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US20110059116A1 - Laser-based vaccine adjuvants - Google Patents

Laser-based vaccine adjuvants Download PDF

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Publication number
US20110059116A1
US20110059116A1 US12/754,081 US75408110A US2011059116A1 US 20110059116 A1 US20110059116 A1 US 20110059116A1 US 75408110 A US75408110 A US 75408110A US 2011059116 A1 US2011059116 A1 US 2011059116A1
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vaccine
laser
cells
subject
exposure
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Sergey B. ONIKIENKO
Alexander V. ZEMLYANOI
Boris A. MARGULIS
Irina V. GUZHOVA
Anna A. PIMENOVA
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NPT MBP Gormezis OOO
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NPT MBP Gormezis OOO
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Publication of US20110059116A1 publication Critical patent/US20110059116A1/en
Priority to US13/428,275 priority Critical patent/US20130078265A1/en
Priority to US14/255,931 priority patent/US10052376B2/en
Abandoned legal-status Critical Current

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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
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
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    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
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    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
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    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0659—Radiation therapy using light characterised by the wavelength of light used infrared
    • A—HUMAN NECESSITIES
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    • A61N2005/0658—Radiation therapy using light characterised by the wavelength of light used
    • A61N2005/0662—Visible light
    • A—HUMAN NECESSITIES
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    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/067—Radiation therapy using light using laser light
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Aluminum salts (aluminum hydroxide, aluminum phosphate or alum) have been used in vaccine preparations for over 80 years (Glenny et al. 1926). They have the ability to create more antigenic precipitates with some vaccines, enhance the uptake by antigen-presenting cells by increasing the local concentration of antigen at the injection site, and stimulate immunogenicity by direct or indirect stimulation of dendritic cells, activation of complement, and by inducing the release of chemokines (Hogen Esch 2002). While these mechanisms can be relevant in stimulating type II (antibody-mediated) immune responses, they do not induce cytotoxic T-cell or cell-mediated immune responses. Aluminum salts also cause side effects in a certain percentage of the population that receives such vaccinations.
  • MPL Monophosphoryl lipid A
  • FENDRIX® GaxoSmithKline
  • Corixa a vaccine against hepatitis B.
  • the EMEA also recently approved the oil-in-water emulsion adjuvant MF59 for use with FOCETRIA®, a Novartis' pandemic influenza vaccine. Neither of these has received market approval by the FDA.
  • the present invention is directed to a vaccine for generating an enhanced immune response in a subject previously exposed to non-destructive laser radiation, as compared to an immune response in a subject previously non-exposed to the non-destructive laser radiation.
  • Such vaccine turns out to have efficacy in the subjects who are immunocompromised or non-responsive to the vaccine without the previous exposure to the laser radiation.
  • the vaccine of the present invention can comprise an amount of HSP 70 for generating an enhanced immune response.
  • the vaccine of the present invention generates the enhanced response from the subject with increased HSP 70 concentration due to the previous exposure to laser radiation.
  • the vaccine generates the enhanced response the subject with an increased Langerhans cell concentration due to the previous exposure to laser radiation.
  • the referenced enhanced immune response comprises an increase in an antibody titer specific to the vaccine.
  • Another kind of an enhanced immune response generated by the vaccine is an increased resistance to a condition, which for, example, can be a decrease in an infection rate upon exposure to a pathogen specific to the vaccine, or it can be a decrease in mortality in response to an exposure to the pathogen specific to the vaccine.
  • the present invention is also directed to the use of a composition
  • a composition comprising a vaccine for use in combination with laser radiation for generating an enhanced immune response from a subject, as compared to an immune response without the use of laser radiation.
  • the composition can further comprise an amount of HSP 70.
  • the referenced enhanced immune response can be generated by the vaccine in a subject who is immunocompromised or who is non-responsive to the vaccine without the previous exposure to laser radiation.
  • the invention contemplates methods of administering a vaccine to a subject including exposing an area of an epidermal layer of skin of the subject to a laser wherein laser exposure does not cause significant or irreversible damage cells in the exposed area, and contacting the subject with the antigen at the site of laser exposure, thereby administering the vaccine.
  • Irreversible damage to the cells includes cell death, either upon exposure to the laser, or as a direct result of laser exposure.
  • Significant damage is damage to at least 1%, 0.5%, 0.25%, 0.1%, 0.01%, or 0.001% of cells exposed to the laser.
  • the invention further provides methods to increase HSP 70 concentration and/or Langerhans cell concentration at or near the site of laser exposure as compared to a site not exposed to laser by exposing an epidermal layer of the skin to laser wherein laser exposure does not cause significant or irreversible damage cells in the exposed area.
