WO2008148164A1 - Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire - Google Patents

Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire Download PDF

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WO2008148164A1
WO2008148164A1 PCT/AU2008/000811 AU2008000811W WO2008148164A1 WO 2008148164 A1 WO2008148164 A1 WO 2008148164A1 AU 2008000811 W AU2008000811 W AU 2008000811W WO 2008148164 A1 WO2008148164 A1 WO 2008148164A1
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cells
vaccine
vaccines
phages
phage
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Ian Andrew Ferguson
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Ian Andrew Ferguson
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Priority to CA2727126A priority Critical patent/CA2727126A1/fr
Priority to EP08756895A priority patent/EP2167110A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14042—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14045—Special targeting system for viral vectors

Definitions

  • This invention is in the fields of medicine, biochemistry, and vaccines.
  • it relates to vaccines that can be manufactured very rapidly and in huge quantities by using specialized viruses that grow in bacteria (rather than requiring bird eggs or other eukaryotic cells for viral incubation), and that can be administered by spraying a mist into the nasal cavities, without requiring needles or syringes.
  • vaccines function by presenting foreign antigens, to cells that function as part of a mammalian immune system. This process allows an immune system to lay the groundwork for accelerated formation of antibodies that will help block and kill invading pathogens, if a need ever arises in the future. Beyond that basic level, the immune system is quite complex, and uses multi-step sequences involving numerous different types of cells. Chapter-length descriptions that provide good overviews are available in books such as Alberts et al, Molecular Biology of the Cell, or Guyton and Hall,
  • Thomas et al 2005 reviews genetically-engineered vaccines, while articles such as Gaubin et al 2003, Clark et al 2004, Wang et al 2004, and Miedzybrodzki et al 2005 focus on bacteriophages as vaccine carriers or delivery vehicles.
  • Various materials also are available via the Internet. For example, the materials for numerous college or medical school courses on immunology are available (one example is at http://uhaweb.hartford.edu/BUGL/immune.htm). Glossaries also are available, such as at http://users.path.ox.ac.uk/ ⁇ scobbold/tig/gloss.html. Accordingly, background information herein on the immune system is intended only as an introduction and overview, and more detailed information is available from other sources.
  • nasal and other mucosal vaccines have not reached a point where they are as effective as injected vaccines, and they have not been highly successful.
  • the obstacles that must be overcome by nasal vaccines, and the progress they have made in recent years, are discussed in articles such as Eriksson et al 2002, Kiyono et al 2004, and Mestecky et al 2005.
  • Mucoadherents also called “absorption enhancers” or similar terms
  • a nasal spray or similar formulation will cause a nasal spray or similar formulation to either: (i) cling to the nasal sinuses (or other mucous membranes) for a longer period of time, or (ii) penetrate through a mucous membrane more rapidly, and in higher quantities. Either of those effects can increase the likelihood that an antigen in a vaccine will be noticed and recognized as foreign, by the immune system, in ways that will provoke a desired immune response. Accordingly, mucoadherents can be used with nasal vaccines as disclosed herein.
  • Examples include chitosan and certain types of cyclodextrins, phospholipids, and other "bioadhesive" compounds, described in articles such as Davis et al 2003 and Zuercher 2003, and powders that convert into gels when they become wet, such as a compound from aloe vera plants sold by DelSite Biotechnologies under the trademarks GELVAC and GELSITE
  • an adjuvant is an agent or substance which, while not having any specific antigenic effect in itself, can stimulate an immune system in a manner that increases a response to a vaccine.
  • an adjuvant is an agent or substance which, when combined with an antigen, reduces the amount of antigen that is needed in order to stimulate an effective immune response.
  • an adjuvant can increase both the speed and the magnitude of an immune response to a vaccine.
  • FCA Freund's complete adjuvant
  • mycobacteria pathogenic microbes
  • FCA tends to provoke painful inflammatory responses, as part of its mode of action. Therefore, it is not used in human medicine, and when laws were passed in most countries (mostly in the 1980's) requiring that any pain and suffering imposed on lab animals must be minimized, other adjuvants began to be actively developed, as described in Eriksson et al 2002 (which focuses on mucosal adjuvants) and Guy et al 2005.
  • Yuki et al 2003 and Lycke 2004 describe adjuvants in which a cholera toxin protein fragment is combined with an E. coli toxin fragment or a Staphylococcus aureus toxin fragment.
  • CT Cholera toxin
  • HT heat- labile E. coli toxin
  • antigen trafficking (collectively referred to as "antigen trafficking"), which raises questions about whether an immune response to a vaccine would properly prepare a host to withstand an actual pathogenic infection; and, (ii) they provoked inflammation in the nasal tract.
  • adjuvants offer the promise of incorporating a polypeptide sequence having adjuvant activity, into a "disarmed" (nonpathogenic) viral particle or fragment (or into an engineered chimeric protein) that also carries an antigenic polypeptide sequence. Therefore, in modern vaccines, adjuvants can be agents (such as known polypeptide sequences that have adjuvant-like activity) that are incorporated into the same particle or protein that also carries an antigen (such as a polypeptide sequence derived from a surface protein of a pathogen).
  • adjuvants can be divided into two classes, based on their mechanism of action.
  • Delivery adjuvants increase immune responses by means that increase the amount of contact between antigens, and “antigen-presenting cells” (APCs), using mechanisms such as described above.
  • APCs antigen-presenting cells
  • immunological adjuvants activate the immune system by stimulating certain types of cells to release cytokines (i.e., hormone-type messenger molecules that trigger various types of cellular responses), or by other similar actions as described in articles such as Hunter (2002).
  • mucosal vaccines One field that requires particular attention involves mucosal vaccines. This includes vaccines that are administered topically to a "mucous membrane", also called an “epithelial” membrane. Such membranes include the mouth, nasal sinuses, vagina, rectum, etc.
  • epidermal cells The type of skin that covers most of the body and the limbs is made of epidermal cells. These cells are created by a "budding” process, in which an underlying layer of cells continuously creates a set of “partial” offspring cells. These cells become dehydrated and flat ("squamous") as they move closer to the outer skin surface, and by the time they reach a subsurface layer called the “stratum corneum”, they are "enucleated” (i.e, they no longer contain a nucleus or chromosomes). By the time they reach the outer epidermal surface, they are effectively dehydrated and nonviable, and cannot support viral replication. This makes them ideal as an outer protective layer. They typically last for only a few days on the surface, then they peel and flake off, in the form of dried single cells or tiny clusters that usually are too small to be seen by the naked eye.
  • epithelial cells In contrast to dry epidermal skin, mucous membranes are covered by epithelial cells. Instead of providing a protective coating of flattened and dehydrated dead or dying cells, epithelial cells remain hydrated, to sustain their viability. After they reach an outer surface of a mucous membrane, they have a relatively short lifespan (typically about 4 to about 8 days), and during that period, they are much more active than epidermal cells, and generally are capable of supporting viral infections, especially if any lesions, abrasions, or other breaches in a mucous membrane enable viruses to penetrate through the outermost layers.
  • mucosal vaccines can avoid any need for needles or syringes, which pose various problems (including hazardous waste disposal, theft by drug users, accidents that can infect healthcare workers, etc.), and which offend or intrude on the cultural norms of some societies.
  • mucosal vaccines that are incorporated into genetically engineered plants offer the potential for helping reduce animal-related and food-related diseases.
  • phage is a shortened form of bacteriophage, derived from the Greek words for "eats bacteria". Most phages will kill and destroy their bacterial hosts. However, a special class of viruses was discovered that can infect bacteria and then emerge, in large numbers, without killing the host cells. These specialized viruses are long and thin "filaments" which, after reproducing in a cell, are thin enough to emerge through pores or other outlets in a cell's membranes, without damaging the cell. Even though these types of filamentous viruses do not "eat” or kill their hosts, they were nevertheless called bacteriophages, or simply phages.
  • phages Since they do not kill their hosts, they can be grown in bacterial cell culture at enormously high rates, so long as the bacteria are cultured with shaking or stirring, to prevent the phages from smothering the bacteria by sheer volume and bulk. [00025 ] Several types of phages have been manipulated in ways that render them highly useful for genetic engineering. The most popular phages, used in laboratories around the world, usually combine all of the following traits:
  • the DNA they carry is small enough to be easily manipulated; (ii) they allow small or moderate-sized foreign polypeptide sequences to be inserted into segments of a first "coat" protein that is present in more than a thousand copies in each phage particle, without disrupting the ability of the modified phages to replicate in bacteria;
  • One form is a preparation containing numerous copies of single specific phage carrying a single foreign gene sequence (or "insert").
  • This type of preparation often called a clonal or monoclonal phage, can be used for various purposes, such as for making vaccines.
  • clonal phages containing a protein sequence from the human beta-amyloid protein (which forms beta-amyloid plaques, in the brains of people suffering from Alzheimer's disease) have been created by Beka Solomon, Dan Frenkel, and their coworkers at Tel Aviv University.
  • the cerebral inflammation may have been due to either or both of two factors: (1) an autoimmune disorder may have arisen, when the immune systems of those patients began attacking a protein that occurs naturally in the blood and brain; and/or (2) beta-amyloid plaques may function as a type of "plumber's putty", which controls and reduces the leakage of blood out of capillary walls that have become thin and fragile, in the brains of elderly people; accordingly, if the sticky deposits that control that type of leakage are dissolved, blood leakage into the brain tissue may increase, leading to edema, inflammation, and other problems.
  • phage display library also called a phage library
  • This type of preparation is a mixture containing millions or billions of phages that carry different foreign polypeptide sequences.
  • phage libraries enable "screening" tests that allow certain specific phages to be selected and isolated, using one or more cellular or physiological processes.
  • the phages that respond in a certain way, in a screening test can be isolated, reproduced, and analyzed, to determine the foreign DNA and/or polypeptide sequence that caused those particular phages to behave in a certain way, in that particular screening test.
  • phage display libraries contain foreign DNA sequences with either random or controlled nucleotide sequences, which will be expressed into random or controlled variations in the coat proteins of phages.
  • That invention can be regarded as comprising two main components, which interact with each other to provide a complete operative embodiment.
  • the first portion can be briefly summarized as follows:
  • BBB-straddling neurons include, for example, olfactory receptor neurons (which have tips that are accessible in the nasal sinuses), and various neurons with tips that can be reached by liquids injected into the tongue, or into various muscles.
  • the gene vectors have certain types of properly-selected proteins on their surfaces (proper selection involves the second part of the invention, described below), they will be taken into the accessible tips of the BBB-straddling neurons.
  • This type of transport into the tips of neurons, uses "endocytotic" receptors on the exposed and accessible tips of the neurons. Endocytosis is a well-known process, and a number of endocytotic receptors that appear on the surfaces of certain types of BBB-straddling neurons have been identified and fully sequenced.
  • the trick to activating and driving both: (i) entry into the neuronal tips, using endocytotic receptors, and (ii) internal transport to reach the main cell bodies of the neurons, is to find and use an effective "transport” protein (which can also be called a delivery, carrier, or locomotive protein, or similar terms) that will activate and drive two different processes, which are endocytotic uptake into the neuron, and retrograde transport within the neuron.
  • transport protein which can also be called a delivery, carrier, or locomotive protein, or similar terms
  • a method for screening and identifying such proteins, using a phage display library that provided billions of candidate protein sequences, and that used a screening test to select, identify, and isolate a few such proteins which actually function in the desired manner, is described below, in the discussion of the second major component of this invention.
  • the gene carried by the vector can be expressed into proteins, and the proteins can be provided with a "leader sequence", which can enable two functions (i) transport of the protein molecules by neuronal fibers that travel deeper into the brain; and.
  • the "transport” or “locomotive” proteins that are exposed on the surfaces of the BBB-penetrating genetic vectors must be able to activate and drive two different cellular processes, which are: (1) uptake into neuronal fibers, followed by (2) transport through the neuronal fibers, toward the main cell bodies of the neurons.
  • the Inventor developed an in vivo screening process, which initially used the sciatic nerve bundle, and which later was extended to olfactory neurons.
  • the sciatic nerve is the main nerve bundle that travels from the base of the spinal cord, through the hip and the leg, to the foot.
  • a "ligature” was created, by surgically placing and then tightening a loop made from a suture strand, around the sciatic nerve bundle near the knee of a rat. This created a form of stress which caused the affected neurons to "upregulate” (i.e., increase the expression and placement of) certain types of cell surface receptors, including the so-called "p75" receptor, which attempts to help nerve cells recover from injuries.
  • the phage harvesting site (near the hip) was more than a centimeter away from the placement site (near the knee), the only phages that could reach the harvesting site were phages that had been taken inside a neuronal cell fiber, and that had been transported toward the spinal cord. Since those phages, traveling inside the nerve bundle, could not cross the constriction created by the tightened loop of suture thread near the hip, the fluid that was attempting to flow through the nerve bundle, toward the spinal cord, caused the phages to cluster and crowd into the nerve region immediately "distal" to the ligature (i.e., on the "downstream" side of the ligature, distant from the spinal cord).
  • phages of interest were harvested, by isolating and removing a short segment of the sciatic nerve bundle, immediately downstream from the ligature at the hip.
  • the phages were removed from the neuronal fiber segments, by using a buffer and enzyme mixture to digest the cell membranes (which are made of lipids) but not the virus particles (which are covered by proteins).
  • the phages were then replicated in bacterial cells, to provide an "amplified" population.
  • the resulting mixture of phages from one round of screening was then screened again, using the same process.
  • the screening procedure can be repeated any number of times, using an "enriched" phage population from a prior screening cycle as the starting material for a subsequent screening cycle.
  • Fluorescent labels can be identified and tracked visually, when thin slices of tissue from a sacrificed animal are analyzed under ultraviolet or similar light. However, in human use, tissue sections will not be available, so other types of labels must be used, such as (for example) short-lived isotopes that will show up in non-invasive imaging methods, such as single positron emission computerized tomography (SPECT) scans, computerized axial tomography (CAT) scans, magnetic resonance imaging, etc.
  • SPECT single positron emission computerized tomography
  • CAT computerized axial tomography
  • NALT tissue a specialized type of tissue that belongs to the immune system, and to differences between the "innate” immune system (which does not generate antibodies), and the “adaptive” immune system (which generates antibodies).
  • NALT refers to "nasopharyngeal-associated lymphoid tissue".
  • Neso- refers to the nose, including the nasal sinuses.
  • Pharyngeal refers to the pharynx, which is a transitional region in the back of the throat, below or behind the mouth, but above the esophagus. Therefore, “naso-pharyngeal tissue” includes tissues located in the nasal sinuses, in the rear portion of the mouth, and/or in the upper throat region.
  • Lymph refers to watery fluids and cells that permeate out of blood vessels and then travel through the soft tissues. Instead of containing cells packed closely or tightly together, soft tissues contain a substantial fraction of extracellular water (typically about 1/6, by volume), in a "gel” matrix held together by proteins.
  • the extracellular fluid in tissue gel allows nutrition to reach cells that are not directly adjacent to a blood vessel, and it also allows certain types of immune cells to permeate and travel through soft tissue, as described in more detail below. Those immune cells slowly travel to lymph nodes, which are highly important in the immune system. Accordingly, the term “lymphoid” has two distinct but heavily connected and interrelated meanings. One meaning refers to the watery fluids that slowly pass and permeate through soft tissues; the other meaning refers to specialized immune cells and tissues that use, handle, or travel in lymph fluids.
  • NALT nasopharyngeal-associated lymphoid tissue
  • tissue the acronym can be used as a noun; however, it is also commonly used as an adjective, so “NALT cells” is correct usage, and "NALT tissue”, although not truly proper, should be politely tolerated.
  • NALT is sometimes referred to as nose or nasal tissue, but that definition might exclude the tonsils or adenoids, which are NALT cells, so it is not used herein.
  • GALT gut-associated lymphoid tissue, which includes specialized intestinal tissues called “Peyer's patches”
  • MALT mucosa-associated lymphoid tissue, which includes both GALT and NALT cells.
  • M cells epithelial membrane cells
  • M cells are sometimes also referred to as mucosal cells and/or as microvilli or microfold cells.
  • a layer of M cells rests on top of an underlying cluster or nodule of tissue called a "subepithelial lymphoid follicle".
  • the M cells contain finger-like and/or heavily folded protrusions, often called microvilli or microfolds, which increase their surface areas and their ability to contact and interact with molecules that are being inhaled, or that are passing through the digestive tract. They began to receive serious attention in the 1980's, and over a hundred review articles describe their structures, activities, and roles in the immune system, as well as various efforts to exploit and use them for vaccines or other research or medical purposes. Such reviews include Kuper et al 1992, Ermak et al 1998,
  • M cells are regarded as a component of NALT or GALT tissues, and as NALT or GALT cells. Because of certain embryo logical factors, which focus on the origins of cells rather than their functions, some authors may regard or refer to M cells as being a cooperating but distinct layer, which is not a part of the actual immune system. Such distinctions are merely semantic, so long as a reader understands that M cells provide a surface layer of cells that have active and crucially important sampling and transport roles, resting on top of an underlying layer, cluster, or follicle of immunoactive cells.
  • NALT cells and tissues are highly important to the immune and allergic systems, since many pathogens, allergens, and other compounds are inhaled, and their first contact in a mammalian body occurs in the nasal cavities. Therefore, mammals evolved with certain types of specialized cells that are exposed on the surfaces of the mucous membranes in the nasal sinuses and mouths, which are active components of the immune systems. When these cells encounter a protein and recognize it as foreign, they effectively "grab" the protein and help deliver it to a lymph node, so that other cells of the immune system can process it, and can create antibodies that will help the body defend against the foreign antigen, if appropriate.
  • NALT tissues offers a potential for transport activities triggered by foreign proteins using immune cells that can travel in the fluid drainage system provided by lymph. That is a very different mechanism, compared to neurons having long fibers that will transport nerve growth factor or other therapeutic proteins into brain tissues, by means of endocytosis followed by retrograde flow within a neuronal axon.
  • a complete mammalian immune system actually comprises two different subsystems, which are called the innate immune system, and the adaptive immune system.
  • the innate system is "hard-wired” and works very rapidly, while the adaptive system takes much more time to generate a response. This is analogous to the way a nervous system enables both: (1) hard- wired and very rapid "reflex" responses, such as withdrawing a finger immediately, when a hot surface is touched; and, (2) learning, which takes longer, but which can accomplish much more complex and sophisticated tasks.
  • the adaptive system requires and uses antibodies, which require participation by several different types of white blood cells, including B cells, T cells, helper T cells, killer cells, etc.
  • This response takes several days to complete, and any delay lasting that long would allow most microbes (which can reproduce many times faster than mammalian cells) to generate huge numbers of invading microbes, before a complete antibody response can move into action. That delay period explains why vaccines, if prepared and administered in ways that allow an animal's immune system to partially get ready, in advance, before an infection even begins, can make a huge difference in how severe a disease or infection will become.
  • That delay period i.e., the fact that several days will pass, before an animal's adaptive immune system can respond fully
  • This "innate” system can respond almost immediately, to help the body fight and slow down a set of invading microbes, while reinforcements (which are analogous to heavy artillery, including antibodies, B cells, T cells, killer cells, etc.) are being prepared, produced, and moved into position.
  • reinforcements which are analogous to heavy artillery, including antibodies, B cells, T cells, killer cells, etc.
  • the innate immune system responds and acts rapidly, as a first line of defense, without having to wait for days; then, while the innate immune system is slowing down the invaders, an adaptive (antibody) response is planned, organized, and made ready.
