EP4637809A2 - Systems, methods, and compositions for preparing vaccines comprising inactivated/attenuated pathogens - Google Patents

Systems, methods, and compositions for preparing vaccines comprising inactivated/attenuated pathogens

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Publication number
EP4637809A2
EP4637809A2 EP23908307.4A EP23908307A EP4637809A2 EP 4637809 A2 EP4637809 A2 EP 4637809A2 EP 23908307 A EP23908307 A EP 23908307A EP 4637809 A2 EP4637809 A2 EP 4637809A2
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EP
European Patent Office
Prior art keywords
vaccine
mycobacterium
attenuated
pathogen
disease
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EP23908307.4A
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German (de)
French (fr)
Inventor
Raymond P. Goodrich
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Colorado State University Research Foundation
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Colorado State University Research Foundation
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Publication of EP4637809A2 publication Critical patent/EP4637809A2/en
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • 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
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing 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
    • A61K2039/55583—Polysaccharides
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/12011—Asfarviridae
    • C12N2710/12061—Methods of inactivation or attenuation
    • C12N2710/12063—Methods of inactivation or attenuation by chemical treatment
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • C12R2001/325—Mycobacterium avium
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the inventive technology is directed to the design, production, and use of vaccines, and in particular the design, production, and use of novel vaccines comprising an attenuated pathogen particle that have further been inactivated.
  • Vaccination is the most effective countermeasure for infection by pathogen in humans and various animal species.
  • Traditional vaccine production methods have included the use of live attenuated vaccines, as well as inactivated (killed) vaccines.
  • Both attenuated and inactivated vaccines each include certain technical and practical advantages, as well as disadvantages that limits their efficient deployment to combat infectious disease.
  • attenuated vaccines more closely mimic the natural infection cycle in a target host and activate all phases of the immune system. They can induce humoral IgG and local IgA, and raise immune responses to many protective antigens.
  • Attenuated vaccines provide a more durable immunity and are more cross- reactive. Moreover they are low-cost and they provide a quick immunity in the majority of cases.
  • Attenuated vaccines have several key limitations that prevent their effectiveness. Principally, it is often difficult to establish the appropriate level of attenuation that maintains the efficacy of the vaccine, while still being safe for use in humans and animals. In addition, all attenuated vaccines carry the possibility of reversion to their native virulence thereby raising the potential to cause, rather than prevent an outbreak of disease.
  • Inactivated vaccines have also been a mainstay of vaccinology for decades. Inactivated vaccines generally provide sufficient humoral immunity if boosters are given, and do not carry the risk of mutation or reversion. Inactivated vaccines can be used with immuno-deficient patients, and are generally considered safe. Examples of inactivated vaccines include constructs for influenza, cholera, bubonic plague and polio. Inactivated viral vaccines are typically made by exposing virulent pathogen, such as a virus to chemical or physical agents, for example, formalin or P -propiolactone, in order to destroy infectivity. However, exposure to such harsh chemical and/or physical agents may destroy viral epitopes, thus reducing or even destroying immunogen content and integrity. This reduces the efficacy of these vaccine preparations and requires greatly increased levels of product to induce immune effective immune responses against the native pathogen.
  • virulent pathogen such as a virus
  • chemical or physical agents for example, formalin or P -propiolactone
  • ASF African Swine Fever
  • ASFV African Swine Fever Virus
  • ASFV infections in swine are often fatal and are characterized by fever, hemorrhages, and ataxia.
  • the course of infection varies, ranging from highly lethal to sub-clinical, depending on host characteristics and the particular virus strain. At present, there is no vaccine available for ASFV and disease outbreaks are controlled by animal quarantine and slaughter.
  • the invention is directed to inactivated pathogen preparations which are created from strains of which have been attenuated for infectivity or pathogenicity by genetic manipulation or serial passage to isolate less infectious clones of the pathogen.
  • the preparation of inactivated/attenuated pathogens such a viral and/or bacterial pathogens, allows for vaccine preparations using starting materials that are not classified as high containment biol ogicals because they are not as infectious as the wild-type strains. This makes vaccine manufacturing much more facile.
  • the use of genetically modified strains can allow for removal of gene products that pose issues in inducing undesirable side effects such as antibody dependent enhancement (ADE)of disease manifestation.
  • AD antibody dependent enhancement
  • the present invention relates to inactivated/attenuated viral vaccines and methods for preparing the same. More specifically, in one preferred embodiment the present invention relates to methods for inactivation of attenuated viral particles, including ASFV viral particles, using a photosensitizer such as riboflavin in combination with UV light.
  • a photosensitizer such as riboflavin in combination with UV light.
  • Figure 1 shows a generalize schematic of the inactivation of an exemplary viral particle using UV light in the presence of riboflavin to disrupts the virus’ nucleic acid constituents in one embodiment thereof.
  • Figure 2 shows a plurality of ASFV particle samples that have been treated or treated to UV radiation, and cycle threshold (Ct mean) and viral quantity after treatment of 80 and 120 Joules, respectively.
  • Figure 3 shows UV treated and untreated ASFV particle samples showing hemadsorption or lack thereof. No hemadsorption in cultured ASFV at energy dose of 120 joules delivered in 1 minute treatment time. By PCR Amplification and quantification, ASFV treated at 120 Joules was detected in cells, though at lower quantities and with no increase upon culture. These data is consistent with virus entry into the cells, but without the ability of the virus to replicate at dose of 120 Joules.
  • Figure 4 show transcriptomics data of ASFV showing activation of Pl 3k pathway associated with viral binding and entry into cells by inactivated virus.
  • Figure 5 show a challenge study utilizing a mix of inactivated-ASF vaccine candidates with and without adjuvants.
  • the candidates were administered to pig models with 6 per group. 20 mg Advax Adjuvant was administered in the “Adjuvant Group.”
  • the pigs were inoculated with 10 4 median hemadsorption unit (HAD 50 ⁇ equivalents for vaccination.
  • HAD 50 median hemadsorption unit
  • Figure 6 shows results of ASFV in tissues post-challenge.
  • qPCR targeting p72 gene set up to detect ASFV in all tissues 6 days post infection.
  • ASFV was detected in all tissues analysed with values averaged per group of 6.
  • Figure 7 shows results of post-challenge serological analysis. These data show that competitive ELISA assay showed no antibodies for P72, except in PB310 (Control group) at day 32 (4 days post challenge). A virus neutralization assay demonstrated that Number of animals show negative neutralization (possibly ADE). Prime boost with adjuvant (G3) has highest number of animals showing virus neutralization (4/6) compared to other groups (1/6).
  • Post-challenge observations include: 1) Group 3 (Inactivated Virus + Boost + Adjuvant) showed lowest levels of tissue viremia; 2) Group 3 (Inactivated Virus + Boost + Adjuvant) showed highest levels of neutralizing antibody; 3) Gross pathologies in the vaccinated animals were more generally characteristic of inflammatory responses and not classical ASF pathology; 4) Control animals displayed classical signs of ASF infection in gross pathologies.
  • Figure 8 shows transcriptomic data demonstrating differentially expressed gene across different challenge groups.
  • Figure 9 shows transcriptomics analysis of the overrepresented gene ontologies in each group reveals.
  • the activation patterns demonstrated several key observations relative to T-cell activation pathways, specifically, for vaccinated groups, Gl, G2, G3, genes were activated for: 1) Cytokine Binding or Cytokine Receptor Binding; 2) Inflammatory Response; 3) Cytokine Production; and 4) Immune Effector Processes. None of these genes were upregulated in the placebo group (Group 4).
  • the present invention inactivated attenuated pathogens, and method of making the same.
  • inactivating a pathogen that has already been attenuated for its ability to induce disease allows for the production of vaccines under less stringent biocontainment conditions, preserves the benefits of having an attenuated pathogen while also preventing their reversion to their native form which can cause of negative disease outcomes.
  • inactivated/attenuated viruses have the advantage of not being able to replicate, thus shutting down the potential that the attenuated viruses could undergo recombination events and re-gain of function via genetic exchange that results in the return of pathogenicity and infectivity.
  • Attenuated pathogens which have been engineered to remove or alter problematic gene constructs that result in increased infectivity and undesirable disease side effects, are easier to handle in production environments due to decreased likelihood of disease spread. These agents can, however, still undergo replication and hence have the potential to re-gain function which returns native pathogenicity and disease outcomes.
  • Using an inactivation method which retains the desired characteristics of pathogen phenotype and antigen content and structure while completely removing the ability of these agents to replicate, could afford a means to create, safer and more effective vaccine candidates for viral, bacterial and parasitic agents.
  • the provides methods of inactivating attenuated pathogens, such as viral, microbial and/or parasitic pathogens using photochemical technology.
  • pathogens including viral and bacterial particles can be inactivated ultra-violet (UV) light in the presence of a photosensitizer, such as riboflavin.
  • UV ultra-violet
  • This method generally being referred to herein as the SolaVax method, disrupts the pathogen’s nucleic acid content without modification to proteins or antigen structure, can better maintain immunogenicity of the inactivated particles.
  • the photochemical disruption of nucleic acids of an attenuated pathogen is achieved using photosensitizers that can act as electron transfer agents.
  • photosensitizers that can act as electron transfer agents.
  • the application of photosensitizer agents that can be placed into an excited state in proximity to a guanine base in DNA or RNA constructs allows for selective modification (e.g., oxidation, crosslinking, fragmentation, deamination) of these bases. Because electron chemistry can only occur over short distances, the photosensitizer agent must be bound or associated with (e g., intercalated with) the nucleic acid in order to carry out the desired chemistry.
  • Attenuated pathogens included viruses, bacteria and parasites could be utilized in this approach of combining attenuation with inactivation using a method that preserves viral protein and antigen structure and function to the same degree as seen in attenuated pathogens alone, but without the potential for replication and hence re-gain of function.
  • vaccines for diseases such as ASF and can be produced. These are diseases which have been known to engage the use of attenuated viruses but where use of these constructs poses issues with re-gain of virulence.
  • compositions and methods for producing inactivated/attenuated vaccines including vaccines for viral, bacterial and parasitic pathogens.
  • the present inventions describes compositions and methods for producing inactivated/attenuated vaccine directed to ASFV.
  • the compositions and methods disclosed herein take advantage of a unique property of the photosensitizer riboflavin and UV light to selectively inactivate virus particles by directed damage to nucleic acids while preserving the integrity of the proteins and other viral antigens.
  • the nature of the photosensitizer riboflavin provides for low toxicity and thus easy handling, distribution and processing under even austere conditions.
  • compositions disclosed herein have several advantages compared to compositions and methods currently used in the art.
  • riboflavin is inexpensive, nontoxic and does not pose safety or environmental concerns, allowing for vaccine production in an austere environment with minimal infrastructure and personnel training needs.
  • the disclosed inactivation methods are more likely to preserve labile and sensitive antigen profdes that could be destroyed by the methods commonly used in the art.
  • the attenuated pathogen is inactivated in situ in its native form, and being inactivated cannot revert to its more virulent form.
  • the processes described herein inactivate nucleic acid replication without requiring additional processing steps to eliminate the replication potential of the attenuated pathogen, and do not require extended protein stability throughout processing.
  • compositions comprise, consist essentially of, or consist of attenuated viral particles or fragments or derivatives thereof, that are further inactivated, preferably by using photochemical technology, and optionally in combination with an adjuvant.
  • methods of viral attenuation are known in the art, or would be easily discernable by those of ordinary skill in the art.
  • the attenuated viral particles are inactivated by modifying their DNA and/or RNA, rendering them incapable of causing disease.
  • the modification of the attenuated viral DNA and/or RNA renders the attenuated virus incapable of replicating but does not kill the attenuated virus.
  • the inactivation process does not substantially alter the structure of antigens (e.g., protein and/or lipid antigens) on the attenuated viral particles, when administered to a subject in need thereof, the vaccines comprising the inactivated attenuated particles present antigenic targets to the subject’s immune system that are substantially identical to those present on a pathogenic, replication-competent virus.
  • Administration of the vaccine compositions to a subject in need thereof stimulates an immune response to the virus, therefore preventing or treating a viral infection in the subject.
  • the inactivated viral vaccine comprises, consists essentially of, or consists of attenuated viral particles inactivated using a photochemical process to inactivate the disease-causing ability of the virus while preserving protein structure and phenotype.
  • the DNA and/or RNA of the inactivated attenuated viruses comprises modified bases.
  • the DNA or RNA of the inactivated attenuated viruses may comprise modified guanine bases, such as oxidized guanine bases.
  • the inactivated attenuated viral particles are adenovirus particles, adeno-associated virus (AAV) particles, lentivirus particles, coronavirus particles or retrovirus particles.
  • the inactivated attenuated viral particles are inactivated ASFV particles.
  • the inactivated attenuated viral particles are chikungunya particles, SARS-CoV2 or MERS-coV particles.
  • the inactivated attenuated viral particles are porcine reproductive and respiratory syndrome (PRRS) particles.
  • PRRS reproductive and respiratory syndrome
  • the inactivated attenuated viral particles are polio particles.
  • the inactivated attenuated viral particles are influenza particles.
  • the inactivated attenuated viral particles are Dengue, Zika, Influenza (e.g., A, B, C), Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS- CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes (e.g., 1, 2, 6, 7, 8), Hepatitis (e.g., A, B, C, D, E), Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, or Vesicular Stomatitis particles.
  • Influenza e.g., A, B, C
  • Marburg Rabies
  • Human Immunodeficiency Virus HIV
  • Smallpox Hantavirus
  • Rotavirus SARS-CoV
  • the attenuated pathogen of the invention may include an attenuated vaccine compositions.
  • the attenuated pathogen may be an attenuated live virus, bacteria, parasite or fungus.
  • the he attenuated pathogen may be an attenuated live that is used and/or approved for use as a live attenuated vaccine which may be selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow
  • the attenuated pathogen of the invention may include an attenuated ASFV vaccine compositions.
  • the he attenuated ASFV may be selected from: (1) ASFV-G-AI177L (See Tran X.H., et al., Transbound Emerg Dis. 2022 Jul;69(4); (2) ASFV-G-A9GL/AUK (See Arias M, et. al., Vaccines (Basel). 2017 Oct 7;5(4):35.); and (3) ASFV-G-AI177LALVR (Id.)#
  • the attenuated pathogen of the invention may include an attenuated vaccine compositions for the treatment of one or more diseases or conditions in animals, which may be selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitio
  • an inactivated attenuated viral particle (e.g., an attenuated ASFV particle) comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more modified guanine bases, such as oxidized guanine bases, in its genome.
  • an attenuated ASFV particle comprises about 9 modified guanine bases in its genome.
  • an attenuated ASFV particle comprises about 16 modified guanine bases in its genome.
  • an attenuated ASFV particle comprises about 17 modified guanine bases in its genome. In some embodiments, an attenuated ASFV article comprises about 18 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 19 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 20 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 21 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 22 modified guanine bases in its genome. In some embodiments, a an attenuated ASFV particle comprises about 23 modified guanine bases in its genome. In some embodiments, a an attenuated ASFV particle comprises about 24 modified guanine bases in its genome.
  • the inactivated attenuated viral vaccine composition further comprises an adjuvant.
  • the effect of the adjuvant is to boost the immunological response.
  • the adjuvant modifies monocyte function.
  • the adjuvant promotes a Thl-type response over a Th2-type response.
  • Non-limiting examples of adjuvants capable of promoting a Thl-type response over a Th2-type response include CpG and/or AS01 .
  • Exemplary adjuvants are provided in Goodrich et al., (PCT/US2021/021190), such adjuvants being specifically incorporated herein by reference.
  • the adjuvant of the invention may include Advax®, which describes an adjuvant derived from inulin, a natural plant- derived polysaccharide that when crystallized in the delta polymorphic form, becomes immunologically active.
  • the vaccine composition comprises an adjuvant.
  • the adjuvant is capable of promoting a Thl-type immune response.
  • the adjuvant is capable of limiting a Th2-type response.
  • the adjuvant is CpG and/or AS01.
  • the adjuvant is a phosphorothioate oligonucleotide comprising about 15 to about 30 nucleotides.
  • the adjuvant comprises a nucleic acid that comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1).
  • the adjuvant comprises a nucleic acid that comprises the sequence 5'-TCCATGACGTTCCTGATGCT-3' (SEQ ID NO: 2). In some embodiments, the adjuvant is ODN 1668. In some embodiments, the adjuvant is CpG 1018. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient.
  • Suitable adjuvants include saponin formulations, virosomes, virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides (e.g., an immunostimulatory oligonucleotide containing a CpG motif), mineral containing compositions, oil-emulsions, polymers, micelle-forming adjuvants (e g., a liposome), immunostimulating complex matrices (e.g., ISCOMATRIX), particles, squalene, phosphate, cationic liposome-DNA complexes (CLDC), DDA, DNA adjuvants, gamma-insulin, ADP- ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund’s complete adjuvant, Freund’s incomplete adjuvant, muramyl dipeptides, monophosphoryl Lipid A (MPL), poly IC
  • a CpG ODN is a class A, class B or a class C CpG ODN.
  • a CpG ODN is CPG 7909 (InvivoGen, San Diego, CA).
  • Other suitable adjuvants include TLR agonists, NOD agonists, and lipid-DNA agonist complexes.
  • the inactivated attenuated viral vaccine composition further comprises one or more agonists or antagonists.
  • the agonist is a Toll-Like Receptor (TLR) agonist.
  • TLR Toll-Like Receptor
  • the TLR agonist is an agonist of one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, or TLR12.
  • the agonist is a TLR3 and/or a TLR9 agonist.
  • the antagonist is a C-C chemokine receptor type 2 (CCR2) antagonist.
  • the antagonist is an angiotensin receptor blocker (ARB), such as losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, or valsartan.
  • ARB angiotensin receptor blocker
  • the ARB is administered at a dose of between about 5 and about 100 mg/kg, for example about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg/kg.
  • the inactivated attenuated virus vaccine comprises at least one of (i.e., one of, two of, or all three of) a TLR agonist, a CCR2 antagonist and an ARB.
  • the agonist or antagonist e.g., a TLR3 and/or a TLR9 agonist
  • a liposome is contained within or coupled to a liposome.
  • Liposomes are spherical, self-enclosed vesicles composed of amphipathic lipids. Liposomes may be unilamellar, having one lipid bilayer membrane, or multilamellar, having two or more concentrically arranged bilayers. Suitable liposomes may have a selected mean particle size diameter of about 200-500 nm.
  • Various methods of preparing liposomes and encapsulation of therapeutic agents therein are well documented (see, for example, U.S. Pat. Nos.
  • Lipids for use in forming the liposomes described herein include vesicle-forming lipids having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PA phosphatidic acid
  • PI phosphatidylinositol
  • SM sphingomyelin
  • the selection of lipids and proportions can be varied to achieve a desired degree of fluidity or rigidity, to control stability, and/or to control the rate of release of an entrapped agent.
  • a suitable amount of a relatively unsaturated lipid such as PC
  • a relatively unsaturated lipid such as PC
  • at least 45- 50 mol % of the lipids used to form the liposome are PC.
  • the liposomes may also include lipids derivatized with a hydrophilic polymer such as polyethylene glycol (PEG).
  • Suitable hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, and hydrophilic peptide sequences.
  • Methods of preparing lipids derivatized with hydrophilic polymers are known (see e.g., U.S. Pat. No, 5,395,619, which is incorporated herein by reference).
  • the inactivated attenuated viral vaccine comprises cationic liposome-DNA complexes (CLDC).
  • the inactivated attenuated viral vaccine further comprises a photosensitizer such as riboflavin (vitamin B2).
  • a photosensitizer such as riboflavin (vitamin B2).
  • the inactivated attenuated viral vaccine is substantially free of photosensitizer.
  • the inactivated attenuated viral vaccine composition further comprises a carrier.
  • the cells and/or the photosensitizer are suspended in the carrier.
  • the carrier comprises normal saline (e.g., 0.9% sodium chloride), dextrose saline (e.g., dextrose 5% in 0.9% sodium chloride), phosphate buffered saline (e.g., 137 mmol/L NaCl, 2.7 mmol/L KC1, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4).
  • the inactivated attenuated viral vaccine composition further comprises one or more additional pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
  • a vaccine composition comprises an inactivated attenuated ASFV viral particle; wherein the composition comprises about 15 to about 50 picograms of viral protein (e.g., about 35 picograms of viral protein) and an adjuvant.
  • a vaccine composition comprises an inactivated attenuated ASFV viral particle; wherein the genome of the ASFV viral particle comprises about 1 to about 30 oxidized guanine residues (e.g., about 9 or about 20 oxidized guanine residues); wherein the structure of antigens on the viral particle is not substantially altered compared to ASFV viral particle that has not been attenuated and/or inactivated.
  • a vaccine composition comprises an inactivated attenuated ASFV viral particles from multiple ASFV strains.
  • a vaccine composition comprises an inactivated attenuated ASFV particle from a first strain, and a second inactivated attenuated ASFV particle from a separate strain.
  • a vaccine composition comprises inactivated attenuated ASFV particles from at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different attenuated ASFV strains.
  • the inactivated attenuated viral vaccines described herein are produced using an innocuous chemical agent in a selective process that prevents cellular replication processes while preserving antigenic protein structure. More specifically, the viral vaccines are produced by the combined application of a photosensitizer and light for rendering attenuated viral particles unable to cause disease i.e., inactivated.
  • the process for producing the inactivated attenuated viral vaccines of the disclosure is described in detail below.
  • the attenuated viral particles may be recombinant attenuated viral particles, or wild-type attenuated viral particles, that have, for example undergone serial passage to select for less virulent strains.
  • the attenuated viral particles are inactivated using photochemical technology.
  • photosensitizers that can act as electron transfer agents.
  • the application of photosensitizer agents that can be placed into an excited state in proximity to a guanine base in DNA or RNA constructs allows for selective modification (e.g. oxidation, cross-linking, fragmentation, deamination) of these bases.
  • the photosensitizer is a flavin, for example riboflavin (Vitamin B2), flavin mononucleotide, or flavin adenine dinucleotide.
  • the photosensitizer is a tertiary aliphatic amine (e.g., l,4-diazabicyclo(2,2,2)octane), a piperazine, (e.g., N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1,4-dimethylpiperazine), an amino acid (e g., tyrosine, tryptophan, histidine, methionine), an enzyme (e.g., superoxide dismutase) or EDTA (ethylenediaminetetraacetic acid).
  • the photosensitizer is riboflavin.
  • the attenuated viral particles are added to a solution containing the photosensitizer (e.g., riboflavin), or the photosensitizer is added to a solution containing the attenuated viral particles.
  • the photosensitizer e.g., riboflavin
  • the concentration of photosensitizer used during inactivation is about 10 pM to about 100 pM, such as about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 200 pM, about 300 pM, about 500 pM, or more.
  • the solution contains the photosensitizer at a concentration of about 1 pM to about 50 pM, such as about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 100 pM , about 200 pM, about 300 pM, about 500 pM, or more.
  • the photosensitizer concentration is less than about 10 pM, such as less than about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, or about 1 pM.
  • the solution containing the photosensitizer and the attenuated viral particles is then subjected to light treatment.
  • the light treatment comprises treatment with visible light, ultraviolet light, and/or infrared light.
  • the light treatment inactivates a nucleic acid (e.g., DNA and/or RNA) in the viral particles by modifying bases of these nucleic acids.
  • guanine bases are selectively modified.
  • guanine bases are selectively oxidized. Oxidized guanine bases cannot be repaired by natural enzymatic and cell repair mechanisms. As such, there is no possibility for reversion of the induced change to a form that would restore the ability of the treated viral particles to cause disease, or revert to their non-attenuated form.
  • the light treatment comprises treatment with ultraviolet (UV) light.
  • the UV light may be UV-A, UV-B, or UV-C light.
  • the UV light may have a wavelength of 170 to 400 nm, including all ranges and subranges therebetween.
  • the UV light has a wavelength of 315 to 400 nm, 310 to 320 nm, 280 to 360 nm, 280 to 315 nm, or 180 to 280 nm.
  • the UV light may be provided by UV light sources known in the art, such as the Mirasol® PRT Illumination device (TerumoBCT, Lakewood, Colorado).
  • the attenuated viral particles may be treated with multiple wavelengths of light simultaneously.
  • UV light having a wavelength of 310 to 320 nm is used. Without being bound by any theory, it is believed that this wavelength prevents riboflavin from reacting in free solution, which would result in production of undesirable oxygen free radicals. At these wavelengths, riboflavin will react only when intercalated with nucleic acid.
  • the dose of the UV light may vary depending on the volume of solution being treated. For example, the dose of the UV light may be between 200-400 Joules (e.g., 300 Joules) for a volume of about 170 to 370 mis of solution, while in alternative embodiments the dose may be between 80 to 120 Joules. As will be understood by those of skill in the art, the dosage may be adjusted up or down if the volume to be treated is above or below this range.
  • the dose of UV light may be from less than 50 Joules, about 50 to 100 Joules, about 100 Joules to about 200 Joules, about 200 Joules to about 600 Joules, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 Joules.