  • the frequency of the laser is about 1 to about 20 kilohertz
  • the power of the laser is about 0.5 to about 10 watts, about 1 watt to 8 watts, about 1 watt to about 5 watts, about 1 watt to about 3 watts, or about 1.5 watts
  • the pulse duration is about 1 to about 1000 nanoseconds, about 1 to 500 nanoseconds, about 1 to 100 nanoseconds, about 1 to 50 nanoseconds, about 5 to 20 nanoseconds, or about 10 nanoseconds.
  • the laser exposure takes place as close as possible to the time of the vaccine, either before or after administration of the vaccine, to within about an hour, within about 45 minutes, within about 30 minutes, within about 15 minutes, to within about 10 minutes of vaccine administration.
  • the methods of the invention include administering a vaccine to a laser exposed area of an epidermal layer of skin of the subject to provide an increase in a detectable response to the vaccine as compared to administering a vaccine to an epidermal layer of skin area of a subject not exposed to laser.
  • an increase in a detectable response can include an increase in antibody titer specific to the vaccine, an increase in resistance to a condition as demonstrated, for example by a decrease in infection rate upon exposure to a pathogen specific to the vaccine, a decrease in mortality in response to exposure to a pathogen specific to the vaccine, and/or a decrease in time to detect a response to the vaccine.
  • the methods of the invention can further include detecting a response to the vaccine.
  • the invention can also include identifying a subject in need of vaccination.
  • the subject is immunocompromised.
  • the invention further includes co-administration of the vaccine with heat shock protein 70.
  • the invention provides methods that can be practiced using any laser that provides a wavelength that can stimulate an immune response to an antigen (see, e.g., Table 2).
  • Lasers for use in the methods of the invention include, but are not limited to a copper bromide laser and a neodymium-doped yttrium aluminium garnet; (Nd:YAG) laser.
  • Laser wavelengths for use in the methods of the invention include, but are not limited to 510 nm and 578 nm.
  • Laser beam sizes for use in the method of the invention include, but are not limited to 1-10 mm, 2-8 mm, 2-7 mm, or 3-5 mm.
  • the invention provides methods for contacting a subject with a vaccine at a site of laser exposure by injection.
  • the invention provides methods for injection including, but not limited to intradermal, subcutaneous, and intramuscular injection.
  • FIG. 1 shows a schematic of the optimal conditions for the laser-based immune enhancement methods of the invention of the skin at a specific wavelength of irradiation.
  • FIG. 2 shows a graph of the rates of accumulation of damage in tissue based on the rate coefficients of Henriques. Times associated with dotted lines indicate the number of seconds to achieve threshold damage for a step rise in tissue temperature.
  • FIG. 3 is a schematic showing the relationship between power density and pulse duration in the generation of phase explosion of water in tissues using a laser.
  • FIG. 4 is a graph showing the logarithmic relationship between temperature and exposure time for induction of a heat shock response in tissue.
  • FIG. 5 shows tissue sections stained to detect the presence of Langerhans cells in the skin before and after laser treatment.
  • FIG. 6 shows changes in the amount of heat shock protein 70 in murine ear sheets after a one or two minute exposure to laser irradiation.
  • FIG. 7 shows immunoblotting results for HSP70 in epidermal cells derived from the ears of mice showing changes in total HSP in response to different combinations of laser light wavelengths and durations of exposure.
  • treating refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that the extent of the disease is decreased or prevented. For example, treating results in the reduction of at least on sign or symptom of the disease or condition.
  • Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, in combination with exposure to a laser as described herein, and may be performed either prophylactically or subsequent to the initiation of a pathologic event. Treatment can require administration of an agent and/or treatment more than once.
  • preventing as it refers to a condition means that a subject in which the occurrence of a condition is prevented, will show no detectable symptoms of a condition of interest. No detectable symptoms means less than 10%, 5%, 1.0%, 0.5%, 0.1% or 0.01% of the level of detectable symptoms of a subject that has been diagnosed with a condition of interest.
  • a “condition” includes any abnormality that can occur in a subject including any disease, infection, disorder, tumor, cancer, inflammatory condition, or cellular structure associated with disordered function.
  • To “prevent the occurrence of a condition” also means to stop or delay the occurrence of a condition of interest. Prevention can require administration of an agent and/or treatment more than once.
  • Irreversible damage is understood as causing harm to the cells which directly leads to death of the cells exposed to the laser in the methods of the invention.