  • the innate immune system also is regarded and sometimes referred to as a "primitive" immune system. Since most invertebrate animals do not have full immune systems with antibodies, their only line of defense against microbes is their innate immune system.
  • PRRs pattern recognition receptors
  • PAMPs pathogen-associated molecular patterns
  • PAMPs pathogen-associated molecular patterns
  • flagella As a second example, animal cells do not have or use certain types of protein complexes that are highly conserved in "flagella", which are the moving whip-like strands that E. coli and various other types of bacteria use to move about, in a liquid. Therefore, flagellar proteins from bacteria became another pathogen-associated molecular pattern.
  • the genes of vertebrates evolved in ways that have two general traits: (i) methyl groups are gradually bonded to cytosine nucleotides, as an animal ages; and, (ii) there tend to be relatively few cytosine and guanidine nucleotides positioned immediately next to each other, in a DNA strand of a higher animal. Therefore, strands of DNA having unusually high numbers of unmethylated cytosine and guanidine nucleotides, adjacent to each other, in the body of an animal larger than a rodent, indicates that a microbial invasion probably is occurring. Therefore, DNA strands having large numbers of unmethylated cytosine-guanine dinucleotide pairs (this pattern is referred to as a "CpG motif) can activate an important class of "toll receptors", discussed below.
  • PAMPs pathogen-associated molecular patterns
  • PRRs pattern recognition receptors
  • receptors are not limited to the standard types of cell-surface receptors found in mammals; instead, they also include other types of molecules that can effectively latch onto (or otherwise respond to) one or more pathogen- associated molecular patterns that are present in various important classes of pathogenic microbes. This leads to a discussion of "toll receptors”.
  • toll receptors An important subclass of "pattern recognition receptors" in animals is called “toll receptors”. These were first seen in Drosophila (i.e., small fruit flies that are widely used in genetic research). They were called “toll” receptors not because of any particular function, but because "toll” is a German word for "amazing”. Since their definitions and boundaries are not always clear, especially as the DNA and amino acid sequences for toll receptors in mice are used to search for "homologous" sequences in humans, they are often referred to as “toll-like receptors", using the acronym "TLR” followed by a number, such as TLR4 or TLR9.
  • toll-like receptors At least 11 types have been identified in humans; in addition, two other types are believed to exist in mice, and there is heavy overlap between human and mice TLR's. Each toll-like receptor type is associated with at least one type of microbial "ligand" that will activate that subclass of TLRs.
  • the known TLR types, and the ligands which activate them, are listed and described in literature that can be downloaded at no charge from Imgenex (www.imgenex.com), a company that sells ligands, antibodies, and other compounds used in research on toll receptors.
  • TLRs function in the same way, and they can be grouped into two main classes, in terms of location.
  • TLRl which includes TLRl, TLR2, TLR4, TLR5, TLR6, and TLRlO
  • the receptors straddle the outer membrane of an animal cell, with an extra-cellular portion, and an intra-cellular portion. If the extra-cellular portion binds to a microbial pathogen (or to an artificially-administered PAMP ligand), changes are triggered in the intra-cellular domain.
  • TLR9 receptors are associated with membrane-enclosed organelles (also called vesicles) located inside a cell.
  • TLR9 receptors are of particular interest herein, since they are located inside macrophage cells (described below), and since they have been studied extensively. TLR9 ligands are fairly well understood, and are described in articles such as Krieg 2002 and Klinman et al 2004.
  • toll receptor activation of a toll receptor will trigger a series of reactions that will lead to movement (translocation) of certain types of DNA transcription factors. These initial responses lead to genes being expressed that will generate and/or trigger the activation or release of certain cytokines, which will then trigger a cellular or multicellular reaction, such as an inflammatory response, or cellular release of various antimicrobial agents.
  • toll receptors recently have been recognized as important "gatekeepers", with functions that can be regarded as comparable to sensors. They can play major roles in determining whether: (i) a complete antibody immune response will be launched, in response to an apparent invasion by a pathogenic microbe; or, (ii) only a localized and/or allergic or tolerance reaction will be commenced.
  • phage vectors as disclosed herein can be modified so that they will specifically target certain types of toll receptors that are located entirely inside immune cells, such as TLR9 receptors. This can be very advantageous, since it reduces and minimizes the risks of triggering system-wide shock, a "cytokine storm", or other undesired responses that would be more likely to occur if toll receptors on the external surfaces of macrophages were being targeted.
  • a vaccine might generate either an undesired allergic reaction, or a desired antibody- forming response.
  • a vaccine might generate either an undesired allergic reaction, or a desired antibody- forming response.
  • some vaccines used to treat specific patients suffering from cancer or other diseases are designed to provoke "cell-mediated immune responses", which involved specialized cytokines (signaling molecules) and activated T cells, without involving B cells or creating antibodies.
  • an important goal in vaccine development is to identify and use vaccine components and formulations that will maximize the likelihood of a desired response (such as antibody formation), while minimizing the risk of unwanted responses (such as allergic reactions).
  • a desired response such as antibody formation
  • unwanted responses such as allergic reactions.
  • MHC-I receptors there are two different classes of “major histocompatibility complex” (MHC) receptors; these are known as MHC-I receptors (present on nearly all cell types) and
  • MHC-2 receptors (present only on certain types of immune cells);
  • cytotoxic T cells which will directly kill animal cells that harbor pathogenic microbes
  • helper T cells which secrete messenger molecules such as lymphokines, interleukins, or cytokines, which activate other cells in the immune system
  • helper T cells are further subdivided into two important classes, which are called THl cells (which secrete interleukin-2 and gamma-interferon), and TH2 cells (which secrete interleukin-4 and interleukin-5);
  • immunoglobulin molecules there are five different classes of immunoglobulin molecules, which are: (1) IgM globulins, which are the initial forms of globulins sometimes bound to the cell membranes; (2) IgG molecules, which are the classic Y-shaped antibodies that are secreted by B cells to fight invading microbes; (3) IgA globulins, which are secreted by mucous membranes, and which latch onto airborne or other arriving pathogens in an effort to inactivate them and deliver them to the stomach or secrete them from the body; (4) IgD globulins, which are membrane-bound antibodies produced by B cells early in their life cycle; and, (5) IgE globulins, which are involved in allergic reactions.
  • IgM globulins which are the initial forms of globulins sometimes bound to the cell membranes
  • IgG molecules which are the classic Y-shaped antibodies that are secreted by B cells to fight invading microbes
  • MHCl and ThI components work together to promote "cell-mediated" responses (also called cytotoxic responses), which involve cytokines and other signaling molecules and helper T cells, without requiring antibodies.
  • MHC2 and Th2 components work together to promote antibody formation.
  • Those two types of responses are not mutually exclusive, and they can reinforce and supplement each other, to produce exceptionally strong and powerful immune responses. Accordingly, if a single vaccine preparation can provoke both type of responses, it may be able to provide better protection against various types of diseases. However, issues of timing may also be important, in such matters.
  • monocytes have surface molecules that grip the interior walls of capillaries and then permeate through the capillary walls, causing monocytes to leave the circulating blood and enter the tissue itself. After they leave a blood vessel, the monocytes swell to a larger size. When that occurs, the enlarged cells become macrophages; as described below, they are also called phagocytes or phagocytic cells, because of how they surround and engulf small particles.
  • Macrophages travel slowly through soft tissues, within the lymph fluid.
  • soft tissues contain extracellular watery fluid (typically about 1/6 by volume) in a "gel” form that is held together by thick bundles of collagen, and by protein filaments coated with sugar groups, called proteoglycans.
  • the slow travel of macrophages, through lymph fluid is analogous to a policeman walking a beat rather than riding in a car; instead of being in a hurry to get somewhere, the policeman is mostly just looking around, to see if anything is present that should not be there.
  • Neutrophil cells should also be mentioned briefly. They act in a manner similar to monocytes, but important differences exist. Neutrophils normally remain in circulating blood until they receive a signal (such as from a cytokine, lymphokine, or other signaling molecule) indicating that an infection is occurring in a certain location. In response to such signals, neutrophils undergo a transformation, pass through a capillary wall, and enter the lymph fluid in the infected area of tissue.
  • a signal such as from a cytokine, lymphokine, or other signaling molecule
  • the neutrophils then swell to a larger size, and begin attacking the foreign particles, hi some cases, they can engage in the classical form of phagocytosis (as described below), but more commonly, a neutrophil will trigger an "oxidative burst” when it senses that it has reached and contacted an invading pathogen.
  • This "oxidative burst” creates and releases large number of "free radicals", which are molecules having unstable and aggressively reactive oxygen atoms with unpaired electrons. Those radicals will attack and help destroy invading microbes, and they often kill the neutrophil as well.
  • the whitish material called pus in infected tissues, often consists largely of neutrophil cell remains.
  • That defensive process, by neutrophils, can be referred to as phagocytosis, but not all authors include or refer to it as actual phagocytosis, which focuses upon engulfing a foreign particle and taking it inside a defending cell.
  • phagocytosis that defensive process, by neutrophils, can be referred to as phagocytosis, but not all authors include or refer to it as actual phagocytosis, which focuses upon engulfing a foreign particle and taking it inside a defending cell.
  • phagocytosis By way of analogy, if a man is using a knife and fork to cut apart a piece of meat, some people would refer to that preparatory action as eating, while others would say that eating does not actually begin until the person puts the food into his mouth.
  • Those types of semantic distinctions are not important, so long as one understands the overall process.
  • phagocytosis Since viruses are particles rather than cells, the word “phagocytosis” has come to refer to cellular ingestion of any type of relatively large particle (including, for example, particles made of starch or plastic, which can be fluorescently labeled in ways that enable the rates of phagocytosis to be easily measured, using automated machines such as flow cytometers). Phagocytosis is distinct from a similar process called “pinocytosis”, which refers to the ingestion of very small particles and/or liquids. In general, phagocytosis is regarded as the cellular equivalent of eating, while pinocytosis is the cellular equivalent of drinking.
  • pathogens have positive charges on their surfaces. This helps them rapidly grasp and infect a mammalian cell, and that type of rapid action is hugely important for pathogenic microbes.
  • the same positive surface charges that help pathogenic microbes attach to and infect mammalian cells also helps macrophages recognize and destroy the foreign invaders.
  • certain other cell types also can perform phagocytosis, including Schwann cells, certain types of glial cells, and various classes of dendritic cells.
  • phagocyte and phagocytic are also used inconsistently for another reason. Some people use those terms to refer to macrophages and other phagocytic cells at all times, while others use those terms only when cells are actively engaged in phagocytosis.
  • phagocytosis involved in apoptosis i.e., the replacement of aging cells in soft tissues
  • phagocytosis involved in apoptosis does not lead to antigen presentation, even though macrophages are involved, potential conflicts between those different uses of the same term should be noted.
  • a macrophage when a macrophage encounters a foreign object, it changes shape, by extending projections (which can also be called fingers, "pseudopods", or other terms) out from the main cell body. These projections begin extending around a foreign particle, in a way that enables the projections to meet and merge on the far side of the particle, effectively engulfing the particle and taking it inside the macrophage.
  • This type of shape-changing is similar in several respects to the motions of amoeba and certain other types of microbes, when they encounter and engulf smaller microbes. In its earliest forms, phagocytosis enabled single-cell microbes such as amoebae to obtain nutrition.
  • a macrophage When a particle-engulfing process begins, a macrophage is sometimes called a dendrite, or a dendritic cell. Those terms are derived from a Greek word for branching, or treelike. Accordingly, the three cell types that are commonly referred to, in the literature, as “professional” phagocytes are macrophages, neutrophils, and dendrites. The terms “dendrite” and “dendritic cells” are not preferred herein, since they are more commonly used in medicine to refer to branching structures that occur among neurons, which use fibers to establish connections with other neurons.
  • phagocytic receptors When carried out by macrophages, the phagocytic engulfing process is activated by cell-surface receptors, usually called “phagocytic receptors” or “phagosome receptors". These receptors are important to this invention, and they are discussed in more detail below, since phage vaccine cassettes have been identified herein that have been selected for their ability to activate and drive the process of phagocytosis, by reacting with phagocytic receptors.
  • phagocytic receptor types include various subclasses of lectin receptors (e.g., McGreal et al 2004), Fc receptors (e.g., Swanson et al 2004), and complement receptors (e.g., Ishibashi et al 1990).
  • lectin receptors e.g., McGreal et al 2004
  • Fc receptors e.g., Swanson et al 2004
  • complement receptors e.g., Ishibashi et al 1990.
  • the cell commences a series of steps, in which the engulfing cell extends two or more finger-like projections, around the sides of the particle that has become bound to the phagocytic receptor.
  • Other cellular "organelles” that have their own membranes (including endosomes, and endoplasmic reticulum) are recruited to assist, and they begin contributing all or parts of their own membranes, to the rapidly- growing membrane "pocket” that is being formed around the particle that is being engulfed by the cell.
  • the newly-formed membrane pocket detaches from the outer membrane of the cell, thereby creating a separate bubble-like component, often called a "vesicle", with its own membrane, floating inside the cytoplasmic liquid inside the cell that has engulfed the particle. After that vesicle detaches from the cell's outer membrane, it is called ⁇ .phagosome.
  • a phagosome will then merge and interact with another class of cellular vesicles, called lysosomes. After a phagosome merges with one or more lysosomes, the combined vesicle is still called a phagosome, or it can be called a phagolysosome.
  • Lysosomes are the main digestive components in eukaryotic cells; their internal fluids are very acidic, and they contain strong digestive enzymes, usually called lysozymes, hydrolyzing enzymes, or hydrolases. Accordingly, lysosomes contribute acids and digestive enzymes to phagosomes, and the resulting mixture kills and digests the foreign particle.
  • phagosomes When a phagosome contains an ingested microbe, then (in at least some cases) the digestive process will not completely digest the microbe (i.e., it will not break apart the microbe all the way to the level of single amino acids, nucleotides, and other building blocks, which would thereby become nutrients for the cell). Instead, macrophages partially digest invading microbes, in ways that lead to presentation of antigenic polypeptide fragments (from a microbe, or from a vaccine particle) on the "tips" of specialized types of cellular "fingers" created by the macrophage.
  • this invention has enabled phage libraries to be screened in ways have identified certain particular phages (from among a huge number of candidate phages, in a phage display library) which happen to be carrying surface polypeptide sequences that can potently activate and drive not just one or two important steps, but an entire sequence and series of steps that will potently activate a desired type of immune response.
  • the necessary transport and delivery steps include each and all of the following: (1) cellular uptake (or entry, intake, ingestion, endocytosis, or similar terms) of the phage vaccine "cassette” particles, into NALT tissues that are exposed and/or accessible inside the nasal, mouth, and throat membranes (this includes both an outer layer of M cells, and NALT cells positioned beneath that surface layer); (2) transport (or delivery, presentation, or similar terms) of the phage vaccine
  • cassette particles to the surfaces of macrophages formed from monocytes
  • phagocytosis occurs.
  • One important class involves a type of "cellular eating” that plays a major role in a process called “apoptosis”. In that process, dying or dead cells are engulfed, broken apart, and digested by macrophages, leading to the release and recycling of their building blocks, which are used to form new cells. Apoptosis enables soft tissues to continually replace old cells with new cells.
  • phagocytosis is unrelated to how the immune system responds to microbes or vaccines, and to antibody formation. Accordingly, to avoid confusing this invention (involving vaccines and immune responses) with apoptosis and cell replacement (which is not relevant herein), the term "macrophage” as used herein is limited to cells that can be converted, under proper triggering conditions, into "antigen-presenting cells” (abbreviated as APC, with the plural forms APCs or APCs).
  • APC antigen-presenting cells
  • Monocytes i.e., pre-macrophage cells that have not yet left the circulating blood
  • macrophages that can be converted into APCs
  • dendrites or dendritic cells that have commenced the process of engulfing and swallowing a microbe or vaccine, all fall within the definition of APCs, if they are involved in the immune system. More information on antigen-presenting cells can be found in numerous reviews, such as Trombetta et al 2005.
  • phagocytosis and phagocytes
  • terms such as “phagocytosis” and “phagocytes”, as used herein, are limited to the types of “particle swallowing” processes that lead to antigen presentation, rather than to the killing and recycling of aging cells that occurs in apoptosis and tissue renewal.
  • a macrophage that has engulfed a foreign particle generally will respond to one or more signals that will lead it down one of several diverging pathways. Accordingly, a macrophage that has encountered a microbe or vaccine particle must effectively choose between several options.
  • One set of options involves a choice as to whether it will stay at the site and engulf more particles (for example, some macrophages reportedly have engulfed as many as 100 bacteria), or whether it will begin traveling rapidly toward a lymph node, to alert other cells in the immune system that a problem has been detected so that a full response can be prepared as rapidly as possible.
  • a second set of options involves whether the macrophage will commit to generating either: (1) an antibody reaction, also called a "humeral” response; or, (2) a cell-mediated immune response, which will involved specialized activated T cells without involving B cells or antibodies.
  • a macrophage commits to helping form a systemic response (which can be either an IgG antibody response, or a cell-mediated response), it will begin moving rapidly toward a lymph node, as though responding to signals indicating that an emergency has arisen, and it must hurry, before more invaders can multiply and cause more problems.
  • the macrophage begins converting (or “maturing") into an "antigen-presenting cell” (APC).
  • APC antigen-presenting cell
  • help activate other types of immune cells including B cells, T cells, and "helper T cells", which will begin performing the next steps in the systemic response.
  • a macrophage that has been activated in this manner will secrete certain types of messenger molecules, such as interleukin-1. These molecules, also called “co-stimulatory” molecules, help activate other types of immune cells.
  • macrophages occupy and perform a gatekeeper role, at a crossroads where the adaptive immune system (in vertebrates) branches out from the innate immune system (which originally formed in primitive animals, and which is the only immune system most invertebrates have). Most invertebrates have cells that can effectively act as macrophages, which will engulf and eat foreign invading pathogens. However, invertebrates do not have any additional components (including antibodies, B cells, and T cells) that can provide more sophisticated adaptive immune responses.
  • macrophages in vertebrate animals stand at a point where two different pathways split and went in different directions.
  • a macrophage encounters a foreign invader, it must either commit to a primitive-type localized response (mainly involving phagocytosis of foreign or damaged cells and debris), or it must commit to a more complex, sophisticated, and time-consuming response to recruit and train an entire team of cells that will defend the organism.
  • vaccines refer to the types of vaccines that will help an animal develop a strong IgG-type and/or IgA-type antibody (or “humeral") and/or a cell-mediated (or “cytotoxic”) immune response (or a combination both types of responses). Most such vaccines are designed to help humans or animals resist infections by pathogens; however, a number of vaccines are being tested in the hope that they will be able to help patients fight non-infective diseases, such as cancer, Alzheimer's disease, etc. Those types of vaccines are discussed in more detail below, and more information on them is available in numerous other sources.
  • one object of this invention is to disclose a new approach to developing and manufacturing vaccines, using nonpathogenic bacteriophages that contain foreign polypeptide sequences that have been screened and shown to actively trigger NALT uptake, association, or other processing.
  • Another object of this invention is to disclose a new approach to developing and manufacturing vaccines, using nonpathogenic bacteriophages containing genes and coat protein polypeptides that can promote delivery of the vaccines (and/or antigenic proteins carried by the vaccines) to antigen-presenting cells, and/or to phagosome components within antigen-presenting cells.