  • the volume of viral preparations for illumination may be from about 200 ml to about 600 ml, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 ml.
  • the dose of UV light may be from about 0.01 Joules/ml to about 1.0 Joules/ml. In some embodiments, the dose of UV light may be from about 0.5 Joules/ml to about 3.0 Joules/ml.
  • the dose of UV light may be about 0.1, about 0.2 about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0 Joules/ml.
  • the dose of UV light may be about 0.1 Joules/ml.
  • the dose of UV light may be from about 1 Joules/ml to about 10 Joules/ml, such as about 1.87 Joules/ml, about 3.74 Joules/ml, or about 6.24 Joules/ml.
  • the attenuated viral particles may be treated with UV light for about 1 minute to about 60 minutes, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes.
  • the attenuated viral particles are treated with UV light for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, or about 1 minute to about 3 minutes.
  • the attenuated viral particles are pre-incubated for a predetermined period of time in the solution containing the photosensitizer (e.g., riboflavin) before subjecting the viral particles to the light treatment.
  • the photosensitizer e.g., riboflavin
  • the attenuated viral particles are not subjected to any additional purification or modification steps after light treatment.
  • the inactivated attenuated viral particles are purified after the light treatment.
  • the inactivated attenuated viral particles are concentrated after the light treatment.
  • the inactivated attenuated viral particles are combined with one or more additional pharmaceutically acceptable carriers or ingredients as described herein after light treatment. In some embodiments, the inactivated attenuated viral particles are combined with a solution comprising an adjuvant after light treatment.
  • the inactivated attenuated viral particles generated using this method are incapable of replication processes or reversion to their virulent form, but substantially maintain and preserve the antigen and epitope profile of the original, native attenuated viral particle or antigen. In some embodiments, the inactivation process does not substantially change the phenotype or structure of the attenuated viral particles.
  • the inactivated attenuated viral particles that are produced by this process provide an improved source for antigen presentation.
  • a method for inactivating an attenuated ASFV viral particle comprises contacting the attenuated ASFV viral particle with a dose of UV light in the presence of riboflavin; wherein the dose of UV light is about 50 Joules to about 1000 Joules; wherein the method comprises selectively oxidizing about 1 to about 30 guanine bases (e.g., about 9 or about 30 guanine bases) in a nucleic acid (e.g., an RNA or a DNA) of the attenuated viral particle; and wherein the method does not comprise substantially altering the structure of antigens on the viral particle.
  • a dose of UV light is about 50 Joules to about 1000 Joules
  • the method comprises selectively oxidizing about 1 to about 30 guanine bases (e.g., about 9 or about 30 guanine bases) in a nucleic acid (e.g., an RNA or a DNA) of the attenuated viral particle; and wherein the method does not
  • compositions comprising the inactivated microbes disclosed herein.
  • the compositions comprise inactivated attenuated microbes, or fragments or derivatives thereof.
  • the compositions comprise inactivated attenuated microbes of more than one species.
  • methods of attenuating microbial pathogens are known in the art, or would be easily discernable to those of ordinary skill. Examples are described by Hajra, D., et al.,. (2021). Attenuation Methods for Live Vaccines.
  • the inactivated attenuated microbes substantially maintain and preserve the antigen and epitope profile, microbe integrity, and protein and/or lipid structure of the original, attenuated or native microbe or antigen. In some embodiments, the inactivated attenuated microbes are not capable of replicating or reverting to their more virulent form. In some embodiments, the inactivated attenuated microbes have modified genome (that is, modified DNA and/or RNA). In some embodiments, the DNA and/or RNA of the inactivated microbes comprises modified bases. In some embodiments, the modification of the microbial DNA and/or RNA renders the attenuated microbe incapable of replicating.
  • the modification of the microbial DNA and/or RNA does not kill the microbe.
  • the DNA or RNA of the inactivated attenuated microbes may comprise modified guanine bases, such as oxidized guanine bases.
  • an inactivated microbe comprises about 1 to about 100,000 modified guanine bases, for example about 10, about 100, about 1000, about 5000, about 10,000, about 15,000, about 20,000, about 50,000, about 75,000, about 100,000 or more modified guanine bases, including all subranges and values that lie therebetween, in its genome.
  • compositions disclosed herein do not comprise any adjuvant.
  • the compositions further comprise an adjuvant.
  • the adjuvant boosts the immunological response.
  • the adjuvant modifies monocyte function.
  • adjuvants includes saponin formulations, virosomes, microbe-like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides (e.g., an immunostimulatory oligonucleotide containing a CpG motif), mineral containing compositions, oil-emulsions, polymers, micelle-forming adjuvants (e g., a liposome), immunostimulating complex matrices (e.g., ISCOMATRIX), particles, squalene, phosphate, cationic liposome-DNA complexes (CLDC), DDA, DNA adjuvants, gammainsulin, ADP- ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund’s complete adjuvant, Freund’s incomplete adjuvant, muramyl dipeptides, monophosphoryl Lipider, advant, gambal
  • a CpG ODN is a class A, class B or a class C CpG ODN.
  • a CpG ODN is CpG 7909 (InvivoGen, San Diego, CA).
  • Other suitable adjuvants include TLR agonists, NOD agonists, and lipid-DNA agonist complexes.
  • the adjuvant is GLA-SE, which is a synthetic toll-like receptor 4 agonist (see, e.g., Behzad, H. et al, J Infect Dis., 2021, 205(3): 466- 473).
  • the adjuvant comprises GLA-SE in a squalene emulsion.
  • the inactivated attenuated microbial vaccine composition further comprises one or more agonists or antagonists as described above.
  • the agonist or antagonist e.g., a TLR3 and/or a TLR9 agonist
  • the inactivated attenuated microbial vaccine comprises cationic liposome- DNA complexes (CLDC).
  • the inactivated attenuated microbial vaccine further comprises a photosensitizer such as riboflavin (vitamin B2). In some embodiments, the inactivated attenuated microbial vaccine is substantially free of photosensitizer.
  • the inactivated attenuated microbial vaccine composition further comprises a carrier.
  • the cells and/or the photosensitizer are suspended in the carrier.
  • the carrier comprises normal saline (e.g., 0.9% sodium chloride), dextrose saline (e.g., dextrose 5% in 0.9% sodium chloride), phosphate buffered saline (e.g, 137 mmol/L NaCl, 2.7 mmol/L KC1, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4).
  • the inactivated attenuated microbial vaccine composition further comprises one or more additional pharmaceutically acceptable ingredients as described above.
  • an inactivated attenuated microbe is associated with and/or contained within a liposome, virosome, ISCOM (immunostimulatory complex) or virus-like particle.
  • ISCOM immunonostimulatory complex
  • virus-like particle a liposome, virosome, ISCOM (immunostimulatory complex) or virus-like particle.
  • an inactivated attenuated microbe is associated with and/or contained within a nanoparticle.
  • an inactivated microbe is associated with and/or contained within a Gantrez Nanoparticle (GNP).
  • GNPs Gantrez Nanoparticles
  • GNPs comprise a copolymer of methyl vinyl ether and maleic anhydride, which readily reacts with amino groups, making it easy to load or link different proteins thereto. Methods for production of GNPs are known, such as a solvent displacement method.
  • the inactivated microbe may be incorporated into the GNP during manufacture (i.e., embedded in the nanoparticle), or after the preparation of the GNP (i.e., coating the nanoparticle).
  • the nanoparticle is a poly(anhydride) nanoparticle. See, e.g., Irache et al, Frontiers in Bioscience S2, 876-890, 2010.
  • the nanoparticle has a diameter in the range of about 50 to about 500 nm, such as about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, or about 500 nm.
  • the nanoparticle further comprises an adjuvant.
  • an inactivated attenuated microbe is associated with and/or contained within a microparticle. In some embodiments, an inactivated attenuated microbe is associated with and/or contained within a poly-e-caprolactone (PCL) microparticle. See, e.g., Roban, B.S. et al, Clin Exp Allergy. 2007 Feb;37(2):287-95.
  • PCL poly-e-caprolactone
  • the microparticle has a diameter of about 1 to about 3 pm, such as about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9 or about 3.0 pm.
  • the microparticle further comprises an adjuvant.
  • an inactivated attenuated microbe is associated with a polymer.
  • an inactivated attenuated microbe is encapsulated by a polymer.
  • the polymer may be, for example, a polymer that enhances the stimulation of mucosal immunity.
  • the polymer is a biodegradable polymer.
  • the biodegradable polymer may be a copolymer of methyl vinyl ether and maleic anhydride (PVM/MA).
  • the copolymer has a molecular weight between 100 and 2400 kDa, such as between 200 and 2000 kDa or between 1880 and 250 kDa.
  • Illustrative polymers are described in US Pat. Nos. 8,628,801, 9,522,197 and 10,933,025, which are incorporated by reference in their entireties.
  • the polymer is a mucoadhesive polymer.
  • the polymer is a chitosan-based mucoadhesive polymer such as chitosan-cysteine, chitosan-4-thio- butylamidine, chitosan-thioglycolic acid, chitosan-glutathione, chitosan-6-mercaptonicotinic acid, chistoan-N-acetylcysteine, chitosan-4-mercaptobenzoic acid, or chitosan-N-acetylpenicillamine.
  • chitosan-based mucoadhesive polymer such as chitosan-cysteine, chitosan-4-thio- butylamidine, chitosan-thioglycolic acid, chitosan-glutathione, chitosan-6-mercaptonicotinic acid, chistoan-
  • the polymer is a eudragit-based mucoadhesive polymer.
  • the polymer is a hybrid polymer, such as poly (lactic-co-glycolic acid) (PLGA).
  • PLGA poly (lactic-co-glycolic acid)
  • the polymer may allow for more efficient delivery of the inactivated attenuated microbe using an oral or nasal route, as compared to a microbe that is not encapsulated by a polymer.
  • a vaccine composition may comprise an inactivated attenuated microbe and a PLGA copolymer, such as poly(lactide) homopolymer (PLA) or poly(lactic-co- glycolic acid) copolymer (PLGA).
  • PLGA poly(lactide) homopolymer
  • PLGA poly(lactic-co- glycolic acid) copolymer
  • the inactivated attenuated microbe is comprised in a PLGA nanoparticle.
  • the disclosure provides methods of inactivating attenuated microbes using photochemical technology.
  • the methods comprise establishing an attenuated microbe, and further contacting the attenuated microbe with a dose of light in the presence of a photosensitizer.
  • the attenuated microbes are added to a solution containing the photosensitizer, or the photosensitizer is added to a solution containing the attenuated microbes.
  • the attenuated microbes are cultured, grown, or stored in the presence of the photosensitizer.
  • the photosensitizer is added to the growth media of the microbes.
  • the photosensitizer is added to the storage media of the attenuated microbes.
  • a solution containing the photosensitizer and the attenuated microbes (optionally, in media) is subjected to light treatment.
  • the attenuated microbes are preincubated for a predetermined period of time in the solution containing the photosensitizer before subjecting the attenuated microbes to the light treatment.
  • the attenuated microbes are cultured for a predetermined period of time in media containing the photosensitizer before subjecting the attenuated microbes to the light treatment.
  • the methods further comprise a step of breaking up clumps or aggregates of attenuated microbial cells, and/or of preventing clumping or aggregation of attenuated microbial cells.
  • Clumping may be prevented and/or disrupted using any physical technique known in the art, for example, by sonicating, vortexing, or pipetting; using any chemical means known in the art, such as using chelators such as EDTA (ethylenediaminetetraacetic acid) and citrate; and/or using any biological means known in the art, such as using an enzyme (e.g., a peptidase, for example, trypsin).
  • the step of breaking up clumps of attenuated microbial cells, and/or preventing clumping of microbial cells may be performed before and/or after the addition of the photosensitizer. In some embodiments, the step of breaking up clumps of attenuated microbial cells, and/or to preventing clumping of attenuated microbial cells may be performed in the presence of the photosensitizer.
  • the solution comprising attenuated microbial cells further comprises an additive that protects cells from hydrodynamic damage.
  • the additive is a detergent.
  • the type of detergent is not limited, and may be any detergent that is used in the presence of cells.
  • the detergent is a non-ionic detergent.
  • the detergent is Pluronic® F-68.
  • the additive is polyethylene glycol (PEG).
  • the photosensitizer is a flavin, for example riboflavin (Vitamin B2), flavin mononucleotide, or flavin adenine dinucleotide.
  • the photosensitizer is a tertiary aliphatic amine (e.g., l,4-diazabicyclo(2,2,2)octane), a piperazine, (e.g., N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1,4-dimethylpiperazine), an amino acid (e.g., tyrosine, tryptophan, histidine, methionine), an enzyme (e.g., superoxide dismutase) or EDTA.
  • the photosensitizer is riboflavin.
  • the concentration of photosensitizer used during inactivation is about 10 pM to about 1 mM, for example, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, or about 1 mM, including all values and subranges therebetween.
  • the solution or media containing the photosensitizer contains the photosensitizer at a concentration of about 1 pM to about 50 pM, for example, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM, including all values and subranges therebetween.
  • the photosensitizer concentration in the solution or media is less than about 10 pM, for example, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM, including all values and subranges therebetween.
  • the light treatment comprises treatment with visible light, ultraviolet light, and/or infrared light.
  • the light treatment inactivates a nucleic acid e.g., DNA and/or RNA) in the microbes by modifying bases of these nucleic acids.
  • guanine bases are selectively modified.
  • guanine bases are selectively oxidized. Oxidized guanine bases cannot be repaired by natural enzymatic and cell repair mechanisms. As such, there is no possibility for reversion of the induced change to a form that would restore the ability of the microbes to cause disease.
  • the light treatment comprises treatment with ultraviolet (UV) light.
  • the UV light may be UV-A, UV-B, or UV-C light.
  • the UV light may have a wavelength of 170 to 400 nm, including all values and subranges therebetween.
  • the UV light has a wavelength of 315 to 400 nm, 310 to 320 nm, 280 to 360 nm, 280 to 315 nm, or 180 to 280 nm.
  • the UV light may be provided by UV light sources known in the art, such as the Mirasol® PRT Illumination device (TerumoBCT, Lakewood, Colorado).
  • the microbes may be treated with multiple wavelengths of light simultaneously.
  • UV light having a wavelength of 310 to 320 nm is used. Without being bound by any theory, it is believed that this wavelength prevents riboflavin from reacting in free solution, which would result in production of undesirable oxygen free radicals. At these wavelengths, riboflavin will react only when intercalated with nucleic acid.
  • the dose of the UV light may vary depending on the volume of solution being treated.
  • the dose of the UV light is in the range of less than 50 Joules, about 50 to 100 Joules, about 100 Joules to about 200 Joules, about 100 Joules to about 50,000 Joules, for example, about 200 Joules, about 300 Joules, about 400, about 500, about 600 Joules, about 700 Joules, about 800 Joules, about 900 Joules, about 1000 Joules, about 2000 Joules, about 3000 Joules, about 4000 Joules, about 5000 Joules, about 6000 Joules, about 7000 Joules, about 8000 Joules, about 9000 Joules, about 10,000 Joules, about 11,000 Joules, about 12,000 Joules, about 13,000 Joules, about 14,000 Joules, about 15,000 Joules, about 16,000 Joules, about
  • the volume of attenuated microbial preparations for illumination is in the range of about 200 ml to about 600 ml, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 ml, including all values and subranges that lie therebetween.
  • the volume of microbial preparations for illumination is in the range of about 170 mL to about 370 mL of solution. In some embodiments, the volume of microbial preparations for illumination is about 300 mL.
  • the dose of UV light is in the range of about 0.3 Joules/ml to about 170 Joules/ml, for example, about 0.4, about 1.0, about 2.0, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 Joules/ml, including all values and subranges that lie therebetween.
  • the dose of UV light may be about 30 Joules/ml. In some embodiments, the dose of UV light may be about 60 Joules/ml.
  • the attenuated microbes may be treated with UV light for about 1 minute to about 60 minutes, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes, including all values and subranges that lie therebetween.
  • the attenuated microbes are treated with UV light for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, or about 1 minute to about 3 minutes.
  • the solution being treated comprises about 10 attenuated microbial cells to about 10 12 attenuated microbial cells, for example, about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about IO 10 , about 10 11 , or about 10 12 attenuated microbial cells, including all values and subranges that lie therebetween.
  • the solution being treated comprises about 10 4 attenuated microbial cells to about 10 6 attenuated microbial cells.
  • the solution being treated comprises about IO 3 attenuated microbial cells.
  • the microbe is a bacterium, a fungus, or a parasite. In some embodiments, the microbe is capable of infecting a subject, for example, a human or animal subject.
  • the attenuated microbe is an attenuated bacterium.
  • the attenuated bacterium is selected from the group consisting of Actinomyces israelii, Acinetobacter baumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difficule, Clostridium neoformans, Clostridium perfringens, Clo
  • the attenuated microbe is a fungus.
  • the attenuated fungus is selected from the group consisting of Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladiella mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium
  • the attenuated microbe is an attenuated parasite.
  • the attenuated parasite is selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coli, Blastocystis hominis, Cyclospora cayetanen
  • the attenuated microbe belongs to the genus Mycobacterium. In some embodiments, the microbe belongs to the Mycobacterium tuberculosis complex. In some embodiments, the attenuated microbe is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, and Mycobacterium mungi. In some embodiments, the attenuated microbe is Mycobacterium tuberculosis.
  • the microbe is the Erdman strain, the H37Rv strain, or the CDC1551 strain of Mycobacterium tuberculosis . In some embodiments, the microbe is the H37Rv strain of Mycobacterium tuberculosis.
  • the microbe is Mycobacterium leprae, Mycobacterium lepromatosis, or nontub er culous mycobacteria (NTM). In some embodiments, the microbe belongs to the Mycobacterium avium complex. In some embodiments, the microbe is selected from the group consisting of Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare.
  • the microbe is resistant to an antibiotic.
  • the antibiotic is penicillin, isoniazid, clarithromycin, fluoroquinolone, amikacin, kanamycin, capreomycin, and/or rifamycin.
  • the photosensitizer enters the microbial cell in a passive manner.
  • the photosensitizer is imported into the microbes by pinocytosis, or clathrin-mediated endocytosis.
  • the microbe actively imports the photosensitizer.
  • the microbe is genetically engineered to import an amount of photosensitizer that is higher than the amount of photosensitizer imported into a wildtype microbial cell under the same conditions. In some embodiments, the microbe is genetically engineered to overexpress one or more receptors of the photosensitizer. In some embodiments, the microbe comprises a polynucleotide encoding one or more receptors of the photosensitizer.
  • the attenuated microbe is genetically engineered to overexpress one or more receptors of riboflavin.
  • the attenuated microbe comprises a polynucleotide encoding one or more receptors of riboflavin.
  • the receptor of riboflavin is not limited, and may be any receptor known in the art, or discovered in the future, to import riboflavin into a microbial cell.
  • the receptor is RibM (GenBank Accession No: K4REQ6), RibN (GenBank Accession No: Q1MIM3), RfuABCD (GenBank Accession No: Q56328), RibU (GenBank Accession No: Q5M614), ImpX (GenBank Accession No: D5RAW5), RfnT (GenBank Accession No: A6X7E7), or RibV (GenBank Accession No: Q6F0N9), or a functional ortholog or variant thereof.
  • the receptor is any one of the bacterial riboflavin receptors described in Gutierrez-Preciado A, et al., PLoS One. 2015; 10(5), which is incorporated herein by reference in its entirety.
  • the attenuated microbes are not subjected to any additional purification or modification steps after light treatment.
  • the inactivated attenuated microbes are purified after the light treatment.
  • the inactivated attenuated microbes are concentrated after the light treatment.
  • the photosensitizer is removed from the solution containing the inactivated attenuated after light treatment.
  • the inactivated attenuated microbes are combined with one or more additional pharmaceutically acceptable carriers after light treatment. In some embodiments, the inactivated attenuated microbes are combined with a solution comprising an adjuvant after light treatment.
  • the method comprises selectively oxidizing guanine bases in a nucleic acid of the attenuated microbe.
  • the inactivation of the attenuated microbes is, at least in part, associated with, caused by, or results from the selective oxidation of guanine bases in a nucleic acid of the attenuated microbe.
  • the method comprises selectively oxidizing at least about 1 to at least about 100,000 guanine bases in a nucleic acid (e.g., a DNA or an RNA) of the microbe, for example at least 10, at least 100, at least 1000, at least 5000, at least 10,000, at least 15,000, at least 20,000, at least 50,000, at least 75,000, at least 100,000 or more guanine bases, including all subranges and values that lie therebetween.
  • a nucleic acid e.g., a DNA or an RNA
  • the inactivation process does not substantially change the phenotype or structure of the attenuated microbes, or the phenotype or structure of the antigens on the surface of the attenuated microbes.
  • the method comprises contacting an attenuated strain of Mycobacterium tuberculosis H37Rv cells with about 8,640 J of UV light in the presence of about 50 pM of riboflavin in a volume of about 300 mb. In some embodiments, the method comprises contacting an attenuated strain of Mycobacterium tuberculosis H37Rv cells with about 17,280 J of UV light in the presence of about 50 pM of riboflavin in a volume of about 300 mb In some embodiments, the method further comprises sonication of the attenuated strain of Mycobacterium tuberculosis cells.
  • the inactivated attenuated pathogen vaccine compositions described herein can be used as vaccine agents or stimulants for immune system priming and recognition that foster immune responses in subjects.
  • the disease may be any disease or infection caused by any one of the pathogens described herein.
  • the inactivated attenuated viral vaccine may be administered to a subject to treat or prevent a viral infection, such as a ASF infection.
  • the subject is assessed for immune function and immune status prior to administration of the vaccine.
  • assessments may include, but are not limited to, DTH skin testing, blood tests, lymph node aspirate tests, tumor tissue tests, and/or determination of whether the subject is anergic, B cell responsive, etc.
  • the subject is not assessed for immune function and immune status prior to administration of the vaccine.
  • the subject is immunocompetent. In some embodiments, the subject is immunocompromised.
  • the vaccine may be used in combination with genetic testing to quantify the degree of immune-responders, or immune non-responders.
  • the vaccine composition comprises about 1 x 10 3 , about 1 x 10 4 , about 1 x IO 5 , about 1 x 10 6 , about 1 x 10 7 , about 1 x 10 8 , about 1 x 10 9 , or about 1 x 10 10 inactivated attenuated pathogen particles.
  • a inactivated attenuated vaccine composition comprises about 1 x 10 5 to about 1 x 10 8 pathogen particles.
  • an inactivated attenuated vaccine dose further comprises an adjuvant.
  • the adjuvant is CpG.
  • the CpG is CpG 7909.
  • a vaccine dose comprises from about 25 pg to about 750 pg CpG, or from about 50 pg to about 500 pg CpG.
  • a inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg CpG.
  • the adjuvant is CpG 1018.
  • a vaccine dose comprises from about 25 pg to about 750 pg CpG 1018, or from about 50 pg to about 500 pg CpG 1018.
  • an inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg CpG 1018.
  • the adjuvant is ODN 1668.
  • an inactivated attenuated vaccine dose comprises from about 25 pg to about 750 pg ODN 1668, or from about 50 pg to about 500 pg ODN 1668.
  • an inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg ODN 1668.
  • about IxlO 5 to about IxlO 8 inactivated attenuated pathogen particles are administered to a subject in each administration.
  • about 1x10’, about 5xl0 5 , about IxlO 6 , about 5xl0 6 , about IxlO 7 , about 5xl0 7 , or about 1x108 inactivated attenuated pathogen particles may be administered to a subject per administration.
  • the administered dose is a split dose, wherein the total number of inactivated attenuated pathogen particles for administration is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sub-doses.
  • about 10 to about 100 micrograms of inactivated attenuated pathogen protein are administered to a subject in each administration.
  • about 1 to about 100 picograms of pathogen protein are administered to a subject in each administration.
  • about 15 to about 50 picograms, or about 30 to about 40 picograms of pathogen protein are administered to a subject in each administration.
  • about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 picograms of pathogen protein may be administered to a subject in each administration.
  • about 10 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 11 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 12 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 13 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 14 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 15 picograms of pathogen protein are administered to a subject in each administration. Tn some embodiments, about 16 picograms of pathogen protein are administered to a subject in each administration.
  • the administered dose is a split dose, wherein the total amount of pathogen protein for administration is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sub-doses.
  • One or more sub-dose may be administered to the subject peripherally, at different locations on the subject’s body.
  • Each sub-dose may be administered at approximately the same time, or administration of the sub-doses may be staggered.
  • sub-doses may be administered at intervals of 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 3 hours.
  • the inactivated pathogen vaccine is administered once, or more than once to the subject. In some embodiments, the inactivated attenuated vaccine is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times to the subject.
  • the inactivated attenuated pathogen vaccine may be administered to the subject every day, about every 3 days, about every 7 days, about every fourteen days, about once per month, or about once per year.
  • the inactivated attenuated vaccine is administered at least once per week, at least every two weeks, or at least once every six months.