  • Irreversible damage to a cell means that the function of the cell is negatively altered or compromised and cannot return to normal. Irreversible damage can occur when the cell is destroyed, physically removed from its environment, or when the function of the cell is compromised, for example by irreversible disruption of the membrane or destruction or damage to one or more of the organelles or processes within the cell.
  • Significant damage refers to the number of cells that are killed, i.e., irreversibly damaged, either as a ratio (percent) of the cells exposed to the laser, as a defined number of cells per laser exposure, or a defined area of cells. Significant damage is damage to at least 1%. 0.5%, 0.25%. 0.1%, 0.001%, or 0.0001% of the cells expose to the laser.
  • adjuvant is understood as an aid or contributor to increase the efficacy or potency of a vaccine or in the prevention, amelioration, or cure of disease by increasing the efficacy or potency of a therapeutic agent as compared to a vaccine or agent administered without the adjuvant.
  • An increase in the efficacy or potency can include a decrease in the amount of vaccine or agent to be administered, a decrease in the frequency and/or number of doses to be administered, or a more rapid or robust response to the agent or vaccine (i.e., higher antibody titer).
  • An adjuvant can be an agent or laser.
  • subject refers to a mammal.
  • a human subject can be known as a patient.
  • mammal refers to any mammal including but not limited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.
  • non-human mammal refers to any mammal that is not a human.
  • exposing means treating with a laser for an exposure time useful to the invention.
  • exposing means to expose a subject to a laser applied in a pulse, wherein the pulse is applied for a particular duration.
  • the range of pulse durations are in the hundreds of picoseconds to tens of nanoseconds (for example, about 100, 200, 300, 400, 500, 600, 700, 800, 900 picoseconds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nanoseconds). It is understood that the actual pulse length will vary somewhat based on the limitations of the laser and the switching rate/shutter speed.
  • “exposing” means to expose a subject to a laser of a particular pulse frequency.
  • Optimal frequencies range from about 1 kHz to about 20 kHz (for example, 1, 5, 10, 15, 20 kHz), with typical pulse frequencies in the 5, 6, 7, 8, 9, or 10 kHz frequency. It is understood that the actual frequency will vary somewhat based on the limitations of the laser and the switching rate/shutter speed.
  • “exposing” means to expose a subject to a laser of a particular wavelength where the range of wavelengths can range from the visible light to the near infrared portion of the electromagnetic spectrum (approximately 400 nm to 1400 nm, for example, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, and 1400 nm), and are typically about 500-1100 nm.
  • “exposing” means to expose a subject to a laser with a particular peak power, where the range of power is 0.1 to 10 Kwatts (for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 Kwatts) and are typically about 1-5 Kwatts.
  • “expose” means to expose a subject to a laser for a particular length of time. The range of exposures can be about 5 seconds to about 600 seconds (for example, about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 seconds).
  • “expose” means to expose a particular area of the subject. Typical exposure areas are about 1-100 mm 2 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mm 2 ).
  • the exposure to a laser occurs prior to administration of said agent.
  • the exposure to said laser occurs about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 seconds, about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours before administration of said agent.
  • the exposure to said laser occurs about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 seconds, about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours after administration of said agent.
  • a “laser” refers to an electronic-optical device that emits coherent light radiation.
  • a typical laser emits light in a narrow, low-divergence monochromatic (single-colored, if the laser is operating in the visible spectrum), beam with a well-defined wavelength.
  • laser light is in sharp contrast with such light sources as the incandescent light bulb, which emits light over a wide area and over a wide spectrum of wavelengths.
  • a “laser” includes any laser that is currently available or may become available that can provide the appropriate pulse duration, power, and pulse frequency required by the methods of the instant invention.
  • Currently available lasers that can be used in the methods of the invention include, but are not limited to, a copper bromide laser such as the Norseld DualYellow copper bromide laser (511 and 578 nm) or the Asclepion ProYellow+copper (511 and 578 nm), or a neodymium-doped yttrium aluminium garnet (Nd:YAG) lasers such as a Q-switched Yag laser such as the RMI 15 Q-Switched Diode-Pumped Solid State Laser with an output at either 532 nm or 1064 nm.
  • a copper bromide laser such as the Norseld DualYellow copper bromide laser (511 and 578 nm) or the Asclepion ProYellow+copper (511 and 578 nm)
  • administering refers to any method according to the invention including but not limited to injection, subcutaneous, transcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, administration directly to a blood vessel, including artery, vein or capillary, intravenous drip, ingestion via the oral route, inhalation, transepithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced agent or may, instead, comprise cells that produce and secrete the therapeutic agent or topical application. Additional methods of administration are provided herein below in the section entitled “Dosage and Administration.” As used herein, “administering” can also refer to exposure of a subject to a laser, preferably for a therapeutic use.