  • Another object of this invention is to disclose new types of vaccines (which can use bacteriophages, other types of viral particles such as glycosylated viruses that infect eukaryotic cells, or cellular microbes) that have been enhanced by incorporating into them a "targeting-and-delivery" polypeptide sequence that will potently trigger and drive (i) intake into NALT and/or GALT cells and tissues, followed by (ii) phagocytic intake and processing by macrophages or other antigen-presenting cells.
  • Another object of this invention is to disclose new types of vaccine "cassettes” that can be used to insert any desired antigenic polypeptide sequences into a highly efficient delivery system that already contains: (i) a NALT-targeting, APC-targeting, and/or phagosome-targeting peptide sequence which will help ensure delivery of completed "cassette” vaccines into specific targeted cells or sites of the immune system; and, (ii) a toll receptor ligand that will help such vaccines reliably provoke desired immune responses, rather than allergic, tolerance or localized reactions, such as by activating one or more toll-like receptors.
  • Another object of this invention is to disclose new types of particulate phage vaccines, with at least one a NALT-targeting, APC-targeting, and/or phagosome-targeting peptide sequence and at least one selected antigen sequence, that can provoke strong and rapid antibody and other immune responses of the type that can subsequently help a host animal resist a pathogenic infection.
  • Another object of this invention is to disclose new types of particulate phage vaccines, with at least one component (which may comprise, for example, a DNA strand that contains CpG motifs) that functions as a toll receptor ligand, to help provoke strong systemic immune responses in inoculated animals and patients.
  • at least one component which may comprise, for example, a DNA strand that contains CpG motifs
  • toll receptor ligand to help provoke strong systemic immune responses in inoculated animals and patients.
  • Another object of this invention is to disclose new methods to identify and isolate, from phage display libraries, ligands that will potently activate phagosome receptors.
  • Another object of this invention is to disclose uses for NALT-targeting and/or phagosome-targeting phage particles, in targeting the delivery of diagnostic and/or therapeutic payloads (such as DNA gene expression plasmids, tracking and/or imaging reagents, etc.) into selected phagocytic cells.
  • diagnostic and/or therapeutic payloads such as DNA gene expression plasmids, tracking and/or imaging reagents, etc.
  • Another object of this invention is to disclose new types of vaccines that have been modified in ways that enable the phage particles to provide potent adjuvant activity while carrying antigenic protein sequences and/or nucleotide segments that function as toll receptor ligands.
  • Another object of this invention is to disclose new types of vaccines that can be manufactured very rapidly, in large quantities, and at low cost, using bacteria (or other cells that can be grown in stirred cell culture) as the host cells for incubation.
  • bacteriophages i.e., nonpathogenic viruses that infect bacteria such as E. coli
  • mucosal vaccines which can be administered without requiring needles, such as via nasal sprays.
  • the coat proteins of phage vaccines for nasal usage must contain foreign polypeptide sequences that will cause the phage particles to bind to and activate nasopharyngeal-associated lymphoid tissue (NALT) cells.
  • NALT nasopharyngeal-associated lymphoid tissue
  • Such phages have been and can be identified and isolated from a phage display library containing billions of candidate phages, by means of an in vivo screening process in which a phage display library is administered nasally to a lab animal, which is later sacrificed so that tissue samples can be harvested and treated, to extract phages that were taken in and transported by NALT cells. Additional screening tests on such "enriched" NALT-targeted phage populations have been and can be screened by additional screening tests, to identify subsets of any such NALT-targeting phages that will also potently drive phagocytic intake and processing, by macrophages or other antigen- presenting cells (APCs).
  • APCs antigen- presenting cells
  • the DNA sequence which encodes that polypeptide can be used to prepare a "cassette" vector, which can receive and hold any additional foreign gene sequence, inserted into one or more surface proteins (such as one or more coat proteins, if filamentous phages are used as the vaccine particles).
  • the completed vector will contain, in an exposed and accessible location, one or more antigen sequences that will provoke an antibody response that will help animals fight an invading pathogen, such as viruses or other pathogens.
  • selected antigens can help a patient's immune system attack and destroy cancer cells, beta-amyloid plaques in Alzheimer patients, or other cells or materials that cause or aggravate noninfective and/or nonmicrobial disorders.
  • the resulting vaccines will contain a combination of useful components and traits, including the following: 1. they will incorporate and use "targeting" polypeptides that have been screened and selected for high levels of binding to, and transport through, specialized epithelial cells (often referred to as "M” cells that provide the surfaces of "nasopharyngeal-associated lymphoid tissue” (NALT) or similar mucous membrane surfaces; 2.
  • the NALT-targeting polypeptides that will enable and drive the first step in the desired transport sequence will also be selected and screened to ensure that they will stimulate active phagocytosis by macrophage cells (which also can be called dendrite cells once they begin the process of phagocytosis), thereby driving and promoting a second crucial step that will create a desired immune (rather than allergic or tolerance) response; 3.
  • macrophage cells which also can be called dendrite cells once they begin the process of phagocytosis
  • the "cassette" system provided by the NALT-active engineered phages can also be used to provide one or more components (such as CpG motifs) that will actively stimulate one or more targeted toll-like receptors (which can be located entirely within the interiors of targeted immune cells, such as TLR9 receptors), in ways that will further promote a desired immune response rather than an unwanted allergic, tolerance or localized response;
  • one or more components such as CpG motifs
  • targeted toll-like receptors which can be located entirely within the interiors of targeted immune cells, such as TLR9 receptors
  • the phage particles will contain both (i) toll receptor activating and/or other adjuvant-type components, and (ii) a selected antigenic polypeptide sequence, preferably in integral and unitary particles that will hold together, rather than in emulsions or other mixtures that may become separated in ways that can render them less effective; 5.
  • these "cassette"-type phage vaccines will be in particulate form, and will have sizes that are ideal for stimulating phagocytosis by immune cells, which is another important step that can promote desired immune responses rather than unwanted allergic or tolerance reactions; and,
  • cassette phages can incorporate a first foreign protein sequence that has either a small or moderate size, and a second protein sequence having a substantially larger size if needed; either sequence can provide the NALT-active transport sequence that will initiate uptake, transport and processing by NALT cells, while the other sequence can provide an antigenic sequence that will trigger a desired antibody formation response.
  • cassette system enables the creation and use of divalent, trivalent, or multivalent vaccines, such as flu vaccines having several different antigenic sequences from different strains of flu; alternately, mixtures of different phage vaccines, each one carrying a specific antigenic sequence (these types of vaccine preparations are often called "subunit" vaccines), can be administered in a vaccine mixture;
  • the vaccines and vaccine cassettes disclosed herein can provide optimized delivery and adjuvant activities, and can be used with any antigenic polypeptide sequence to provide potent and effective vaccines that can be administered via nasal sprays or similar means.
  • FIGURE 1 is a flowchart that shows the major steps for screening a display library containing a very large number of phages, in a manner that will select and isolate those particular phages which demonstrate: (i) efficient binding to NALT cells that are accessible in the nasal sinuses, and (ii) efficient intake and transport of those particular phages, by NALT cells in the nasal sinuses.
  • FIGURE 2 shows the time-dependent passage of two different populations of phages into olfactory bulbs in the forebrains of mice, after administration by nasal spray.
  • the open circles indicate that "wild-type" phages (with no foreign DNA sequences from a display library) passed through the olfactory bulbs with a single early peak.
  • the dark circles indicate olfactory bulb concentrations of phages that contained polypeptide sequences from single-chain variable fractions (scFv) of human antibodies.
  • scFv single-chain variable fractions
  • FIGURES 3 A and 3B show the time-dependent passage of two different populations of phages into circulating blood, in mice.
  • FIG. 3 A shows the transport of: (1) wild-type phages without any foreign scFv sequences ("single-chain variable fraction" sequences, isolated from human antibodies), indicated by open circles; and (2) phages containing scFv sequences, indicated by black triangles.
  • FIG. 3B (which uses a vertical scale that is flattened by a factor of 1Ox) compares the same types of wild- type phages, against phages carrying scFv peptide sequences that had been selected by four successive rounds of in vivo nasal-to-blood transport screening.
  • FIGURE 4 shows a screening test for selecting NALT-targeting phages from a phage display library containing random 15-mer foreign polypeptide sequences in a coat protein.
  • phages that lingered in the olfactory bulb were harvested, grown in bacterial hosts, and used as the starting population for second-round screening.
  • phages were harvested from NALT cells in an animal's windpipe.
  • phages selected by the two prior rounds were incubated with mammalian white blood cells, such as peripheral blood mononuclear cells (PBMCs), to identify phages that bind to the immune cells
  • mammalian white blood cells such as peripheral blood mononuclear cells (PBMCs)
  • PBMCs peripheral blood mononuclear cells
  • phages selected by the third round were isolated from the "phagosomes" of mammalian PBMCs, to identify and isolate phages carrying peptide sequences that drove efficient "phagocytosis" (i.e., intake and processing) by the immune cells.
  • the phages that were selected by the successive screening steps listed above are analyzed, to determine the gene and amino acid sequences of the polypeptide fragments carried by those particular phages. Those genes and polypeptide sequences can efficiently drive both: (i) intake and transport by NALT cells, and (ii) phagocytosis and antibody- related processing by white blood cells.
  • FIGURES 5-10 are photographs or color drawings that will not reproduce well in a published patent application. Therefore, the corresponding pictures have been posted and and publicly available on a website, at www.tetraheed.net/ferguson.
  • FIGURE 5 is a photomicrograph showing fluorescent-labeled phages that were transported into NALT tissues in the nasal regions of mice. These tissue samples were taken 30 minutes after nasal . administration.
  • FIGURE 6 is a photomicrograph showing fluorescent-labeled NALT-targeting phages that were transported into immune tissues in lymph nodes in the neck, and into antigen-presenting cells (APCs), two hours after nasal administration.
  • APCs antigen-presenting cells
  • FIGURE 7 is a photomicrograph showing fluorescent-labeled NALT-targeting phages that intensely labeled a minority of the cells in a preparation of human lymphocytes.
  • [000142 JFIGURE 8 contains panels 8A, 8B, and 8C, which display the results of fluorescent-activated cell sorting of human lymphocyte cells that had been incubated with NALT-targeting phages that had been selected in prior screening tests.
  • FIGURE 9 contains panels 9A, 9B, and 9C, which are photomicrographs created by using fluorescent-labeled antibodies that bind to MHC-I protein sequences, showing that screened and selected NALT-targeting phages efficiently triggered the clustering of MHC-I proteins on human lymphocytes. This result indicates that the cells have committed to a desirable cross-presentation of_antigen, rather than triggering an undesired allergic, tolerance or localized response.
  • FIGURE 10 is a photograph of the results of a gel-shift assay showing a reduction in electrophoretic mobility, when plasmid DNA was coated onto the surfaces of cationised phage. Since DNA is negatively charged, it clung to the cationised phages (i.e., phage particles that had been treated to create positive electrical charges on their surfaces), creating neutralized DNA/phage complexes with little if any net electrical charge. Since the DNA/phage complexes had little if any electrical charge, they were not driven through the gel by an applied voltage.
  • FIGURE 11 depicts the results of a purification method that used ceramic hydroxyapatite in an affinity column to purify phages.
  • concentration of sodium biphosphate was increased, during a series of elution steps, phages emerged in purified form, in the peak that includes elution fractions 61 through 71.
  • FIGURE 12 is a drawing depicting a phage particle that carries a NALT-targeting polypeptide in one type of coat protein, and an antigen sequence derived from a microbial pathogen (or from cancerous cells) in a different coat protein.
  • Exposed DNA strands with a CpG motif (a known type of "pathogen-associated molecular pattern” (PAMP) that can activate TLR9 toll-like receptors) cling to the surface of the cationised phage particles, to help ensure a desired immune response rather than an undesired allergic or tolerance reaction.
  • PAMP pathogen-associated molecular pattern
  • Amino acid sequence data also is disclosed herein, for polypeptide sequences that were demonstrated by screening tests to potently drive both: (i) NALT-targeting, intake, and transport activity; and (ii) phagocytic intake and processing by antigen- presenting cells (APCs).
  • APCs antigen- presenting cells
  • this invention discloses methods for developing and using modified virus particles (which includes phages as well as eukaryotic viruses) having specialized "transport" polypeptide sequences (also called targeting-and-delivery sequences, as described below), as potent vaccines that can be administered to mucosal surfaces of animals, such as in the nasal sinuses and/or the mouth and throat, without requiring needles.
  • modified virus particles which includes phages as well as eukaryotic viruses
  • transport polypeptide sequences also called targeting-and-delivery sequences, as described below
  • mammals includes humans, and can refer to either an individual or a population, and the term “inoculated” refers to any animal or human (or any population) that has received a vaccine, regardless of the route or mode of administration.
  • a vaccine preparation must be suited for use in at least one animal species, and does not require activity in all species (for example, a vaccine intended for humans does not require activity or efficacy in any other species, and a vaccine intended for chickens does not require activity or efficacy in any other species).
  • a vaccine preparation for delivering antigens to an immune system of at least one type of animal includes and covers both: (i) a batch of vaccine material in bulk, such as in a bulk container being transported from a manufacturing site to a packaging, distribution, and/or inoculation site, and (ii) a batch or aliquot of vaccine material that is packaged in some type of single-dosage or multiple-dosage form.
  • the invention also discloses three closely-related compositions of matter, all of which can be created by using the methods disclosed herein.
  • the first composition of matter comprises vaccine "cassettes", which have been optimized so that the viral genome is ready to have a foreign DNA or RNA sequence inserted into a target insertion site, as described below.
  • This type of "vaccine cassette” particle or preparation does not yet carry an antigenic DNA or RNA sequence that will trigger the production of antibodies; instead, a "cassette” is designed and optimized to receive and handle any antigenic sequence that a vaccine-manufacturing company chooses to insert into a cassette.
  • this invention also covers complete vaccine particles, in which a foreign (or heterologous, exogenous, passenger, payload, etc.) DNA or RNA sequence has been spliced into a target (or insertion) site in the viral genome.
  • the foreign DNA or RNA sequence will encode an antigenic polypeptide sequence, such as a sequence derived from a surface protein of a pathogenic microbe.
  • an antigenic polypeptide sequence such as a sequence derived from a surface protein of a pathogenic microbe.
  • surface protein refers to microbial proteins that are exposed and accessible (usually in multiple copies) on one or more surfaces of a virus or other microbe.
  • Coat proteins of phages are a class of surface proteins; other types of viruses are surrounded by membrane-type envelopes (usually made of lipid bilayers, taken from one or more membranes of a host cell), and the surface proteins in such viruses are embedded in, or otherwise affixed to, the envelope.
  • the third composition of matter disclosed herein comprises vaccines that contain "targeting-and-delivery" polypeptide sequences (referred to in the claims as "transport” sequences) that were identified by screening of a phage display library, regardless of whether the vaccine particles are, or are not, phage particles.
  • transport sequences referred to in the claims as "transport” sequences
  • a potent targeting-and- delivery polypeptide After a potent targeting-and- delivery polypeptide has been identified (such as the transport polypeptide disclosed herein with sequence data), it can be inserted into various types of viruses or other vaccine components other than bacteriophages, to make enhanced vaccines.
  • a potent "target- and-deliver" polypeptide sequence can be inserted into surface proteins of vaccine viruses that are manufactured by culturing them in eukaryotic cells, such as in bird eggs, caterpillars, insects, mammalian cells, etc.
  • eukaryotic cells such as in bird eggs, caterpillars, insects, mammalian cells, etc.
  • This approach can enable a transport sequence to be incorporated into "glycosylated” viruses and other types of viruses that are formed and/or processed by eukaryotic cells, in ways that bacteria cannot accomplish. This approach is discussed in more detail below, under the heading, "NALT-Targeting Polypeptides hi Eukaryotic Viruses".
  • the types of "target-and-deliver" polypeptide sequences disclosed herein also can be inserted into surface proteins in vaccines made from cells, such as killed or disarmed pathogenic microbes that are used to create various types of vaccines. This option also is discussed in more detail below.
  • a vaccine cassette (and a vaccine cassette preparation) becomes useful, valuable, and lawful for use as a vaccine, only after a "clonal" isolate has been identified, isolated, and sequenced, using screening tests such as described below, and has been proved and demonstrated to carry a polypeptide sequence that will actively promote the specialized "target-and-deliver" functions described herein. Accordingly, selection and use of suitable screening and isolation steps, leading to identification and purification of clonal isolates that will deliver antigen sequences in vaccines to targeted immune cells, is crucial to this invention.
  • clonal and “monoclonal” are used interchangeably, and refer to a population of viruses (which can include eukaryotic viruses, bacteriophages, virions, etc.) that have descended from a single ancestor virus, with all members of the population presumably being genetically identical.
  • Clonal (or monoclonal) colonies of viruses (and of virus-infected host cells) can be obtained by known methods. Typically, a dilute preparation of host cells, briefly incubated with phages or other viruses, are inoculated at moderate density onto one edge of a solid (agar or gel) nutrient that contains an antibiotic, in a shallow dish.
  • the inoculated area is then "streaked" at high speed (creating low density) across the remaining area of the plate, using a tiny wire loop. Since the only cells that can grow on the nutrient in the culture plate are cells that contain an antibiotic-resistance gene carried by the viruses, clonal colonies (isolated from each other, and typically having small round shapes that grow larger as time passes) will arise. A small sample of virus-carrying cells from a clonal colony can be harvested from the culture plate, and those cells can be grown (in very large numbers, but still in clonal form) in liquid or other media. These and other methods for isolating clonal populations are well-known and conventional.
  • phage display libraries that contained millions or billions of candidate phages, all having different "inserts” containing foreign polypeptide sequences. Any such phage display library may contain several (or possibly dozens or even hundreds) of phages that may function, with varying levels of moderate, good, or excellent potency, as a vaccine cassette as disclosed herein.
  • a safe and effective targeting-and-delivery system for a vaccine can be created only by identifying, isolating, and reproducing one or more specific phages which happen to carry specific foreign polypeptide sequences that will activate and drive a series of desired immune cell responses, as described herein. Accordingly, the claims below contain phrases referring to, for example, compositions of matter such as "a clonal virus (or phage) preparation", which is distinct and very different from a phage display library.
  • Other components may include carriers and/or diluents (which can be either a liquid or a powder); microbicides or other preservatives or stabilizers; an acid, alkali, or salt; adjuvant-type additives; and, if a vaccine is to be administered to a mucous membrane, one or more mucoadherents.
  • diluents which can be either a liquid or a powder
  • microbicides or other preservatives or stabilizers an acid, alkali, or salt
  • adjuvant-type additives and, if a vaccine is to be administered to a mucous membrane, one or more mucoadherents.
  • some viral vaccines contain mixtures of two or three different types of vaccine particles (these are often referred to as divalent or trivalent vaccines), and some bacterial vaccines have even more substituents (for example, two vaccines against pneumonia are referred to as 7-valent and 23-valent).
  • a vaccine preparation (or a clonal population of phages, other viruses, or cellular microbes, or an intermediate preparation that is created during a manufacturing process) is deemed to be "purified” if the vaccine particles have been processed by one or more purification steps of a type used in vaccine manufacture.
  • Such purification methods used in manufacturing vaccines and other pharmaceutical or biochemical compounds, are well-known, and include (as non-limiting examples) filtering (which can remove a population of viruses from a population of host cells), centrifugation and other physical methods, methods that use affinity binding, and methods that exploit differing travel rates, settling positions, or other factors that can be exploited by using gels, matrices, or other semi-permeable media (often used in conjunction with voltages, ionic gradients, etc.).