  • the vaccine is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, twelve times, fifteen times, twenty times, or twenty-five times in a year.
  • a first dose e.g., a prime dose
  • a second dose e.g., a boost dose
  • the second dose is administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after the first dose.
  • the amount of pathogen protein in the first dose is greater than the amount of pathogen protein in the second dose.
  • the amount of pathogen protein may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% greater than the amount of pathogen protein in the first dose.
  • the amount of pathogen protein in the first dose is less than the amount of pathogen protein in the second dose.
  • the amount of pathogen protein may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than the amount of pathogen protein in the first dose.
  • the amount of pathogen protein in the first dose is about the same as the amount of pathogen protein in the second dose.
  • a first inactivated attenuated pathogen vaccine and a second inactivated attenuated pathogen vaccine are administered to the subject.
  • the second inactivated attenuated pathogen vaccine is administered after the first vaccine to boost the immune response.
  • immune response in the subject is monitored between administration of the first vaccine and the second vaccine.
  • the second vaccine is administered when it is determined that the subject has not exhibited a satisfactory immune response following administration of the first vaccine.
  • the inactivated attenuated pathogen vaccine may be delivered to the subject intramuscularly, intramucosally, intranasally, subcutaneously, intratumorally, intradermally, transdermally, intravaginally, intraperitoneally, intrarectally, intra-articularly or intra- lymphatically, orally or intravenously.
  • administration may be by sublingual, buccal, intra-organ (e g., intrasplenic), or inhaled routes.
  • the vaccine composition may be in the form of a parenterally acceptable aqueous solution which has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
  • the vaccine is administered peripherally to the subject. In some embodiments, multiple aliquots of the vaccine are administered peripherally to the subject, in different locations.
  • the inactivated attenuated vaccine is administered simultaneously or sequentially (either before or after) with a vaccine-enhancing agent.
  • the vaccine-enhancing agent is an angiotensin receptor blocker (ARB) or a beta blocker (BB).
  • Exemplary vaccine-enhancing agents include losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, valsartan, propranolol, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol.
  • the vaccine-enhancing agent is selected from the group consisting of losartan and propranolol.
  • the inactivated attenuated vaccine-enhancing agent is losartan.
  • the vaccineenhancing agent is propranolol.
  • a method for vaccinating a subject comprises administering an inactivated attenuated pathogen vaccine composition comprising inactivated attenuated pathogen particles, and a potent adjuvant comprising TLR3 and/or TLR9 agonists attached to liposomes, and also comprises sequential or simultaneous administration of a vaccine-enhancing agent (e.g., losartan), which is given at or around the time of vaccination and reduces recruitment of immune suppressive myeloid cells.
  • a vaccine-enhancing agent e.g., losartan
  • a method for vaccinating a subject comprises administering an inactivated attenuated pathogen vaccine composition comprising inactivated attenuated pathogen particles to a subject in need thereof.
  • An adjuvant may optionally be administered at the time of vaccination.
  • an adjuvant is administered after vaccination to boost the immune response, for example about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after vaccination.
  • the adjuvant comprises liposomes, e.g., CLDC.
  • a vaccine-enhancing agent such as losartan may be administered at or around the time of the vaccination.
  • a vaccine-enhancing agent such as losartan may be administered after vaccination, for example, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after vaccination.
  • a vaccine-enhancing agent such as losartan may be administered to the subject daily for an effective number of days, optionally beginning on the day that the vaccine is administered.
  • the vaccineenhancing agent e.g., losartan
  • the vaccineenhancing agent is administered at a dose of between about 5 and about 100 mg/kg, for example about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg/kg.
  • the inactivated attenuated pathogen vaccine may elicit an immune response in the subject.
  • the immune response may include one or more of the following: (i) upregulation of immunoglobulin expression (e.g., IgG, IgM), (ii) T-cell activation, (iii) modulation of innate immune cells (e.g., myeloid cells), and (iv) revival of “exhausted” T-Cell populations.
  • Suitable subjects include both avians and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets, and the like.
  • mammals as used herein includes, but is not limited to, humans, nonhuman primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.
  • Human subjects include neonates, infants, juveniles, adults and geriatric subjects.
  • kits for treating and/or preventing a ASF infection in a subject in need thereof comprise administering to the subject an effective amount of one or more inactivated attenuated vaccine compositions described herein.
  • the subject may be, for example, a mammal such as a pig.
  • the inactivated attenuated vaccine is orally or administered intramuscularly. In some embodiments, the inactivated attenuated vaccine is administered subcutaneously. In some embodiments, the methods comprise administering a booster dose of the vaccine composition.
  • the booster dose may be administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after administering the vaccine composition.
  • the number of ASFV particles in the first vaccine composition is greater than the number of ASFV particles in the second vaccine composition.
  • the number of ASFV particles in the first vaccine composition is less than the number of ASFV particles in the second vaccine composition.
  • the number of ASFV particles in the first vaccine composition is about the same as the number of ASFV particles in the second vaccine composition.
  • the second vaccine composition may be administered, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after the first vaccine composition.
  • An immunogen is a molecule capable of eliciting an immune response in a subject.
  • compositions described herein can be used to provoke an immune response in a host.
  • a composition comprising an inactivated attenuated pathogen is administered to a host, in order to provoke an immune response in the host.
  • the immune response may comprise, for example, production of antibodies against various different antigens on the inactivated microbe.
  • an immunogen may be used to stimulate a subject’s immune system to produce antibodies against a pathogen (e.g., ASFV). Because an intact inactivated attenuated pathogen is used, the subject’s immune system will produce antibodies directed against a wide array of pathogen antigens (i.e., polyclonal antibodies).
  • polyclonal antibodies refers to antibodies that are secreted by different B cell lineages.
  • a monoclonal antibody comes from a single-cell lineage.
  • a polyclonal antibody comprises a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope.
  • the host produces a polyclonal antibody against the microbe that was administered.
  • the polyclonal antibody will typically include a mixture of pathogen produced by different B cell lineages, and each antibody may identify a different epitope.
  • a method of producing a polyclonal antibody that binds to a pathogen comprises i) generating an inactivated attenuated pathogen by contacting the microbe with a dose of UV light in the presence of riboflavin; ii) administering the inactivated attenuated pathogen to a host, wherein the host produces a polyclonal antibody; and iii) recovering the polyclonal antibody.
  • the microbe is tuberculosis.
  • the antibodies can be harvested and purified and can be used therapeutically to treat subjects with a disease (e.g., ASF).
  • the subject may be, for example, a mammal or an avian, as described above.
  • the subject is a pig.
  • the subject is a human.
  • an inactivated attenuated pathogen e.g., ASFV
  • ASFV inactivated attenuated pathogen
  • the immune response may comprise, for example, production of antibodies that bind to and/or neutralize the pathogen.
  • an immunogenic composition comprises a polyclonal antibody that binds to a pathogen, wherein the polyclonal antibody is produced by administering to a host an inactivated attenuated pathogen, wherein the inactivation is performed by contacting the attenuated pathogen with a dose of UV light in the presence of riboflavin as described herein.
  • the attenuated pathogen is an attenuated viral particle selected from: adenovirus particle, an adeno-associated virus (AAV) particle, a lentivirus particle, a coronavirus particle, or a retrovirus particle.
  • the viral particle is a ASFV particle.
  • the inactivated attenuated viral particle is a Dengue, Zika, African Swine Fever, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, or Vesicular Stomatitis particle.
  • HCV Human Immunodeficiency Virus
  • CMV Cytomegalovirus
  • Ebola Epstein-Barr
  • Herpes Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus
  • the composition comprises an adjuvant.
  • the adjuvant is capable of promoting a Thl-type response.
  • the composition comprises a pharmaceutically acceptable carrier or excipient.
  • the host is a mammal. In some embodiments, the host is a nonhuman primate, a bovine, an ovine, a caprine, an equine, a feline, a canine, a rodent or a lagomorph. In some embodiments, the host is a pig or human. In some embodiments, the host is an avian. In some embodiments, the host is a chicken, a duck, a goose, a quail, a turkey, a pheasant, a parrot, or a parakeet.
  • the inactivated attenuated viral particle is ASFV
  • the disease or disorder is ASF
  • the immunogenic composition is administered intravenously to the subject. In some embodiments, the immunogenic composition is administered intramuscularly to the subject. In some embodiments, the subject is a human or a pig.
  • a method of producing a polyclonal antibody that binds to a pathogen particle comprises generating an inactivated attenuated pathogen particle by contacting the particle with a dose of UV light in the presence of riboflavin; administering the inactivated attenuated viral particle to a host, wherein the host produces a polyclonal antibody; and recovering the polyclonal antibody.
  • the pathogen is an inactivated attenuated ASFV particle.
  • the host is a chicken, and the polyclonal antibody is recovered from an egg produced by the host.
  • the host is a pig.
  • the polyclonal antibody is recovered from the blood of the host.
  • the polyclonal antibody is recovered from B cells of the host.
  • polyclonal antibodies produced by the methods described herein.
  • the viral particle is a ASFV particle.
  • the biological sample is whole blood, serum, plasma, urine, saliva, lymph fluid, bile, cerebrospinal fluid, nasal mucus, or stool.
  • the polyclonal antibody is conjugated to a substrate.
  • the substrate is a bead, a chip, a slide or a dish.
  • the detecting step comprises contacting the polyclonal antibody with a secondary antibody that is conjugated to an enzyme or to a fluorophore.
  • polyclonal antibodies produced the immunogenic compositions as described herein can be used to treat or prevent a disease in a subject in need thereof.
  • a polyclonal antibody generated in a host may be administered to a subject to treat or prevent a viral infection, such as a ASFV infection.
  • a method for treating or preventing a disease or disorder in a subject in need thereof comprises administering to a subject in need thereof an effective amount of a polyclonal antibody as described herein.
  • a method for treating or preventing a ASF in a subject in need thereof comprises administering to a subject in need thereof an effective amount of a polyclonal antibody as described herein. The effective amount may be determined according to standard practices.
  • the titer of circulating virus may be evaluated, and the dose of antibody required to achieve a specific neutralization of the virus may be established through Plaque Reduction Neutralization Tests in vitro. This information may subsequently be used to provide a suitable dose estimate for therapy.
  • the subject is assessed for immune function and immune status prior to administration of the composition. Such assessments may include, but are not limited to, DTH skin testing, blood tests, lymph node aspirate tests, tumor tissue tests, and/or determination of whether the subject is anergic, B cell responsive, etc.
  • Attenuated pathogens inactivated according to the methods disclosed herein may be used in diagnostic compositions and methods.
  • the inactivated attenuated pathogens for example, ASFV
  • ASFV may be used to detect the presence of an anti-pathogen antibody (e.g, a neutralizing antibody) in a biological sample.
  • the biological sample may be, for example, blood (e.g., whole blood, serum, or plasma), stool, urine, saliva, or swab specimens of the nostril, throat, cervix, or urethra. Because intact inactivated attenuated pathogens are used, antibodies that bind to many different targets (i.e., different microbial epitopes) may be detected using a single assay.
  • an inactivated attenuated pathogen (e.g., a ASFV particle) is coupled to and/or immobilized on a substrate.
  • the substrate may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, fdms, plates, slides, etc.
  • the substrate may have any convenient shape, such as a disc, square, sphere, and a circle.
  • the substrate may, in some embodiments, form a rigid support. In some embodiments, the substrate and its surface may be chosen to provide appropriate light-absorbing characteristics.
  • the substrate may be a polymerized Langmuir Blodgett fdm, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, or any one of a wide variety of polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, or combinations thereof.
  • the substrate may be a bead, a resin, a membrane, a fiber, a polymer, a matrix, a chip, a microplate or a tissue culture vessel.
  • the substrate is pre-treated before the inactivated attenuated pathogen is coupled thereto.
  • the substrate may be treated with an enzyme, such as a DNAse, RNAse or protease.
  • the substrate may be coated with a polymer or a carbohydrate.
  • the substrate may be coated with a protein, such as an antibody, antibody fragment (e.g., Fab), or antibody derivative (e.g., a single chain variable fragment (scFv)).
  • the inactivated attenuated pathogen particle may be coupled to the substrate using various different methods.
  • the inactivated attenuated pathogen particle may be reversibly or irreversibly coupled to the substrate.
  • the inactivated attenuated pathogen particle is coupled to the substrate via a linker.
  • the linker may be a chemical or a protein linker.
  • the chemical linker may be, for example, a carbohydrate linker, a polyether linker, a fatty acid linker, or a lipid linker.
  • the protein linker may comprise about 1 to about 50 amino acids.
  • the protein linker is a flexible linker (i.e., a linker that comprises small polar amino acids, including threonine, serine, and/or glycine). In some embodiments, the protein linker is not flexible (i.e., a linker that comprises one or more proline residues).
  • the inactivated attenuated pathogen particles described herein may be used in a method for detecting the presence of an antibody in a biological sample.
  • the antibody may be an antibody that binds to the inactivated attenuated pathogen particle, such as a neutralizing antibody.
  • the method for detecting an antibody in a biological sample comprises contacting the biological sample with a virus particle (e.g., a ASFV particle) that is coupled to a substrate.
  • the antibody binds to the pathogen particle that is coupled to the substrate, thereby immobilizing the antibody.
  • the method further comprises contacting the immobilized antibody with a second antibody, such as a detection antibody.
  • the detection antibody may be coupled to, for example, a fluorophore, or to an enzyme (e g., horseradish peroxidase (HRP)) capable of cleaving a substrate (e g., a fluorescent substrate).
  • HRP horseradish peroxidase
  • the inactivated attenuated pathogen particles described herein may be used in an ELISA-based assay.
  • An ELISA enzyme-linked immunosorbent assay
  • An antigen is typically immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection may be accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product.
  • ELISAs There are several different types of ELISAs commonly used, including direct ELISAs, indirect ELISAs, sandwich ELISAs, and competitive ELISAs.
  • an inactivated attenuated ASFV particle is coupled to a substrate.
  • the inactivated attenuated ASFV particle is contacted with a biological sample comprising an antibody that binds to the S inactivated attenuated ASFV particle.
  • a complex is formed between the antibody and to the virus particle that is coupled to the substrate, thereby immobilizing the antibody.
  • the biological sample including any unbound antibodies
  • the antibody is contacted with a detection antibody.
  • the binding of the antibody to the ASFV particle is measured, for example, by detecting the amount of detection antibody bound.
  • the detection antibody is conjugated to an enzyme (e.g., an HRP)
  • binding of the antibody to the ASFV particle may be measured by measuring the amount of a fluorophore produced when an appropriate substrate is added to the sample.
  • the term “about” as used herein when referring to a measurable value such as an amount, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • the terms “reduce,” “reduces,” “reduction” and similar terms mean a decrease of at least about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% or more.
  • the terms “enhance,” “enhances,” “enhancement” and similar terms indicate an increase of at least about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more.
  • the term “vaccine” is typically understood to be a prophylactic or therapeutic material providing at least one antigen or antigenic function.
  • the antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response.
  • the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal.
  • the mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human.
  • African swine fever virus is the causative agent of African swine fever (ASF).
  • ASF African swine fever
  • the virus causes a hemorrhagic fever with high mortality rates in pigs, but persistently infects its natural hosts, warthogs, bushpigs with no disease signs. It also infects soft ticks of the Oniithodoros genus, which are thought to be used as a vector.
  • ASFV replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family. ASFV is endemic to sub-Saharan Africa and exists in the wild through a cycle of infection between ticks and wild pigs, bushpigs and warthogs.
  • ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an “emerging infection.”
  • ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least 150 genes. The number of genes differs slightly between different isolates of the virus.
  • ASFV has similarities to the other large DNA viruses, . ., poxvirus iridovirus and mimivirus.
  • the main target cells for replication are those of monocyte, macrophage lineage.
  • Exemplary nucleotide and amino acid sequences for its constituent parts from select ASFV strains can selected from the following: GenBank Accession: (MK 128995); GenBank Accession: (AY261362); GenBank Accession: (NC_044956); GenBank Accession: (MN913970); GenBank Accession: (MW856068); GenBank Accession: (LR812933); GenBank Accession: (NC 044959.2); GenBank Accession: (MK645909); GenBank Accession: (LR813622); GenBank Accession: (AY261365); GenBank Accession: (AY261366): GenBank Accession: (AY261364); GenBank Accession: (AY261361); GenBank Accession: (NC_044946); GenBank Accession:(MH025920); GenBank Accession: MW856067); GenBank Accession: (NC 044945.1); GenBank Accession: LR899131); GenBank Accession: (MT956648); GenBank Accession: (AY26
  • ASFV genotypes (I-XXII) have been identified. All ASFV p72 genotypes have been circulating in eastern and southern Africa. Genotype I has been circulating in Europe, South America, the Caribbean and western Africa, Genotype VIII is confined to four East African countries. Examples of strains from some of the genotypes are given below:
  • Genotype I OURT88/3; Brazil/79; Lisbon/60; BA715; Pret; Benin 97/1; IC/1/96; IC/576;
  • CAM/82 Madrid/62; Malta/78; ZAR85; Katange63; Togo; Dakar59; Ourt88/1; BEN/1/97; Dom Rep; VAL/76; IC/2/96; Awoshie/99; NIG/1/99; NIG/1/98; ANG/70; BEL/85; SPEC 120; Portugal/57; ASFV-Warm; GHA/1/00; GAM/1/00; Ghana; HOL/86; NAM/1/80; NUR/90/1; CAM/4/85; ASFV-Teng; Tegani; ASFV-E75.
  • Genotype III BOT 1/99
  • Genotype IV ASFV-War; RSA/1/99NV
  • Genotype VI MOZ 94/1
  • Genotype VIII NDA/1/90; KAL88/1; ZAM/2/84; JON89/13; KAV89/1; DEZda; AFSV-
  • Genotype X BUR/1/84; BUR/2/84; BUR/90/1; UGA/3/95; TAN/Kwhl2; Hindell; ASFV- Ken; Virulent Kenya 65.
  • ASFV contains five multi-gene families which are present in the left and right variable regions of the genome.
  • the MGFs are named after the average number of codons present in each gene: MGF100, 110, 300, 360 and 505/530.
  • the N-terminal regions of members of MGFs 300, 360 and 505/530 share significant similarity with each other.
  • Several genes in MGF360 and 505/530 determine host range and virulence.
  • the five multigene families of the ASFV genome (MGF 100, MGF 110, MGF 360 and MGF 505/530) are located within the left hand 35 kb or right hand 15 kb terminal variable regions.
  • the MGFs constititute between 17 and 25% of the total coding capacity of the ASFV genome. They lack similarity to other known genes.
  • MGF 505 genes (1R, 2R) are deleted and the MGF 505R 3R gene is truncated in the OURT88/3 genome. These genes are present in the genomes of all eight other pathogenic isolates of ASFV that have been sequenced.
  • the genome contains the MGF 505 3R gene but lacks the other seven MGF genes and in addition also has the MGF 3609L gene truncated (total deletion of 8250 bp).
  • MGF 360 genes 9L, 10L, 1 IL, 12L, 13L and 14L
  • MGF 505 genes 1R and 2R
  • This deletion markedly reduced viral growth in primary macrophage cell cultures by 100- to 1000-fold (Zsak et al 2001, as above) and led to attenuation of the virus (cited as unpublished results, Afonso et al 2004 J. Virol 78: 1858-1864).
  • the Pr4 deletion mutant was not protective and induced a chronic form of the disease.
  • the term “inactivated” refers to a pathogen, such as a virus or microbe, or a vaccine comprising microbes, that has been treated and/or modified so that the pathogen do not have disease-producing capacity.
  • an inactivated vaccine comprises microbes that have been killed by physical and/or chemical processes.
  • the inactivated microbes do not replicate in a subject.
  • the inactivated pathogen are alive, while in some embodiments, the inactivated microbes are dead.
  • the inactivated pathogen is generated by the SolaVAX process.
  • the term “attenuated,” the term “live vaccine” or “attenuated vaccine” refers to a vaccine containing a live viral active ingredient.
  • “attenuation” means that the toxicity of a living pathogen is artificially weakened, and it does not cause disease in the body by mutating genes involved in the essential metabolism of pathogens, but only stimulates the immune system to induce immunity.
  • Viral attenuation can be achieved by irradiation, chemical treatment, or n by high-order continuous subculture in vitro, a.k.a serial passage. Attenuation can also be achieved by making clear genetic changes, for example by specific deletions of viral sequences known to provide toxicity or by insertion of sequences into the genome.
  • the term “inactivated/attenuated” or alternatively “attenuated/inactivated” refers to a pathogen, such as an ASFV particle, that has been attenuated and further inactivated, preferably through a SolaVAX process.
  • SolaVAX refers to a vaccine comprising a pathogen particle, including a viral, bacterial or pathogen or fragments thereof, which have been inactivated using riboflavin and UV light as described herein.
  • the systems, methods and processes that encompass SolaVAX are described by Goodrich et al., PCT/US2021/021190 and PCT/US2021/053160, entitled, the entire specifications, claims and figures related to the inactivation of pathogen particles using UV radiation in the presence of riboflavin is hereby being incorporated by reference.
  • pre-SolaVAX and post-SolaVAX refer to pathogen preparations before and after inactivation, respectively, using riboflavin and UV light.
  • the pathogen is attenuated prior to inactivation by application of SolaVAX treatment.
  • treat By the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • prevent refers to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the disclosure.
  • the prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s).
  • the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present disclosure.
  • Effective amount refers to an amount that, when administered to a subject for treating or preventing a disease (e.g., a viral infection), or at least one of the clinical symptoms of a disease, is sufficient to affect such treatment or prevention of the disease or symptom thereof.
  • the “effective amount” may vary depending, for example, on the disease and/or symptoms of the disease, severity of the disease and/or symptoms of the disease or disorder, the age, weight, and/or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance may be ascertained by those skilled in the art or capable of determination by routine experimentation.
  • the term “pig” refers to an animal in the Suidae family of even-toed ungulates.
  • the term pig includes a domestic pig and its ancestor, the common Eurasian wild boar (Sus scrofa), Palawan bearded pig, Bornean bearded pig, Heude's pig or Vietnamese warty pig, Visayan warty pig, Celebes warty pig, Flores warty pig, Mindoro warty pig, Philippine warty pig, Java warty pig, babirusa and warthog.
  • viral protein refers to the total amount of viral protein measured in a vaccine preparation using, for example, a standard assay to measure protein content.
  • T cell epitope refers to an epitope that can be recognized by the immune system after intracellular processing of an antigen. After processing, a T cell epitope becomes bound to at least one MHC molecule and is expressed on the surface of the antigen presenting cell as a MHC -peptide complex.
  • T cell epitopes presented by MHC class I molecules are typically between 8 and 11 amino acids in length, whereas MHC class II molecules may present peptides of about 12-25 amino acids, preferably about 13-17 amino acids, in length.
  • adjuvant is typically understood not to comprise agents which confer immunity by themselves.
  • An adjuvant assists the immune system unspecifically to enhance the antigenspecific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response.
  • an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Thl-based antigen specific response or vice versa. Accordingly, an adjuvant may favorably modulate cytokine expression/secretion, antigen presentation, type of immune response etc.
  • Th1 lymphocytes secrete interleukin (IL)-2, interferon-y, and lymphotoxin-a and stimulate type 1 immunity, which is often characterized by intense phagocytic activity.
  • Th2 cells secrete IL-4, IL-5, IL-9, IL-10, and IL-13 and stimulate type 2 immunity, which is often characterized by high antibody titers.
  • Non-limiting examples of adjuvants capable of promoting a Thl-type response over a Th2-type response include CpG and/or AS01, CpG 1018, ODN 1688, or AdVaxTM.
  • AdVaxTM comprises delta inulin, specifically delta inulin of highly specific particle size and morphology (See, e.g., Petrovsky, N., et al., Vaccine; 33(44): 5920-5926 (2015)).
  • Not all adjuvants can promote a Thl-type immune response.
  • MontanideTM is one type of adjuvant that does not promote a Thl-type immune response (See, e.g., van Doorn, E., et al., Hum Vaccin Immunother; 12(1): 159-169 (2016)).
  • the immune system may protect organisms from infection. If a pathogen breaks through a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts contains so called humoral and cellular components.
  • Immune response may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response).
  • the invention relates to the core to specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g. ASFV. However, this specific response can be supported by an additional unspecific reaction (innate immune response). Therefore, the invention also relates to a compound for simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient adaptive immune response.
  • the adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth.
  • the adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered.
  • the system is highly adaptable because of somatic hypermutation (a process of increased frequency of somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte.
  • Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions.
  • Adaptive immune response The adaptive immune response is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by “memory cells”.
  • the first step of an adaptive immune response is the activation of naive antigen- specific T cells or different immune cells able to induce an antigen-specific immune response by antigen- presenting cells.
  • Cell types that can serve as antigen- presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g.
  • Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules.