  • contacting means exposing a subject to, for example by any of the methods of administration described herein. “Contacting” refers to exposing a subject to, for example, an agent for a duration of about 1, 5, 10, 20, 30, 40, 50 minutes, about 1, 2, 5, 10, 20, 24 hours, about 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. In one embodiment, “contacting” refers to exposing a subject more than once, for example about 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.
  • an “agent” refers to any protein, recombinant protein, small molecule, DNA, RNA, antigen, parasite, virus, bacteria, or other prokaryotic or eukaryotic cells, either whole cells or fragments thereof, or combination thereof.
  • An “agent” also includes a vaccine.
  • a used herein, a “vaccine” is a preparation which is used to increase immunity of a subject to a particular condition or antigen.
  • an “antigen” is understood as any compound that can be used to stimulate a specific immune response.
  • An antigen can be an isolated or purified protein, nucleic acid, carbohydrate, small molecule, and the like.
  • an antigen can be a complex mixture, naturally or artificially generated including a mixture of one or more of protein, nucleic acid, carbohydrate, small molecule optionally in the form of a pathogen, particularly a killed or attenuated pathogen.
  • Antigens include self- and non-self antigens.
  • an antigen can be a protein that is not normally present in a subject, e.g., a cancer cell.
  • An antigen can also be a contraceptive protein (e.g., riboflavin carrier protein).
  • nucleic acid therapeutic or “nucleic acid antagonist” can be any nucleic acid (DNA, RNA, or a combination thereof) or an analog thereof (e.g., PNA) optionally including one or more modifications (see, e.g., U.S. Pat. Nos. 7,015,315 and 6,670,461, incorporated herein by reference) to modulate pharmacokinetic or pharmacodynamic properties of the nucleic acid.
  • Nucleic acid antagonists can be antisense oligonucleotides (see, e.g., U.S. Pat. No. 5,366,878; or 6,921,812, both incorporated herein by reference), small interfering (si)RNA (see, e.g., U.S.
  • a “small molecule” is understood as is meant a compound having a molecular weight of no more than about 1500 daltons, 1000 daltons, 750 daltons, 500 daltons.
  • a small molecule is not a nucleic acid or polypeptide.
  • analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • “increase” as it refers to a response means elevating the level of response (for example, cell membrane permeability, production or secretion of Heat Shock Proteins (HSPs), increased concentration, maturation and/or activation of Langerhans cells at the site of laser exposure) following administration of an agent or antigen by at least about 2-fold (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%), in a subject that has been exposed to a laser, as compared to an untreated subject.
  • HSPs Heat Shock Proteins
  • a “heat shock protein” refers to a group of proteins whose expression is increased when cells are exposed to elevated temperatures or other stress.
  • the HSPs are named according to their molecular weights. For example, Hsp60, Hsp70 and Hsp90 (the most widely-studied HSPs) refer to families of heat shock proteins on the order of 60, 70 and 90 kilodaltons in size, respectively.
  • the small 8 kilodalton protein ubiquitin which marks proteins for degradation, also has features of a heat shock protein.
  • a “detectable response” includes a discernable, preferably a measurable level of a response that occurs in a subject that has been exposed to a laser, as described herein, but not in a subject that has not been exposed to a laser.
  • a “response” that is detected includes, but is not limited to, one or more of an increase in immunogenicity, an increase in an immune response, an increase in the concentration of Langerhans cells at the site of irradiation, an increase in the maturation or activation of Langerhans cells, an increase in the number of mature Langerhans cells, an increase in the production of Heat Shock Proteins (HSPs), an increase in the secretion of HSPs, an increase in cell membrane permeability, an increase in the level of one or more cytokine or chemokine, an increase in antibody titer to an agent of interest, an increase in lymphocyte cytotoxic activity, an increase in response to antigen challenge, or an increase in viability in response to pathogen challenge.
  • HSPs Heat Shock Proteins
  • a “detectable response” means a response that is at least about 0.01%, 0.5%, 1%, 10%, 20%, 30% or more than the response of a subject that has not been exposed to a laser.
  • a “detectable response” also means a response that is at least about 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000-fold or more greater than the response of a subject that has not been exposed to a laser.
  • detecting a response means performing an assay to determine if a response has occurred.
  • the response can be zero or below the limit of detection of the assay method.
  • immunogenicity refers to the ability, for example the ability of an agent, to induce humoral and/or cell-mediated immune responses in a subject.