  • vaccine preparations are not limited to final and complete formulations. Instead, a "raw" or unfinished preparation containing clonal virus particles is a vaccine preparation, if it will be passed through one or more purification, processing, blending, or other manufacturing steps that will render it suited for use as a vaccine.
  • progeny phages will inherit and contain a "foreign" insert from their ancestor does not make an insert "native" or less foreign, with respect to those viruses; instead, if a polypeptide sequence is "foreign" to wild-type phages, then it also is foreign to any progeny in a clonal population.
  • promote when applied to polypeptide-driven uptake (or intake, transport, delivery, or similar terms) of particles by NALT cells and/or phagocytic antigen-presenting cells (which includes macrophages), requires a level of activity (or efficacy, potency, or similar terms) that allows otherwise identical particles (such as bacteriophages having an assortment of different foreign polypeptide inserts) to be cleanly and clearly separated, isolated, and identified, by means of actual performance in screening tests that rely upon intake of such particles into NALT cells and/or into the phagosomes of antigen-presenting cells.
  • level of activity or efficacy, potency, or similar terms
  • Such screening tests provide a straightforward and functional method that will enable anyone skilled in this art to determine whether (and how strongly) a certain polypeptide sequence can and will promote the intake of such particles, into such cells.
  • the results of such tests can be quantified, if desired, by various methods, such as competitive screening tests (comparable to competitive binding assays), in which a polypeptide sequence of interest can be pitted and tested against another polypeptide sequence, such as in a test that can, if desired, use a mixture of two different phages, at identical concentrations, tested in a single animal or cell culture.
  • the other polypeptide sequence (usually called a comparison or control sample, or similar terms) can be provided by, for example: (i) a random and unsorted population of phages, such as in a phage display library; or, (ii) the polypeptide sequence disclosed herein.
  • an arbitrary "benchmark" level of potency is hereby established, at a level of at least 50% of the cell-intake efficacy of the polypeptide sequence disclosed herein, when measured using NALT-related cells or tissues (which may include measurements of "downstream” tissues in animals), or in monocyte, macrophage, or other appropriate cell cultures.
  • This type of "benchmark" standard can be measured by inoculating a population of animals (preferably with at least six mice, rats, or chickens per sample, to obtain statistically-significant results) or cells, with a 50:50 mixture that contains both: (i) a first phage preparation carrying the foreign polypeptide sequence disclosed herein, and (ii) a second phage preparation carrying a second candidate foreign polypeptide sequence that is being tested and measured.
  • the second candidate foreign polypeptide sequence should be regarded as being capable of promoting cellular intake into NALT cells and/or antigen-presenting cells.
  • cassette systems (or vaccine cassettes, or similar terms) uses a term that is well-known in genetic engineering.
  • Such "cassettes” usually involve plasmids, phages, or other vectors that have been manipulated in ways that allow them to be readily and conveniently modified, by inserting additional strands or segments of DNA or RNA into one or more predetermined locations in such vectors.
  • the term "cassette” arose in the pre-digital era of music, when a cassette player could play a tape containing any musical selection the owner happened to have.
  • Cassettes had standard sizes and mechanisms (several types became popular, initially for audio recordings, and subsequently for video recordings). To use a cassette, an owner merely inserted a tape containing the desired music or video (or a tape that was ready to be recorded onto), into an accommodating machine. Furthermore, the cassettes themselves could be loaded with any audio or video content that the owner or operator chose.
  • cassette systems are somewhat different but well-understood. Their principal feature is that they are designed to receive, accommodate, and work with essentially any DNA or RNA sequence that is inserted into them, so long as the insert has a compatible format, as will be understood by those skilled in the art.
  • DNA vectors and cassettes sometimes are used, and are widely discussed in the prior art, DNA vectors generally are preferred, for a number of reasons.
  • DNA vectors generally are preferred, for a number of reasons.
  • One important set of reasons arise from two facts: (i) DNA is more chemically stable than
  • RNA Ribonucleic acid
  • mRNA messenger RNA
  • RNA vectors and cassettes Another major factor that leads to a preference for DNA vectors and cassettes arises from that fact that whenever an RNA vector is used, some type of "reverse transcription" step (i.e., creation of DNA, from the RNA strand carried by the vector) is almost always required, to create a lasting and inheritable transformation. If an additional required step must be inserted into an already-complex process, it creates another set of potential problems, and reduces the likelihoods and rates of desired outcomes. Therefore, it usually is easier and more reliable to use DNA vectors, unless strong reasons indicate that an RNA vector is better suited for some particular task.
  • the filamentous phages described herein carry DNA, rather than RNA, and that approach is usually preferred, for uses such as described herein.
  • DNA vectors tend to be easier to work with than RNA vectors, because there is a broader (and more adaptable and useful) assortment of "restriction endonuclease” enzymes that can be used to manipulate DNA vectors, compared to RNA vectors.
  • restriction endonucleases that can cleave double-stranded DNA are very useful, in genetic vectors, since they enable a cassette to be provided with several unique insertion sites, allowing foreign DNA sequences to be inserted into specific targeted locations without disrupting any important genes in a vector.
  • viruses carry RNA rather than DNA, in their genomes; examples include the human immunodeficiency viruses (HIV) that cause AIDS, and the coronaviruses that cause severe acute respiratory syndrome (SARS).
  • HIV human immunodeficiency viruses
  • SARS severe acute respiratory syndrome
  • Restriction sites usually require four to six nucleotides, in an exact sequence; as one example, the restriction sequence for BamHl is G/GATC/C, where the slash marks indicate the cleavage locations on the double-helix strands of the DNA.
  • the BamHl sequence is symmetric, since the sequence of bases on the other strand of the double helix is the reverse, CCTAGG.
  • the BamHl enzyme will leave a 4-base "sticky end" on each of the two resulting “cut ends” of DNA, when it cleaves double-stranded DNA.
  • a foreign DNA segment can be given accommodating "sticky ends", to promote insertion of the foreign DNA sequence into the cleaved vector.
  • Cassette vectors usually are designed to have several unique restriction sites (such as one site that can be cleaved by EcoRl, another site cleaved by BamHl, and a third site cleaved by Hindlll), clustered together in a location that enables a foreign DNA sequence to be inserted into a targeted location without disrupting any genes or other sequences that are important to functioning of the vector. If several such cleavage sites are available, at least one endonuclease almost always will be available that will not inadvertently cleave a foreign DNA sequence being inserted into the cassette. If all of the desired restriction sequences are present in a foreign DNA sequence, the foreign DNA sequence usually can be altered, by replacing one or more 3-letter codons in the foreign
  • any gene (or other sequence of interest) in a cassette vector can be flanked by one or more unique restriction sites, to enable the gene to be removed from the cassette vector at an appropriate time.
  • viral vectors usually contain an antibiotic resistance gene (such as a gene that encodes an enzyme that will inactivate ampicillin or tetracycline) or some other type of "selectable marker" gene, to make it easier to isolate and reproduce the vector in bacterial cells.
  • an antibiotic resistance gene such as a gene that encodes an enzyme that will inactivate ampicillin or tetracycline
  • selectable marker such as a gene that encodes an enzyme that will inactivate ampicillin or tetracycline
  • any such marker gene can be removed, after the basic research has been completed and a vaccine candidate is approaching final testing and actual use.
  • At least one and preferably several candidate DNA insertion sites preferably should be located in the coding portion of a gene that encodes "coat protein 3" (also referred to as pill, cpIII, or cp3), which is present (in several copies) at one end of each phage, as illustrated in FIG. 11. This will cause the foreign polypeptide sequence to be inserted into (or added to one end of) the amino acid sequence of coat protein 3, which can carry relatively large foreign protein sequences.
  • coat protein 3 also referred to as pill, cpIII, or cp3
  • the phage cassettes also should contain insertion sites in the gene that encodes "coat protein 8" (also referred to as pVIII, cpVIII, or cp8). Over 2000 copies of that coat protein are present in the cylindrical outer shells of filamentous phages.
  • two different genes that encode coat protein 8 can be present in the viral genome, and only one of those two genes, under the control of a relatively weak and/or inducible promoter, will be provided with DNA insertion sites.
  • This can create phage particles with a few hundred copies of a foreign protein sequence, while most of the coat protein 8 subunits have unmodified sequences. This will reduce the "burden" (or passenger load, payload bulk or weight, or similar terms) that the resulting vaccine particles must carry. If all copies of the cp8 subunit in a modified phage vector carry foreign polypeptide sequences, the foreign sequence often must be limited to less than about 10 altered amino acid residues, to avoid hindering reproduction of the phages.
  • the insert often can be substantially longer, such as up to about 15 to 20 amino acids, and possibly more. In either case, the exact length of a tolerable insert will vary, depending on the specific amino acid sequence of the insert.
  • the phage vaccine cassettes disclosed herein can be regarded as comprising a targeting and/or delivery system. Because of the nature of its functions and components, it also can be referred to by other terms that imply an intentionally-designed delivery system (such as, for example, a transport, transfer, carrier, vehicle, ferry, uptake, intake, conveyance, or homing system).
  • an intentionally-designed delivery system such as, for example, a transport, transfer, carrier, vehicle, ferry, uptake, intake, conveyance, or homing system.
  • a DNA sequence that is inserted into the genome of a phage cassette, and the foreign polypeptide sequence that will be encoded by the foreign DNA (which will appear in one of the coat proteins of the modified phage) also can be referred to by various terms.
  • such DNA sequences (and the polypeptide sequences they encode) can be referred to as passenger or payload sequences (or components), or as antigen or antigenic sequences.
  • Inserts for vaccines must be antigenic, because of the nature of vaccines; however, non-antigenic sequences can be inserted into a phage cassette for other uses, if desired.
  • a foreign DNA sequence encoding a polypeptide sequence which is not antigenic, but which binds tightly to a known monoclonal antibody preparation or to a particular surface molecule on certain types of cells can be useful in research or in diagnostic, imaging, or similar work.
  • an inserted DNA and/or amino acid sequence is referred to as foreign, heterologous, exogenous, or similar terms, such terms imply that the inserted sequence is not present in the phage, regardless of whether the "foreign" sequence might be present in an animal or human treated by a vaccine.
  • a polypeptide sequence that is "foreign" to a phage cassette can contain a polypeptide sequence that appears on the surfaces of cancer cells, in cancer patients who will be treated by the vaccine, in a vaccine designed to trigger the formation of antibodies (and/or the activation of cytotoxic T cells) that will kill the cancer cells.
  • vaccine cassettes as disclosed herein can be identified and isolated for any such set of mucosal immune cells, merely by screening for cells that are actively taken into any such cluster or class of immune cells.
  • nasal administration can be enhanced and speeded up, merely by inhaling a nasal spray; it does not require any removal of clothes or other time-consuming preparatory actions; and, if a major pandemic emerges, a long line of people can be treated very rapidly (without even requiring any delays for creating or keeping records, if local public health officials deem it prudent to speed up mass distribution as much as possible).
  • NALT-targeting activity are used for convenience, and are intended to be exemplary rather than limiting, to refer to activity in triggering uptake (also referred to as intake, entry into, etc.) by one or more types of specialized immune cells that are exposed and accessible on one or more types of mucosal surfaces.
  • specialized bactericidal nozzles for nasal sprays can be used.
  • the surfaces of certain metals can increase the microbicidal potency of alcohol and certain other disinfectants.
  • nasal-spray nozzles coated with silver or other metals can be rapidly and efficiently disinfected by wiping them with alcohol, between uses.
  • a spray nozzle (coupled to a supply tube from a trigger-operated dispensing unit) can be designed with a placement component that would be pressed against the epidermal skin, above the upper lip and below the nose. This would enable two thin tubes at the upper tip of the spray nozzle, spaced roughly a centimeter apart, to inject a small pressurized jet or spray of liquid into both nostrils, without touching anything inside the nostrils.
  • the cassette approach of this invention can enable the screening and development of various different sets of phage cassettes, with each set being optimized for a particular species.
  • the first set of NALT-intake screening tests described herein used mice.
  • a second set of phage cassettes can be screened and isolated in rats, using the same or similar screening approaches; a third set of phage cassettes, screened and isolated in chickens, for use in chickens; and so on.
  • Those types of screening tests can be repeated in any species of interest (such as in species that are important in farming or food supply, in veterinary or medical use, etc.), up to and including humans.
  • these types of phage cassettes can be optimized for various wild species that are being endangered by viral or other epidemics.
  • phage cassettes that are screened and isolated in mice are likely to function efficiently in rats, rabbits, and other rodents; phage cassettes that are isolated in chickens are likely to function efficiently in turkeys or other poultry; and phage cassettes that are isolated in monkeys or chimpanzees are likely to function efficiently in humans and other primates.
  • phage cassettes such as disclosed herein, and adaptations of the screening methods disclosed herein, will provide immunology researchers with powerful tools for analyzing and quantifying various types and degrees of overlap and cross-reactivity, between various components and cell types of the immune systems of different types of mammals.
  • phage cassettes will show high levels of vaccine-type potency among different classes of mammals (such as between mice and humans), while other phage cassettes will show lower levels of cross-reactivity.
  • isolation of a preferred phage cassette for use in a particular species of interest may be able to provide even more potent and efficient carrier phages, for use in a particular species or other ancestral group.
  • phage cassettes While some phage cassettes are likely to emerge that can provide good vaccine potency among all humans, other more specialized phage cassettes may be able to provide even higher levels of potency among people whose ancestors lived and evolved among a semi-localized and particularized set of pathogens.
  • mice since solid tissues can be harvested easily from mice, the screening tests described herein initially used mice, to screen for phage intake into NALT tissues, and for transfer from NALT tissues into other tissue types (such as olfactory bulb tissue).
  • monocyte cells white blood cells which are the precursors of macrophage cells
  • human cells were used, partly because it is easier to obtain large quantities of white blood cells from humans than from mice, and also because the goal of this research is to move rapidly toward vaccines that can and will be used in humans.
  • the first concept is this: if a properly screened and isolated vaccine cassette can accomplish even a single crucial targeting-and-delivery step, it can provide an important and useful advance over the prior art. Accordingly, any such advance merits recognition and coverage for what it has accomplished, and some of the claims below focus on phage vaccine cassettes that were screened and isolated because they were able to accomplish a single specific step that is useful or crucial in provoking a desired immune response to a vaccine.
  • targeting-and-delivery phage vaccines should (and ultimately must) be identified and isolated, not just by using a single round of screening, but by using a succession of several different screening tests, where the starting population for each screening test is obtained from candidates that performed well in other types of screening tests. This has been accomplished by the methods disclosed herein, and if desired, it can be repeated in screening tests that are limited to one particular species (such as humans, for example), to isolate phage vaccine cassettes that will be truly optimized for that particular species.
  • this test identified and isolated phages that successfully completed each and all of the following steps: (i) intake into NALT cells, presumably via endocytotic or other cell-surface receptors; (ii) release of the phage by the receptor, after a phage/receptor complex had entered a NALT cell; (iii) secretion of the phages by the NALT cells, into some type of cellular junction that delivered the phages to one or more types of "downstream” cells; and, (iv) intake of phages that had passed through NALT cells, into one or more types of "second stage” cells or tissues.
  • the "second stage” tissue was harvested, and the membranes of the cells were dissolved, using a detergent-type "lysis buffer” that dissolves lipid membranes of cells, without damaging the coat proteins of viruses. That step released the contents of the cells, allowing the phages to be extracted. The isolated phages were then reproduced in E. coli bacteria, to provide a starting population for subsequent screening tests.
  • a detergent-type "lysis buffer” that dissolves lipid membranes of cells, without damaging the coat proteins of viruses. That step released the contents of the cells, allowing the phages to be extracted.
  • the isolated phages were then reproduced in E. coli bacteria, to provide a starting population for subsequent screening tests.
  • a second screening test (which used "enriched" populations of phages that had been screened for NALT intake and transport, as described above) was used to isolate phages that triggered binding to (and/or intake into) macrophage cells.
  • macrophage cells as used herein includes monocyte cells, which are precursor cells that become macrophage cells after they pass through a capillary wall, leave the circulating blood, and enter the lymph fluid in soft tissue.
  • This screening test began with sampled human blood.
  • the red blood cells were removed, and the semi-purified white blood cells were processed by a surface-binding step, to isolate monocyte (macrophage) cells, which have unusual surface-adhering molecules that enable monocytes to grip a capillary wall and permeate through the capillary wall, to reach the lymph fluid.
  • the purified monocyte (macrophage) cells were incubated with phages that had been fluorescently labeled, and the cell/phage mixture was processed by "cell sorting", using a machine called a flow cytometer, to isolate monocyte (macrophage) cells that were strongly labeled by fluorescent phages. This was done by setting the controls of the machine so that only the top 3% of the cell population was isolated, based on strength and intensity of the fluorescent signal from a cell/phage complex.
  • the membranes of the isolated highly- fluorescent monocyte (macrophage) cells were dissolved, viable phages were extracted, and the harvested phages were reproduced in E. coli, to provide starting populations for subsequent screening tests. 3.
  • the third screening test (which used phages that had already been selected by the screening tests described above) isolated phages that triggered phagocytosis (i.e., entry into macrophage cells). This test was carried out by using several stages of centrifugation, to isolate phagosomes (i.e., intra-cellular compartments enclosed within their own membranes) from macrophage cells, obtained from human blood by the surface-binding selection process mentioned above.
  • the cells were incubated with phages that had passed the prior screening tests, and were then processed using a mechanical homogenizer.
  • the homogenizer broke apart cells, without breaking the phagosomes (which are much smaller than cells).
  • Intact phagosomes were isolated by (i) a first mild centrifugation, which formed a pellet of intact cells and nuclei, which were discarded; and, (ii) a second stronger centrifugation, which pelletized the phagosomes.
  • the phagosome pellet was resuspended in liquid, then the membranes were dissolved by a lysis buffer, and phages contained within the phagosomes were harvested, and reproduced in E. coli.
  • the isolated phages had to activate and participate properly in three sequential cellular processes: (i) binding (as a "ligand") to a phagocytic receptor, on a surface of a macrophage cell; (ii) intake of the bound receptor/ligand complex into the macrophage; and (iii) separation of a completed and functional phagosome, from the outer cell membrane.
  • the phage cassettes disclosed herein can include (and/or can be supplemented by) enhancing components that can promote additional desired responses.
  • Such cellular responses might include, for example: (i) activation of one or more types of toll-like receptors (TLR's), to help ensure that an immune response leads to desired immune response, rather than an undesired allergic or tolerance reaction; (ii) activation of a desired ThI and/or Th2 response; and, (iii) activation of a desired MHC-I and/or MHC-2 response.
  • TLR's toll-like receptors
  • enhancements can enable vaccines to be optimized for either of two different usages: (1) inducing an antibody-producing "humeral” response, for fighting off microbial pathogens; or, (2) inducing other cell-mediated responses, such as for killing cancer cells, or for removing other types of cells or materials (such as, for example, beta- amyloid plaques in the brains of people suffering from Alzheimer's disease).
  • phage cassettes Some of those enhancements and/or adjuvant features, activities, and advantages of these vaccines arise from the inherent properties of the phage cassettes. As one example, these phages appear to have an ideal size for triggering intake into NALT cells, and into phagosomes within macrophages.