  • the unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • effector T cells The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Thl cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Thl cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • Cellular immunity/cellular immune response relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
  • cellular immunity is not related to antibodies but to the activation of cells of the immune system.
  • a cellular immune response is characterized e.g.
  • cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
  • Humoral immunity refers typically to antibody production and the accessory processes that may accompany it.
  • a humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation.
  • Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
  • the innate immune system also known as non-specific immune system, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
  • the innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g.
  • PAMP pathogen-associated molecular patterns
  • TLRs Tolllike receptors
  • auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL- 9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN- gamma, GM-CSF, G-CSF, M-CSF, LT- beta, TNF-alpha, growth factors, and hGH, TLR
  • a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and/or acting as a physical and chemical barrier to infectious agents.
  • Immunostimulatory RNA in the context of the invention may typically be an RNA that is able to induce an innate immune response itself. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an innate immune response e.g. by binding to a specific kind of Toll-like- receptor (TLR) or other suitable receptors. However, of course also mRNAs having an open reading frame and coding for a peptide/protein (e.g. an antigenic function) may induce an innate immune response.
  • TLR Toll-like- receptor
  • Antigen refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells.
  • an antigen may be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein.
  • fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigen.
  • an antigen may preferably be an antigen related to the ASFV.
  • T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • an antigen may preferably be an epitope related to one or more antigens of the ASFV.
  • Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides, and preferably from ASFV.
  • antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three- dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • expression refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein.
  • Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein.
  • Gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof.
  • Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
  • nucleic acid or “nucleic acid molecules” include single- and double- stranded forms of DNA; single- stranded forms of RNA; and double-stranded forms of RNA (dsRNA).
  • dsRNA double-stranded forms of RNA
  • nucleotide sequence or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.
  • RNA is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA).
  • RNA is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA).
  • deoxyribonucleic acid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids.
  • nucleic acid segment and “nucleotide sequence segment,” or more generally “segment,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encoded or may be adapted to encode, peptides, polypeptides, or proteins.
  • gene refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner.
  • a gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).
  • structural gene as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide. It should be noted that any reference to a SEQ ID, or sequence specifically encompasses that sequence, as well as all corresponding sequences that correspond to that first sequence. For example, for any amino acid sequence identified, the specific specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa.
  • peptide or “protein” is a polymer of amino acid monomers. Usually the monomers are linked by peptide bonds. The term “peptide” does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.
  • “Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of the disclosed compound(s) together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19th Edition).
  • a pharmaceutical compositions of the invention may include a quantity of HIV- 1, and a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable excipient or carriers
  • compositions/formulations are useful for administration to a subject, in vivo or ex vivo.
  • Pharmaceutical compositions and formulations include carriers or excipients for administration to a subject.
  • pharmaceutically acceptable and “physiologically acceptable” mean a biologically compatible formulation, gaseous, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact.
  • Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or nonaqueous), emulsions (e g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules, and crystals.
  • Supplementary active compounds can also be incorporated into the compositions.
  • the formulations may, for convenience, be prepared or provided as a unit dosage form. In general, formulations are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • a tablet may be made by compression or molding. Compressed tablets may be prepared by compressing, in a suitable machine, an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Molded tablets may be produced by molding, in a suitable apparatus, a mixture of powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.
  • compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18. sup. th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12. sup.
  • compositions can optionally be formulated to be compatible with a particular route of administration.
  • Exemplary routes of administration include administration to a biological fluid, an immune cell (e.g., T or B cell) or tissue, mucosal cell or tissue (e.g., mouth, buccal cavity, labia, nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina, rectum, or colon), neural cell or tissue (e.g., ganglia, motor or sensory neurons) or epithelial cell or tissue (e.g., nose, fingers, ears, cornea, conjunctiva, skin or dermis).
  • an immune cell e.g., T or B cell
  • mucosal cell or tissue e.g., mouth, buccal cavity, labia, nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina, rectum, or colon
  • neural cell or tissue e.g., ganglia, motor or sensory neurons
  • epithelial cell or tissue
  • compositions include carriers (excipients, diluents, vehicles, or filling agents) suitable for administration to any cell, tissue, or organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by various routes and delivery, locally, regionally, or systemically.
  • carriers excipients, diluents, vehicles, or filling agents
  • Exemplary routes of administration for contact or in vivo delivery of a target inhibitor is a dosage of the compound that is sufficient to achieve a desired therapeutic effect, such as can optionally be formulated include inhalation, respiration, intubation, intrapulmonary instillation, oral (buccal, sublingual, mucosal), intrapulmonary, rectal, vaginal, intrauterine, intradermal, topical, dermal, parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epidural), intranasal, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, ophthalmic, optical (e.g., corneal), intraglandular, intraorgan, and intralymphatic.
  • parenteral e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epidural
  • parenteral e.g., subcutaneous, intramuscular,
  • isolated or substantially purified pathogens such as virions, bacterium or constituents thereof.
  • isolated or purified ASFV virions or constituents thereof are substantially or essentially free from components that normally accompany or interact with ASFV virions or constituents thereof as found in its naturally occurring environment.

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Abstract

Provided herein are methods for inactivating an attenuated pathogen, the methods comprising contacting the microbe with UV light in the presence of riboflavin such that the integrity of the antigenic proteins of the pathogen is preserved In some embodiments, the viral pathogen is a African Swine Fever Virus (ASFV). Vaccine compositions comprising attenuated pathogen, and methods of use thereof, are also provided.

Description

SYSTEMS, METHODS, AND COMPOSITIONS FOR PREPARING VACCINES COMPRISING INACTIVATED/ATTENUATED PATHOGENS
CROSS-REFERENCE TO RELATED APPLICATIONS
This International PCT application claims the benefit of and priority to U.S. Provisional Application No. 63/434,296, filed December 21, 2022, the specification, claims and drawings of which are incorporated herein by reference in their entirety.
STATEMENT OF GOVERNMENT INTEREST
This invention was made with Government support under grant number PR180446 awarded by the Department of Defense - Congressionally Directed Medical Research Program (CDMRP). The U.S. Government has certain rights in this invention.
TECHNICAL FIELD
The inventive technology is directed to the design, production, and use of vaccines, and in particular the design, production, and use of novel vaccines comprising an attenuated pathogen particle that have further been inactivated.
BACKGROUND
Vaccination is the most effective countermeasure for infection by pathogen in humans and various animal species. Traditional vaccine production methods have included the use of live attenuated vaccines, as well as inactivated (killed) vaccines. Both attenuated and inactivated vaccines each include certain technical and practical advantages, as well as disadvantages that limits their efficient deployment to combat infectious disease. For example, attenuated vaccines more closely mimic the natural infection cycle in a target host and activate all phases of the immune system. They can induce humoral IgG and local IgA, and raise immune responses to many protective antigens. Attenuated vaccines provide a more durable immunity and are more cross- reactive. Moreover they are low-cost and they provide a quick immunity in the majority of cases. However, attenuated vaccines have several key limitations that prevent their effectiveness. Principally, it is often difficult to establish the appropriate level of attenuation that maintains the efficacy of the vaccine, while still being safe for use in humans and animals. In addition, all attenuated vaccines carry the possibility of reversion to their native virulence thereby raising the potential to cause, rather than prevent an outbreak of disease.
Inactivated vaccines have also been a mainstay of vaccinology for decades. Inactivated vaccines generally provide sufficient humoral immunity if boosters are given, and do not carry the risk of mutation or reversion. Inactivated vaccines can be used with immuno-deficient patients, and are generally considered safe. Examples of inactivated vaccines include constructs for influenza, cholera, bubonic plague and polio. Inactivated viral vaccines are typically made by exposing virulent pathogen, such as a virus to chemical or physical agents, for example, formalin or P -propiolactone, in order to destroy infectivity. However, exposure to such harsh chemical and/or physical agents may destroy viral epitopes, thus reducing or even destroying immunogen content and integrity. This reduces the efficacy of these vaccine preparations and requires greatly increased levels of product to induce immune effective immune responses against the native pathogen.
As such, there is a long-felt need for improved vaccine compositions and methods for producing vaccines that incorporate the advantages of attenuation as well as inactivation, which disrupts a attenuated pathogen’s ability to replicate while preserving the potency and integrity of the antigenic proteins thereon. In particular, there is a need in the art for compositions and methods for producing an inactivated viral vaccine against viral particles, as well as bacterial pathogens, among others.
One example for which the present invention can be applied is the development of an inactivated/attenuated vaccine directed to the prevention of African Swine Fever (ASF) which is a contagious hemorrhagic disease of swine. The causative agent, African Swine Fever Virus (ASFV), is a large, enveloped DNA virus containing a double-stranded DNA genome of approximately 190 kilo base pairs. ASFV infections in swine are often fatal and are characterized by fever, hemorrhages, and ataxia. However, the course of infection varies, ranging from highly lethal to sub-clinical, depending on host characteristics and the particular virus strain. At present, there is no vaccine available for ASFV and disease outbreaks are controlled by animal quarantine and slaughter.
Attempts to vaccinate animals using infected cell extracts, supernatants of infected pig peripheral blood leukocytes, purified and inactivated virions, infected glutaraldehyde-fixed macrophages, or detergent-treated infected alveolar macrophages failed to induce protective immunity. Homologous protective immunity does develop in pigs surviving viral infection. Pigs surviving acute infection with moderately virulent or attenuated variants of ASFV develop longterm resistance to homologous, but rarely to heterologous, virus challenge. Based on the recent acceleration of fatal infections with ASFV in China, across South Asia, Russia and Europe, there is a long-felt need for an effective vaccine to control ASFV infection and prevent spread of the disease in swine populations. To be effective a vaccine should trigger B- and T-cell responses to prevent the fast developing infection with ASFV. As described below, the present inventors demonstrate the production of an effective inactivated/attenuate vaccine for ASF.
SUMMARY OF THE INVENTION
In one aspect, the invention is directed to inactivated pathogen preparations which are created from strains of which have been attenuated for infectivity or pathogenicity by genetic manipulation or serial passage to isolate less infectious clones of the pathogen. The preparation of inactivated/attenuated pathogens, such a viral and/or bacterial pathogens, allows for vaccine preparations using starting materials that are not classified as high containment biol ogicals because they are not as infectious as the wild-type strains. This makes vaccine manufacturing much more facile. In addition, the use of genetically modified strains (by natural means or genetically engineered manipulations) can allow for removal of gene products that pose issues in inducing undesirable side effects such as antibody dependent enhancement (ADE)of disease manifestation.
The present invention relates to inactivated/attenuated viral vaccines and methods for preparing the same. More specifically, in one preferred embodiment the present invention relates to methods for inactivation of attenuated viral particles, including ASFV viral particles, using a photosensitizer such as riboflavin in combination with UV light.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows a generalize schematic of the inactivation of an exemplary viral particle using UV light in the presence of riboflavin to disrupts the virus’ nucleic acid constituents in one embodiment thereof.
Figure 2 shows a plurality of ASFV particle samples that have been treated or treated to UV radiation, and cycle threshold (Ct mean) and viral quantity after treatment of 80 and 120 Joules, respectively.
Figure 3 shows UV treated and untreated ASFV particle samples showing hemadsorption or lack thereof. No hemadsorption in cultured ASFV at energy dose of 120 joules delivered in 1 minute treatment time. By PCR Amplification and quantification, ASFV treated at 120 Joules was detected in cells, though at lower quantities and with no increase upon culture. These data is consistent with virus entry into the cells, but without the ability of the virus to replicate at dose of 120 Joules. Figure 4 show transcriptomics data of ASFV showing activation of Pl 3k pathway associated with viral binding and entry into cells by inactivated virus.
Figure 5 show a challenge study utilizing a mix of inactivated-ASF vaccine candidates with and without adjuvants. The candidates were administered to pig models with 6 per group. 20 mg Advax Adjuvant was administered in the “Adjuvant Group.” In this study, the pigs were inoculated with 104 median hemadsorption unit (HAD50^equivalents for vaccination. In this challenge study, no adverse vaccination effects observed in the animals. No site reactions, elongated fevers or lameness or other clinical observations were observed.
Figure 6 shows results of ASFV in tissues post-challenge. In this experiment, qPCR targeting p72 gene set up to detect ASFV in all tissues 6 days post infection. ASFV was detected in all tissues analysed with values averaged per group of 6.
Figure 7 shows results of post-challenge serological analysis. These data show that competitive ELISA assay showed no antibodies for P72, except in PB310 (Control group) at day 32 (4 days post challenge). A virus neutralization assay demonstrated that Number of animals show negative neutralization (possibly ADE). Prime boost with adjuvant (G3) has highest number of animals showing virus neutralization (4/6) compared to other groups (1/6). Post-challenge observations include: 1) Group 3 (Inactivated Virus + Boost + Adjuvant) showed lowest levels of tissue viremia; 2) Group 3 (Inactivated Virus + Boost + Adjuvant) showed highest levels of neutralizing antibody; 3) Gross pathologies in the vaccinated animals were more generally characteristic of inflammatory responses and not classical ASF pathology; 4) Control animals displayed classical signs of ASF infection in gross pathologies.
Figure 8 shows transcriptomic data demonstrating differentially expressed gene across different challenge groups.
Figure 9 shows transcriptomics analysis of the overrepresented gene ontologies in each group reveals. The activation patterns demonstrated several key observations relative to T-cell activation pathways, specifically, for vaccinated groups, Gl, G2, G3, genes were activated for: 1) Cytokine Binding or Cytokine Receptor Binding; 2) Inflammatory Response; 3) Cytokine Production; and 4) Immune Effector Processes. None of these genes were upregulated in the placebo group (Group 4). These data evidence the: 1) ability of virus to bind to and be taken up into target cells by qPCR and HADso analysis; 2) ability to stimulate gene pathways associated with ASF virus entry into cells (P13k-AKT signaling pathway); and 3) ability of vaccine to induce gene ontologies associated with T-cell stimulation and response, distinct from what is seen in placebo group following exposure to live virus.
DETAILED DESCRIPTION OF INVENTION
The present invention inactivated attenuated pathogens, and method of making the same. As described below, inactivating a pathogen that has already been attenuated for its ability to induce disease allows for the production of vaccines under less stringent biocontainment conditions, preserves the benefits of having an attenuated pathogen while also preventing their reversion to their native form which can cause of negative disease outcomes. As generally described herein, inactivated/attenuated viruses have the advantage of not being able to replicate, thus shutting down the potential that the attenuated viruses could undergo recombination events and re-gain of function via genetic exchange that results in the return of pathogenicity and infectivity. Attenuated pathogens which have been engineered to remove or alter problematic gene constructs that result in increased infectivity and undesirable disease side effects, are easier to handle in production environments due to decreased likelihood of disease spread. These agents can, however, still undergo replication and hence have the potential to re-gain function which returns native pathogenicity and disease outcomes. Using an inactivation method which retains the desired characteristics of pathogen phenotype and antigen content and structure while completely removing the ability of these agents to replicate, could afford a means to create, safer and more effective vaccine candidates for viral, bacterial and parasitic agents.
In a preferred embodiment, the provides methods of inactivating attenuated pathogens, such as viral, microbial and/or parasitic pathogens using photochemical technology. As described by described by Goodrich et al., (PCT/US2021/021190 and PCT/US2021/053160, pathogens, including viral and bacterial particles can be inactivated ultra-violet (UV) light in the presence of a photosensitizer, such as riboflavin. This method, generally being referred to herein as the SolaVax method, disrupts the pathogen’s nucleic acid content without modification to proteins or antigen structure, can better maintain immunogenicity of the inactivated particles. In a preferred embodiment, the photochemical disruption of nucleic acids of an attenuated pathogen is achieved using photosensitizers that can act as electron transfer agents. The application of photosensitizer agents that can be placed into an excited state in proximity to a guanine base in DNA or RNA constructs allows for selective modification (e.g., oxidation, crosslinking, fragmentation, deamination) of these bases. Because electron chemistry can only occur over short distances, the photosensitizer agent must be bound or associated with (e g., intercalated with) the nucleic acid in order to carry out the desired chemistry. A variety of attenuated pathogens included viruses, bacteria and parasites could be utilized in this approach of combining attenuation with inactivation using a method that preserves viral protein and antigen structure and function to the same degree as seen in attenuated pathogens alone, but without the potential for replication and hence re-gain of function. For example, in one embodiment, vaccines for diseases such as ASF and can be produced. These are diseases which have been known to engage the use of attenuated viruses but where use of these constructs poses issues with re-gain of virulence.
Also described herein are compositions and methods for producing inactivated/attenuated vaccines, including vaccines for viral, bacterial and parasitic pathogens. In a preferred embodiment, the present inventions describes compositions and methods for producing inactivated/attenuated vaccine directed to ASFV. As noted above, the compositions and methods disclosed herein take advantage of a unique property of the photosensitizer riboflavin and UV light to selectively inactivate virus particles by directed damage to nucleic acids while preserving the integrity of the proteins and other viral antigens. The nature of the photosensitizer (riboflavin) provides for low toxicity and thus easy handling, distribution and processing under even austere conditions.
The methods and compositions disclosed herein have several advantages compared to compositions and methods currently used in the art. For example, riboflavin is inexpensive, nontoxic and does not pose safety or environmental concerns, allowing for vaccine production in an austere environment with minimal infrastructure and personnel training needs. Additionally, the disclosed inactivation methods are more likely to preserve labile and sensitive antigen profdes that could be destroyed by the methods commonly used in the art. Also, the attenuated pathogen is inactivated in situ in its native form, and being inactivated cannot revert to its more virulent form. The processes described herein inactivate nucleic acid replication without requiring additional processing steps to eliminate the replication potential of the attenuated pathogen, and do not require extended protein stability throughout processing.
METHODS OF PRODUCING INACTIVATED ATTENUATED VIRAL VACCINES
Provided herein are inactivated attenuated viral vaccine compositions. The compositions comprise, consist essentially of, or consist of attenuated viral particles or fragments or derivatives thereof, that are further inactivated, preferably by using photochemical technology, and optionally in combination with an adjuvant. As noted above, methods of viral attenuation are known in the art, or would be easily discernable by those of ordinary skill in the art.
The attenuated viral particles are inactivated by modifying their DNA and/or RNA, rendering them incapable of causing disease. In some embodiments, the modification of the attenuated viral DNA and/or RNA renders the attenuated virus incapable of replicating but does not kill the attenuated virus. Because the inactivation process does not substantially alter the structure of antigens (e.g., protein and/or lipid antigens) on the attenuated viral particles, when administered to a subject in need thereof, the vaccines comprising the inactivated attenuated particles present antigenic targets to the subject’s immune system that are substantially identical to those present on a pathogenic, replication-competent virus. Administration of the vaccine compositions to a subject in need thereof stimulates an immune response to the virus, therefore preventing or treating a viral infection in the subject.
In some embodiments, the inactivated viral vaccine comprises, consists essentially of, or consists of attenuated viral particles inactivated using a photochemical process to inactivate the disease-causing ability of the virus while preserving protein structure and phenotype. In some embodiments, the DNA and/or RNA of the inactivated attenuated viruses comprises modified bases. For example, in some embodiments, the DNA or RNA of the inactivated attenuated viruses may comprise modified guanine bases, such as oxidized guanine bases.
In some embodiments, the inactivated attenuated viral particles are adenovirus particles, adeno-associated virus (AAV) particles, lentivirus particles, coronavirus particles or retrovirus particles. In some embodiments, the inactivated attenuated viral particles are inactivated ASFV particles. In some embodiments, the inactivated attenuated viral particles are chikungunya particles, SARS-CoV2 or MERS-coV particles. In some embodiments, the inactivated attenuated viral particles are porcine reproductive and respiratory syndrome (PRRS) particles. In some embodiments, the inactivated attenuated viral particles are polio particles. In some embodiments, the inactivated attenuated viral particles are influenza particles. In some embodiments, the inactivated attenuated viral particles are Dengue, Zika, Influenza (e.g., A, B, C), Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS- CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes (e.g., 1, 2, 6, 7, 8), Hepatitis (e.g., A, B, C, D, E), Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, or Vesicular Stomatitis particles.
In certain embodiment, the attenuated pathogen of the invention may include an attenuated vaccine compositions. In one embodiment, the attenuated pathogen may be an attenuated live virus, bacteria, parasite or fungus. In certain other embodiment, the he attenuated pathogen may be an attenuated live that is used and/or approved for use as a live attenuated vaccine which may be selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow fever vaccine; Zoster/shingles vaccine; Enterotoxigenic Escherichia coli vaccine; Shigella vaccine, Vibrio cholera vaccine; Tick-borne encephalitis vaccine; COVID-19 vaccine, Avian influenza virus H5N1 and H5N3 subtypes and Foot-and- mouth disease, FMD-LL3B3D A24.#
In certain embodiment, the attenuated pathogen of the invention may include an attenuated ASFV vaccine compositions. In certain other embodiment, the he attenuated ASFV may be selected from: (1) ASFV-G-AI177L (See Tran X.H., et al., Transbound Emerg Dis. 2022 Jul;69(4); (2) ASFV-G-A9GL/AUK (See Arias M, et. al., Vaccines (Basel). 2017 Oct 7;5(4):35.); and (3) ASFV-G-AI177LALVR (Id.)#
In certain embodiments, the attenuated pathogen of the invention may include an attenuated vaccine compositions for the treatment of one or more diseases or conditions in animals, which may be selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitiosa; Infection with Bonamia ostreae; Infection with Marteilia refringens; Infection with Microcytos mackini; Infection with Perkinsus marinus; West-Nil e-Virus; Infectious Hematopoietic Necrosis of Salmonids; Ovine Epididymitis (Brucella ovis); Koi Herpesvirus Infection of Carp; Lumpy-skin-Disease (Dermatitis nodularis); Bovine Pleuropneumonia; Foot-and-Mouth-Disease; Anthrax; Newcastle Disease; Avian Influenza; Peste des Petits Ruminants; Equine Encephalomyelitis; Sheep and Goat Pox; Blackleg; Rift Valley Fever; Rinderpest; Glanders; Bovine Salmonellosis; Swine Fever; Vesicular Stomatitis; Taura Syndrome; Rabies; Transmissible Spongiform Encephalopathies (including Bovine Spongiform Encephalopathy (BSE) and Scrapie); Bovine Trichomonosis; Bovine Tuberculosis (Mycobacterium bovis und Mycobacterium caprae); Swine Vesicular Disease; Bovine Genital Campylobacteriosis; Viral Hemorrhagic Septicemia of Salmonids; White Spot Disease of Crustaceans; Vibriosis; Shigellosis; Hepatopancreatic Acute Necrosis Syndrome; and Yellowhead Disease.
In some embodiments, an inactivated attenuated viral particle (e.g., an attenuated ASFV particle) comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or more modified guanine bases, such as oxidized guanine bases, in its genome. In some embodiments, an attenuated ASFV particle comprises about 9 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 16 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 17 modified guanine bases in its genome. In some embodiments, an attenuated ASFV article comprises about 18 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 19 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 20 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 21 modified guanine bases in its genome. In some embodiments, an attenuated ASFV particle comprises about 22 modified guanine bases in its genome. In some embodiments, a an attenuated ASFV particle comprises about 23 modified guanine bases in its genome. In some embodiments, a an attenuated ASFV particle comprises about 24 modified guanine bases in its genome.
In some embodiments, the inactivated attenuated viral vaccine composition further comprises an adjuvant. The effect of the adjuvant is to boost the immunological response. In some embodiments, the adjuvant modifies monocyte function. In some embodiments, the adjuvant promotes a Thl-type response over a Th2-type response. Non-limiting examples of adjuvants capable of promoting a Thl-type response over a Th2-type response include CpG and/or AS01 . Exemplary adjuvants are provided in Goodrich et al., (PCT/US2021/021190), such adjuvants being specifically incorporated herein by reference. In one embodiment, the adjuvant of the invention may include Advax®, which describes an adjuvant derived from inulin, a natural plant- derived polysaccharide that when crystallized in the delta polymorphic form, becomes immunologically active.
In some embodiments, the vaccine composition comprises an adjuvant. In some embodiments, the adjuvant is capable of promoting a Thl-type immune response. In some embodiments, the adjuvant is capable of limiting a Th2-type response. In some embodiments, the adjuvant is CpG and/or AS01. In some embodiments, the adjuvant is a phosphorothioate oligonucleotide comprising about 15 to about 30 nucleotides. In some embodiments, the adjuvant comprises a nucleic acid that comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ ID NO: 1). In some embodiments, the adjuvant comprises a nucleic acid that comprises the sequence 5'-TCCATGACGTTCCTGATGCT-3' (SEQ ID NO: 2). In some embodiments, the adjuvant is ODN 1668. In some embodiments, the adjuvant is CpG 1018. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient.
