if increasing the valency of the hFcγRI targeting component in the FP is superior to the proven bivalent FP; 2) To determine if adding an FcRn targeting component to the FP will further increase the potency of the bivalent or trivalent FP; 3) To determine if a hFcγRI-targeted DNA vaccine is efficacious vs. non-targeted DNA vaccine; 4) To determine if a hFcγRI-targeted DNA prime-FP boost regimen is superior to DNA or FP regimens; 5) To determine if a multipathogen (combination) vaccine platform is efficacious by combining optimized Sp and flu vaccine platforms. Once NHP studies are completed demonstrating the efficacy of this approach in this model, the final platform formulation will be put into large-scale production. However, prior to initiating large scale production and clinical studies, the PspA and HA Ags used will be re-evaluated to determine if in the interim, superior Ags more appropriate for clinical application against these pathogens have been identified. Also, protective Ags against biodefense pathogens such as Ft may have been identified at this point, which could be used with this platform, expanding the potential for clinical trials with Category A-C biodefense pathogens, in addition to those pathogens utilized in these studies.
Coxiella burnetti (Q fever)
Brucella species
Burkholderia mallei
The contents of each of the below-identified references are hereby incorporated by reference.
This application claims priority to U.S. Provisional Application No. 61/938,607, filed Feb. 11, 2014, the entire content of which are hereby incorporated by reference.
This invention was made with government support under Grant No. 5R01 AI076408-04 awarded by the National Institute of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/015507 | 2/11/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/123359 | 8/20/2015 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6277375 | Ward | Aug 2001 | B1 |
| 7378504 | Graziano | May 2008 | B2 |
| Entry |
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| Number | Date | Country | |
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| 20170035878 A1 | Feb 2017 | US |
| Number | Date | Country | |
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| 61938607 | Feb 2014 | US |