  • immune response refers to a response made by the immune system of an organism to a substance, which includes but is not limited to foreign or self proteins. There are three general types of “immune response” including, but not limited to mucosal, humoral, and cellular “immune responses.”
  • a “mucosal immune response” results from the production of secretory IgA (sIgA) antibodies in secretions that bathe all mucosal surfaces of the respiratory tract, gastrointestinal tract and the genitourinary tract and in secretions from all secretory glands (McGhee, J. R. et al., 1983, Annals NY Acad. Sci. 409).
  • sIgA antibodies act to prevent colonization of pathogens on a mucosal surface (Williams, R. C. et al., Science 177, 697 (1972); McNabb, P. C. et al., Ann. Rev. Microbiol.
  • sIgA can be stimulated either by local immunization of the secretory gland or tissue or by presentation of an antigen to either the gut-associated lymphoid tissue (GALT or Peyer's patches) or the bronchial-associated lymphoid tissue (BALT; Cebra, J. J. et al., Cold Spring Harbor Symp. Quant. Biol. 41, 210 (1976); Bienenstock, J. M., Adv. Exp. Med. Biol. 107, 53 (1978); Weisz-Carrington, P. et al., J. Immunol.
  • GALT gut-associated lymphoid tissue
  • BALT bronchial-associated lymphoid tissue
  • M cells cover the surface of the GALT and BALT and may be associated with other secretory mucosal surfaces.
  • M cells act to sample antigens from the luminal space adjacent to the mucosal surface and transfer such antigens to antigen-presenting cells (dendritic cells and macrophages), which in turn present the antigen to a T lymphocyte (in the case of T-dependent antigens), which process the antigen for presentation to a committed B cell.
  • B cells are then stimulated to proliferate, migrate and ultimately be transformed into an antibody-secreting plasma cell producing IgA against the presented antigen.
  • an “immune response” can be measured using a technique known to those of skill in the art.
  • serum, blood or other secretions may be obtained from an organism for which an “immune response” is suspected to be present, and assayed for the presence of a of the above mentioned immunoglobulins using an enzyme-linked immuno-absorbent assay (ELISA; U.S. Pat. No. 5,951,988; Ausubel et al., Short Protocols in Molecular Biology 3rd Ed. John Wiley & Sons, Inc. 1995).
  • ELISA enzyme-linked immuno-absorbent assay
  • an antigen can be said to stimulate an “immune response” if the quantitative measure of immunoglobulins in an animal treated with an antigen detected by ELISA is statistically different from the measure of immunoglobulins detected in an animal not treated with the antigen, wherein the immunoglobulins are specific for the antigen.
  • a statistical test known in the art may be used to determine the difference in measured immunoglobulin levels including, but not limited to ANOVA, Student's T-test, and the like, wherein the P value is at least ⁇ 0.1, ⁇ 0.05, ⁇ 0.01, ⁇ 0.005, ⁇ 0.001, and even ⁇ 0.0001.
  • an “immune response” can be measured using other techniques such as immunohistochemistry using labeled antibodies which are specific for portions of the immunoglobulins raised during the “immune response”.
  • Tissue from an animal to which an antigen has been administered according to the invention may be obtained and processed for immunohistochemistry using techniques well known in the art (Ausubel et al., Short Protocols in Molecular Biology 3rd Ed. John Wiley & Sons, Inc. 1995).
  • Microscopic data obtained by immunohistochemistry can be quantitated by scanning the immunohistochemically stained tissue sample and quantitating the level of staining using a computer software program known to those of skill in the art including, but not limited to NIH Image (National Institutes of Health, Bethesda, Md.).
  • an antigen of the present invention can be said to stimulate an “immune response” if the quantitative measure of immunohistochemical staining in an animal treated with an antigen is statistically different from the measure of immunohistochemical staining detected in an animal not treated with the same antigen, wherein said histochemical staining requires binding specific for that protein.
  • a statistical test known in the art may be used to determine the difference in measured immunohistochemical staining levels including, but not limited to ANOVA, Student's T-test, and the like, wherein the P value is at least ⁇ 0.1, ⁇ 0.05, ⁇ 0.01, ⁇ 0.005, ⁇ 0.001, and even ⁇ 0.0001.
  • “Langerhans cells” refers to bone marrow-derived, antigen-processing and—presenting cells that are involved in a variety of T cell responses. These cells have dark nuclei and pale, or clear cytoplasm, no desmosomes attaching to neighboring cells, no tonofilament bundles and no melanosomes. However, they do contain smooth vesicles, multivesicular bodies, and lysosomes, but most characteristic are the Birbeck granules.