Examples of other suitable adjuvants include saponin formulations, virosomes, virus like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides (e.g., an immunostimulatory oligonucleotide containing a CpG motif), mineral containing compositions, oil-emulsions, polymers, micelle-forming adjuvants (e g., a liposome), immunostimulating complex matrices (e.g., ISCOMATRIX), particles, squalene, phosphate, cationic liposome-DNA complexes (CLDC), DDA, DNA adjuvants, gamma-insulin, ADP- ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund’s complete adjuvant, Freund’s incomplete adjuvant, muramyl dipeptides, monophosphoryl Lipid A (MPL), poly IC, CpG oligodeoxynucleotides (ODNs), imiquimod, adjuvant system AS01, adjuvant system AS02, adjuvant system AS03, MF59® and aluminum or aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide). In some embodiments, a CpG ODN is a class A, class B or a class C CpG ODN. In some embodiments, a CpG ODN is CPG 7909 (InvivoGen, San Diego, CA). Other suitable adjuvants include TLR agonists, NOD agonists, and lipid-DNA agonist complexes.
In some embodiments, the inactivated attenuated viral vaccine composition further comprises one or more agonists or antagonists. In some embodiments, the agonist is a Toll-Like Receptor (TLR) agonist. In some embodiments, the TLR agonist is an agonist of one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, or TLR12. In some embodiments, the agonist is a TLR3 and/or a TLR9 agonist. In some embodiments, the antagonist is a C-C chemokine receptor type 2 (CCR2) antagonist. In some embodiments, the antagonist is an angiotensin receptor blocker (ARB), such as losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, or valsartan. In some embodiments, the ARB is administered at a dose of between about 5 and about 100 mg/kg, for example about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg/kg.
In some embodiments, the inactivated attenuated virus vaccine comprises at least one of (i.e., one of, two of, or all three of) a TLR agonist, a CCR2 antagonist and an ARB.
In some embodiments, the agonist or antagonist (e.g., a TLR3 and/or a TLR9 agonist) is contained within or coupled to a liposome. Liposomes are spherical, self-enclosed vesicles composed of amphipathic lipids. Liposomes may be unilamellar, having one lipid bilayer membrane, or multilamellar, having two or more concentrically arranged bilayers. Suitable liposomes may have a selected mean particle size diameter of about 200-500 nm. Various methods of preparing liposomes and encapsulation of therapeutic agents therein are well documented (see, for example, U.S. Pat. Nos. 3,932,657, 4,311,712, and 5,013,556, all of which are incorporated herein by reference). Known methods include the reverse phase evaporation method as described in U.S. Pat. No. 4,235,871, which is incorporated herein by reference.
Lipids for use in forming the liposomes described herein include vesicle-forming lipids having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. The selection of lipids and proportions can be varied to achieve a desired degree of fluidity or rigidity, to control stability, and/or to control the rate of release of an entrapped agent. Where more than one type of lipid is used, a suitable amount of a relatively unsaturated lipid (such as PC), may be used in order to form stable liposomes. In one embodiment, at least 45- 50 mol % of the lipids used to form the liposome are PC. The liposomes may also include lipids derivatized with a hydrophilic polymer such as polyethylene glycol (PEG). Suitable hydrophilic polymers include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, polyaspartamide, and hydrophilic peptide sequences. Methods of preparing lipids derivatized with hydrophilic polymers are known (see e.g., U.S. Pat. No, 5,395,619, which is incorporated herein by reference). In some embodiments, the inactivated attenuated viral vaccine comprises cationic liposome-DNA complexes (CLDC).
In some embodiments, the inactivated attenuated viral vaccine further comprises a photosensitizer such as riboflavin (vitamin B2). In some embodiments, the inactivated attenuated viral vaccine is substantially free of photosensitizer.
In some embodiments, the inactivated attenuated viral vaccine composition further comprises a carrier. In some embodiments, the cells and/or the photosensitizer are suspended in the carrier. In some embodiments, the carrier comprises normal saline (e.g., 0.9% sodium chloride), dextrose saline (e.g., dextrose 5% in 0.9% sodium chloride), phosphate buffered saline (e.g., 137 mmol/L NaCl, 2.7 mmol/L KC1, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4).
In some embodiments, the inactivated attenuated viral vaccine composition further comprises one or more additional pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
In some embodiments, a vaccine composition comprises an inactivated attenuated ASFV viral particle; wherein the composition comprises about 15 to about 50 picograms of viral protein (e.g., about 35 picograms of viral protein) and an adjuvant.. In some embodiments, a vaccine composition comprises an inactivated attenuated ASFV viral particle; wherein the genome of the ASFV viral particle comprises about 1 to about 30 oxidized guanine residues (e.g., about 9 or about 20 oxidized guanine residues); wherein the structure of antigens on the viral particle is not substantially altered compared to ASFV viral particle that has not been attenuated and/or inactivated.
In some embodiments, a vaccine composition comprises an inactivated attenuated ASFV viral particles from multiple ASFV strains. For example, in some embodiments, a vaccine composition comprises an inactivated attenuated ASFV particle from a first strain, and a second inactivated attenuated ASFV particle from a separate strain. In some embodiments, a vaccine composition comprises inactivated attenuated ASFV particles from at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different attenuated ASFV strains.
The inactivated attenuated viral vaccines described herein are produced using an innocuous chemical agent in a selective process that prevents cellular replication processes while preserving antigenic protein structure. More specifically, the viral vaccines are produced by the combined application of a photosensitizer and light for rendering attenuated viral particles unable to cause disease i.e., inactivated. The process for producing the inactivated attenuated viral vaccines of the disclosure is described in detail below.
Initially, attenuated viral particles are provided. The attenuated viral particles may be recombinant attenuated viral particles, or wild-type attenuated viral particles, that have, for example undergone serial passage to select for less virulent strains.
Next, the attenuated viral particles are inactivated using photochemical technology. This is achieved using photosensitizers that can act as electron transfer agents. The application of photosensitizer agents that can be placed into an excited state in proximity to a guanine base in DNA or RNA constructs allows for selective modification (e.g. oxidation, cross-linking, fragmentation, deamination) of these bases.
In some embodiments, the photosensitizer is a flavin, for example riboflavin (Vitamin B2), flavin mononucleotide, or flavin adenine dinucleotide. In some embodiments, the photosensitizer is a tertiary aliphatic amine (e.g., l,4-diazabicyclo(2,2,2)octane), a piperazine, (e.g., N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1,4-dimethylpiperazine), an amino acid (e g., tyrosine, tryptophan, histidine, methionine), an enzyme (e.g., superoxide dismutase) or EDTA (ethylenediaminetetraacetic acid). In some embodiments, the photosensitizer is riboflavin.
In some embodiments, the attenuated viral particles are added to a solution containing the photosensitizer (e.g., riboflavin), or the photosensitizer is added to a solution containing the attenuated viral particles.
In some embodiments, the concentration of photosensitizer used during inactivation is about 10 pM to about 100 pM, such as about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 200 pM, about 300 pM, about 500 pM, or more. In some embodiments, the solution contains the photosensitizer at a concentration of about 1 pM to about 50 pM, such as about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 100 pM , about 200 pM, about 300 pM, about 500 pM, or more. In some embodiments, the photosensitizer concentration is less than about 10 pM, such as less than about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, or about 1 pM.
The solution containing the photosensitizer and the attenuated viral particles (optionally, in media) is then subjected to light treatment. In some embodiments, the light treatment comprises treatment with visible light, ultraviolet light, and/or infrared light. The light treatment inactivates a nucleic acid (e.g., DNA and/or RNA) in the viral particles by modifying bases of these nucleic acids. In some embodiments, guanine bases are selectively modified. In some embodiments, guanine bases are selectively oxidized. Oxidized guanine bases cannot be repaired by natural enzymatic and cell repair mechanisms. As such, there is no possibility for reversion of the induced change to a form that would restore the ability of the treated viral particles to cause disease, or revert to their non-attenuated form.
In some embodiments, the light treatment comprises treatment with ultraviolet (UV) light. The UV light may be UV-A, UV-B, or UV-C light. The UV light may have a wavelength of 170 to 400 nm, including all ranges and subranges therebetween. For example, in some embodiments, the UV light has a wavelength of 315 to 400 nm, 310 to 320 nm, 280 to 360 nm, 280 to 315 nm, or 180 to 280 nm. The UV light may be provided by UV light sources known in the art, such as the Mirasol® PRT Illumination device (TerumoBCT, Lakewood, Colorado). In some embodiments, the attenuated viral particles may be treated with multiple wavelengths of light simultaneously.
In some embodiments wherein riboflavin is used as a photosensitizer, UV light having a wavelength of 310 to 320 nm is used. Without being bound by any theory, it is believed that this wavelength prevents riboflavin from reacting in free solution, which would result in production of undesirable oxygen free radicals. At these wavelengths, riboflavin will react only when intercalated with nucleic acid. The dose of the UV light may vary depending on the volume of solution being treated. For example, the dose of the UV light may be between 200-400 Joules (e.g., 300 Joules) for a volume of about 170 to 370 mis of solution, while in alternative embodiments the dose may be between 80 to 120 Joules. As will be understood by those of skill in the art, the dosage may be adjusted up or down if the volume to be treated is above or below this range.
In some embodiments, the dose of UV light may be from less than 50 Joules, about 50 to 100 Joules, about 100 Joules to about 200 Joules, about 200 Joules to about 600 Joules, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 Joules. In some embodiments, the volume of viral preparations for illumination may be from about 200 ml to about 600 ml, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 ml. In some embodiments, the dose of UV light may be from about 0.01 Joules/ml to about 1.0 Joules/ml. In some embodiments, the dose of UV light may be from about 0.5 Joules/ml to about 3.0 Joules/ml. For example, the dose of UV light may be about 0.1, about 0.2 about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0 Joules/ml. In some embodiments, the dose of UV light may be about 0.1 Joules/ml. In some embodiments, the dose of UV light may be from about 1 Joules/ml to about 10 Joules/ml, such as about 1.87 Joules/ml, about 3.74 Joules/ml, or about 6.24 Joules/ml.
The attenuated viral particles may be treated with UV light for about 1 minute to about 60 minutes, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes. In some embodiments, the attenuated viral particles are treated with UV light for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, or about 1 minute to about 3 minutes.
In some embodiments, the attenuated viral particles are pre-incubated for a predetermined period of time in the solution containing the photosensitizer (e.g., riboflavin) before subjecting the viral particles to the light treatment.
In some embodiments, the attenuated viral particles are not subjected to any additional purification or modification steps after light treatment. In other embodiments, the inactivated attenuated viral particles are purified after the light treatment. In some embodiments, the inactivated attenuated viral particles are concentrated after the light treatment.
In some embodiments, the inactivated attenuated viral particles are combined with one or more additional pharmaceutically acceptable carriers or ingredients as described herein after light treatment. In some embodiments, the inactivated attenuated viral particles are combined with a solution comprising an adjuvant after light treatment.
In some embodiments, the inactivated attenuated viral particles generated using this method are incapable of replication processes or reversion to their virulent form, but substantially maintain and preserve the antigen and epitope profile of the original, native attenuated viral particle or antigen. In some embodiments, the inactivation process does not substantially change the phenotype or structure of the attenuated viral particles.
In some embodiments, because the specificity of the chemistry preserves the antigen profile and viral particle integrity, and maintains viral protein and/or lipid structure in its native state, the inactivated attenuated viral particles that are produced by this process provide an improved source for antigen presentation.
In some embodiments, a method for inactivating an attenuated ASFV viral particle comprises contacting the attenuated ASFV viral particle with a dose of UV light in the presence of riboflavin; wherein the dose of UV light is about 50 Joules to about 1000 Joules; wherein the method comprises selectively oxidizing about 1 to about 30 guanine bases (e.g., about 9 or about 30 guanine bases) in a nucleic acid (e.g., an RNA or a DNA) of the attenuated viral particle; and wherein the method does not comprise substantially altering the structure of antigens on the viral particle.
METHODS OF PRODUCING INACTIVATED ATTENUATED MICROBIAL VACCINES The disclosure provides the inactivated attenuated microbes produced by any one of the methods disclosed herein, and compositions comprising the inactivated microbes disclosed herein. In some embodiments, the compositions comprise inactivated attenuated microbes, or fragments or derivatives thereof. In some embodiments, the compositions comprise inactivated attenuated microbes of more than one species. As noted above, methods of attenuating microbial pathogens are known in the art, or would be easily discernable to those of ordinary skill. Examples are described by Hajra, D., et al.,. (2021). Attenuation Methods for Live Vaccines. In: Pfeifer, B.A., Hill, A. (eds) Vaccine Delivery Technology. Methods in Molecular Biology, vol 2183. Humana, New York, NY., and include attenuation by serial passaging, attenuation by mutagenesis, transposon mutagenesis, microRNA-based viral attenuation, codon usage for viral attenuation. The entire disclosure, being incorporated herein in its entirety.
In some embodiments, the inactivated attenuated microbes substantially maintain and preserve the antigen and epitope profile, microbe integrity, and protein and/or lipid structure of the original, attenuated or native microbe or antigen. In some embodiments, the inactivated attenuated microbes are not capable of replicating or reverting to their more virulent form. In some embodiments, the inactivated attenuated microbes have modified genome (that is, modified DNA and/or RNA). In some embodiments, the DNA and/or RNA of the inactivated microbes comprises modified bases. In some embodiments, the modification of the microbial DNA and/or RNA renders the attenuated microbe incapable of replicating. In some embodiments, the modification of the microbial DNA and/or RNA does not kill the microbe. For example, in some embodiments, the DNA or RNA of the inactivated attenuated microbes may comprise modified guanine bases, such as oxidized guanine bases. In some embodiments, an inactivated microbe comprises about 1 to about 100,000 modified guanine bases, for example about 10, about 100, about 1000, about 5000, about 10,000, about 15,000, about 20,000, about 50,000, about 75,000, about 100,000 or more modified guanine bases, including all subranges and values that lie therebetween, in its genome.
In some embodiments, the compositions disclosed herein do not comprise any adjuvant. In some embodiments, the compositions further comprise an adjuvant. In some embodiments, the adjuvant boosts the immunological response. For example, in some embodiments, the adjuvant modifies monocyte function. As noted above, a non-limiting list of adjuvants includes saponin formulations, virosomes, microbe-like particles, non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), immunostimulatory oligonucleotides (e.g., an immunostimulatory oligonucleotide containing a CpG motif), mineral containing compositions, oil-emulsions, polymers, micelle-forming adjuvants (e g., a liposome), immunostimulating complex matrices (e.g., ISCOMATRIX), particles, squalene, phosphate, cationic liposome-DNA complexes (CLDC), DDA, DNA adjuvants, gammainsulin, ADP- ribosylating toxins, detoxified derivatives of ADP-ribosylating toxins, Freund’s complete adjuvant, Freund’s incomplete adjuvant, muramyl dipeptides, monophosphoryl Lipid A (MPL), poly IC, CpG oligodeoxynucleotides (ODNs), imiquimod, adjuvant system AS01, adjuvant system AS02, adjuvant system AS03, MF59® (i.e., an oil-in-water emulsion containing metabolizable oil squalene and the surfactants Tween 80 and sorbitan trioleate Span 85) and aluminum or aluminum salts (e.g., alum, aluminum phosphate, aluminum hydroxide). In some embodiments, a CpG ODN is a class A, class B or a class C CpG ODN. In some embodiments, a CpG ODN is CpG 7909 (InvivoGen, San Diego, CA). Other suitable adjuvants include TLR agonists, NOD agonists, and lipid-DNA agonist complexes. In some embodiments, the adjuvant is GLA-SE, which is a synthetic toll-like receptor 4 agonist (see, e.g., Behzad, H. et al, J Infect Dis., 2021, 205(3): 466- 473). In some embodiments, the adjuvant comprises GLA-SE in a squalene emulsion.
In some embodiments, the inactivated attenuated microbial vaccine composition further comprises one or more agonists or antagonists as described above.
In some embodiments, the agonist or antagonist (e.g., a TLR3 and/or a TLR9 agonist) is contained within or coupled to a liposome as described above. In some embodiments, the inactivated attenuated microbial vaccine comprises cationic liposome- DNA complexes (CLDC).
In some embodiments, the inactivated attenuated microbial vaccine further comprises a photosensitizer such as riboflavin (vitamin B2). In some embodiments, the inactivated attenuated microbial vaccine is substantially free of photosensitizer.
In some embodiments, the inactivated attenuated microbial vaccine composition further comprises a carrier. In some embodiments, the cells and/or the photosensitizer are suspended in the carrier. In some embodiments, the carrier comprises normal saline (e.g., 0.9% sodium chloride), dextrose saline (e.g., dextrose 5% in 0.9% sodium chloride), phosphate buffered saline (e.g, 137 mmol/L NaCl, 2.7 mmol/L KC1, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4). In some embodiments, the inactivated attenuated microbial vaccine composition further comprises one or more additional pharmaceutically acceptable ingredients as described above.
In some embodiments, an inactivated attenuated microbe is associated with and/or contained within a liposome, virosome, ISCOM (immunostimulatory complex) or virus-like particle. These types of delivery systems offer numerous advantages, including one or more of the following: i) protection of the inactivated microbe against enzymes, ii) increased stability of the inactivated microbe during vaccine manufacturing, transport, and storage, iii) increased efficacy of presentation of antigens on the inactivated microbe to antigen presenting cells (APCs), and iv) increased half-life of the inactivated microbe after administration to a subject.
In some embodiments, an inactivated attenuated microbe is associated with and/or contained within a nanoparticle. In some embodiments, an inactivated microbe is associated with and/or contained within a Gantrez Nanoparticle (GNP). See, e.g., Gomez et al, Journal of Immunological Methods 348 (2009) 1-8; Gomez et al, Vaccine 25 (2007) 5263-5271. GNPs comprise a copolymer of methyl vinyl ether and maleic anhydride, which readily reacts with amino groups, making it easy to load or link different proteins thereto. Methods for production of GNPs are known, such as a solvent displacement method. The inactivated microbe may be incorporated into the GNP during manufacture (i.e., embedded in the nanoparticle), or after the preparation of the GNP (i.e., coating the nanoparticle). In some embodiments, the nanoparticle is a poly(anhydride) nanoparticle. See, e.g., Irache et al, Frontiers in Bioscience S2, 876-890, 2010. In some embodiments, the nanoparticle has a diameter in the range of about 50 to about 500 nm, such as about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, or about 500 nm. In some embodiments, the nanoparticle further comprises an adjuvant.
In some embodiments, an inactivated attenuated microbe is associated with and/or contained within a microparticle. In some embodiments, an inactivated attenuated microbe is associated with and/or contained within a poly-e-caprolactone (PCL) microparticle. See, e.g., Roban, B.S. et al, Clin Exp Allergy. 2007 Feb;37(2):287-95. In some embodiments, the microparticle has a diameter of about 1 to about 3 pm, such as about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9 or about 3.0 pm. In some embodiments, the microparticle further comprises an adjuvant. In some embodiments, an inactivated attenuated microbe is associated with a polymer. In some embodiments, an inactivated attenuated microbe is encapsulated by a polymer. The polymer may be, for example, a polymer that enhances the stimulation of mucosal immunity. In some embodiments, the polymer is a biodegradable polymer. For example, the biodegradable polymer may be a copolymer of methyl vinyl ether and maleic anhydride (PVM/MA). In some embodiments, the copolymer has a molecular weight between 100 and 2400 kDa, such as between 200 and 2000 kDa or between 1880 and 250 kDa. Illustrative polymers are described in US Pat. Nos. 8,628,801, 9,522,197 and 10,933,025, which are incorporated by reference in their entireties.
In some embodiments, the polymer is a mucoadhesive polymer. In some embodiments, the polymer is a chitosan-based mucoadhesive polymer such as chitosan-cysteine, chitosan-4-thio- butylamidine, chitosan-thioglycolic acid, chitosan-glutathione, chitosan-6-mercaptonicotinic acid, chistoan-N-acetylcysteine, chitosan-4-mercaptobenzoic acid, or chitosan-N-acetylpenicillamine. In some embodiments, the polymer is a eudragit-based mucoadhesive polymer. In some embodiments, the polymer is a hybrid polymer, such as poly (lactic-co-glycolic acid) (PLGA). In some embodiments, the polymer may allow for more efficient delivery of the inactivated attenuated microbe using an oral or nasal route, as compared to a microbe that is not encapsulated by a polymer.
In some embodiments, a vaccine composition may comprise an inactivated attenuated microbe and a PLGA copolymer, such as poly(lactide) homopolymer (PLA) or poly(lactic-co- glycolic acid) copolymer (PLGA). In some embodiments, the inactivated attenuated microbe is comprised in a PLGA nanoparticle.
The disclosure provides methods of inactivating attenuated microbes using photochemical technology. In some embodiments, the methods comprise establishing an attenuated microbe, and further contacting the attenuated microbe with a dose of light in the presence of a photosensitizer. In some embodiments, the attenuated microbes are added to a solution containing the photosensitizer, or the photosensitizer is added to a solution containing the attenuated microbes. In some embodiments, the attenuated microbes are cultured, grown, or stored in the presence of the photosensitizer. In some embodiments, the photosensitizer is added to the growth media of the microbes. In some embodiments, the photosensitizer is added to the storage media of the attenuated microbes. In some embodiments, a solution containing the photosensitizer and the attenuated microbes (optionally, in media) is subjected to light treatment. In some embodiments, the attenuated microbes are preincubated for a predetermined period of time in the solution containing the photosensitizer before subjecting the attenuated microbes to the light treatment. In some embodiments, the attenuated microbes are cultured for a predetermined period of time in media containing the photosensitizer before subjecting the attenuated microbes to the light treatment.
In some embodiments, the methods further comprise a step of breaking up clumps or aggregates of attenuated microbial cells, and/or of preventing clumping or aggregation of attenuated microbial cells. Clumping may be prevented and/or disrupted using any physical technique known in the art, for example, by sonicating, vortexing, or pipetting; using any chemical means known in the art, such as using chelators such as EDTA (ethylenediaminetetraacetic acid) and citrate; and/or using any biological means known in the art, such as using an enzyme (e.g., a peptidase, for example, trypsin). In some embodiments, the step of breaking up clumps of attenuated microbial cells, and/or preventing clumping of microbial cells may be performed before and/or after the addition of the photosensitizer. In some embodiments, the step of breaking up clumps of attenuated microbial cells, and/or to preventing clumping of attenuated microbial cells may be performed in the presence of the photosensitizer. In some embodiments, the solution comprising attenuated microbial cells further comprises an additive that protects cells from hydrodynamic damage. In some embodiments, the additive is a detergent. The type of detergent is not limited, and may be any detergent that is used in the presence of cells. In some embodiments, the detergent is a non-ionic detergent. In some embodiments, the detergent is Pluronic® F-68. In some embodiments, the additive is polyethylene glycol (PEG).
In some embodiments, the photosensitizer is a flavin, for example riboflavin (Vitamin B2), flavin mononucleotide, or flavin adenine dinucleotide. In some embodiments, the photosensitizer is a tertiary aliphatic amine (e.g., l,4-diazabicyclo(2,2,2)octane), a piperazine, (e.g., N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid and 1,4-dimethylpiperazine), an amino acid (e.g., tyrosine, tryptophan, histidine, methionine), an enzyme (e.g., superoxide dismutase) or EDTA. In some embodiments, the photosensitizer is riboflavin.
In some embodiments, the concentration of photosensitizer used during inactivation is about 10 pM to about 1 mM, for example, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM, about 75 pM, about 80 pM, about 85 pM, about 90 pM, about 95 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, or about 1 mM, including all values and subranges therebetween. In some embodiments, the solution or media containing the photosensitizer contains the photosensitizer at a concentration of about 1 pM to about 50 pM, for example, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM, including all values and subranges therebetween. In some embodiments, the photosensitizer concentration in the solution or media is less than about 10 pM, for example, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM, including all values and subranges therebetween.
In some embodiments, the light treatment comprises treatment with visible light, ultraviolet light, and/or infrared light. The light treatment inactivates a nucleic acid e.g., DNA and/or RNA) in the microbes by modifying bases of these nucleic acids. In some embodiments, guanine bases are selectively modified. In some embodiments, guanine bases are selectively oxidized. Oxidized guanine bases cannot be repaired by natural enzymatic and cell repair mechanisms. As such, there is no possibility for reversion of the induced change to a form that would restore the ability of the microbes to cause disease. In some embodiments, the light treatment comprises treatment with ultraviolet (UV) light. The UV light may be UV-A, UV-B, or UV-C light. The UV light may have a wavelength of 170 to 400 nm, including all values and subranges therebetween. For example, in some embodiments, the UV light has a wavelength of 315 to 400 nm, 310 to 320 nm, 280 to 360 nm, 280 to 315 nm, or 180 to 280 nm. The UV light may be provided by UV light sources known in the art, such as the Mirasol® PRT Illumination device (TerumoBCT, Lakewood, Colorado). In some embodiments, the microbes may be treated with multiple wavelengths of light simultaneously.
In some embodiments wherein riboflavin is used as a photosensitizer, UV light having a wavelength of 310 to 320 nm is used. Without being bound by any theory, it is believed that this wavelength prevents riboflavin from reacting in free solution, which would result in production of undesirable oxygen free radicals. At these wavelengths, riboflavin will react only when intercalated with nucleic acid.