  • adjuvant, adjuvant-like, or other enhancing components can be provided in any of several ways, such as by using one or more of the following approaches: (i) adjuvant, adjuvant-like, or other enhancing components can designed and incorporated into phage cassettes, so that such components will be integral and inseparable features of all vaccine particles; (ii) adjuvant or other enhancing components can be covalently or non-covalently bonded to phage particles (which presumably will occur after final assembly of completed vaccine particles that carry inserted foreign DNA sequences and antigenic polypeptide sequences), so that the adjuvant or enhancing components will remain securely bonded to the vaccine particles, until phagocytic or other cellular or enzymatic processes take over and begin dismantling a vaccine particle; and/or, (iii) adjuvant or other enhancing components can be coupled to the phage cassettes by other means, such as by ionic or hydrogen bonding
  • adjuvant As used herein, terms such as adjuvant, adjuvant-like activity, or enhancing components are intended to refer to any component, activity, or other trait or feature that will either: (i) increase the likelihood (or rates, probabilities, yields, etc.) of triggering desirable immune responses, in members of a large treated population; and/or, (ii) reduce the amount of a vaccine preparation that must be administered, to provoke desired immune responses in some target fraction or percentage of a treated population.
  • some of the vaccines disclosed herein will be designed to help an organism defend itself against an infection, while other vaccines will be designed to help fight cancer or kill other unwanted cells, or to dissolve or neutralize plaques or other unwanted materials.
  • TLRs toll-like receptors
  • Quality control will become even more difficult yet critical, if a need arises to manufacture hundreds of millions (or even billions) of dosages very rapidly, as may be required if a "bird flu" strain emerges that can be transmitted human-to-human, or if a strain of HIV/ AIDS emerges that can be transmitted by insects.
  • phage vaccines that can be manufactured in huge quantities in a matter of hours, using in vitro culture of bacterial cells (or other types of eukaryotic, glycosylating, or other cells that can be grown in cell culture), can make quality control issues and problems simpler and more manageable, compared to prior manufacturing methods, such as incubating vaccines in bird eggs (which normally must incubate for weeks).
  • the coat proteins of phage vaccines as disclosed herein can be chemically treated (or modified, enhanced, or similar terms), to enable them to carry or deliver additional antigens, adjuvants, or other useful molecules.
  • phage particles are "cationised” (i.e., treated with agents that will impart a positive electrical charge to their surfaces)
  • ODN' s relatively short strands of DNA
  • DNA segments having (for example) "CpG motif sequences will be affixed to the surfaces of the phages.
  • CpG motif sequences As described in the Background section, in DNA strands with CpG motifs, large numbers of unmethylated cytosine residues are positioned adjacent to guanidine residues. These are recognized by mammalian immune systems as a "pathogen-associated molecular pattern" (PAMP), which can activate toll-like receptors, to promote immune response rather than allergic or tolerancejesponses.
  • PAMP pathogen-associated molecular pattern
  • DNA strands having CpG motifs can be synthesized and affixed to the surfaces of "cationised " or otherwise treated phages, using ionic and/or hydrogen bonding, to increase the efficacy and potency of the resulting vaccine particles.
  • certain amino acid residues in proteins can be chemically treated, by known reagents, in ways that will covalently crosslink other compounds to the proteins.
  • the side chain of each lysine residue in a protein has a reactive primary amine group, at the end of a four-carbon chain.
  • the accessibility and hydrophilic (water-soluble) nature of such amine groups allows certain known reagents (such as isothiocyanate, or bis(sulfosuccinimidyl)suberate) to react with lysine residues, in ways that will create covalent crosslinking bonds with other compounds. Since covalent bonding generally is stronger than ionic and/or hydrogen bonding, such reagents can be used to covalently bond adjuvants, secondary antigens, or other potentially useful compounds to the surfaces of phage particles in vaccines, if desired.
  • phage vaccines as described herein can be used to carry, into the phagosomes of targeted antigen-presenting cells, molecules or substances (which can be referred to by terms such as payloads, passengers, supplements, enhancers, adjuncts, etc.) that normally would not be efficiently internalized by such cells, or that normally would not potently activate a desired immune response in a large fraction of a treated population.
  • molecules or substances which can be referred to by terms such as payloads, passengers, supplements, enhancers, adjuncts, etc.
  • Such molecules or substances include: (1) small antigenic epitope molecules; (2) labeling or imaging molecules (also called trackers, tracers, or similar terms), which can be useful in research, diagnostic medicine, and other situations; (3) small and/or soluble antigen molecules that are only weakly immunogenic unless attached to a larger molecule; (4) adjuvant molecules, such as short DNA strands having CpG motifs, or other agents that can activate toll-like receptors; and, (5) plasmid DNA, which in some cases may be able to provoke gene expression, leading to useful polypeptides within (or possibly secreted by) targeted cells.
  • Those are non-limiting examples of compounds that can be useful if incorporated into, or affixed to, phage vaccines having targeting-and- delivery capabilities as disclosed herein.
  • This screening method required that any phages that were actually selected, in the screening test that was used, had to actually and effectively perform both of those two different and distinct functions, by emplacing candidate phages at a first location near the knee, and by harvesting phages from a different location near the hip.
  • an analogous type of in vivo screening test using a phage display library administered by nasal spray, can be used to identify, select, and isolate certain particular phages (out of millions or billions of candidate phages, in a display library) that will efficiently target and enter NALT cells in the nasal and throat region.
  • mice tests are not claimed or asserted to be optimal; instead, certain steps were taken because the inventor herein had done similar work for other purposes previously in his career, and therefore was already familiar with certain types of tests. Nevertheless, the complete set of steps that emerged and evolved, during the course of this research, were shown to work effectively. Now that this approach have been disclosed, experts and researchers who study these disclosures can adapt and improve these particular screening steps, for use with various species.
  • any in vitro screening tests that only require white blood cells can be completed first, and enriched populations of phages identified by the in vitro tests can then be screened by in vivo tests, using intact animals.
  • the screening tests developed and used for in vivo testing in mice used the following steps: 1.
  • the inventor had identified various reagents and methods that helped this work proceed more rapidly and efficiently. Two such reagents are worth noting.
  • the inventor used a compound called FITC, which uses fluorescein as a label and isothiocyanate as a linker, which will form a covalent bond that links a fluorescein label to an amine group (such as on a lysine residue) on a protein.
  • the FITC labeling agent enabled rapid analysis of tissue slices under a fluorescent microscope (i.e., a microscope that uses ultraviolet or other short-wavelength light as a light source). It also eliminated the need for expenses and delays that would have occurred if other types of labeling had been used that required incubation with antibodies, specialized enzymes, or other reagents.
  • mice Since he had seen prior results and reports (involving other, earlier research) indicating that some types of compounds, when inhaled by mice, will be taken into the olfactory bulbs, and will pass through that portion of the brain before being taken elsewhere, he decided to include olfactory bulbs as one of the tissue types that were tested for phage concentrations, at various time intervals after nasal administration.
  • the initial tracking and timing tests were performed using fluorescent-labeled "wild type" phages with no foreign gene sequences.
  • the results indicated by the open circles in the graph in FIG. 2, indicated a peak of high phage concentration in olfactory bulb tissue at the earliest testing time, in mice sacrificed 5 minutes after phage administration. That peak passed fairly quickly, as indicated in FIG. 2.
  • a second series of tests using olfactory bulb tissues was carried out using a phage display library (prepared by Cambridge Antibody Technology) containing scFv gene sequences from human antibody genes, inserted into coat protein 3 (which is present in several copies at one end of a phage particle).
  • the results indicated by the dark circles in FIG. 2, indicated an early first peak, which coincided in time with the single early peak of the wild-type phages.
  • a second peak was seen at 2 hours after phage administration, which had not been present at 1 hour or at earlier test times.
  • a first round of in vivo screening tests was carried out, using nasal administration of a phage library containing a diverse set of inserted DNA sequences encoding random 15-mer polypeptide sequences in coat protein 8 (which is present in over a thousand copies, in the cylindrical shells of the phages).
  • Tissue was harvested from the olfactory bulb portion of the brain, and the cells were incubated with a lysis buffer, which dissolved the cell membranes and released viable phages. The harvested phages were reproduced in bacteria, with the help of a tetracycline-resistance gene carried elsewhere in the phage genome (the scFv phage library similarly contains an ampicillin resistance gene). Samples from both sets of animals were pooled together, to provide an enriched starting population for the second round of screening.
  • NALT screening process was repeated, using phages selected in the first round of NALT screening as a starting population, to further enrich for NALT-targeting phages.
  • the ability of those screened and selected phages to actually target and bind to NALT cells, and subsequently to be transported by the immune system into lymph nodes, is confirmed by the photomicrographs in FIGS. 5 and 6.
  • Phage preparations that were screened and isolated as described above were replicated, and fluorescently labeled using FITC.
  • the labeled phages were then administered to mice, in nasal sprays. After 30 minutes or 2 hours, some of the mice were sacrificed, and perfused with a mixture of parabenzoquinone and paraformaldehyde.
  • Tissue sections were obtained, and photographed with a fluorescent microscope.
  • the large picture in FIG. 5 indicates the region of tissue that was photographed.
  • the two smaller pictures (which show up more clearly in color photographs posted on a website, www.tetraheed.net/ferguson) clearly indicated the presence of various clusters of fluorescent-labeled phages that had been transported to NALT tissues.
  • phagocytosis i.e., the intake of a solid particle into a cell.
  • phagocytosis involves four sequential steps, all of which must occur for the complete process to succeed.
  • a pathogen or vaccine must bind to (and activate) a phagocytic receptor, on the surface of a macrophage.
  • a phagocytic receptor any molecule that will bind to and activate such a receptor is called a "ligand”.
  • the in vitro screening steps described herein were used to identify specific phages, from among a large library of candidate phages, which happen to carry polypeptide sequences that act as "phagocytic receptor ligand" sequences.
  • the receptor-binding reaction must activate a membrane- altering process, to trigger formation of finger- like membrane extensions by the cell. These membrane extensions will flank and then surround the receptor/ligand complex, enabling the receptor/ligand complex to be taken into the cell.
  • the membrane "pocket” containing a receptor/ligand complex grows larger, and is drawn deeper into the cell, with the aid of additional "organelles” having their own membranes (such as endosomes and lysosomes).
  • the membranes of those organelles merge with the membrane of the phagosomal pocket that is being formed, to enlarge the phagosomal membrane and speed up the process.
  • the phagosome disengages from the outer membrane of the cell, to form a cxomplete and intact phagosome (i.e., a discrete compartment with its own membrane), which encloses a particle such as a microbe or a vaccine.
  • the macrophage uses specialized processes and enzymes to partially digest the microbe or vaccine, in a way that creates relatively short polypeptide sequences, typically about 15 amino acids long. Those short polypeptide sequences, from a partially-digested microbe or vaccine, are "mounted” on either MHCl or MHC2 proteins, to form an antigen/MHC complex, which is transported to the external surface of the macrophage cell. When that occurs, the macrophage becomes an "antigen-presenting cell” (APC). It will deliver its surface-mounted antigen polypeptide (from the microbe or vaccine particle) to a "B cell", which will perform the next steps in creating antibodies that will bind to microbes having the same antigenic polypeptide sequence.
  • APC antigen-presenting cell
  • phagocytosis is a multi-step process
  • two different screening tests were used, to ensure that the screened and selected phages would be highly potent and efficient, in initiating and enabling all of the steps in the process.
  • the first phagocytosis-related screening test might be eliminated, since: (i) the second screening test will screen for the desired final result, and (ii) successful completion of a desired final result implies and even requires that any necessary earlier steps also must have been completed, successfully.
  • the initial screening test is not difficult; it merely requires an initial incubation, followed by flow cytometry using an automated machine.
  • steps should be taken to ensure that the most potent and efficient candidate phage(s), from a large phage library, will indeed be identified and selected. Therefore, if an additional screening test can increase the likelihood that the most potent and efficient targeting-and-delivery phage (for use in vaccine cassettes) will be identified and isolated, then that screening test should be regarded as useful and desirable, even if not strictly necessary.
  • a "first phagocytotic" screening test was performed, using a phage population that already had been screened and selected for NALT-targeting activity, as described above.
  • human blood was sampled and loaded into tubes, on top of a buffer solution called “Lymphoprep” (sold by Nycomed Pharma, Oslo, Norway).
  • Lymphoprep a buffer solution
  • RBCs will pass through the buffer and can be removed and discarded, while WBCs will remain on top of it.
  • the white cells were harvested, washed twice in Dulbecco's phosphate-buffered saline (PBS), and placed in plastic tissue culture flasks.
  • PBS Dulbecco's phosphate-buffered saline
  • the white blood cells of interest are called "monocytes" when they circulate in blood. They have special surface-adhering molecules that cause them to grip and cling to the internal surfaces of capillary walls. That clinging process is crucial, in enabling these cells to pass through a capillary wall and enter the lymph fluid (the watery fluid that moves slowly through soft tissues). After a monocyte cell leaves the circulating blood and enters the lymph, it swells to a larger size, and is called a macrophage.
  • the monocyte/phage mixture was processed by "fluorescent-activated cell sorting" (FACS).
  • FACS fluorescent-activated cell sorting
  • a photo-detector (which detected the emission wavelength of the FITC label on the phages) was used to activate a tiny jet of liquid, injected into the flow passageway, each time a highly fluorescent cell passed through the glass tube. That control mechanism caused highly fluorescent cells to be sent to a special collection chamber, while cells with lower levels of fluorescence were sent to a discard bin. The controls on the flow cytometer were adjusted so that only 3% of the monocytes were selected; this is indicated by the rectangle on the right side of FIG. 8 A. [000235 ] In this "first phagocytotic" screening test, it did not matter whether phages entered the cell interiors, or merely clung to the cell surfaces. However, the test did required substantial binding to occur. Therefore, this screening test selected phages that happened to be carrying polypeptide sequences that function as ligands that bind tightly (with high affinity) to phagocytotic receptors on the surfaces of monocyte (macrophage) cells.
  • the selected monocytes (and their associated phages) were treated by lysis buffer, to dissolve the cell membranes and release the phages.
  • the phages were replicated in bacteria, and were used as the starting population in yet another round of screening, as described below.
  • FIG. 5 (which includes FIGS. 5 A and 5B) provides visual confirmation that the flow cytometry screening test performed as intended.
  • phages selected by the flow cytometry screening were incubated with a preparation of white blood cells.
  • the white cell population contained a relatively small number of monocytes and macrophages, mixed with other non-macrophage lymphocytes.
  • FIG. 5 A shows large numbers of white blood cells, under normal visible light (the smaller cells are mainly platelets).
  • FIG. 5B is a photograph of the same cells in the same pattern and position, at a fluorescent wavelength.
  • PBMCs that adhered to the plastic walls of culture flasks was prepared, as described above. These cells were contacted by a population of phages that already had been screened for NALT uptake, and for macrophage surface binding. After incubation with the monocyte cells, unbound phages were washed off and removed, and a liquid that contained trypsin (a protein-digesting enzyme) and ethylamine-diamine-tetra-acetate (EDTA, which enhances trypsin activity) was added, to detach the cells from the plastic surfaces of the flasks.
  • trypsin a protein-digesting enzyme
  • EDTA ethylamine-diamine-tetra-acetate
  • This trypsin treatment step also served to digest and render non-infective any phage adhering to the outer surfaces of the cell, thereby reducing any risk that phages isolated from phagosomes might be contaminated by phages that did not promote phagocytosis.
  • the harvested cells were suspended in fresh media, centrifuged, and resuspended.
  • the cell/phage preparation was then homogenized, using 10 strokes of a mechanical plunger device known as a Dounce glass-glass tissue homogenizer. Breakage of the cells by the homogenizer ruptured the outer membranes of the cells, in a way that did not destroy the phagosomes and other organelles (which are much smaller) contained inside the cells.
  • the homogenate was centrifuged under conditions that pelletized the cell nuclei and unbroken cells, while leaving the phagosomes (with any uptaken phages) in the " supernatant. The supernatant was then centrifuged at a higher speed for a longer time, to pelletize the phagosomes.
  • phage display library can become a very useful tool in studying, analysing, and utilizing various aspects of phagocytosis, including identification of new and additional classes of phagocytic receptors. While a number of classes of phagocytic receptors are already known (such as lectin receptors, Fc receptors, and complement receptors, as mentioned in the Background section and as reviewed in articles such as Aderem and Underhill 1999, Jutras and Desjardins 2005, and Blander 2007), other classes and types are likely to exist, and merit attention.
  • the methods described herein can be used to screen phage display libraries (including phage display libraries that may not have been previously screened at all, or that may have been screened using methods other then the NALT-related screening described herein), to identify additional receptor types of receptors that can trigger phagocytosis.
  • identification of phagosome receptor ligands for specialized cell types may be used to target efficient delivery of payloads to particular cells, or to stimulate or otherwise modulate various types of phagocytic processes that are important in development, disease, or other processes or problems.
  • stimulation of enhanced phagocytosis of beta-amyloid peptides by microglial cells, in brain tissue may be able to provide or improve various types of research and/or therapy, in Alzheimer's disease; this concept is discussed in more detail in articles such as Gelinas et al 2004.
  • Schwann cells phagocytose the myelin proteins that form the sheaths of neuronal fibers, (e.g., Hirata et al 2002); and, olfactory-ensheathing glial cells phagocytose the axons of olfactory cells that have been replaced (e.g., Wewetzer et al 2005).
  • a vaccine preparation can activate one or more types of "toll receptors" (also referred to interchangeably as toll-like receptors, abbreviated as TLR' s), on or in macrophage cells, the vaccine can be much more efficient in provoking a desired immune response. Accordingly, the phage cassette vaccines disclosed herein can be designed in ways that will strongly activate one or more types of toll receptors.
  • toll receptors also referred to interchangeably as toll-like receptors, abbreviated as TLR' s
  • phage cassette vaccines can be designed and assembled in ways that will activate specific toll receptors that are present inside macrophage cells, rather than on the cell surfaces. This can help increase the safety of vaccines derived from such vectors, since it can avoid unintended activation of toll receptors on the surfaces of macrophage cells, which otherwise might increase the risks of triggering or aggravating allergic or other unwanted responses in some recipients.
  • Cell-internal toll receptors that are known at the present time are believed to include TLR3, TLR7, TLR8, and TLR9.
  • TLR3, TLR7, TLR8, and TLR9 A large and rapidly growing body of published reports have identified DNA sequences containing CpG motifs (mentioned above) as among the agents that are known to activate internal TLR9 class of toll receptors. Accordingly, DNA sequences contain CpG motifs can be "woven into" the single-stranded DNA genome of phage cassettes as described herein.
  • oligo-deoxy-nucleotides can be affixed to the surfaces of phage particles, using either covalent or ionic/hydrogen bonding as described above, to trigger the activation of TLR9 receptors.
  • this type of controllable targeting of specific types of toll receptors is believed to be capable of creating vectors that can drive and steer the immune system toward creating either: (i) a "humeral” response, involving antibodies and B cells; or, (ii) a "cell-mediated” response, involving activated T cells without any substantial involvement by antibodies or B cells. It is believed and anticipated that this type of "guiding" system that targets certain types of toll receptors can be used, in phage cassette systems as disclosed herein, to create two different but important classes of vaccines:
  • vaccines that will provoke antibody-producing humeral responses, for fighting off microbial pathogens; or,
  • vaccines that will provoke specifically targeted cell-mediated responses, for killing cancerous cells and for dissolving or otherwise removing or moderating certain other types of harmful or dangerous cells or materials, such as plaques or other deposits.