  • Langerhans cells are present in the basal, spinous and granular layers of the skin, but are also found in other squamous epithelia, including the oral cavity, esophagus, and vagina, as well as in lymphoid organs such as the spleen, thymus, and lymph node.
  • an “activated Langerhans cell” refers to Langerhans cells that have phagocytosed antigens and are increased their expression of major histocompatibility complex class I or II and costimulatory molecules and will migrate to T cell areas of draining lymph nodes. Activated Langerhans cells undergo a maturation process that allows them to become stimulators of T cell immunity. As used herein a “mature Langerhans cell” refers to cells that have gained potent immunogenic capacity, including the ability to secrete T-cell attracting chemokines and to interact with T-cells. They are characterized as having the properties of being CD24+, CD25+, CD69 ⁇ , CD80 (B7-1)+, CD83+, CD122+ and CCR7+.
  • identifying refers detecting indicators that a subject that is susceptible to a condition refers to the process of assessing a subject and determining that the subject is at risk of developing or could develop a condition.
  • a subject can be susceptible to a condition due to environmental conditions (e.g., population density, availability of clean water, hospitalization), genetic predisposition (e.g., heritable immune deficiency), the presence of other condition (e.g., burns or other injury in which the skin is disrupted, cystic fibrosis, AIDS), or the treatment of conditions with drugs that result in immunodeficiency (e.g., cancer chemotherapeutic agents).
  • environmental conditions e.g., population density, availability of clean water, hospitalization
  • genetic predisposition e.g., heritable immune deficiency
  • the presence of other condition e.g., burns or other injury in which the skin is disrupted, cystic fibrosis, AIDS
  • drugs that result in immunodeficiency e.
  • selecting refers to the process of determining that an identified subject will receive an agent to prevent or treat the occurrence of a condition. Selecting can be based on an individuals susceptibility to a particular disease or condition due to, for example, family history, lifestyle, age, ethnicity, or other factors.
  • “Measuring” means detecting or determining the amount, for example, any one of an increase or a decrease in immunogenicity, an increase in an immune response, an increase in the concentration of Langerhans cells at the site or irradiation, an increase in the maturation or activation of Langerhans cells, an increase in the number of mature Langerhans cells, an increase in the production of Heat Shock Proteins (HSPs), an increase in the secretion of HSPs, an increase in cell membrane permeability, an increase in the level of one or more cytokines or chemokines, an increase in antibody titer to an agent, or an increase in lymphocyte cytotoxic activity, according to the methods described herein. Measuring is the steps taken to determine if an increase or decrease in a level of the material to be detected. Measuring may indicate a level that is zero or below the level of detection, or greater than the linear detection limit of the method used for measuring.
  • Measureping is performed in vitro or in vivo, for example in the skin at the site of laser exposure, in serum or in blood or other biological sample, tissue or organ.
  • “Measuring” also means detecting a change that is either an increase or decrease in the response (for example an increase in immunogenicity, an increase in an immune response, an increase in the concentration of Langerhans cells at the site or irradiation, an increase in the maturation or activation of Langerhans cells, an increase in the number of mature Langerhans cells, an increase in the production of Heat Shock Proteins (HSPs), an increase in the secretion of HSPs, an increase in cell membrane permeability, an increase in the level of one or more cytokine or chemokine, an increase in antibody titer to an agent, or an increase in lymphocyte cytotoxic activity, a decrease in the development of disease, or a decrease in the death rate in response to a pathogen challenge) of a subject to administration of an agent, according to the methods described herein.
  • HSPs Heat Shock Proteins
  • “Measuring” is performed in a subject wherein an agent has been administered and wherein said subject has been exposed to a laser. “Measuring” is also performed in a control subject, for example a subject that has not received an agent and that has not been exposed to a laser, or a subject that has received an agent but has not been exposed to a laser.
  • a “decrease” as it refers to a diminution in the level of a response as defined herein means a response that is at least about 2-fold (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%), less than the level of response of an untreated subject, for example a subject that has not received an agent and has not been exposed to a laser or a subject that has received an agent but has not been exposed to a laser subject.
  • 2-fold for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%
  • phrases “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds used in the methods described herein to subjects, e.g., mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • suitable carriers to be administered prior to exposure to the laser preferably do not include chromophores or other compounds that alter the absorption of heat by the tissue, or result in chemical reactions that can cause significant and/or irreversible damage to the cells or tissue.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as e
  • therapeutically effective amount or a “therapeutically effective dose” of a compound is the amount necessary to or sufficient to provide a detectable improvement in of at least one symptom associated or caused by the state, disorder or disease being treated.