The dose of the UV light may vary depending on the volume of solution being treated. In some embodiments, the dose of the UV light is in the range of less than 50 Joules, about 50 to 100 Joules, about 100 Joules to about 200 Joules, about 100 Joules to about 50,000 Joules, for example, about 200 Joules, about 300 Joules, about 400, about 500, about 600 Joules, about 700 Joules, about 800 Joules, about 900 Joules, about 1000 Joules, about 2000 Joules, about 3000 Joules, about 4000 Joules, about 5000 Joules, about 6000 Joules, about 7000 Joules, about 8000 Joules, about 9000 Joules, about 10,000 Joules, about 11,000 Joules, about 12,000 Joules, about 13,000 Joules, about 14,000 Joules, about 15,000 Joules, about 16,000 Joules, about 17,000 Joules, about 18,000 Joules, about 19,000 Joules, about 20,000 Joules, about 25,000 Joules, about 30,000 Joules, about 35,000 Joules, about 40,000 Joules, about 45,000 Joules, or about 50,000 Joules, including all values and subranges that lie therebetween. In some embodiments, the dose of the UV light is about 17,000 Joules. In some embodiments, the dose of the UV light is about 8,640 Joules. In some embodiments, the dose of the UV light is about 17,280 Joules.
In some embodiments, the volume of attenuated microbial preparations for illumination is in the range of about 200 ml to about 600 ml, for example about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, or about 600 ml, including all values and subranges that lie therebetween. In some embodiments, the volume of microbial preparations for illumination is in the range of about 170 mL to about 370 mL of solution. In some embodiments, the volume of microbial preparations for illumination is about 300 mL. In some embodiments, the dose of UV light is in the range of about 0.3 Joules/ml to about 170 Joules/ml, for example, about 0.4, about 1.0, about 2.0, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 Joules/ml, including all values and subranges that lie therebetween. In some embodiments, the dose of UV light may be about 30 Joules/ml. In some embodiments, the dose of UV light may be about 60 Joules/ml.
The attenuated microbes may be treated with UV light for about 1 minute to about 60 minutes, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, or about 60 minutes, including all values and subranges that lie therebetween. In some embodiments, the attenuated microbes are treated with UV light for about 1 minute to about 10 minutes, about 1 minute to about 5 minutes, or about 1 minute to about 3 minutes. In some embodiments, the solution being treated comprises about 10 attenuated microbial cells to about 1012 attenuated microbial cells, for example, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about IO10, about 1011, or about 1012 attenuated microbial cells, including all values and subranges that lie therebetween. In some embodiments, the solution being treated comprises about 104 attenuated microbial cells to about 106 attenuated microbial cells. In some embodiments, the solution being treated comprises about IO3 attenuated microbial cells. In some embodiments, the microbe is a bacterium, a fungus, or a parasite. In some embodiments, the microbe is capable of infecting a subject, for example, a human or animal subject.
In some embodiments, the attenuated microbe is an attenuated bacterium. In some embodiments, the attenuated bacterium is selected from the group consisting of Actinomyces israelii, Acinetobacter baumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difficule, Clostridium neoformans, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Francisella tularensis, vancomycin-resistant Enterococci (VRE), Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, Leptospira mayottensis, Leptospira noguchii, Leptospira santar osai, Leptospira weilii, Listeria monocytogenes, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma pneumonia, Nocardia farcinica, Neisseria meningitides, Neisseria gonorrhoeae, Nocardia asteroids, Pseudomonas aeruginosa, Propionibacterium acnes, Rickettsia rickettsia, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella sp., Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Staphylococcus aureus, methicillin- susceptible Staphylococcus aureus (MSSA), methicillin- resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B, Streptococcus Group C, Treponema pallidum, Vibrio cholera, Yersinia pestis and Yersinia pseudotuberculosis.
In some embodiments, the attenuated microbe is a fungus. In some embodiments, the attenuated fungus is selected from the group consisting of Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladiella mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Exophiala jeanselmei, Exophiala dermatitidis, Curvularia lunata, Bipolaris spicifera, Bipolaris hawaiiensis, Bipolaris cynodontis, Alternaria alternata, Histoplasma capsulatum, Histoplasma duboisii, Histoplasma farciminosum, Lacazia loboi, Mucor indicus, Malassezia furfur, Trichosporon asahii, Pneumocystis jirovecii, Pneumocystis carinii, Paracoccidioides brasiliensis, Pseudoalle scher ia boydii, Paracoccidioides lutzii, Penicillium marneffei, Penicillium aurantiogriseum, Sporothrix schenkii, Stachybotrys chartarum, Saccharomyces cerevisiae, Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globose .
In some embodiments, the attenuated microbe is an attenuated parasite. In some embodiments, the attenuated parasite is selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coli, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Fasciola hepatica, Fasciola magna, Fasciola gigantic, Giardia lamblia, Isospora belli, Leishmania donovani, Leishmania infantum, Leishmania mexicana, Enter obius vermicular is, Ascaris lumbricoides, Trichur is trichur ia, Necator americanus, Naegleria fowleri, Onchocerca volvulus, Dracancuhis medinensis, Trichinella spiralis, Strongyloides steroralis, Clonorchis sinensis, Paragonimus westermani, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium know le si, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongy, Taenia saginata, Taenia solium, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii and Wuchereria bancrofti.
In some embodiments, the attenuated microbe belongs to the genus Mycobacterium. In some embodiments, the microbe belongs to the Mycobacterium tuberculosis complex. In some embodiments, the attenuated microbe is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, and Mycobacterium mungi. In some embodiments, the attenuated microbe is Mycobacterium tuberculosis. In some embodiments, the microbe is the Erdman strain, the H37Rv strain, or the CDC1551 strain of Mycobacterium tuberculosis . In some embodiments, the microbe is the H37Rv strain of Mycobacterium tuberculosis.
In some embodiments, the microbe is Mycobacterium leprae, Mycobacterium lepromatosis, or nontub er culous mycobacteria (NTM). In some embodiments, the microbe belongs to the Mycobacterium avium complex. In some embodiments, the microbe is selected from the group consisting of Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare.
In some embodiments, the microbe is resistant to an antibiotic. In some embodiments, the antibiotic is penicillin, isoniazid, clarithromycin, fluoroquinolone, amikacin, kanamycin, capreomycin, and/or rifamycin. In some embodiments, the photosensitizer enters the microbial cell in a passive manner. In some embodiments, the photosensitizer is imported into the microbes by pinocytosis, or clathrin-mediated endocytosis. In some embodiments, the microbe actively imports the photosensitizer. In some embodiments, the microbe is genetically engineered to import an amount of photosensitizer that is higher than the amount of photosensitizer imported into a wildtype microbial cell under the same conditions. In some embodiments, the microbe is genetically engineered to overexpress one or more receptors of the photosensitizer. In some embodiments, the microbe comprises a polynucleotide encoding one or more receptors of the photosensitizer.
In some embodiments, the attenuated microbe is genetically engineered to overexpress one or more receptors of riboflavin. In some embodiments, the attenuated microbe comprises a polynucleotide encoding one or more receptors of riboflavin. The receptor of riboflavin is not limited, and may be any receptor known in the art, or discovered in the future, to import riboflavin into a microbial cell. In some embodiments, the receptor is RibM (GenBank Accession No: K4REQ6), RibN (GenBank Accession No: Q1MIM3), RfuABCD (GenBank Accession No: Q56328), RibU (GenBank Accession No: Q5M614), ImpX (GenBank Accession No: D5RAW5), RfnT (GenBank Accession No: A6X7E7), or RibV (GenBank Accession No: Q6F0N9), or a functional ortholog or variant thereof. In some embodiments, the receptor is any one of the bacterial riboflavin receptors described in Gutierrez-Preciado A, et al., PLoS One. 2015; 10(5), which is incorporated herein by reference in its entirety.
In some embodiments, the attenuated microbes are not subjected to any additional purification or modification steps after light treatment. In other embodiments, the inactivated attenuated microbes are purified after the light treatment. In some embodiments, the inactivated attenuated microbes are concentrated after the light treatment. In some embodiments, the photosensitizer is removed from the solution containing the inactivated attenuated after light treatment.
In some embodiments, the inactivated attenuated microbes are combined with one or more additional pharmaceutically acceptable carriers after light treatment. In some embodiments, the inactivated attenuated microbes are combined with a solution comprising an adjuvant after light treatment.
In some embodiments, the method comprises selectively oxidizing guanine bases in a nucleic acid of the attenuated microbe. Without being bound by any theory, it is believed that the inactivation of the attenuated microbes is, at least in part, associated with, caused by, or results from the selective oxidation of guanine bases in a nucleic acid of the attenuated microbe. In some embodiments, the method comprises selectively oxidizing at least about 1 to at least about 100,000 guanine bases in a nucleic acid (e.g., a DNA or an RNA) of the microbe, for example at least 10, at least 100, at least 1000, at least 5000, at least 10,000, at least 15,000, at least 20,000, at least 50,000, at least 75,000, at least 100,000 or more guanine bases, including all subranges and values that lie therebetween. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 40%, about 60%, about 80%, or about 100% of the guanine bases in the genome of the microbe are oxidized. In some embodiments, the inactivation process does not substantially change the phenotype or structure of the attenuated microbes, or the phenotype or structure of the antigens on the surface of the attenuated microbes.
In some embodiments, the method comprises contacting an attenuated strain of Mycobacterium tuberculosis H37Rv cells with about 8,640 J of UV light in the presence of about 50 pM of riboflavin in a volume of about 300 mb. In some embodiments, the method comprises contacting an attenuated strain of Mycobacterium tuberculosis H37Rv cells with about 17,280 J of UV light in the presence of about 50 pM of riboflavin in a volume of about 300 mb In some embodiments, the method further comprises sonication of the attenuated strain of Mycobacterium tuberculosis cells.
METHODS OF TREATMENT
The inactivated attenuated pathogen vaccine compositions described herein can be used as vaccine agents or stimulants for immune system priming and recognition that foster immune responses in subjects. The disease may be any disease or infection caused by any one of the pathogens described herein. In some embodiments, the inactivated attenuated viral vaccine may be administered to a subject to treat or prevent a viral infection, such as a ASF infection.
In some embodiments, the subject is assessed for immune function and immune status prior to administration of the vaccine. Such assessments may include, but are not limited to, DTH skin testing, blood tests, lymph node aspirate tests, tumor tissue tests, and/or determination of whether the subject is anergic, B cell responsive, etc. In some embodiments, the subject is not assessed for immune function and immune status prior to administration of the vaccine.
In some embodiments, the subject is immunocompetent. In some embodiments, the subject is immunocompromised. Optionally, the vaccine may be used in combination with genetic testing to quantify the degree of immune-responders, or immune non-responders.
It will be appreciated by one of skill in the art that appropriate number of inactivated attenuated pathogen particles in the vaccine composition can vary. In some embodiments, the vaccine composition comprises about 1 x 103, about 1 x 104, about 1 x IO5, about 1 x 106, about 1 x 107, about 1 x 108, about 1 x 109, or about 1 x 1010 inactivated attenuated pathogen particles. In some embodiments, a inactivated attenuated vaccine composition comprises about 1 x 105 to about 1 x 108 pathogen particles.
In some embodiments, an inactivated attenuated vaccine dose further comprises an adjuvant. In some embodiments the adjuvant is CpG. In some embodiments, the CpG is CpG 7909. In some embodiments, a vaccine dose comprises from about 25 pg to about 750 pg CpG, or from about 50 pg to about 500 pg CpG. In some embodiments, a inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg CpG. In some embodiments, the adjuvant is CpG 1018. In some embodiments, a vaccine dose comprises from about 25 pg to about 750 pg CpG 1018, or from about 50 pg to about 500 pg CpG 1018. In some embodiments, an inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg CpG 1018. In some embodiments the adjuvant is ODN 1668. In some embodiments, an inactivated attenuated vaccine dose comprises from about 25 pg to about 750 pg ODN 1668, or from about 50 pg to about 500 pg ODN 1668. In some embodiments, an inactivated attenuated vaccine dose comprises about 50 pg or about 500 pg ODN 1668.
In some embodiments, about IxlO5 to about IxlO8 inactivated attenuated pathogen particles are administered to a subject in each administration. For example, about 1x10’, about 5xl05, about IxlO6, about 5xl06, about IxlO7, about 5xl07, or about 1x108 inactivated attenuated pathogen particles may be administered to a subject per administration. In some embodiments, the administered dose is a split dose, wherein the total number of inactivated attenuated pathogen particles for administration is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sub-doses.
In some embodiments, about 10 to about 100 micrograms of inactivated attenuated pathogen protein are administered to a subject in each administration. In some embodiments, about 1 to about 100 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 15 to about 50 picograms, or about 30 to about 40 picograms of pathogen protein are administered to a subject in each administration. For example, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 picograms of pathogen protein may be administered to a subject in each administration. In some embodiments, about 10 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 11 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 12 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 13 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 14 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 15 picograms of pathogen protein are administered to a subject in each administration. Tn some embodiments, about 16 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 17 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, about 35 picograms of pathogen protein are administered to a subject in each administration. In some embodiments, the administered dose is a split dose, wherein the total amount of pathogen protein for administration is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 sub-doses.
One or more sub-dose may be administered to the subject peripherally, at different locations on the subject’s body. Each sub-dose may be administered at approximately the same time, or administration of the sub-doses may be staggered. For example, sub-doses may be administered at intervals of 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 3 hours.
In some embodiments, the inactivated pathogen vaccine is administered once, or more than once to the subject. In some embodiments, the inactivated attenuated vaccine is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times to the subject.
The inactivated attenuated pathogen vaccine may be administered to the subject every day, about every 3 days, about every 7 days, about every fourteen days, about once per month, or about once per year. In some embodiments, the inactivated attenuated vaccine is administered at least once per week, at least every two weeks, or at least once every six months. In some embodiments, the vaccine is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, twelve times, fifteen times, twenty times, or twenty-five times in a year.
In some embodiments, a first dose (e.g., a prime dose) of inactivated attenuated pathogen vaccine is administered, followed by a second dose (e.g., a boost dose) to the subject. In some embodiments, the second dose is administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after the first dose. In some embodiments, the amount of pathogen protein in the first dose is greater than the amount of pathogen protein in the second dose. For example, the amount of pathogen protein may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% greater than the amount of pathogen protein in the first dose. In some embodiments, the amount of pathogen protein in the first dose is less than the amount of pathogen protein in the second dose. For example, the amount of pathogen protein may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than the amount of pathogen protein in the first dose. In some embodiments, the amount of pathogen protein in the first dose is about the same as the amount of pathogen protein in the second dose.
In some embodiments, a first inactivated attenuated pathogen vaccine and a second inactivated attenuated pathogen vaccine are administered to the subject. In some embodiments, the second inactivated attenuated pathogen vaccine is administered after the first vaccine to boost the immune response. In some embodiments, immune response in the subject is monitored between administration of the first vaccine and the second vaccine. In some embodiments, the second vaccine is administered when it is determined that the subject has not exhibited a satisfactory immune response following administration of the first vaccine.
The inactivated attenuated pathogen vaccine may be delivered to the subject intramuscularly, intramucosally, intranasally, subcutaneously, intratumorally, intradermally, transdermally, intravaginally, intraperitoneally, intrarectally, intra-articularly or intra- lymphatically, orally or intravenously. In some embodiments, administration may be by sublingual, buccal, intra-organ (e g., intrasplenic), or inhaled routes. For intravenous, cutaneous or subcutaneous injection, or injection at the site of an infection, the vaccine composition may be in the form of a parenterally acceptable aqueous solution which has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.
In some embodiments, the vaccine is administered peripherally to the subject. In some embodiments, multiple aliquots of the vaccine are administered peripherally to the subject, in different locations.
In some embodiments, the inactivated attenuated vaccine is administered simultaneously or sequentially (either before or after) with a vaccine-enhancing agent. In some embodiments, the vaccine-enhancing agent is an angiotensin receptor blocker (ARB) or a beta blocker (BB). Exemplary vaccine-enhancing agents include losartan, telmisartan, irbesartan, azilsartan, candesartan, eprosartan, olmesartan, valsartan, propranolol, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol, sotalol, timolol. In some embodiments, the vaccine-enhancing agent is selected from the group consisting of losartan and propranolol. In some embodiments, the inactivated attenuated vaccine-enhancing agent is losartan. In some embodiments, the vaccineenhancing agent is propranolol.
In some embodiments, a method for vaccinating a subject comprises administering an inactivated attenuated pathogen vaccine composition comprising inactivated attenuated pathogen particles, and a potent adjuvant comprising TLR3 and/or TLR9 agonists attached to liposomes, and also comprises sequential or simultaneous administration of a vaccine-enhancing agent (e.g., losartan), which is given at or around the time of vaccination and reduces recruitment of immune suppressive myeloid cells.
In some embodiments, a method for vaccinating a subject comprises administering an inactivated attenuated pathogen vaccine composition comprising inactivated attenuated pathogen particles to a subject in need thereof. An adjuvant may optionally be administered at the time of vaccination. In some embodiments, an adjuvant is administered after vaccination to boost the immune response, for example about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after vaccination. In some embodiments, the adjuvant comprises liposomes, e.g., CLDC. In some embodiments, a vaccine-enhancing agent such as losartan may be administered at or around the time of the vaccination. In some embodiments, a vaccine-enhancing agent such as losartan may be administered after vaccination, for example, about 6 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after vaccination. In some embodiments, a vaccine-enhancing agent such as losartan may be administered to the subject daily for an effective number of days, optionally beginning on the day that the vaccine is administered. In some embodiments, the vaccineenhancing agent (e.g., losartan) is administered at a dose of between about 5 and about 100 mg/kg, for example about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mg/kg.
The inactivated attenuated pathogen vaccine may elicit an immune response in the subject. In some embodiments, the immune response may include one or more of the following: (i) upregulation of immunoglobulin expression (e.g., IgG, IgM), (ii) T-cell activation, (iii) modulation of innate immune cells (e.g., myeloid cells), and (iv) revival of “exhausted” T-Cell populations. Suitable subjects include both avians and mammals. The term “avian” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets, and the like. The term “mammals” as used herein includes, but is not limited to, humans, nonhuman primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects.
METHODS FOR TREATING AND/OR PREVENTING ASF INFECTION
Also provided herein are methods for treating and/or preventing a ASF infection in a subject in need thereof. In some embodiments, the methods comprise administering to the subject an effective amount of one or more inactivated attenuated vaccine compositions described herein. The subject may be, for example, a mammal such as a pig.
In some embodiments, the inactivated attenuated vaccine is orally or administered intramuscularly. In some embodiments, the inactivated attenuated vaccine is administered subcutaneously. In some embodiments, the methods comprise administering a booster dose of the vaccine composition. The booster dose may be administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after administering the vaccine composition.
Also provided is a method for treating and/or preventing a ASF in a subject in need thereof, the method comprising administering to the subject a first vaccine composition comprising an effective amount of an inactivated attenuated ASFV vaccine composition described herein; and a second vaccine composition, comprising an effective amount of the inactivated attenuated ASFV vaccine composition. In some embodiments, the number of ASFV particles in the first vaccine composition is greater than the number of ASFV particles in the second vaccine composition. In some embodiments, the number of ASFV particles in the first vaccine composition is less than the number of ASFV particles in the second vaccine composition. In some embodiments, the number of ASFV particles in the first vaccine composition is about the same as the number of ASFV particles in the second vaccine composition. The second vaccine composition may be administered, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year after the first vaccine composition.
INACTIVATED ATTENUATED PATHOGENS AS IMMUNOGENS Attenuated pathogen inactivated according to the methods disclosed herein, and compositions comprising the same, may be used as immunogens. An immunogen is a molecule capable of eliciting an immune response in a subject.
The compositions described herein, including the immunogenic compositions, can be used to provoke an immune response in a host. In some embodiments, a composition comprising an inactivated attenuated pathogen is administered to a host, in order to provoke an immune response in the host. The immune response may comprise, for example, production of antibodies against various different antigens on the inactivated microbe. For example, an immunogen may be used to stimulate a subject’s immune system to produce antibodies against a pathogen (e.g., ASFV). Because an intact inactivated attenuated pathogen is used, the subject’s immune system will produce antibodies directed against a wide array of pathogen antigens (i.e., polyclonal antibodies). As used herein, the term “polyclonal antibodies” refers to antibodies that are secreted by different B cell lineages. In contrast, a monoclonal antibody comes from a single-cell lineage. A polyclonal antibody comprises a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. Typically, when a pathogen is administered to a host, the host produces a polyclonal antibody against the microbe that was administered. The polyclonal antibody will typically include a mixture of pathogen produced by different B cell lineages, and each antibody may identify a different epitope.
In some embodiments, a method of producing a polyclonal antibody that binds to a pathogen comprises i) generating an inactivated attenuated pathogen by contacting the microbe with a dose of UV light in the presence of riboflavin; ii) administering the inactivated attenuated pathogen to a host, wherein the host produces a polyclonal antibody; and iii) recovering the polyclonal antibody. In some embodiments, the microbe is tuberculosis.
As noted above, the antibodies can be harvested and purified and can be used therapeutically to treat subjects with a disease (e.g., ASF). The subject may be, for example, a mammal or an avian, as described above. In some embodiments, the subject is a pig. In some embodiments, the subject is a human. In some embodiments, an inactivated attenuated pathogen (e.g., ASFV), or a composition comprising the same, is administered to a subject in order to elicit immune response in the subject. The immune response may comprise, for example, production of antibodies that bind to and/or neutralize the pathogen. As noted above, an immunogenic composition comprises a polyclonal antibody that binds to a pathogen, wherein the polyclonal antibody is produced by administering to a host an inactivated attenuated pathogen, wherein the inactivation is performed by contacting the attenuated pathogen with a dose of UV light in the presence of riboflavin as described herein. In some embodiments, the attenuated pathogen is an attenuated viral particle selected from: adenovirus particle, an adeno-associated virus (AAV) particle, a lentivirus particle, a coronavirus particle, or a retrovirus particle. In some embodiments, the viral particle is a ASFV particle. In some embodiments, the inactivated attenuated viral particle is a Dengue, Zika, African Swine Fever, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, or Vesicular Stomatitis particle.
In some embodiments, the composition comprises an adjuvant. In some embodiments, the adjuvant is capable of promoting a Thl-type response. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments, the host is a mammal. In some embodiments, the host is a nonhuman primate, a bovine, an ovine, a caprine, an equine, a feline, a canine, a rodent or a lagomorph. In some embodiments, the host is a pig or human. In some embodiments, the host is an avian. In some embodiments, the host is a chicken, a duck, a goose, a quail, a turkey, a pheasant, a parrot, or a parakeet.
Also provided here in are methods for treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of an immunogenic composition as described herein. In some embodiments, the inactivated attenuated viral particle is ASFV, and the disease or disorder is ASF
In some embodiments, the immunogenic composition is administered intravenously to the subject. In some embodiments, the immunogenic composition is administered intramuscularly to the subject. In some embodiments, the subject is a human or a pig.
In some embodiments, a method of producing a polyclonal antibody that binds to a pathogen particle comprises generating an inactivated attenuated pathogen particle by contacting the particle with a dose of UV light in the presence of riboflavin; administering the inactivated attenuated viral particle to a host, wherein the host produces a polyclonal antibody; and recovering the polyclonal antibody. In some embodiments, the pathogen is an inactivated attenuated ASFV particle. In some embodiments, the host is a chicken, and the polyclonal antibody is recovered from an egg produced by the host. In some embodiments, the host is a pig. In some embodiments, the polyclonal antibody is recovered from the blood of the host. In some embodiments, the polyclonal antibody is recovered from B cells of the host.
Also provided are polyclonal antibodies produced by the methods described herein.
Also provided are methods for treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a polyclonal antibody described herein.
Also provided are methods for detecting the presence of a pathogen particle in a subject in need thereof, the method comprising contacting a biological sample of the subject with a polyclonal antibody as described herein; and detecting binding between the polyclonal antibody and the pathogen particle. In some embodiments, the viral particle is a ASFV particle.
In some embodiments, the biological sample is whole blood, serum, plasma, urine, saliva, lymph fluid, bile, cerebrospinal fluid, nasal mucus, or stool.
In some embodiments, the polyclonal antibody is conjugated to a substrate. In some embodiments, the substrate is a bead, a chip, a slide or a dish.
In some embodiments, the detecting step comprises contacting the polyclonal antibody with a secondary antibody that is conjugated to an enzyme or to a fluorophore.