  • FIG. 12 is a schematic depiction of a cassette-type phage particle 40, with various elements indicated by callout numbers.
  • Callout number 41 indicates a NALT-targeting peptide that will stimulate phagocytosis by human monocytes; callout number 41 indicates another attached molecule that has a different desired function, such as a fluorescent label, a compound that will provoke toll receptor activation, etc.
  • Callout number 43 is a DNA strand carried by the phage, which can contain (for example) a strong CpG motif, to activate toll receptor 9.
  • Callout number 44 depicts an antigenic protein that has been incorporated into coat protein 3 (CP3) of the phage; several copies of the CP3 proteins are present at the end of a phage filament, and at least one (and presumably all) of those copies have been modified to include a relatively large foreign polypeptide sequence.
  • CP3 coat protein 3
  • toll receptors With regard to toll receptors, it should be noted that the use of CpG motifs, in DNA strands carried inside filamentous phages, is believed to represent an advance in methods and reagents for exploiting toll receptor activation.
  • a phagocytic cell internalises and processes a bacteriophage DNA sequences that were carried and hidden inside the phage will be exposed, and can begin to stimulate TRL9 receptors, in ways that can initiate and/or increase an immunostimulatory cascade that will lead to an enhanced immune and antibody response.
  • Filamentous phages provide a different technology platform for developing and delivering CpG sequences, as immune adjuvants, compared to other candidate delivery vehicles, hi other settings, ODNs typically must be synthesized to be resistant to DNAase activity by the host cells.
  • immunostimulatory CpG motifs carried within the phage filament will be shielded and protected from DNAase inactivation, by the phage coat proteins. However, those phage coat proteins will be removed, after a phage vector has entered a macrophage or other phagocytic cell.
  • phage cassettes as disclosed herein will enable researchers to determine whether the presence of two or more such patterns can lead to additive or even synergistic levels of potency and efficacy, when incorporated into vaccines.
  • cassette systems will be able to receive and incorporate selected foreign DNA sequences (such as, but not limited to, sequences that encode antigenic proteins from pathogenic microbes), and they will be able to deliver the foreign DNA sequences to specific targeted classes of phagocytic cells, in ways that have not been possible under the prior art.
  • selected foreign DNA sequences such as, but not limited to, sequences that encode antigenic proteins from pathogenic microbes
  • the phage cassette will provide components that can be referred to as carrier, vehicle, transport, targeting, or delivery components, or by similar terms.
  • the cassettes must provide, in an exposed surface-accessible location, at least one polypeptide sequence that has been shown to provide at least one (and preferably all) of the following activities: a. targeting-and-delivery activity that will promote intake of vaccine particles into specialized cells that are exposed and accessible on one or more mucosal surfaces.
  • NALT cells in the nasal and throat region
  • other mucosal routes involving vaginal, rectal, or other surfaces
  • NALT-targeting activity are used for convenience, and are exemplary rather than limiting, and refer to activity in triggering uptake by one or more types of specialized immune cells that are exposed and accessible on one or more types of mucosal surfaces.
  • targeting-and-delivery activity that will promote the intake of vaccine particles into phagosomes, inside macrophage cells.
  • phagocytic processing by macrophage cells is a crucial step in generating a desired antibody response to a vaccine.
  • a coat protein of a phage cassette preferably should contain one or more exposed and accessible polypeptide sequences that will actively trigger binding to (and activation of) phagocytic receptors, on the surfaces of macrophage cells.
  • polypeptide sequences can be referred to as phagocytic (or phagocytotic) ligands.
  • the phage cassettes disclosed herein can contain DNA sequences having CpG motifs (either carried within the genomes of the phages, or affixed to the surfaces of the phage particles), which can activate targeted toll-like receptors in ways that will increase the likelihood of a desired immune response (which will depend on the type of vaccine that is being administered).
  • One or more foreign gene sequences will be inserted into the genome of a phage cassette, into targeted insertion sites that are properly positioned within the coding sequence for a viral coat protein. These insertion sites will contain unique sequences that can be recognized and cleaved by at least one (and preferably several) restriction endonuclease(s).
  • the inserted foreign DNA sequence will encode an antigenic protein sequence that is normally found on the surface of the pathogen.
  • antigenic protein sequences By placing such antigenic protein sequences into phage cassettes that will specifically target and deliver the antigenic polypeptide sequences to NALT cells and then to macrophage cells, increased efficacy and potency for such vaccines can be achieved, and nasal administration of these vaccines can offer an effective route of administration, not just for humans, but for large numbers of animals as well (notably including poultry).
  • microparticles can be used to deliver, into a mammalian body, DNA strands that can be expressed into foreign polypeptides, by means of normal cellular processes. This approach, called "DNA vaccination", is reviewed in articles such as Jilek et al 2004. Under the prior art, it has not been highly efficient, and it has not become a widely-used method of vaccination.
  • single-stranded DNA segments such as from phages that carry ssDNA
  • double-stranded DNA segments such as from bacterial plasmids
  • covalent bonding or ionic/hydrogen bonding
  • strands of ssDNA and/or dsDNA can be affixed to the surfaces of particles that can be manufactured inexpensively in large quantities, and that can potently and efficiently deliver surface-affixed DNA strands into specific targeted cells, such as: (i) into NALT cells and macrophages, if the phages that were identified and isolated by screening tests such as disclosed herein; or, (ii) into other cell types, if the phages were identified and isolated by screening tests that select for uptake into those particular cell types.
  • ThI Th2 cells
  • ThI and Th2 cells There are several ways to distinguish between ThI and Th2 cells, depending on how they are activated, and what they do after they are activated. Importantly, those known differences also offer potential ways to manipulate and control vaccines, in ways that can "steer” or “direct” T helper cells in either of those two directions when desired.
  • ThI cells The distinctions between ThI cells versus Th2 cells are described in review articles such as Moingeon 2002, Knutson et al 2005, and Burrows 2005. While other articles such as Rosloniec et al 2002 point out that the distinctions are not always entirely clear, and that apparently paradoxical responses are sometimes observed, relevant reports generally indicate and agree upon the following:
  • T helper cells commit to the ThI pathway when a messenger molecule called interleukin 12 (IL- 12) is present.
  • the ThI cells then begin producing gamma interferon, interleukin 2, and tumor necrosis factor alpha.
  • T helper cells commit to the Th2 pathway when IL-4 is present.
  • the Th2 cells then begin producing more IL-4, as well as IL-5 and IL-10.
  • ThI cells are involved in what are often called “cytotoxic T cell” (CTL) responses, also called “cell-mediated” responses. These can be important in fighting cancer, and in fighting some types of chronic, lingering microbial diseases. However, cell-mediated responses also are involved in autoimmune diseases, and can create severe problems in some cases.
  • CTL cytotoxic T cell
  • Th2 cells are involved in systemic responses that use B cells to create antibodies (these are also called humoral responses).
  • ThI versus Th2 pathways are important differences between ThI versus Th2 pathways into account, and by also considering other articles and teachings in this field, it is believed and anticipated that steps can be taken that will enable phage cassette vaccines as described herein to be manipulated in ways that will trigger either: (1) Th2 responses, when desired, such as for creating antibodies that will fight off microbial pathogens that typically cause short-term infections, such as flu viruses; or, (2) ThI responses, when desired, such as in vaccines for treating cancer or other disorders, or for treating some types of lingering infections.
  • Th2 responses when desired, such as for creating antibodies that will fight off microbial pathogens that typically cause short-term infections, such as flu viruses
  • ThI responses when desired, such as in vaccines for treating cancer or other disorders, or for treating some types of lingering infections.
  • DNA sequences with CpG motifs that will stimulate ThI (“cell-mediated") responses are described in articles such as Krieg et al 1998, while other DNA sequences with "suppressive” CpG motifs that will steer the immune system toward Th2 (antibody-generating) responses are described in articles such as Ho et al 2003 and Shirota et al 2004. Accordingly, these types of relatively short DNA sequences will merit attention, as enhancers and adjuvants that can be incorporated into, or affixed to the surfaces of, phage cassette vaccines as described herein.
  • timed coadministration of selected interleukin molecules or certain other cytokine molecules, and/or other types of adjuvants as described in articles such as Guy et al 2005, can also be used to help steer an immune response in a desired ThI or Th2 direction.
  • a primary reason to vaccinate is to "prime” the immune system, so it can recognize a future invasion and respond as rapidly as possible.
  • the immune system launches a Th2 type of response, it generates "memory" B cells that effectively "remember” the vaccine antigens.
  • This process involves a complex mechanism, wherein a large variety of responsive B cells with reshuffled short DNA sequences are generated, which will encode a variety of newly-created variable fragments that are incorporated into new types of antibody molecules.
  • the immune system identifies particular B cells that happen to be making antibodies that efficiently bind to the invading microbe.
  • Those particular B cells are stimulated to reproduce rapidly, causing the enlarged population of selected B cells to secrete large numbers of their antibodies. Then, after an infection recedes, the number of those particular B cells drops off greatly. However, a few of those B cells remain in the system for years or even decades, and if a need arises, they can be stimulated to cause them to rapidly reproduce again, so that they and their progeny cells can rapidly begin making a renewed supply of the antibodies that were effective in helping fight off a particular type of microbe.
  • CD8(+) T cells are generated. Like the antibody secreting memory B cells generated by vaccination, these memory CD8(+) T cells are primed to multiply rapidly, if the same antigen subsequently appears in the body. CD8(+) T cells are also known as "killer" T cells, because one of their primary roles is to detect and destroy cells that have become infected by invading viruses.
  • antigen-specific memory CD8(+) T cells can recognize the early signs that a cell has been infected by a virus, and can destroy virus-infected cells, to minimize the number of additional viruses being made by the infected cells.
  • ThI responses and antigen-specific CD(+) T cells, in protecting against viral infections is described in reviews such as Wiley et al 2001, and Wong and Pamer 2003.
  • NALT-targeting phages carrying TRL-related or other PAMP-related components can carry one or more genes that encode the haemagglutinin (HA) and/or neuraminidase (NA) proteins.
  • NALT-targeting phages carrying TRL-related or other PAMP-related components that will trigger a ThI response can carry one or more genes that encode the influenza proteins that take control over a host's cellular machinery, because those proteins are more likely to be present and active, in host cells that have been infected by the viruses and that need to be destroyed by killer T cells.
  • the vaccine can reduce the risk and probability that a single recombination event or mutational shift, in a pathogenic virus, would lead to emergence of a new and virulent strain that cannot be recognized by a vaccinated population, and which therefore might spread more rapidly and cause greater illness, suffering, and deaths.
  • a CpG motif that may be effective for both avian and human vaccines has been identified and disclosed. Work with macrophage cells from poultry has shown that a B/K type CpG motif designated as "ODN 2006", which is known to have strong stimulatory activity in human cells, also strongly stimulates costimulatory molecule expression in avian macrophage cells (Xie et al 2003).
  • the oligonucleotides will act as adjuvants.
  • the CpG adjuvants call attention to the presence of the injected vaccine particles, thereby boosting the immune system and pushing it into high gear.
  • the activated immune system will then create an amplified response to the antigenic influenza protein sequences carried by the flu vaccines.
  • phage cassette vaccines for use in vertebrate animals (e.g., Frenkel et al 2004).
  • Phage genomes can be manipulated, in ways that can allow the phage vectors to provide an array and assortment of different CpG motifs. Studies have shown that the response of monocyte cells, from different human donors, to stimulation by DNA sequences having CpG motifs, is not always consistent; no single oligo-deoxy-nucleotide sequence is maximally stimulatory in all human monocytes (e.g., Klinman and Currie 2003, and Leifer et al 2003). Therefore, a mixture of ODN sequences having different sequences and activities can be woven into a phage genome.
  • phage cassettes may be able to induce the desired immune system activation in the widest possible range of recipients, in mixed populations that will have diverse genomes and potentially differing responses to such vaccines.
  • the third composition of matter disclosed herein comprises vaccines that contain targeting-and-delivery polypeptide sequences that were identified by screening of a phage display library, regardless of whether the vaccine particles are, or are not, phage particles. If a certain polypeptide sequence, carried by a phage particle that is one out of millions or billions of phages in a phage display library, has been discovered and shown to be highly potent and effective at triggering both (i) uptake into NALT cells, and (ii) transport into the phagosomes of macrophages, then that "target-and-deliver" polypeptide sequence can be used in ways that are not limited to vaccine particles made by phages.
  • a target-and-deliver polypeptide sequence can be incorporated into various types of viruses that infect eukaryotic cells, rather than infecting bacteria.
  • viruses are widely used in vaccines today, in either "disarmed” forms (which can also be referred to as attenuated, crippled, etc.) or in "killed” (or inactivated, nonviable, etc.) forms.
  • Most "subunit" vaccines which contain an antigenic polypeptide sequence from a pathogen that has been spliced into some other type of carrier-type virus, also use carrier viruses that infect eukaryotic cells, rather than bacteria.
  • glycosylated vaccines can more closely resemble and mimic the surfaces of a pathogenic microbe that a vaccine is designed to defend against. Since bacterial cells normally cannot glycosylate proteins, glycosylated vaccines usually must be manufactured in eukaryotic cells.
  • bacteria cannot perform some types of protein folding (i.e., shaping of a protein strand into a certain three-dimensional shape and conformation), or other types of "post-translational processing" performed by eukaryotic cells.
  • protein folding i.e., shaping of a protein strand into a certain three-dimensional shape and conformation
  • post-translational processing performed by eukaryotic cells.
  • a vaccine must be cultured and reproduced in eukaryotic cells rather than bacteria, this can impede the use of bacteriophage particles as vaccines, since bacteriophages normally infect only certain types of bacteria, and cannot infect animal cells.
  • eukaryotic cells can be genetically manipulated, to give them a foreign gene that will express a new surface protein that will serve as a binding site (or docking site, or similar terms) for phages. This can allow phages to enter eukaryotic cells which carry those proteins on their surfaces.
  • a "target-and-deliver" polypeptide sequence can be incorporated into a coat protein (or other surface protein) of other types of engineered viruses that are used to create vaccines.
  • This approach can be used to create nasally-administered vaccines, using engineered viruses that can be cultured and manufactured in eukaryotic host cells (such as bird eggs, insects or caterpillars, yeast, mammalian cells, etc.) that will perform glycosylation, protein folding, post-translational, or other processing steps that may be necessary to give the vaccine a desired activity and potency.
  • the "target-and-deliver" polypeptide sequence When administered via nasal spray (which can be a liquid, aerosol, powder, etc.), the "target-and-deliver" polypeptide sequence (which initially was discovered in a phage display library, and which later was “transplanted” into a virus that infects eukaryotic cells) will cause the viral vaccine particles to more readily bind to (and enter) NALT cells in inoculated animals, which will then deliver the engineered viral vaccines to macrophages, and possibly to one or more other types of phagocytic antigen-presenting cells.
  • nasal spray which can be a liquid, aerosol, powder, etc.
  • a "target-and-deliver" polypeptide sequence when inserted into a type of vaccine virus that can be manufactured (and glycosylated or otherwise processed) in eukaryotic cells, can increase the potency and efficacy of the resulting vaccine, and can render the vaccine well-suited for administration using a nasal spray, rather than a needle.
  • NALT-targeting polypeptide sequences as disclosed herein can be inserted into vaccines such as influenza vaccines, which are manufactured by culturing viruses in eukaryotic cells, such as bird eggs. Flu viruses are among the most rapidly- mutating viruses known, and new mutants appear each year that can cause severe illness and major epidemics, even among people who have been exposed multiple times to previous flu infections and/or vaccines. Two major surface proteins of influenza are haemagglutinin (HA) and neuraminidase (NA). Therefore, each year, newly-updated vaccine strains must be generated, by using genetic engineering to alter a "master strain" that is attenuated (i.e., effectively crippled, so that it cannot cause major problems or severe illness).
  • HA haemagglutinin
  • NA neuraminidase
  • the teachings herein can be adapted for use within that system, by including (in the final versions of the influenza vaccine viruses) a "target-and-deliver" polypeptide sequence (such as the sequence disclosed herein) that will trigger and promote the two immune cell reactions discussed herein, which are: (i) uptake of the vaccine particles into NALT cells in the nasal and/or throat region, followed by (ii) transport of the vaccine particles into the phagosomes of macrophage cells, which will become antigen-presenting cells.
  • a target-and-deliver polypeptide sequence such as the sequence disclosed herein
  • the preferred insertion site, for such a NALT-targeting polypeptide sequence can be determined by experts working for a company that manufactures such a vaccine. Several companies manufacture such vaccines, and each vaccine is somewhat different; as just one example, an annually-updated, nasally-administered flu vaccine is sold by Medlmmune, under the trademark NASALMIST.
  • the "target-and-deliver" polypeptide sequence (which typically will comprise a stretch of roughly 15 amino acid residues or less) can be used to replace a sequence having a similar length, or added as an "epitope tag", presumably near the N-terminus or C-terminus as appropriate, in a surface protein of the attenuated master strain, so that the size of the modified surface protein will be unaltered, or altered only slightly. This can avoid creating surface proteins with substantially altered sizes, which might hinder assembly of the modified virus particles.
  • the other gene variant can be used to provide copies of: (i) an unmodified surface protein, if desired; or, (ii) a modified protein carrying an antigenic polypeptide sequence from a pathogenic microbe, which has been selected because it will trigger the formation of antibodies that will enable an inoculated host to mount a rapid immune response against the pathogenic microbe.
  • viruses that can infect the upper respiratory system include coronaviruses (which includes SARS-CoV, a variant that causes "severe acute respiratory syndrome", or SARS), picornaviruses, rhinoviruses, adenoviruses, etc. Most of these viruses (other than SARS-CoV) do not cause potentially lethal illnesses, so most of them have not received major attention in terms of vaccine development (a notable exception involves adenoviruses, which have been extensively studied and developed for various types of gene therapy, largely because they can be used with a class of so-called “helper viruses” (also called satellite viruses) that enable various useful techniques and safeguards). However, all of the virus types listed above can trigger immune responses.
  • helper viruses also called satellite viruses
  • any of these types of viruses can be converted into nasally- administered attenuated carriers, which can carry exposed surface proteins that will include: (i) at least one NALT-targeting polypeptide sequence, which will efficiently deliver vaccine particles to antigen-presenting immune cells; and, (ii) at least one antigenic polypeptide sequence from a pathogenic microbe, which will be incorporated into the vaccine in order to trigger the formation of antibodies that will bind to the pathogen.
  • NSV nonsegmented negative-strand viruses
  • nonsegmented means that the entire viral genome is carried in a single molecule, of either single-stranded or double-stranded DNA or RNA (by contrast, some viruses require two different segments of DNA or RNA to be gathered and packaged into each viral particle).
  • negative-strand also called anti-sense strand, or nonsense strand
  • negative-strand means that when a "complementary" copy of the viral DNA or RNA is formed by a cell, the "complementary” strand will be the “sense” strand, with codons that directly encode a viral protein.
  • NNSV viruses are surrounded by lipid membranes, usually called “envelopes”. These usually are obtained from a host cell by means of a “budding" process, in which each virus particle surrounds itself by an initial pocket that enlarges into a bubble-type enclosure, made from one or more membranes of a host cell.
  • viruses that are released by a "membrane budding" process usually spare the lives of infected cells, allowing infected cells to make even more copies of the virus.