  • the therapeutically effective amount can be administered as a single dose or in multiple doses over time. Two or more compounds can be used together to provide a “therapeutically effective amount” to provide a detectable improvement wherein the same amount of either compound alone would be insufficient to provide a therapeutically effective amount.
  • the terms “effective” and “effectiveness” includes both pharmacological effectiveness and physiological safety.
  • Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient.
  • Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment.
  • side-effects the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment.
  • the term “ineffective” indicates that a treatment does not provide sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population.
  • “Less effective” means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.
  • a drug which is “effective against” a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.
  • the term “tolerance” refers to the ability of a patient to accept a treatment, based, e.g., on deleterious effects and/or effects on lifestyle. Frequently, the term principally concerns the patients perceived magnitude of deleterious effects such as nausea, weakness, dizziness, and diarrhea, among others. Such experienced effects can, for example, be due to general or cell-specific toxicity, activity on non-target cells, cross-reactivity on non-target cellular constituents (non-mechanism based), and/or side effects of activity on the target cellular substituents (mechanism based), or the cause of toxicity may not be understood. In any of these circumstances one may identify an association between the undesirable effects and variances in specific genes from the subject to contact with an agent ex vivo.
  • the method of selecting a treatment involves selecting a method of administration of a compound, combination of compounds, or pharmaceutical composition, for example, selecting a suitable dosage level and/or frequency of administration, and/or mode of administration of a compound.
  • the method of administration can be selected to provide better, preferably maximum therapeutic benefit.
  • “maximum” refers to an approximate local maximum based on the parameters being considered, not an absolute maximum.
  • a “suitable dosage level” refers to a dosage level that provides a therapeutically reasonable balance between pharmacological effectiveness and deleterious effects. Often this dosage level is related to the peak or average serum levels resulting from administration of a drug at the particular dosage level.
  • cell membrane permeability refers to a property of cell membranes that allows the passage of solvents and solutes into and out of cells.
  • Cell membrane permeability can be measured by methods well known in the art including but not limited to spectrofluorometry, luminometry, or flow cytometry.
  • cell membrane permeability refers to an increase of at least about 2-fold (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% over the normal permeability of a particular cell (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%), in a subject that has been exposed to a laser, as compared to an untreated subject.
  • 2-fold for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%
  • an increase in cell membrane permeability refers to an increase that persists for a duration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 minutes, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.
  • cytokine refers to a class of signaling proteins and glycoproteins that, like hormones and neurotransmitters, are used extensively in cellular communication. While hormones are secreted from specific organs to the blood, and neurotransmitters are related to neural activity, the cytokines are a more diverse class of compounds in terms of origin and purpose. They are produced by a wide variety of hematopoietic and non-hematopoietic cell types and can have effects on both nearby cells or throughout the organism, sometimes strongly dependent on the presence of other chemicals.
  • the cytokine family consists mainly of smaller, water-soluble proteins and glycoproteins with a mass of between 8 and 30 kDa.
  • Cytokines are critical to the functioning of both innate and adaptive immune responses. They are often secreted by immune cells which have encountered a pathogen as a way to activate and recruit more immune cells and increase the system's response to the pathogen. However, apart from their role in the development and functioning of the immune system, as well as their aberrant modes of secretion in a variety of immunological, inflammatory and infectious diseases, cytokines are also involved in several developmental processes during embryogenesis.
  • a cytokine useful according to the invention includes but is not limited to pro-inflammatory cytokines such as IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-12, as well as growth-promoting cytokines such as granulocyte-macrophage colony stimulating factor or type I and type II interferons.
  • pro-inflammatory cytokines such as IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-12
  • growth-promoting cytokines such as granulocyte-macrophage colony stimulating factor or type I and type II interferons.
  • chemokine refers to a family of small cytokines, or proteins secreted by cells. Proteins are classified as chemokines according to shared structural characteristics such as small size (they are all approximately 8-10 kilodaltons in size), and the presence of four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. Their name is derived from their ability to induce directed chemotaxis in nearby responsive cells; they are chemotactic cytokines. Some chemokines are considered pro-inflammatory and can be induced during an immune response to promote cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development.
  • Chemokines are found in all vertebrates, some viruses and some bacteria, but none have been described for other invertebrates. These proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.
  • a chemokine useful according to the invention includes but is not limited to IL-8, MIP-1, MIP-2, IP-10, RANTES, CCR5, CCR6, CCR7, or CXCR4.