The polyclonal antibodies produced the immunogenic compositions as described herein can be used to treat or prevent a disease in a subject in need thereof. In some embodiments, a polyclonal antibody generated in a host may be administered to a subject to treat or prevent a viral infection, such as a ASFV infection. In some embodiments, a method for treating or preventing a disease or disorder in a subject in need thereof comprises administering to a subject in need thereof an effective amount of a polyclonal antibody as described herein. In some embodiments, a method for treating or preventing a ASF in a subject in need thereof comprises administering to a subject in need thereof an effective amount of a polyclonal antibody as described herein. The effective amount may be determined according to standard practices. For example, the titer of circulating virus may be evaluated, and the dose of antibody required to achieve a specific neutralization of the virus may be established through Plaque Reduction Neutralization Tests in vitro. This information may subsequently be used to provide a suitable dose estimate for therapy. In some embodiments, the subject is assessed for immune function and immune status prior to administration of the composition. Such assessments may include, but are not limited to, DTH skin testing, blood tests, lymph node aspirate tests, tumor tissue tests, and/or determination of whether the subject is anergic, B cell responsive, etc.
DIAGNOSTIC METHODS
Attenuated pathogens inactivated according to the methods disclosed herein may be used in diagnostic compositions and methods. For example, the inactivated attenuated pathogens (for example, ASFV) may be used to detect the presence of an anti-pathogen antibody (e.g, a neutralizing antibody) in a biological sample. The biological sample may be, for example, blood (e.g., whole blood, serum, or plasma), stool, urine, saliva, or swab specimens of the nostril, throat, cervix, or urethra. Because intact inactivated attenuated pathogens are used, antibodies that bind to many different targets (i.e., different microbial epitopes) may be detected using a single assay.
In some embodiments, an inactivated attenuated pathogen (e.g., a ASFV particle) is coupled to and/or immobilized on a substrate. The substrate may be biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, fdms, plates, slides, etc. The substrate may have any convenient shape, such as a disc, square, sphere, and a circle. The substrate may, in some embodiments, form a rigid support. In some embodiments, the substrate and its surface may be chosen to provide appropriate light-absorbing characteristics. For instance, the substrate may be a polymerized Langmuir Blodgett fdm, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, or any one of a wide variety of polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, or combinations thereof. In some embodiments, the substrate may be a bead, a resin, a membrane, a fiber, a polymer, a matrix, a chip, a microplate or a tissue culture vessel.
In some embodiments, the substrate is pre-treated before the inactivated attenuated pathogen is coupled thereto. For example, the substrate may be treated with an enzyme, such as a DNAse, RNAse or protease. In some embodiments, the substrate may be coated with a polymer or a carbohydrate. In some embodiments, the substrate may be coated with a protein, such as an antibody, antibody fragment (e.g., Fab), or antibody derivative (e.g., a single chain variable fragment (scFv)). The inactivated attenuated pathogen particle may be coupled to the substrate using various different methods. For example, the inactivated attenuated pathogen particle may be reversibly or irreversibly coupled to the substrate. In some embodiments, the inactivated attenuated pathogen particle is coupled to the substrate via a linker. The linker may be a chemical or a protein linker. The chemical linker may be, for example, a carbohydrate linker, a polyether linker, a fatty acid linker, or a lipid linker. In some embodiments, the protein linker may comprise about 1 to about 50 amino acids. In some embodiments, the protein linker is a flexible linker (i.e., a linker that comprises small polar amino acids, including threonine, serine, and/or glycine). In some embodiments, the protein linker is not flexible (i.e., a linker that comprises one or more proline residues).
In some embodiments, the inactivated attenuated pathogen particles described herein may be used in a method for detecting the presence of an antibody in a biological sample. The antibody may be an antibody that binds to the inactivated attenuated pathogen particle, such as a neutralizing antibody. In some embodiments, the method for detecting an antibody in a biological sample comprises contacting the biological sample with a virus particle (e.g., a ASFV particle) that is coupled to a substrate. In some embodiments, the antibody binds to the pathogen particle that is coupled to the substrate, thereby immobilizing the antibody. In some embodiments, the method further comprises contacting the immobilized antibody with a second antibody, such as a detection antibody. The detection antibody may be coupled to, for example, a fluorophore, or to an enzyme (e g., horseradish peroxidase (HRP)) capable of cleaving a substrate (e g., a fluorescent substrate).
In some embodiments, the inactivated attenuated pathogen particles described herein may be used in an ELISA-based assay. An ELISA (enzyme-linked immunosorbent assay) is a platebased assay technique designed for detecting and quantifying proteins, such as antibodies, in a biological sample. In an ELISA, an antigen is typically immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection may be accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. There are several different types of ELISAs commonly used, including direct ELISAs, indirect ELISAs, sandwich ELISAs, and competitive ELISAs.
In some embodiments, an inactivated attenuated ASFV particle is coupled to a substrate. The inactivated attenuated ASFV particle is contacted with a biological sample comprising an antibody that binds to the S inactivated attenuated ASFV particle. A complex is formed between the antibody and to the virus particle that is coupled to the substrate, thereby immobilizing the antibody. After the biological sample (including any unbound antibodies) is washed away, the antibody is contacted with a detection antibody. The binding of the antibody to the ASFV particle is measured, for example, by detecting the amount of detection antibody bound. In some embodiments wherein the detection antibody is conjugated to an enzyme (e.g., an HRP), binding of the antibody to the ASFV particle may be measured by measuring the amount of a fluorophore produced when an appropriate substrate is added to the sample.
The following terms are used in the description herein and the appended claims:
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the detailed description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Furthermore, the term “about” as used herein when referring to a measurable value such as an amount, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
As used herein, the terms “reduce,” “reduces,” “reduction” and similar terms mean a decrease of at least about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% or more.
As used herein, the terms “enhance,” “enhances,” “enhancement” and similar terms indicate an increase of at least about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more.
As used herein, the term “vaccine” is typically understood to be a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response. The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal. The mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human.
As used herein, “African swine fever virus” (ASFV) is the causative agent of African swine fever (ASF). The virus causes a hemorrhagic fever with high mortality rates in pigs, but persistently infects its natural hosts, warthogs, bushpigs with no disease signs. It also infects soft ticks of the Oniithodoros genus, which are thought to be used as a vector. ASFV replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family. ASFV is endemic to sub-Saharan Africa and exists in the wild through a cycle of infection between ticks and wild pigs, bushpigs and warthogs. ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an “emerging infection.” ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least 150 genes. The number of genes differs slightly between different isolates of the virus. ASFV has similarities to the other large DNA viruses, . ., poxvirus iridovirus and mimivirus. In common with other viral haemorrhagic fevers, the main target cells for replication are those of monocyte, macrophage lineage.
Exemplary nucleotide and amino acid sequences for its constituent parts from select ASFV strains can selected from the following: GenBank Accession: (MK 128995); GenBank Accession: (AY261362); GenBank Accession: (NC_044956); GenBank Accession: (MN913970); GenBank Accession: (MW856068); GenBank Accession: (LR812933); GenBank Accession: (NC 044959.2); GenBank Accession: (MK645909); GenBank Accession: (LR813622); GenBank Accession: (AY261365); GenBank Accession: (AY261366): GenBank Accession: (AY261364); GenBank Accession: (AY261361); GenBank Accession: (NC_044946); GenBank Accession:(MH025920); GenBank Accession: MW856067); GenBank Accession: (NC 044945.1); GenBank Accession: LR899131); GenBank Accession: (MT956648); GenBank Accession: (AY261360); GenBank Accession: (MN630494.2); GenBank Accession: (MN641877.2); and GenBank Accession: (MN336500.3)
Further, based on sequence variation in the C-terminal region of the B646L gene encoding the major capsid protein p72, 22 ASFV genotypes (I-XXII) have been identified. All ASFV p72 genotypes have been circulating in eastern and southern Africa. Genotype I has been circulating in Europe, South America, the Caribbean and western Africa, Genotype VIII is confined to four East African countries. Examples of strains from some of the genotypes are given below:
Genotype I: OURT88/3; Brazil/79; Lisbon/60; BA715; Pret; Benin 97/1; IC/1/96; IC/576;
CAM/82; Madrid/62; Malta/78; ZAR85; Katange63; Togo; Dakar59; Ourt88/1; BEN/1/97; Dom Rep; VAL/76; IC/2/96; Awoshie/99; NIG/1/99; NIG/1/98; ANG/70; BEL/85; SPEC 120; Lisbon/57; ASFV-Warm; GHA/1/00; GAM/1/00; Ghana; HOL/86; NAM/1/80; NUR/90/1; CAM/4/85; ASFV-Teng; Tegani; ASFV-E75.
Genotype IE Georgia 2007/1
Genotype III: BOT 1/99
Genotype IV: ASFV-War; RSA/1/99NV
Genotype VI: MOZ 94/1
Genotype VIE VICT/90/1; ASFV-Mku; RSA/1/98
Genotype VIII: NDA/1/90; KAL88/1; ZAM/2/84; JON89/13; KAV89/1; DEZda; AFSV-
Mal; Malawi LIL 20/1
Genotype IX. UGA/1/95
Genotype X: BUR/1/84; BUR/2/84; BUR/90/1; UGA/3/95; TAN/Kwhl2; Hindell; ASFV- Ken; Virulent Uganda 65.
ASFV contains five multi-gene families which are present in the left and right variable regions of the genome. The MGFs are named after the average number of codons present in each gene: MGF100, 110, 300, 360 and 505/530. The N-terminal regions of members of MGFs 300, 360 and 505/530 share significant similarity with each other. Several genes in MGF360 and 505/530 determine host range and virulence. The five multigene families of the ASFV genome (MGF 100, MGF 110, MGF 360 and MGF 505/530) are located within the left hand 35 kb or right hand 15 kb terminal variable regions. The MGFs constititute between 17 and 25% of the total coding capacity of the ASFV genome. They lack similarity to other known genes.
The complete genome sequences of isolate Benin 97/1 (a highly pathogenic virus from West Africa, Group 1), isolate OURT88/3 (non-pathogenic, attenuated virus from Portugal, Group 1) and isolate BA71V (Vero cell tissue culture adapted non-pathogenic virus, Group 1) have been compared (Chapman et al (2008) J. Gen Virol. 89:397-408). The isolate OURT88/3 has a deletion of 10007 bp compared to Benin 97/1 extending from nucleotide number (nt no.) 19448 to nt no. 29525 in the Benin 97/1 genome. In all, five MGF 360 genes (10L, 11L, 12L, 13L and 14L) are deleted from the OURT88/3 genome. Two MGF 505 genes (1R, 2R) are deleted and the MGF 505R 3R gene is truncated in the OURT88/3 genome. These genes are present in the genomes of all eight other pathogenic isolates of ASFV that have been sequenced.
In the attenuated tissue-culture adapted BA71V isolate, the genome contains the MGF 505 3R gene but lacks the other seven MGF genes and in addition also has the MGF 3609L gene truncated (total deletion of 8250 bp).
Previously six MGF 360 genes (9L, 10L, 1 IL, 12L, 13L and 14L) and two MGF 505 genes (1R and 2R) were deleted from the highly pathogenic South African isolate Pr4. This deletion markedly reduced viral growth in primary macrophage cell cultures by 100- to 1000-fold (Zsak et al 2001, as above) and led to attenuation of the virus (cited as unpublished results, Afonso et al 2004 J. Virol 78: 1858-1864). However no experiments were carried out to challenge the recovered pigs to determine if they were protected. In fact, at an African swine fever virus workshop at the Biosecurity Research Institute, Manhattan Kans, in May 2011 it was mentioned that the Pr4 deletion mutant was not protective and induced a chronic form of the disease.
It has also been shown that deletion of three MGF 360 genes (12L, 13L and 14L) and four MGF 505 genes (1R, 2R, 3R and 4R truncation) from the pathogenic virus Malawi ADP71 reduces virus replication in pigs and in addition attenuated the virus (Neilan et al (2002) J. Virol. 76:3095- 3104). However again, no experiments were reported to determine if the recovered pigs were protected against challenge.
As used herein, the term “inactivated” refers to a pathogen, such as a virus or microbe, or a vaccine comprising microbes, that has been treated and/or modified so that the pathogen do not have disease-producing capacity. In some embodiments, an inactivated vaccine comprises microbes that have been killed by physical and/or chemical processes. In some embodiments, the inactivated microbes do not replicate in a subject. In some embodiments, the inactivated pathogen are alive, while in some embodiments, the inactivated microbes are dead. In a preferred embodiment, the inactivated pathogen is generated by the SolaVAX process.
As used herein, the term “attenuated,” the term “live vaccine” or “attenuated vaccine” refers to a vaccine containing a live viral active ingredient. In addition, “attenuation” means that the toxicity of a living pathogen is artificially weakened, and it does not cause disease in the body by mutating genes involved in the essential metabolism of pathogens, but only stimulates the immune system to induce immunity. Viral attenuation can be achieved by irradiation, chemical treatment, or n by high-order continuous subculture in vitro, a.k.a serial passage. Attenuation can also be achieved by making clear genetic changes, for example by specific deletions of viral sequences known to provide toxicity or by insertion of sequences into the genome.
As used herein, the term “inactivated/attenuated” or alternatively “attenuated/inactivated” refers to a pathogen, such as an ASFV particle, that has been attenuated and further inactivated, preferably through a SolaVAX process.
As used herein, the term “SolaVAX” refers to a vaccine comprising a pathogen particle, including a viral, bacterial or pathogen or fragments thereof, which have been inactivated using riboflavin and UV light as described herein. The systems, methods and processes that encompass SolaVAX are described by Goodrich et al., PCT/US2021/021190 and PCT/US2021/053160, entitled, the entire specifications, claims and figures related to the inactivation of pathogen particles using UV radiation in the presence of riboflavin is hereby being incorporated by reference.) The terms “pre-SolaVAX” and “post-SolaVAX” refer to pathogen preparations before and after inactivation, respectively, using riboflavin and UV light. In a preferred embodiment, the pathogen is attenuated prior to inactivation by application of SolaVAX treatment.
By the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and/or delay of the onset of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the disclosure. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset is less than what would occur in the absence of the present disclosure.
“Effective amount,” or “Therapeutically Effective Amounf’as used herein refers to an amount that, when administered to a subject for treating or preventing a disease (e.g., a viral infection), or at least one of the clinical symptoms of a disease, is sufficient to affect such treatment or prevention of the disease or symptom thereof. The “effective amount” may vary depending, for example, on the disease and/or symptoms of the disease, severity of the disease and/or symptoms of the disease or disorder, the age, weight, and/or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance may be ascertained by those skilled in the art or capable of determination by routine experimentation.
As is used herein, the term “pig” refers to an animal in the Suidae family of even-toed ungulates. The term pig includes a domestic pig and its ancestor, the common Eurasian wild boar (Sus scrofa), Palawan bearded pig, Bornean bearded pig, Heude's pig or Vietnamese warty pig, Visayan warty pig, Celebes warty pig, Flores warty pig, Mindoro warty pig, Philippine warty pig, Java warty pig, babirusa and warthog.
When used herein to refer to an amount, the term “viral protein” refers to the total amount of viral protein measured in a vaccine preparation using, for example, a standard assay to measure protein content.
As is used herein, the term “T cell epitope” or “T cell antigen”, refers to an epitope that can be recognized by the immune system after intracellular processing of an antigen. After processing, a T cell epitope becomes bound to at least one MHC molecule and is expressed on the surface of the antigen presenting cell as a MHC -peptide complex. T cell epitopes presented by MHC class I molecules are typically between 8 and 11 amino acids in length, whereas MHC class II molecules may present peptides of about 12-25 amino acids, preferably about 13-17 amino acids, in length. Software programs are available that may predict potential T cell epitopes within proteins on the basis of, for example, amphipathicity profdes of proteins, sequence motifs, quantitative matrices, artificial neural networks, support vector machines, quantitative structure activity relationship and molecular docking simulations [Desai and Kulkarni-Kale, 2014. Methods Mol Biol 1184: 333-64], These programs include IEDB Analysis Resource, ELISpot (PepScan, Lelystad, the Netherlands), RANKPEP [Reche et al., 2004. Immunogenetics 56: 405-19], nHLAPred [Bhasin and Raghava, 2007. J Biosci 32: 31-42], and NetMHC [Lundegaard et al., 2008. Nucleic Acids Res 36: W509-12],
The term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigenspecific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Thl-based antigen specific response or vice versa. Accordingly, an adjuvant may favorably modulate cytokine expression/secretion, antigen presentation, type of immune response etc. More specifically, when used herein to refer to an adjuvant, the term “capable of promoting a Thl-type immune response” refers to those adjuvants that promote a Thl- type response over a Th2-type response. Thl-type and Th2-type immune responses are distinguished by the types of immune cells involved and the cytokines produced thereby. T helper type 1 (Th 1) lymphocytes secrete interleukin (IL)-2, interferon-y, and lymphotoxin-a and stimulate type 1 immunity, which is often characterized by intense phagocytic activity. Conversely, Th2 cells secrete IL-4, IL-5, IL-9, IL-10, and IL-13 and stimulate type 2 immunity, which is often characterized by high antibody titers. Non-limiting examples of adjuvants capable of promoting a Thl-type response over a Th2-type response include CpG and/or AS01, CpG 1018, ODN 1688, or AdVaxTM. AdVaxTM comprises delta inulin, specifically delta inulin of highly specific particle size and morphology (See, e.g., Petrovsky, N., et al., Vaccine; 33(44): 5920-5926 (2015)). Not all adjuvants can promote a Thl-type immune response. For example, MontanideTM is one type of adjuvant that does not promote a Thl-type immune response (See, e.g., van Doorn, E., et al., Hum Vaccin Immunother; 12(1): 159-169 (2016)).
Immune system: The immune system may protect organisms from infection. If a pathogen breaks through a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts contains so called humoral and cellular components.
Immune response: An immune response may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response). The invention relates to the core to specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g. ASFV. However, this specific response can be supported by an additional unspecific reaction (innate immune response). Therefore, the invention also relates to a compound for simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient adaptive immune response.
Adaptive immune system: The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. The system is highly adaptable because of somatic hypermutation (a process of increased frequency of somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the gene rearrangement leads to an irreversible change in the DNA of each cell, all of the progeny (offspring) of that cell will then inherit genes encoding the same receptor specificity, including the Memory B cells and Memory T cells that are the keys to long-lived specific immunity. Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions. Adaptive immune response: The adaptive immune response is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by “memory cells”. Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. In this context, the first step of an adaptive immune response is the activation of naive antigen- specific T cells or different immune cells able to induce an antigen-specific immune response by antigen- presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing. Cell types that can serve as antigen- presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Thl cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Thl cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.
Cellular immunity/cellular immune response: Cellular immunity relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
Humoral immunity /hum oral immune response: Humoral immunity refers typically to antibody production and the accessory processes that may accompany it. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
Innate immune system: The innate immune system, also known as non-specific immune system, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g. Tolllike receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL- 9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN- gamma, GM-CSF, G-CSF, M-CSF, LT- beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), a CpG- DNA, an antibacterial agent, or an anti-viral agent. Typically a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and/or acting as a physical and chemical barrier to infectious agents.
Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the context of the invention may typically be an RNA that is able to induce an innate immune response itself. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an innate immune response e.g. by binding to a specific kind of Toll-like- receptor (TLR) or other suitable receptors. However, of course also mRNAs having an open reading frame and coding for a peptide/protein (e.g. an antigenic function) may induce an innate immune response.
Antigen: In the context of the present invention “antigen” refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. In the sense of the present invention an antigen may be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, also fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigen. In a preferred embodiment, an antigen may preferably be an antigen related to the ASFV.
Epitope (also called “antigen determinant” ): T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule. B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form. In a preferred embodiment, an antigen may preferably be an epitope related to one or more antigens of the ASFV.
Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides, and preferably from ASFV. In this context antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three- dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
The term “expression,” as used herein, or “expression of a coding sequence” (for example, a gene or a transgene) refer to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).
The term “nucleic acid” or “nucleic acid molecules” include single- and double- stranded forms of DNA; single- stranded forms of RNA; and double-stranded forms of RNA (dsRNA). The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex. The term “ribonucleic acid” (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA). The term “deoxyribonucleic acid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. The terms “nucleic acid segment” and “nucleotide sequence segment,” or more generally “segment,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encoded or may be adapted to encode, peptides, polypeptides, or proteins.
The term “gene” or “sequence” refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons). The term “structural gene” as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide. It should be noted that any reference to a SEQ ID, or sequence specifically encompasses that sequence, as well as all corresponding sequences that correspond to that first sequence. For example, for any amino acid sequence identified, the specific specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa. The term “peptide” or “protein” is a polymer of amino acid monomers. Usually the monomers are linked by peptide bonds. The term “peptide” does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.
“Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of the disclosed compound(s) together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (19th Edition). In one embodiment, a pharmaceutical compositions of the invention may include a quantity of HIV- 1, and a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable excipient or carriers
Such pharmaceutical compositions/formulations are useful for administration to a subject, in vivo or ex vivo. Pharmaceutical compositions and formulations include carriers or excipients for administration to a subject. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically compatible formulation, gaseous, liquid, or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery, or contact. Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or nonaqueous), emulsions (e g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) can also be incorporated into the compositions. The formulations may, for convenience, be prepared or provided as a unit dosage form. In general, formulations are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. For example, a tablet may be made by compression or molding. Compressed tablets may be prepared by compressing, in a suitable machine, an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be produced by molding, in a suitable apparatus, a mixture of powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.
Pharmaceutical formulations and delivery systems appropriate for the compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18. sup. th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12. sup. th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) l l .sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315). For example, pharmaceutical compositions can optionally be formulated to be compatible with a particular route of administration. Exemplary routes of administration include administration to a biological fluid, an immune cell (e.g., T or B cell) or tissue, mucosal cell or tissue (e.g., mouth, buccal cavity, labia, nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina, rectum, or colon), neural cell or tissue (e.g., ganglia, motor or sensory neurons) or epithelial cell or tissue (e.g., nose, fingers, ears, cornea, conjunctiva, skin or dermis). Thus, pharmaceutical compositions include carriers (excipients, diluents, vehicles, or filling agents) suitable for administration to any cell, tissue, or organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by various routes and delivery, locally, regionally, or systemically.
Exemplary routes of administration for contact or in vivo delivery of a target inhibitor, is a dosage of the compound that is sufficient to achieve a desired therapeutic effect, such as can optionally be formulated include inhalation, respiration, intubation, intrapulmonary instillation, oral (buccal, sublingual, mucosal), intrapulmonary, rectal, vaginal, intrauterine, intradermal, topical, dermal, parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epidural), intranasal, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, ophthalmic, optical (e.g., corneal), intraglandular, intraorgan, and intralymphatic. The invention encompasses isolated or substantially purified pathogens, such as virions, bacterium or constituents thereof. In one example, “isolated” or “purified” ASFV virions or constituents thereof, are substantially or essentially free from components that normally accompany or interact with ASFV virions or constituents thereof as found in its naturally occurring environment.

Claims

CLAIMS What is claimed is:
1. A method for inactivating an attenuated pathogen, the method comprising contacting the attenuated pathogen with a dose of UV light in the presence of riboflavin, wherein the method does not substantially alter the structure of antigens on the pathogen.
2. The method of claim 1 , wherein the attenuated pathogen comprises a viral pathogen.
3. The method of claim 1, wherein the viral pathogen comprises African Swine Fever Virus (ASFV).
4. The method of claim 3, wherein the ASFV comprises an attenuated ASFV strain selected from: ASFV-G-AI177L, ASFV-G-A9GL/AUK, and ASFV-G-AI177LALVR, or a combination of the same.
5. The method of claim 2, wherein the viral pathogen is an adenovirus particle, an adeno- associated virus (AAV) particle, a lentivirus particle, a coronavirus particle, or a retrovirus particle.
6. The method of claim 2, wherein the viral particle is a Dengue, Zika, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, or Vesicular Stomatitis particle.
7. The method of claim 1, wherein the attenuated pathogen comprises an attenuated vaccine selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow fever vaccine; Zoster/shingles vaccine; Enterotoxigenic Escherichia coli vaccine; Shigella vaccine, Vibrio cholera vaccine; Tick-borne encephalitis vaccine; COVID-19 vaccine, Avian influenza virus H5N1 and H5N3 subtypes, Foot-and-mouth disease vaccine.
8. The method of claim 1, wherein the attenuated pathogen comprises an attenuated bacterial pathogen.
9. The method of claim 7, wherein the attenuated bacterium is selected from the group consisting of: Actinomyces israelii, Acinetobacter haumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difficult, Clostridium neoformans, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterobacter aerogenes, Enterobacler cloacae, Francisella tularensis, vancomycin-resistant Enterococci (VRE), Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, Leptospira mayottensis, Leptospira noguchii, Leptospira santar osai, Leptospira weilii, Listeria monocytogenes, Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma pneumonia, Nocardia farcinica, Neisseria meningitides, Neisseria gonorrhoeae, Nocardia asteroids, Pseudomonas aeruginosa, Propionibacterium acnes, Rickettsia rickettsia, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella sp., Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Staphylococcus aureus, methicillin- susceptible Staphylococcus aureus (MSSA), methicillin- resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B, Streptococcus Group C, Treponema pallidum, Vibrio cholera, Yersinia pestis, and Yersinia pseudotuberculosis.