  • NNSV viruses tend to pose the most rapid, acute, and aggressive pathogenic threats to health. They include, for example, rabies virus, measles and mumps viruses, sendai virus, human parainfluenza viruses, vesicular stomatitis virus, Newcastle disease virus, human respiratory syncytical and metapneumovirus, Ebola and Marburg viruses, and Bora disease virus. NNSV viruses are regarded as posing the greatest threats of bioterrorism, and they have received a great deal of attention and research.
  • NNSV viruses in vaccines
  • articles ' such as Bukreyev et al 2006. Since some types of NNSV viruses can efficiently infect nasal and throat tissues, those NNSV viruses offer promising vectors for creating attenuated or inactivated vaccine vectors, which can be enhanced by inserting NALT-targeting sequences into one or more of their exposed surface proteins. Such NALT-targeting sequences can accelerate and increase the entry of the resulting modified vaccines into NALT cells, and then into the phagosomes of macrophage cells.
  • NNSV vaccines that have been enhanced in that manner are not limited to vaccines for preventing NNSV diseases. Instead, NNSV vaccine vectors can be modified to include antigenic proteins that will immunize recipients against completely different types of pathogens.
  • the potency of at least some vaccines created from NNSV vectors is likely to be increased even more, by also incorporating one or more additional components that will activate one or more types of TLR receptors, or that can otherwise help direct and guide an immune response toward a desired response, such as either an MHC-I or MHC-2 response, or a TH-I or TH-2 response.
  • NALT-targeting polypeptide sequences as disclosed herein also can be incorporated into vaccines made from bacteria or other cellular (rather than viral) microbes.
  • cellular is used conventionally, to exclude viruses while including bacteria, fungi (including yeast cells), mycobacteria, and other microbes that are regarded as "cells" by biologists. In general, viruses do not carry the enzymes necessary to metabolize nutrients, synthesize DNA or proteins, or make new lipid membranes, so viruses must obtain those building blocks from host cells.
  • cellular microbes carry the enzymes necessary to metabolize nutrients, synthesize DNA and proteins, and make lipid membranes.
  • respiratory diseases involve mucosal tissues in the nose and throat
  • respiratory diseases are likely to be of early interest among researchers studying the disclosures herein.
  • respiratory diseases caused by cellular microbes include tuberculosis (caused by Mycobacterium tuberculosis), pneumonia (caused by Streptococcus pneumoniae, also referred to as pneumococcus), and a disease of horses known as "strangles" (caused by Streptococcus equi).
  • Vaccines containing killed or attenuated microbes are available for all three diseases, as described in various articles, government reports, and websites maintained by vaccine manufacturers. However, none of those vaccines are fully optimal, and improved vaccines for any of those diseases could be useful and helpful.
  • NALT-targeting polypeptide sequences are used to incorporate into vaccines against cellular (non-viral) diseases and pathogens.
  • three major routes include: (1) development of phage particle vaccines, which will include NALT-targeting polypeptide sequences identified by screening methods as described herein, combined with antigenic sequences derived from cellular (non- viral) pathogens; (2) development of viral vaccines derived from viruses (presumably glycosylated) that normally infect eukaryotic cells, which will include NALT-targeting polypeptide sequences, combined with antigenic sequences derived from cellular (non- viral) pathogens; and, (3) development of killed or attenuated cellular vaccines, which will incorporate NALT-targeting polypeptide sequences, and antigenic sequences derived from cellular (non-viral) pathogens, into various cell-surface proteins.
  • a candidate vaccine performs well in the safety tests, it can be tested for efficacy against actual pathogens, using known procedures (such as inoculating a large population of people who are at elevated risk of a certain disease, then gathering statistical data on how many inoculated people contracted the disease, compared to how many people contracted the disease in an untreated population of the same size).
  • cancer vaccines have been extensively researched, as described in sources such as Acres et al 2007 and www.cancer.gov/cancertopics/factsheet/cancervaccine. While some types of cancer can be caused by viral infections (such as cervical cancer, caused by papilloma viruses), most cancers have no specific microbial cause, and arise from mutations that can occur when cells reproduce. In addition, even if a cancer is caused by a virus or other microbe, the nature of the disease requires that the cancerous cells must be attacked and destroyed, and the initial causative factor has little or no importance after cancerous cells begin replicating uncontrollably.
  • nonmicrobial disorders that may someday benefit from vaccine therapy include Alzheimer's disease, autoimmune disorders, hormonal or endocrine disorders, and other disorders that arise when native cells, body parts, or metabolites go wrong, rather than arising from infections by microbes.
  • Information on how vaccines might benefit such nonmicrobial disorders can be located easily by searching the U.S. National Library of Medicine database, or various Internet databases.
  • NALT-targeting vaccines as disclosed herein
  • approaches that can extend NALT-targeting vaccines (as disclosed herein) into uses for treating cancer or other nonmicrobial diseases can be summarized as follows:
  • phage particles having a variety of different polypeptide sequences in their coat proteins, can be nasally administered, in the form of a phage display library suspended in a liquid or powder solution;
  • the isolated NALT-targeting phages can be tested, using cell culture methods, to determine whether they activated either: (i) MHCl receptors and THl cells, or (ii) MHC2 receptors and TH2 cells. This can be done, for example, by using assays that can detect interleukin-2 or gamma-interferon proteins (which will indicate that THl cells were stimulated), versus assays that can detect interleukin-4, interleukin-5 or interleukin-10 proteins (which will indicate that TH2 cells were stimulated).
  • NALT-targeting phages that activate MHCl receptors and THl cells can then be used as targeting polypeptides, in vaccine preparations that use clonal phage particles that are ideally sized and suited for such use.
  • anticancer (or similar) vaccines will be used to deliver selected antigen protein sequences to T cell types that, when activated, will begin killing and destroying cancer or other disease-causing cells.
  • those types of cell-mediated immune responses by activated T cells responding to antigenic polypeptides that were presented to them by macrophage cells, can be triggered by a sequence of steps such as the following:
  • a gene that encodes a known antigenic polypeptide sequence which will have the same amino acid sequence as a cancer-related antigen found in large numbers on the surfaces of certain types of cancer cells, is inserted into a NALT-targeting phage cassette that activates MHCl receptors and THl cells preferentially over MHC2 receptors and TH2 cells;
  • the resulting anti-cancer vaccine carrying both (i) a cancer-antigen protein sequence, and (ii) a NALT-targeting polypeptide, is administered to an animal or patient, via a mucosal mode, such as a nasal spray; (3) the phage vaccine particles will be transported into NALT tissue, and it then will be taken into the phagosomes of macrophage cells, because of the target-and-deliver protein sequence inserted into its coat proteins;
  • the macrophages will convert into antigen-presenting cells, which will travel to lymph nodes and "present" the phage antigens to T cells; (5) the phage components that were selected and used, in that particular type of vaccine cassette, because they activate MHCl receptors and THl cells, will do their work, and will steer and guide the immune system into launching a cell-mediated immune response, which uses activated T cells, rather than triggering formation of IgG antibodies via B cells; (6) at least some of the activated T cells will bind to cancer cells that have, on their cell surfaces, the same cancer-related antigenic protein sequence that was placed into the coat proteins of the genetically-engineered anti-cancer vaccine particles;
  • an activated T cell attaches itself to a cancer cell that contains the same antigenic protein sequence that was present in the cancer-fighting vaccine, the activated T cell will inject perforin into the cancer cell; (8) the cancer cell's mitochondrial membranes will become permeable, due to the action of the perforin from the activated T cells; and,
  • NALT-targeting polypeptides can be screened and selected. Particular polypeptide sequences, selected because they performed efficiently in those screening tests, can then be incorporated into the coat proteins of phage vaccines (or other types of vaccine particles, as described above), by genetic engineering methods. This will create "cassette"-type phages, which can then be modified by a final step to turn them into cancer-fighting (or similar) vaccines.
  • cassette-type phage vaccine vehicles can be supplemented, by inserting into the phage genome an additional gene sequence, which will encode (in one of the phage coat proteins) a second foreign polypeptide sequence, which will be a known cancer antigen.
  • the resulting anti-cancer phage vaccine particles will have: (i) a targeting sequence that makes the phage particles efficient in promoting uptake by NALT cells, followed by uptake into the phagosomes of macrophages; (ii) one or more components that will steer and guide the response by the macrophage cells in a manner that activates MHCl receptors and THl cells preferentially, over MHC2 receptors and TH2 cells; and, (iii) an antigen sequences that will cause activated T cells to seek out and destroy cancer cells that have the same antigen proteins on their surfaces.
  • Another class of vaccines that can be enabled and/or enhanced by the disclosures herein involve vaccines that will trigger the production of antibodies that will bind to one or more types of peptide hormones, or hormone receptors.
  • FSH follicle-stimulating hormone
  • LH luteinizing hormone
  • vaccines could be developed that would trigger the production of antibodies that would bind to (and thereby inactivate) FSH and/or LH (or GnRH, which stimulates the pituitary gland to secrete FSH and/or LH), such vaccines might be able to help treat and minimize Alzheimer's disease.
  • Similar examples can be provided for various types of cancer that are "fueled” by certain types of peptide hormones (including prostate cancer, as one example).
  • GnRH gonadotropin release hormone
  • somatotropin is a technical (and marketing) term for “growth hormone”; the root “somato-” refers to body, and “-trop” refers to nutrition, sustenance, and/or growth.
  • the offsetting (growth-inhibiting) hormone is called “somatostatin", where "statin” indicates “unchanging” (as in found in words such as stay, stable, and stationery).
  • vaccines are being developed and tested for birth and population control, in humans and in various animals. Such vaccines are reviewed in Naz et al 2005, and numerous other sources. For example, using the technology disclosed herein, painless nasal administration of a vaccine to dogs or cats may be able to eliminate the need for surgical neutering.
  • the types of screening tests disclosed herein also can be used to identify particular phages, from a display library, that will pass through one or more types of biological membranes.
  • phage display libraries were nasally administered to mice. At various times after intranasal administration, animals were anesthetised, and blood was sampled. The results indicated that phages appeared within the blood circulation within 15 minutes, and were rapidly cleared from the blood.
  • Phages in the blood were reproduced in E. coli, and the resulting phage populations were tested by additional rounds of nasal-to-blood in vivo selection. Increasing numbers of phages were recovered with each selection round, during several rounds of testing. When fully-diverse scFv phage were used, initial nasal-to-blood screening tests yielded highly variable results. When similar tests were performed using scFv phage that previously had been enriched by sciatic nerve screening (as mentioned above, and described in more detail in WO 2003/091387), larger and more consistent numbers of phages were obtained.
  • the phages selected by those types of screening tests were demonstrated to pass through endothelial membranes (i.e., the class of membranes that form capillaries and other blood vessels); and, the polypeptide sequences carried by those phages were shown to efficiently drive the transport of particles through endothelial membranes, into circulating blood.
  • endothelial membranes i.e., the class of membranes that form capillaries and other blood vessels
  • the intestinal tract contains specialized clusters of "lymphoid follicles", including structures called “Peyer's patches”, located mainly in the small intestines (as an indicator of their importance, it has been estimated that 70% of a mammalian immune system, by volume, resides in the digestive tract).
  • M cells As with NALT tissues in the nose and throat, GALT tissues in the gut are covered by "M cells” having specialized microfolds, which create expanded surface areas that enable the M cells to "sample” molecules passing through the intestines, and to transfer those molecules or particles that appear unusual (such as molecules with "pathogen-associated molecular patterns", or PAMPs, described in the Background section) to immune cells that reside or travel beneath the outer layer of M cells.
  • M cells having specialized microfolds, which create expanded surface areas that enable the M cells to "sample” molecules passing through the intestines, and to transfer those molecules or particles that appear unusual (such as molecules with "pathogen-associated molecular patterns", or PAMPs, described in the Background section) to immune cells that reside or travel beneath the outer layer of M cells.
  • NALT-targeting vaccines are like to be more efficient than comparable orally- ingested GALT-targeting vaccines.
  • GALT-targeting vaccines will need to be specially formulated to survive stomach acidity, presumably by placing them in carriers that will release the vaccine particles only after the vaccine reaches the small intestines (one such carrier, developed by students working with Prof. Hai-Quan Mao at Johns Hopkins University, has been announced for a rotavirus vaccine being developed by Aridis Pharamaceuticals, www.aridispharma.com).
  • the vaccine particles will be mixed with relatively large quantities of food that is being digested and converted into feces, in the intestines, and that natural process will inevitably reduce the contact and uptake of the vaccine, compared to a nasally administered vaccine that can be: (i) emplaced directly on NALT surfaces, in the nasal sinuses and (ii) held in position for sustained times by a mucoadherent compound, as described in the Background section.
  • NALT-targeting and/or GALT-targeting transport polypeptide sequences can be merged and combined with such efforts, to utilize the potent and specialized transport activities of such polypeptide sequences.
  • phage library can be fed to lab animals, using a suitable carrier system (such as the carrier mentioned above, for a rotavirus vaccine that will be taken orally).
  • Peyer's patch tissues or similar intestinal tissues, or possibly certain types of "downstream" tissues or cells, such as macrophages in lymph nodes that serve the digestive tract
  • the cell membranes will be lysed, using a buffer that does not damage the coat proteins of the phages (as described for the NALT screening tests), to harvest enriched populations of phages that were preferentially taken into the Peyer's patch or other GALT cells.
  • a phage library can be labeled, using FITC or other labeling agents, to make the tracking and harvesting steps easier or more efficient.
  • That type of screening process can be repeated as many times as desired, and the resulting enriched phage populations can be screened again, using macrophage association and/or phagosomal entry screening tests, as described herein.
  • a sequential combination of screening tests can be used to identify one or more phages, from a phage display library, that will potently drive both steps of a two-step process: (i) intake into GALT cells in the intestines, followed by (ii) phagocytic intake, by antigen-presenting cells.
  • in vitro screening tests can also be used, in one or more assays designed to identify enrich any GALT-targeting polypeptide sequences.
  • O'Mahony et al 2004 describes the use of a tissue culture method for screening a phage display library, using "CACO” cells from a transformed cell line that was initially derived from a colon cancer tumor.
  • CACO cells which are anchorage-dependent
  • CACO cells can be grown into a cohesive layer which will have an "apical" side (which normally would be exposed to semi-digested food passing through the intestines), and a "basal" side.
  • O'Mahony et al contacted the apical sides of their CACO cell layers with phage populations, and selected phages which passed through those cell layers.
  • Any DNA or polypeptide sequence disclosed herein, and any other DNA or polypeptide sequence hereafter discovered to have potent targeting-and-delivery activity for vaccine use can be used as a starting sequence (which can also be called a baseline, initial, or reference sequence, or similar terms), in efforts to find analogs having comparable or even higher potency.
  • an analog is a molecule that resembles a certain designated molecule (which can be called a baseline, starting, initial, reference, or referent molecule or compound, or similar terms), but which has been modified by substituting or altering one or more groups or substituents of the starting molecule.
  • a molecule has a relatively small "moiety" (i.e., an atom or cluster of atoms, such as a hydrogen, sulfur, or halogen atom, or a hydroxyl, amine, methyl, or similar small group) at a specific location on the compound
  • analogs can be formed by replacing that moiety with various other atoms or small groups, or by moving a moiety to a different location on the molecule.
  • saturated bonds can be replaced by unsaturated bonds, and various other limited modifications can be made, to create molecules that are similar but not identical to a starting compound.
  • Such analogs can then be screened, to determine whether any of them have a comparable (or improved) level of a desired activity, compared to the starting molecule. If a particular analog is discovered to offer a significant improvement, it can then be tested and studied more closely, and it also can be used as a new starting or baseline molecule, in subsequent efforts to develop even better analogs.
  • the detector device will analyze each aliquot, in turn, hi some cases, this is done by taking and processing a sample of liquid from each container; in other cases, it is done by nondestructive means, such as by shining a light having a certain wavelength (or range of wavelengths) through each container, to measure one or more factors such as turbidity or color intensity (or to create a graph showing various peaks, as a function of differing wavelengths).
  • nondestructive means such as by shining a light having a certain wavelength (or range of wavelengths) through each container, to measure one or more factors such as turbidity or color intensity (or to create a graph showing various peaks, as a function of differing wavelengths).
  • the results of each test will be recorded in a computer, with identifying numbers to correlate each analytical result with a specific container and aliquot.
  • the computer software can even rank the output data, so that the best-performing analogs can be quickly identified and ranked.
  • Such alterations can take the form of: (i) substituting (or "swapping") one or more specific amino acid residues, without altering the number of residues in a sequence; (ii) inserting one or more additional amino acid residues into a known sequence, in a way that increases the number of amino acid residues in a sequence; or, (iii) deleting one or more amino acid residues, to decrease the number of residues in the sequence.
  • These types of alterations can be created by well- known methods, such as by using automated machinery to create synthetic segments of DNA, which can be spliced into a known restriction site in a gene that encodes a coat protein, in a plasmid and/or phage.
  • the synthetic segments of DNA can have either: (i) exact and known sequences, in “controlled mutagenesis”; or (ii) random and assorted sequences, in “random mutagenesis”.
  • the resulting modified genes can then be expressed into polypeptides (by using phage vectors, microbial fermentation, or other methods), and the resulting phages or polypeptides can be tested, using cell culture, small animals, etc., to determine which particular polypeptides happen to have the strongest levels of activity, using any screening test that is of interest.
  • this invention anticipates that analogs can be prepared and tested, using any DNA or polypeptide sequence that is disclosed herein (or that is hereafter discovered to function potently as a targeting-and-delivery sequence, when used as disclosed herein), as a starting sequence. If some particular analog sequence is found to be more potent for the purposes disclosed herein than the starting sequence it was derived from, then that analog may rise to the level of a patentable improvement; nevertheless, if it is created and tested as an analog of a known sequence, using processes such as controlled or random mutagenesis following by screening tests as disclosed herein, then any such analog that arises from the teachings herein is within the scope of this invention.
  • a phage display library believed to contain 1.3x10 10 individual recombinants, each containing a single chain variable fragment (scFv) gene sequence derived from human B-cells, and a CANTAB6 control phage lacking an scFv insert, were obtained from Cambridge Antibody Technology (United Kingdom). These phages carry an ampicillin resistance gene, as well as a plasmid origin of replication.
  • scFv single chain variable fragment
  • a second type of "peptide display” phage library was obtained from George Smith (University of Missouri, USA). It is believed to contain approximately 10 8 different clonal phages, and is derived from a filamentous phage designated as fd-tet, which carries a tetracycline resistance gene (as a selectable marker), and an origin of replication that allows the phage genome to be manipulated and reproduced in double-stranded DNA form, as a plasmid. These phages also are known as "type 88" phages (Smith 1993), since their genome (9273 bases) carries two different genes that encode coat protein VIII.
  • One of the two protein VIII genes carried by "type 88" phages is a wild- type (unmodified) gene, while the other coat protein VIII gene carries a foreign gene sequence that encodes a "15-mer” polypeptide (i.e., an inserted polypeptide sequence containing 15 amino acid residues, spliced into the normal amino acid sequence of the phage's coat protein VIII).
  • This vector is also referred to as a f88-15mer phage library (GenBank accession number AF246448).
  • the random 15-mer polypeptide sequences typically are expressed at up to about 300 copies per phage.
  • f88-15mer phages that were selected by some particular screening round as described below were reproduced ("amplified") in the K91Kan strain of E.coli, a lamba-derivative of a strain known as K-38. Unless transformed by a phage or plasmid carrying a resistance gene, K91 cells are susceptible to the antibiotics kanamycin (used at 50 micrograms/milliliter, ug/ml) or tetracycline (20 ug/ml).
  • an inducible promoter was placed in front of the gene that encodes coat protein VIII; this allows a compound known as IPTG (1 mM) to be used, when desired, to increase expression levels of recombinant coat proteins containing the various 15-mer foreign inserts that have been inserted into the coat protein VIII coding sequence.
  • the harvested phages (which can be released from the neurons by mechanical or ultrasonic homogenization and/or dissolution of the nerve cell membranes using a detergent) were incubated for 1 hour with a TG-I strain of E. coli, in their log growth phase, in 2TY cell culture medium.
  • the phage- infected E. coli cells were grown overnight in shaker flasks, in 400 ml of 2TY medium containing 100 ug/ml ampicillin.
  • Infected E. coli (carrying phages with ampicillin resistance genes) were pelleted by centrifugation at 3000 rpm for 20 min, then taken up in
  • PEG polyethylene glycol
  • PBS Dulbecco's phosphate buffered saline
  • Phage were stored as PEG precipitates at 4 0 C until subsequent use. Phages from f88-15mer were produced using similar methods, using LB medium with 12.5 ug/ml tetracycline in place of ampicillin, and without using helper phage. Phage-infected host cells in PBS or 2TY medium were stored at -80 0 C after mixing 1:1 with cell freezing medium, which contained 88 g/L glycerol, 12.6 g/L K 2 HPO 4 , 3.6 g/L KH 2 PO 4 , 1.8 g/L (NH 4 ) 2 SO 4 , 0.9 g/L Na 3 citrate, and 0.18 g/L MgSO 4 -7H 2 O).
  • M13KO7 helper phages were added, and allowed to infect the cells for 1 hour.
  • infected host cells were grown in a 2L shaker flask containing ampicillin and kanamycin supplementing 800 ml of medium with a recipe based on Liu et al 2000; in addition to 40 g/L D-sorbitol as a carbon source, this medium contained 5 g/L yeast extract, 5 g/L tryptone, 7g/L NaH 2 PO 4 - 12H 2 O, 4 g/L KH 2 PO 4 , 4 g/L K 2 HPO 4 , 1.2 g/L (NH 4 ) 2 SO 4 , 0.2 g/L NH 4 Cl, 2.4 g/L MgSO 4 -7H 2 O, and 0.02 g/L CaCl 2 .
  • Affinity column purification of phages was carried out using particulate hydroxyapatite (HA), a ceramic material containing calcium and phosphate, purchased from BioRad.
  • HA particulate hydroxyapatite
  • Protein content of the eluted fractions were optically monitored at a light wavelength of 280 nanometers (nm), in part because maleic acid buffer absorbs light strongly, at wavelengths below 280 nm.
  • the elution profiles were generally similar to those reported in Smith and Gingrich 2005, and highly purified phages eluted in fractions 60 through 70, as illustrated in FIG. 10.
  • EDC ethylene dichloride
  • 10 mg EDC was added to 1 ml 40 mM NaH 2 PO 4 (adjusted to pH 7.0 by NaOH) containing 16 mg purified CANTAB6 phages.
  • the mixture was allowed to react for 4 hours at room temperature, then another 10 mg of EDC was added, and the mixture was incubated (with mixing) for another 4 hours.
  • the EDC-crosslinked phage were precipitated overnight using polyethylene glycol, then dissolved in 5 ml of coupling buffer (0.1 M NaHCO 3 , pH 8.3, plus 0.5 M NaCl).
  • 1 gm of CNBr Sepharose 4B was washed with 1 mM HCl, then added to the coupling buffer containing
  • EDC-crosslinked phages The mixtures was incubated for 4 hours, then quenched with 0.1 M Tris (pH 7.6) and incubated overnight. Unbound phage and coat protein components were removed, using 4M MgCl 2 + 50 mM acetate, pH 5.0.
  • ELISA enzyme-linked immuno-sorbent assay
  • ELISA assays to determine the concentration of phages in a liquid being analyzed, involved: (i) a first incubation step, using a body fluid that was being measured to determine its phage concentration, followed by, (ii) a second incubation step, using wild-type phages crosslinked to a peroxidase enzyme. Unbound enzyme-linked phages were rinsed away and removed after the second incubation, and a liquid with a color- forming reagent was added to the plate. The color-forming reagent was converted into a colored compound by the peroxidase enzyme, and its intensity was measured by a spectrophotometer.
  • a weak color change indicated that most of the anti-phage antibodies had become bound and occupied, by a high concentration of phage particles (with no peroxidase enzyme) in the body fluid, before the second incubation was carried out using phages carrying peroxidase enzymes.
  • a strong color change indicated that fewer anti-phage antibodies had been occupied by phage particles from the body fluid, thereby indicating a lower concentration of such phage particles.
  • Tubes were weighed to determine the volume of the sample, and aliquots of the blood-bore phages were added to TG-I E. coli cells in log growth phase (optical density approximately 0.2, at 600 nm). After 1 hour of incubation, the preparation (or a dilution thereof) was spread evenly across the surface of agar plates containing 2% glucose and 100 ug/ml ampicillin (200 ml of agar, in 234 mm x 234 mm Nunc tissue culture plates). The plates were sealed with parafilm and incubated overnight at 30 C. By 18 hours, typical colonies of E. coli cells infected by phages carrying the ampicillin resistance gene had grown to 0.5 to 2 mm diameters, without evidence of secondary colonies.
  • Plates were then refrigerated, if necessary, at 4 0 C, and the number of colonies per plate were counted within 24 hours.
  • the number of phage in each blood sample was determined from at least two titering tests, to calculate the mean number of phage recoverable per animal, when blood samples were tested.
  • mice Sixty minutes after intranasal administration of phages, 10 mice were anesthetized, euthanized, and perfused with saline, and their olfactory bulbs (OB) were removed. Similarly, 45 minutes after phage administration, NALT tissue flanking the windpipe (Asanuma et al 1997) was removed from 9 mice. The OB or NALT tissue was dissected into 100 ul of cell lysing buffer and triturated, pooled, and given two sonication pulses, 1 second each. An equal volume of 2X stock of LB culture medium was added, then a sufficient quantity of LB culture medium carrying K91 E.
  • OB olfactory bulbs
  • coli cells in logarithmic growth phase was added to make 10 mL. After allowing 60 minutes for phage to infect the cells, cell broth was plated onto 22 x 22 cm LB agar with tetracycline, and incubated overnight. Glycerol scrapes of clonal colonies were prepared for production of phage for additional tests, and for storage at -8O 0 C.
  • PBMCs peripheral blood mononuclear cells
  • Tubes were centrifuged at 800 rpm for 25 min.
  • the "buffy coat" layer (approx 3.5 ml) was transferred to a centrifuge tube, diluted to 15 ml with Dulbecco's PBS, and centrifuged at 1,600 rpm for 15 min, to pelletized blood cells. Supernatant was removed by aspiration, and the pellet was resuspended in 15 ml of Dulbecco's PBS and recentrifuged.
  • the washed pellet was resuspended in RPMI 1640, supplemented with 2 mmol/L glutamine, 100 ug/mL penicillin, 100 ug/mL streptomycin, and 10% fetal calf serum (FCS), from GIBCO-BRL (Gaithersburg, MD) at 37 0 C.
  • FCS fetal calf serum
  • the mixture was diluted to 2x10(6) cells/ml. Viability of the cells was >95% as determined by trypan blue exclusion.
  • FITC fluorescein isothiocyanate
  • the other batch contained a subset of the 15-mer phage library that had been screened once for olfactory bulb (OB) targeting, and then screened twice for NALT targeting, by removing and isolating them from OB tissue and then NALT tissue in mice, after nasal administration, as described above; those active test phages were also fluorescently labeled, using FITC.
  • the different test and control mixtures were prepared in microfuge tubes, mixed, and incubated for 30 min at room temp. Each cell/phage mixture was then diluted with 10 ml Dulbecco's PBS, centrifuged at 5 minutes at 2000 rpm, and resuspended in 2 ml Dulbecco's PBS. 5 uL aliquots were then removed and placed on 4% gelatin-coated slides, which were cover-slipped, and examined under a fluorescent microscope. Visual inspection indicated that labeling of about 5 to 20% of the PBMC cells clearly exceeded background levels.
  • FITC-phage-labeled PBMC pellets were resuspended in 2 ml Dulbecco's PBS, and processed through a BD-FACSAria Flow Cytometer (Becton-Dickinson AG, Basel, Switzerland) equipped with 405 nm, 488 ran, and 633 ran lasers.
  • That population of strongly-labeled PBMC cells (approximately 5x 10(4) cells) was suspended in 1.5 ml of culture medium. 1.5 ml of lysis buffer was added and incubated, to digest the cells without damaging the phages. The resulting selected phages were then used to infect K91 E. coli cells in logarithmic growth phase. After allowing 1 hour for infection, cells were pelleted (10 min at 3,500 rpm), resuspended in LB medium, and plated on agar containing tetracycline, to select for cells infected by phages carrying the tetracycline resistance gene. After overnight incubation, numerous colonies were observed. Those colonies of phage-infected E. coli were expanded, and used to produce phage populations which were then screened for phagosome selection, as described below.
  • white blood cells As mentioned in the Background section, an important class of white blood cells passes through series of stages. They are called “monocytes” when circulating in the blood, in relatively compact form. They have special surface molecules that cause them to grip and permeate through capillary walls, causing them to leave the blood and enter the lymph, which slowly moves through soft tissues. In the lymph, they swell to a larger size, and are called macrophages, phagocytes, or phagocytic cells. If they encounter a foreign microbe, they will extend out projections, often called fingers, "pseudopods", dendrites, etc., which will surround the microbe. The cell will partially digest the microbe, using internal organelles called phagosomes.
  • a cell which goes through those stages can be called a monocyte, a macrophage or phagocyte, a dendrite or dendritic cell, and an antigen-presenting cell (APC).
  • APC antigen-presenting cell
  • That attachment process is important, since it arises from the same surface molecules that enable certain monocytes to grip the interior walls of capillaries, and then pass through the capillaries, which is a crucial step in the conversion of some monocytes into macrophages.
  • the culture medium was removed, and the cell layer that adhered to the flask surface was washed 5 times, using 2 ml volumes of Dulbecco's PBS at 37 0 C, to remove and discard any monocytes or other white blood cells that had not adhered to a plate surface.
  • DME/F12/10% FCS which was added to the centrifuge tubes.
  • the cells were centrifuged at 1,600 rpm for 10 min at 4°C. Supernatant was discarded, and the pellet was resuspended in 10 ml DME/F12/10% FCS and centrifuged again. Supernatant was discarded, and cells taken up in 2 ml of 0.25M sucrose buffer with 10 niM HEPES, pH 7.2.
  • the cells were transferred to a 1 ml conical glass-glass homogenizer (Wheaton-USA), and broken apart by 10 strokes of the homogenizer.
  • the homogenate was then transferred to a centrifuge tube; the homogenizer also was rinsed twice, using 2 ml of sucrose solution each time, and the washings were added to the centrifuge tube, making a volume of 6 ml.
  • the homogenate and washings were centrifuged at 900 rpm for 10 min at
  • coli cells were pelleted by centrifugation at 3,500 rpm for 10 min, and the supernatant was discarded. The cells were suspended in 250 ul of LB medium, plated onto agar with tetracycline, and incubated overnight.
  • This process selected phages that, in addition to triggering and driving NALT uptake, also could efficiently trigger and drive uptake and processing by macrophages and antigen-presenting cells.
  • PBMC peripheral blood mononuclear cells
  • Human autologous serum was prepared by collecting 20 ml of donor blood in two serum clot separator tubes, then centrifuging at 4,500 rpm for 10 min at room temperature (autologous serum was used to minimize any risk of activation of PBMC cells by foreign proteins, such as in fetal calf serum).
  • autologous serum was used to minimize any risk of activation of PBMC cells by foreign proteins, such as in fetal calf serum.
  • In a 50 ml Falcon tube was added 5 ml of autologous serum and 45 ml of
  • RPMI/NaHCO 3 /glutamine Washed PBMC from 20 ml blood were taken up in 3 ml of the RPMI mixture with 10% serum, and 1 ml of each was placed in T25 flasks. Wild-type fdTet phages (used as controls), or phages selected by the phagosome isolation process described above, were added to PBMC cells that had adhered to the flask surfaces, at viral loads estimated at 20 ug/ml (7.3x10(10) virions per ml), and incubated for 30 min. The phage-containing medium was then removed, and adhering cells were washed twice with 2 ml of RPMI/10% serum, then incubated overnight in fresh serum.
  • the cells were gently scraped off and placed in a 15 ml centrifuge tube.
  • the flask surface was rinsed with RPMI/10% serum, which also was transferred to the centrifuge tube, which was centrifuged at 1,600 rpm for 5 min at room temperature. The supernatant was discarded, and the cells were resuspended in 250 ul of fresh medium.
  • 10 ul of FITC-labeled antibodies that bind to the MHC-I protein were added, and incubated for 30 minutes.
  • the cells were then diluted to 5 ml with RPMI/serum, and centrifuged.
  • the supernatant was discarded, and cells were taken up in 500 ul of RPMI/ 10% serum on ice. They were then subjected to fluorescence-activated cell sorting (FACS), using a 530 nm laser beam.
  • FACS fluorescence-activated cell sorting
  • an APC cell can present exogenous virus proteins, in ways that involve MHC-I proteins, by means of a recently-described mechanism called “cross-presentation” (Houde et al 2003). hi such macrophages, phagocytosis proceeds by means of endoplasmic reticulum (ER) recruitment at the cell surface.
  • ER endoplasmic reticulum
  • ER-mediated phagocytosis This triggers a process called ER-mediated phagocytosis (Gagnon et al 2002), in which a transient fusing of an endoplasmic reticulum with a phagosome brings an antigen into contact with MHC-I molecules and TLR9 toll-like receptors.
  • Parallel processes involving other cells also leads to expression of co-stimulatory molecules (such as various cytokines), and to robust stimulation of CD8+ and CD4+ receptors on T-cells, as described in Desjardins et al 2005.
  • the cross-presentation process also accounts for the observation that the immunostimulatory effects of CpG-ODN motifs, which is manifested by segments of free DNA, can be greatly amplified (such as 50 to 100 times higher) when a DNA segment having the CpG-ODN motif is conjugated to a proteinous antigen, rather than simply mixed with the antigen in a vaccine formulation, as reviewed in Wagner et al 2004.
  • the FITC labeling reagent was coupled to phages that had been selected by a screening round which isolated them from olfactory bulb tissue. This was done by buffer- exchanging PEG-precipitated phages into PBS (pH 7.4), using a 10 ml Sephadex G-25 column. 1.8 mg of FITC dissolved in 180 ul of DMSO was added to the phage solution, the reaction proceeded for 2 hours at room temperature, or overnight at 4 0 C, with mixing. Phage were PEG-precipitated at least twice, to remove unreacted FITC, and the FITC-labeled phages were nasally administered to mice, as described above.
  • FIGS. 5 and 6 Typical results are presented in FIGS. 5 and 6, which also have been posted (in downloadable versions that provide better resolution) at www.tetraheed.net/ferguson.
  • Other organs such as heart, lung, liver, etc.
  • Other organs also were analyzed using similar methods, and were found to have lower yet significant levels of fluorescently-labeled phages present.
  • HRP horseradish peroxidase
  • EXAMPLE 12 COUPLING OF CATIONS TO PHAGE SURFACES
  • Cationised phage was separated from unreacted reagents by passage through a Sephadex G25 gel (Pharmacia PDlO) into Dulbecco's PBS. Compared to uncationised phage controls, elution of cationised phage from the column was significantly retarded. Emergent fractions that contained purified cationised phage (indicated by spectroscopic monitoring at 280 nm) were collected, pooled, and used. While unmodified phage could be precipitated by adding 2% PEG, precipitation of 1.5 ml of cationised phage required 600 ul of PEG stock.
  • variable sequences in the phages contained in the display library, is in a specific 45 base sequence containing 15 codons, with each variable codon specifying a single amino acid residue that will appear in the 15-mer variable portion of the coat protein VIII polypeptide.
  • Gagnon E et al (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages, Cell 110: 119-131.
  • Gaubin M et al (2003) Processing of filamentous bacteriophage virions in antigen- presenting cells targets both HLA class I and class II peptide loading compartments.
  • Vaccine 17: 1796-803 Liu YC, et al (2000) Cultivation of recombinant Escherichia coli to achieve high cell density with a high level of penicillin G acylase activity. Proc Natl Sci Counc Repub China B 24: 156-60.
  • Villard S et al (2003) Peptide decoys selected by phage display block in vitro and in vivo activity of a human anti-FVIII inhibitor. Blood 102: 949-952.
  • Wagner H, et al (2004) Targeting split vaccines to the endosome improves vaccination.

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Abstract

La présente invention concerne l'identification de séquences polypeptidiques - suite à la réalisation de tests de détection séquentiels multiples sur des banques de présentation du phage - qui constituent de puissants déclencheurs des étapes suivantes : (i) admission dans les cellules immunitaires des muqueuses, y compris les cellules NALT du nez et de la gorge; et (ii) absorption phagocytaire et traitement par des cellules présentant un antigène, telles que des macrophages. De telles séquences polypeptidiques peuvent être utilisées en tant que puissants composants de « ciblage et d'administration » dans des vaccins susceptibles d'être administrés par voie nasale ou sur d'autres muqueuses. Ces vaccins peuvent être fabriqués très rapidement et en grandes quantités, à partir de bactériophages qui porteront également des séquences antigéniques dans leurs protéines de coque ou d'autres composants immunoactifs. En variante, de tels polypeptides de « ciblage et d'administration » peuvent être incorporés dans des vaccins dérivés de virus eucaryotes ou de pathogènes cellulaires. La présente invention concerne également des améliorations, telles que des agents pouvant activer un ou plusieurs types de récepteurs « Toll-like », destinées à renforcer les réponses immunitaires et à les orienter dans les directions souhaitées.
PCT/AU2008/000811 2007-06-08 2008-06-06 Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire WO2008148164A1 (fr)

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CA2727126A CA2727126A1 (fr) 2007-06-08 2008-06-06 Vaccins administres par voie nasale utilisant le ciblage nalt a detection multiple et des sequences de transport de polypeptide phagocytaire
EP08756895A EP2167110A4 (fr) 2007-06-08 2008-06-06 Vaccins administrés par voie nasale utilisant le ciblage nalt à détection multiple et des séquences de transport de polypeptide phagocytaire

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WO2021195039A1 (fr) * 2020-03-27 2021-09-30 Chen Shu Chih Méthodes de traitement ou de prévention d'une infection virale utilisant des bactériophages

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CN113604481B (zh) * 2021-07-07 2023-05-02 江苏农牧科技职业学院 一种t7噬菌体识别的锚定序列、dna疫苗重组质粒及应用

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See also references of EP2167110A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014085297A1 (fr) * 2012-11-27 2014-06-05 Colorado State University Research Foundation Bactériophage multifonctionnel pour l'administration d'agents thérapeutiques et de réactifs d'imagerie
WO2021195039A1 (fr) * 2020-03-27 2021-09-30 Chen Shu Chih Méthodes de traitement ou de prévention d'une infection virale utilisant des bactériophages

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