10. The method of claim 1, wherein the attenuated pathogen comprises an attenuated fungus.
11 . The method of claim 9, wherein the attenuated fungus is selected from the group consisting of: Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladiella mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Exophiala jeanselmei, Exophiala dermatitidis, Curvularia lunata, Bipolar is spicifera, Bipolar is hawaiiensis, Bipolaris cynodontis, Alternaria alternata, Histoplasma capsulatum, Histoplasma duboisii, Histoplasma farciminosum, Lacazia loboi, Mucor indicus, Malassezia furfur, Trichosporon asahii, Pneumocystis jirovecii, Pneumocystis carinii, Paracoccidioides brasiliensis, Pseudoalle scher ia boydii, Paracoccidioides lutzii, Penicillium marnejfei, Penicillium aurantiogriseum, Sporothrix schenkii, Stachybotrys chartarum, Saccharomyces cerevisiae, Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globose.
12. The method of claim 1, wherein the attenuated pathogen comprises an attenuated parasite.
13. The method of claim 11, wherein the attenuated parasite is selected from the group consisting of: Ancylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coli, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Fasciola hepatica, Fasciola magna, Fasciola gigantic, Giardia lamblia, Isospora belli, Leishmania donovani, Leishmania infantum, Leishmania mexicana, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Naegleria fowleri, Onchocerca volvulus, Dracancuhis medinensis, Trichinella spiralis, Strongyloides steroralis, Clonorchis sinensis, Paragonimus westermani, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium know le si, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongy, Taenia saginata, Taenia solium, Trichomonas vaginalis, Trypanosoma hrucei, Trypanosoma cruzi, Toxoplasma gondii and Wuchereria bancrofti.
14. The method of claim 1, wherein the attenuated pathogen comprises an attenuated microbe belonging to the genus Mycobacterium .
15. The method of claim 11, wherein the attenuated Mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Erdman strain of Mycobacterium tuberculosis, H37Rv strain of Mycobacterium tuberculosis, or CDC1551 strain of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare .
16. The method of claim 1, wherein the attenuated pathogen comprises a pathogen that is the causative agent for a disease or condition selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitiosa; Infection with Bonamia ostreae; Infection with Marteilia refringens; Infection with Microcytos mackini; Infection with Perkinsus marinus; West-Nile- Virus; Infectious Hematopoietic Necrosis of Salmonids; Ovine Epididymitis (Brucella ovis); Koi Herpesvirus Infection of Carp; Lumpy- skin-Disease (Dermatitis nodularis); Bovine Pleuropneumonia; Foot-and- Mouth-Disease; Anthrax; Newcastle Disease; Avian Influenza; Peste des Petits Ruminants; Equine Encephalomyelitis; Sheep and Goat Pox; Blackleg; Rift Valley Fever; Rinderpest; Glanders; Bovine Salmonellosis; Swine Fever; Vesicular Stomatitis; Taura Syndrome; Rabies; Transmissible Spongiform Encephalopathies (including Bovine Spongiform Encephalopathy (BSE) and Scrapie); Bovine Trichomonosis; Bovine Tuberculosis (Mycobacterium bovis und Mycobacterium caprae); Swine Vesicular Disease; Bovine Genital Campylobacteriosis; Viral Hemorrhagic Septicemia of Salmonids; White Spot Disease of Crustaceans; Vibriosis; Shigellosis; Hepatopancreatic Acute Necrosis Syndrome; and Yellowhead Disease.
17. The method of any one of claims 1-16, wherein the dose of UV light is about 80 Joules to about 1000 Joules.
18. The method of any one of claims 1-41, wherein the method comprises altering a nucleic acid of the pathogen.
19. The method of claim 18, wherein the method comprises selectively oxidizing guanine bases in the nucleic acid.
20. The method of claim 19, wherein the UV light selectively oxidizes about 1 to about 30 guanine bases in the nucleic acid of the pathogen.
21. The method of claim 20, wherein the UV light selectively oxidizes about 20 guanine bases in the nucleic acid of the pathogen.
22. The method of any one of claims 8-21, wherein the nucleic acid of the viral particle is a DNA or an RNA.
23. The method of any one of claims 1-22, wherein the inactivated attenuated pathogen is not capable of reverting to a non-attenuated form.
24. The method of any one of claims 1-23, wherein the inactivated attenuated pathogen is not capable of replicating.
25. The method of any one of claims 1-24, wherein the inactivated attenuated pathogen is not capable of causing disease in a subject.
26. A vaccine composition comprising an inactivated attenuated pathogen according to any one of claims 1-25.
27. The vaccine composition of claim 26, wherein the composition comprises about 1 to about 100 picograms of pathogen protein.
28. The vaccine composition of claim 27, wherein the composition comprises about 15 to about 50 picograms of pathogen protein.
29. The vaccine composition of claim 28, wherein the composition comprises about 35 picograms of pathogen protein.
30. The vaccine composition of any one of claims 26-29, wherein the composition comprises an adjuvant.
31. The vaccine composition of claim 30, wherein the adjuvant is capable of promoting a Thl- type immune response.
32. The vaccine composition of claim 30 or 31, wherein the adjuvant is capable of limiting a Th2- type response.
33. The vaccine composition of any one of claims 30-32, wherein the adjuvant is CpG and/or AS01.
34. The vaccine composition of any one of claims 30-32, wherein the adjuvant is a phosphorothioate oligonucleotide comprising about 15 to about 30 nucleotides.
35. The vaccine composition of any one of claims 30-32, wherein the adjuvant comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3 '.
36. The vaccine composition of any one of claims 30-32, wherein the adjuvant comprises the sequence 5 -TCC ATGACGTTCCTGATGCT-3 '.
37. The vaccine composition of any one of claims 30-32, wherein the adjuvant is ODN 1668.
38. The vaccine composition of any one of claims 30-32, wherein the adjuvant is CpG 1018.
39. The vaccine composition of any one of claims 26-38, wherein the composition comprises a pharmaceutically acceptable carrier or excipient.
40. A vaccine composition comprising an attenuated ASFV viral particle, that has been inactivated with a dose of UV light in the presence of riboflavin, wherein the method does not substantially alter the structure of antigens on the pathogen, and wherein the ASFV genome comprises one or more oxidized guanine residues.
41. The vaccine composition of claim 40, wherein the attenuated ASFV comprises an attenuated ASFV strain selected from: ASFV-G-AI177L, ASFV-G-A9GL/AUK, and ASFV-G- AI177LALVR, or a combination of the same.
42. The vaccine composition of claim 41, wherein the composition comprises about 15 to about 50-100 picograms of viral protein.
43. The vaccine composition of claim 42, wherein the composition comprises about 35 picograms of viral protein.
44. The vaccine composition of any one of claims 40-43, wherein the composition comprises an adjuvant.
45. The vaccine composition of claim 44, wherein the adjuvant is capable of promoting a Thl- type immune response.
46. The vaccine composition of claim 44 or 45, wherein the adjuvant is capable of limiting a Th2- type response.
47. The vaccine composition of any one of claims 44-46, wherein the adjuvant is CpG and/or AS01.
48. The vaccine composition of any one of claims 44-46, wherein the adjuvant is a phosphorothioate oligonucleotide comprising about 15 to about 30 nucleotides.
49. The vaccine composition of any one of claims 44-46, wherein the adjuvant comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-3
50. The vaccine composition of any one of claims 44-46, wherein the adjuvant comprises the sequence 5 '-TCCATGACGTTCCTGATGCT-3 '.
51. The vaccine composition of any one of claims 44-46, wherein the adjuvant is ODN 1668.
52. The vaccine composition of any one of claims 44-46, wherein the adjuvant is CpG 1018.
53. The vaccine composition of any one of claims 40-52, wherein the attenuated ASFV genome comprises about 1 to about 30 oxidized guanine bases.
54. The vaccine composition of any one of claims 53, wherein the attenuated ASFV genome comprises about 20 oxidized guanine bases.
55. The vaccine composition of any one of claims 40-54, wherein the composition comprises a pharmaceutically acceptable carrier or excipient.
56. A method for treating or preventing a pathogen infection in a subject in need thereof, the method comprising administering to the subject an effective amount of the vaccine composition of any one of claims 26-39 or 40-55.
57. The method of claim 56, wherein the subject is a mammal.
58. The method of claim 57, wherein the subject is a pig.
59. The method of any one of claims 56-58, wherein the pathogen is selected from: African Swine Fever Vims (ASFV), Dengue, Zika, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, Vesicular Stomatitis particle, Actinomyces israelii, Acinetobacter baumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella cants, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difficule, Clostridium neoformans, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Francisella tularensis, vancomycin-resistant Enterococci (VRE), Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, Leptospira mayottensis, Leptospira noguchii, Leptospira santar osai, Leptospira weilii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Erdman strain of Mycobacterium tuberculosis, H37Rv strain of Mycobacterium tuberculosis, or CDC1551 strain of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare, Mycoplasma pneumonia, Nocardia farcinica, Neisseria meningitides, Neisseria gonorrhoeae, Nocardia asteroids, Pseudomonas aeruginosa, Propionibacterium acnes, Rickettsia rickettsia, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella sp., Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus (MSSA), methicillin- resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B, Streptococcus Group C, Treponema pallidum, Vibrio cholera, Yersinia pestis, Yersinia pseudotuberculosis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladi lla mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Exophiala jeanselmei, Exophiala dermatitidis, Curvularia lunata, Bipolaris spicifera, Bipolaris hawaiiensis, Bipolaris cynodontis, Alternaria alternata, Histoplasma capsulatum, Histoplasma duboisii, Histoplasma farciminosum, Lacazia loboi, Mucor indicus, Malassezia furfur, Trichosporon asahii, Pneumocystis jirovecii, Pneumocystis carinii, Paracoccidioides brasiliensis, Pseudoalle scher ia boydii, Paracoccidioides lutzii, Penicillium marneffei, Penicillium aurantiogriseum, Sporothrix schenkii, Stachybotrys chartarum, Saccharomyces cerevisiae, Sporothrix brasiliensis, Sporothrix schenckii, Sporothrix globose, ncylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coll, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Fasciola hepatica, Fasciola magna, Fasciola gigantic, Giardia lamblia, Isospora belli, Leishmania donovani, Leishmania infantum, Leishmania mexicana, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Naegleria fowleri, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Strongyloides steroralis, Clonorchis sinensis, Paragonimus westermani, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium know le si, Schistosoma mansonii, Schistosoma haematobium, Schistosoma Japonicum, Schistosoma mekongy, Taenia saginata, Taenia solium, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii and Wuchereria bancrofti.
60. The method of any one of claims 56-458, wherein the pathogen is an attenuated vaccine selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow fever vaccine; Zoster/shingles vaccine; Enterotoxigenic Escherichia coli vaccine; Shigella vaccine, Vibrio cholera vaccine; Tick-borne encephalitis vaccine; COVID-19 vaccine, Avian influenza virus H5N1 and H5N3 subtypes, Foot-and-mouth disease vaccine.
61. The method of any one of claims 56-8, wherein the pathogen comprises an pathogen that is the causative agent for a disease or condition selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitiosa; Infection with Bonamia ostreae; Infection with Marteilia refringens; Infection with Microcytos mackini; Infection with Perkinsus marinus; West-Nile- Virus; Infectious Hematopoietic Necrosis of Salmonids; Ovine Epididymitis (Brucella ovis); Koi Herpesvirus Infection of Carp; Lumpy- skin-Disease (Dermatitis nodularis); Bovine Pleuropneumonia; Foot-and- Mouth-Disease; Anthrax; Newcastle Disease; Avian Influenza; Peste des Petits Ruminants; Equine Encephalomyelitis; Sheep and Goat Pox; Blackleg; Rift Valley Fever; Rinderpest; Glanders; Bovine Salmonellosis; Swine Fever; Vesicular Stomatitis; Taura Syndrome; Rabies; Transmissible Spongiform Encephalopathies (including Bovine Spongiform Encephalopathy (BSE) and Scrapie); Bovine Trichomonosis; Bovine Tuberculosis (Mycobacterium bovis und Mycobacterium caprae); Swine Vesicular Disease; Bovine Genital Campylobacteriosis; Viral Hemorrhagic Septicemia of Salmonids; White Spot Disease of Crustaceans; Vibriosis; Shigellosis; Hepatopancreatic Acute Necrosis Syndrome; and Yellowhead Disease.
62. A method for producing a vaccine, the method comprising (i) providing a plurality of attenuated pathogen particles, and (ii) inactivating the attenuated pathogen particles by contacting them with UV light in the presence of riboflavin, and wherein the structure of antigens on the attenuated pathogen particle is not substantially altered compared to an attenuated pathogen particle that has not been inactivated.
63. The method of claim 62, wherein the method comprises purifying the inactivated attenuated pathogen particles.
64. The method of claim 62 or 63, wherein the inactivated attenuated pathogen particles are ASFV viral particles, or ASFV viral particles selected from: ASFV-G-AI177L, ASFV-G-A9GL/AUK, and ASFV-G-AI177LALVR, or a combination of the same.
65. The method of claim 62 or 63, wherein the inactivated attenuated pathogen particles are selected from: African Swine Fever Virus (ASFV), Dengue, Zika, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (('MV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, Vesicular Stomatitis particle, Actinomyces israelii, Acinetobacter baumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difflcule, Clostridium neoformans, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Francisella tularensis, vancomycin-resistant Enterococci (VRE), Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, Leptospira mayottensis, Leptospira noguchii, Leptospira santar osai, Leptospira weilii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Erdman strain of Mycobacterium tuberculosis, H37Rv strain of Mycobacterium tuberculosis, or CDC1551 strain of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare, Mycoplasma pneumonia, Nocardia farcinica, Neisseria meningitides, Neisseria gonorrhoeae, Nocardia asteroids, Pseudomonas aeruginosa, Propionibacteriuni acnes, Rickettsia rickettsia, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella sp., Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Stenotrophomonas maltophilia, Staphylococcus aureus, methicillin- susceptible Staphylococcus aureus (MSSA), methicillin- resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B, Streptococcus Group C, Treponema pallidum, Vibrio cholera, Yersinia pestis, Yersinia pseudotuberculosis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladiella mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferation, Exophiala jeanselmei, Exophiala dermatitidis, Curvularia lunata, Bipolaris spicifera, Bipolaris hawaiiensis, Bipolaris cynodontis, Alternaria alternata, Histoplasma capsulatum, Histoplasma duboisii, Histoplasma farciminosum, Lacazia loboi, Mucor indicus, Malassezia furfur, Trichosporon asahii, Pneumocystis jirovecii, Pneumocystis carinii, Paracoccidioides brasiliensis, Pseudoalle scher ia boydii, Paracoccidioides lutzii, Penicillium marneffei, Penicillium aurantiogriseum, Sporothrix schenkii, Stachybotrys chartarum, Saccharomyces cerevisiae, Sporothrix brasiliensis, Sporothrix schenckii, Sporothrix globose, ncylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coli, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Fasciola hepatica, Fasciola magna, Fasciola gigantic, Giardia lamblia, Isospora belli, Leishmania donovani, Leishmania infantum, Leishmania mexicana, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Naegleria fowleri, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Strongyloides steroralis, Clonorchis sinensis, Paragonimus westermani, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium know le si, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongy, Taenia saginata, Taenia solium, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii and Wuchereria bancrofti
66. The method of claim 62 or 63, wherein the inactivated attenuated pathogen particles is an attenuated vaccine selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow fever vaccine; Zoster/shingles vaccine; Enterotoxigenic Escherichia coli vaccine; Shigella vaccine, Vibrio cholera vaccine; Tick- borne encephalitis vaccine; COVID- 19 vaccine, Avian influenza virus H5N1 and H5N3 subtypes, Foot-and-mouth disease vaccine
67. The method of claim 62 or 63, wherein the inactivated attenuated pathogen particles comprises an pathogen that is the causative agent for a disease or condition selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitiosa; Infection with Bonamia ostreae; Infection with Marteilia refringens; Infection with Microcytos mackini; Infection with Perkinsus marinus; West-Nile- Virus; Infectious Hematopoietic Necrosis of Salmonids; Ovine Epididymitis (Brucella ovis); Koi Herpesvirus Infection of Carp; Lumpy-skin-Disease (Dermatitis nodularis); Bovine Pleuropneumonia; Foot-and- Mouth-Disease; Anthrax; Newcastle Disease; Avian Influenza; Peste des Petits Ruminants; Equine Encephalomyelitis; Sheep and Goat Pox; Blackleg; Rift Valley Fever; Rinderpest; Glanders; Bovine Salmonellosis; Swine Fever; Vesicular Stomatitis; Taura Syndrome; Rabies; Transmissible Spongiform Encephalopathies (including Bovine Spongiform Encephalopathy (BSE) and Scrapie); Bovine Trichomonosis; Bovine Tuberculosis (Mycobacterium bovis und Mycobacterium caprae); Swine Vesicular Disease; Bovine Genital Campylobacteriosis; Viral Hemorrhagic Septicemia of Salmonids; White Spot Disease of Crustaceans; Vibriosis; Shigellosis; Hepatopancreatic Acute Necrosis Syndrome; and Yellowhead Disease.
68. A composition comprising an attenuated pathogen, wherein said attenuated pathogen has been inactivated by contacting it with UV light in the presence of riboflavin forming an inactivated attenuated pathogen, and wherein the structure of antigens on the inactivated attenuated pathogen particle is not substantially altered compared to an attenuated or wild-type pathogen that has not been inactivated.
69. The composition of claim 68, wherein the inactivated attenuated pathogen is isolated.
70. The composition of claim 68 or 69, wherein the inactivated attenuated pathogen particles is ASFV, or ASFV strain selected from: ASFV-G-AI177L, ASFV-G-A9GL/AUK, and ASFV-G- AI177LALVR, or a combination of the same.
71. The composition of claim 68 or 69, wherein the inactivated attenuated pathogen is selected from: African Swine Fever Virus (ASFV), Dengue, Zika, Influenza, Marburg, Rabies, Human Immunodeficiency Virus (HIV), Smallpox, Hantavirus, Rotavirus, SARS-CoV, MERS-CoV, Cytomegalovirus (CMV), Ebola, Epstein-Barr, Herpes, Hepatitis, Human Papillomavirus, Mumps, Measles, Rubella, Polio, Varicella Zoster, Respiratory Syncytial Virus (RSV), Semliki Forest, West Nile, Yellow Fever, Vesicular Stomatitis particle, Actinomyces israelii , Acinetobacter baumanii, Bacillus anthracis, Burkholderia cepacia, Bacterioides fragilis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Bordetella pertussis, Bacillus subtilis, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter freundii, Campylobacter jejuni, Clostridium botulinum, Clostridium difficule, Clostridium neoformans, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Enterohacter aerogenes, Enterobacter cloacae, Francisella tularensis, vancomycin-resistant Enterococci (VRE), Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumonia, Legionella pneumophila, Leptospira alexanderi, Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, Leptospira mayottensis, Leptospira noguchii, Leptospira santar osai, Leptospira weilii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium orygis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, Mycobacterium mungi, Erdman strain of Mycobacterium tuberculosis, H37Rv strain of Mycobacterium tuberculosis, or CDC1551 strain of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium hominissuis, Mycobacterium colombiense, Mycobacterium indicus pranii, and Mycobacterium intracellulare , Mycoplasma pneumonia, Nocardia farcinica, Neisseria meningitides, Neisseria gonorrhoeae, Nocardia asteroids, Pseudomonas aeruginosa, Propionibacterium acnes, Rickettsia rickettsia, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella sp., Shigella flexneri, Shigella sonnet, Shigella dysenteriae, Stenotrophomonas maltophilia, Staphylococcus aureus, methicillin- susceptible Staphylococcus aureus (MSSA), methicillin- resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B, Streptococcus Group C, Treponema pallidum, Vibrio cholera, Yersinia pestis, Yersinia pseudotuberculosis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Blastomyces dermatitidis, Basidiobolus ranarum, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida tripicalis, Candida parpasilosis, Cryptococcus neoformans, Cryptococcus gattii, Coccidioides immitis, Coccidioides posadasii, Conidiobolus coronatus, Conidiobolus incongruous, Cladophialphora bantianum, Rhinocladiella mackenziei, Dactylaria gallopava, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum, Exophiala jeanselmei, Exophiala dermatitidis, Curvularia lunata, Bipolaris spicifera, Bipolaris hawaiiensis, Bipolaris cynodontis, Alternaria alternata, Histoplasma capsulatum, Histoplasma duboisii, Histoplasma farciminosum, Lacazia loboi, Mucor indicus, Malassezia fiirfur, Trichosporon asahii, Pneumocystis jirovecii, Pneumocystis carinii, Paracoccidioides brasiliensis, Pseudoalle scher ia boydii, Paracoccidioides lutzii, Penicillium marneffei, Penicillium aurantiogriseum, Sporothrix schenkii, Stachybotrys chartarum, Saccharomyces cerevisiae, Sporothrix brasiliensis, Sporothrix schenckii, Sporothrix globose, ncylostoma ceylanicum, Ancylostoma duodenale, Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba rhysodes, Acanthamoeba divionensis, Acanthamoeba lugdunensis, Acanthamoeba lenticulata, Ancylostoma duodenale, Angiostrongylus cantonensis, Brugia malayi, Balamuthia mandrillaris, Babesia microti, Balantidium coli, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium, Dientamoeba fragilis, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Fasciola hepatica, Fasciola magna, Fasciola gigantic, Giardia lamblia, Isospora belli, Leishmania donovani, Leishmania infantum, Leishmania mexicana, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichuria, Necator americanus, Naegleria fowleri, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Strongyloides steroralis, Clonorchis sinensis, Paragonimus westermani, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium malariae, Plasmodium know le si, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mekongy, Taenia saginata, Taenia solium, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Toxoplasma gondii and Wuchereria bancrofti
72. The composition of claim 68 or 69, wherein the inactivated attenuated pathogen is an attenuated vaccine selected from: live attenuated influenza vaccine (LAIV); Adenovirus vaccine; Anthrax vaccine; Cholera vaccine; Yersinia pestis vaccine; Salmonella vaccine; Tuberculosis vaccine; Typhoid vaccine; Japanese encephalitis vaccine; Measles vaccine; Mumps vaccine; Measles and rubella (MR) vaccine; Measles, mumps, and rubella (MMR) vaccine; Measles, mumps, rubella and varicella (MMRV) vaccine; Polio vaccine; Rotavirus vaccine; Rubella vaccine; Smallpox/monkey pox vaccine; Varicella vaccine; Yellow fever vaccine; Zoster/shingles vaccine; Enterotoxigenic Escherichia coli vaccine; Shigella vaccine, Vibrio cholera vaccine; Tick- borne encephalitis vaccine; COVID- 19 vaccine, Avian influenza virus H5N1 and H5N3 subtypes, Foot-and-mouth disease vaccine
73. The composition of claim 68 or 69, wherein the inactivated attenuated pathogen comprises an pathogen that is the causative agent for a disease or condition selected from: Monkeypox; African Horse Sickness; African Swine Fever (ASF); American Foulbrood; Equine Infectious Anemia (EIA); Infectious Salmon Anemia (ISA); Aujeszky’s Disease; Small Hive Beetle Infestation (Aethina tumida); Tropilaelaps Infestation of Honey Bees; Dourine; Bluetongue Disease (BTV); Bovine Herpesvirus Type 1 Infection; Bovine Viral Diarrhea (BVD); Bovine, Porcine, Ovine and Caprine Brucellosis; Ebola Virus Infection; Enzootic Bovine Leukosis; Epizootic Hemorrhagic Disease (EHD); Epizootic Hematopoietic Necrosis (EHN); Fowl Plague; Infection with Bonamia exitiosa; Infection with Bonamia ostreae; Infection with Marteilia refringens; Infection with Microcytos mackini; Infection with Perkinsus marinus; West-Nile-Virus; Infectious Hematopoietic Necrosis of Salmonids; Ovine Epididymitis (Brucella ovis); Koi Herpesvirus Infection of Carp; Lumpy-skin-Disease (Dermatitis nodularis); Bovine Pleuropneumonia; Foot- and- Mouth-Disease; Anthrax; Newcastle Disease; Avian Influenza; Peste des Petits Ruminants; Equine Encephalomyelitis; Sheep and Goat Pox; Blackleg; Rift Valley Fever; Rinderpest; Glanders; Bovine Salmonellosis; Swine Fever; Vesicular Stomatitis; Taura Syndrome; Rabies; Transmissible Spongiform Encephalopathies (including Bovine Spongiform Encephalopathy (BSE) and Scrapie); Bovine Trichomonosis; Bovine Tuberculosis (Mycobacterium bovis und Mycobacterium caprae); Swine Vesicular Disease; Bovine Genital Campylobacteriosis; Viral Hemorrhagic Septicemia of Salmonids; White Spot Disease of Crustaceans; Vibriosis; Shigellosis; Hepatopancreatic Acute Necrosis Syndrome; and Yellowhead Disease.
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