  • antibody titer means a measurement of how much antibody an organism has produced that recognizes a particular epitope, expressed as the greatest dilution ratio (or its reciprocal) that still gives a positive result. In one embodiment, antibody titers are determined by ELISA assays.
  • lymphocyte cytotoxic activity means the ability of a lymphocyte to kill another cell of a different type, typically by means of elaborating porins. This capability can be measured by co-culturing lymphocytes and target cells.
  • improving the efficiency means increasing, as defined herein, any one of the level of response, the speed with which a response occurs, or the duration of the response, as compared to the response to an agent in a subject that has not been exposed to a laser.
  • An increase in the efficiency refers to a change in the level of a response, the speed with which a response occurs or a duration of the response in a subject that has been exposed to a laser as compared to a subject that has not been exposed to a laser, that is at least about 2-fold more (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% more (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% more), than the efficiency of a response in a subject that has been exposed to a laser, as compared to an untreated subject.
  • a subject that is “immunocompromised” refers to a subject that has an immunodeficiency of any kind
  • An “immunodeficiency” (or immune deficiency) is a state in which the immune system's ability to fight infectious disease is compromised or entirely absent. Most cases of immunodeficiency are acquired (“secondary”) but some people are born with defects in the immune system, or primary immunodeficiency. An immunocompromised person may be particularly vulnerable to opportunistic infections, in addition to normal infections that could affect everyone.
  • a subject that is “immunocompromised” refers to a subject wherein the decrease in the ability of the subject to respond to an infection or infectious disease is at least about 2-fold less (for example about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1000-fold or more) or at least about 2% less (for example, about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%), than the ability of a subject that is not immunocompromised to respond to an infection or an infectious disease.
  • extracellular matrix refers to the extracellular part of tissue that usually provides structural support to the cells in addition to performing various other important functions.
  • the extracellular matrix is the defining feature of connective tissue in mammals.
  • Extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various cells (i.e., in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the ECM.
  • Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest.
  • the extracellular matrix comprises polysaccharides (for example, glycosaminoglycans or cellulose) and proteins (such as collagen) secreted by cells. Examples of extracellular matrix proteins include but are not limited to collagen, laminin, fibronectin, vitronectin, elastin, glycosaminoglycans, proteoglycans, and combinations of some or all of these components.
  • skin cells are cells which make up the epidermis for example, Merkel cells, keratinocytes, melanocytes and Langerhans cells.
  • secretion refers to the process of segregating, elaborating, and releasing a substance from a cell.
  • obtaining is understood as purchase, procure, manufacture, or otherwise come into possession of the desired material.
  • “Providing,” refers to obtaining, by for example, buying or making the, e.g., polypeptide, drug, polynucleotide, probe, antigen, and the like, including libraries of such compounds or libraries of combinations of types of compounds.
  • the material provided may be made by any known or later developed biochemical or other technique.
  • compounds may be derived from natural sources, be chemically synthesized by directed or combinatorial methods, or a collection of known compounds (e.g., compounds approved for therapeutic use in humans).
  • the term “recombinant DNA molecule” as used herein refers to a DNA molecule, which is comprised of segments of DNA joined together by means of molecular biological techniques.
  • a nucleic acid sequence even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends.
  • discrete elements are referred to as being “upstream” or 5′ of the “downstream” or 3′ elements. This terminology reflects the fact that transcription proceeds in a 5′ to 3′ fashion along the DNA strand.
  • the promoter and enhancer elements which direct transcription of a linked gene are generally located 5′ or upstream of the coding region. However, enhancer elements can exert their effect even when located 3′ of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3′ or downstream of the coding region.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • “about” is understood to be relative to the amount of variance typically tolerated in the specific assay, method, or measurement provided. For example, “about” is typically understood to be within about 3 standard deviations of the mean, or two standard deviations of the mean. About can be understood as a variation of 20%, 15%, 12%, 10%, 8%, 5%, 3%, 2%, or 1%, depending upon the tolerances in the particular art, device, assay, or method.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the invention provides a laser-based method of creating both photothermal and photokinetic effects in skin cells leading to enhanced immunostimulatory effects, without causing significant and irreversible cell damage.
  • laser treatment stimulates the extracellular release of HSP70 from cells in the skin. This requires a laser emitting light at a specific combination of wavelength, pulse duration, frequency and fluence to induce production and extracellular release of HSP70 in the epidermal and dermal layer of the skin over a relatively short exposure time without causing concomitant tissue damage.
  • the invention provides methods for laser-based enhancement of vaccine efficacy. Fundamentally, it utilizes a specific type of laser irradiation for an exposure duration and frequency. It is suggested that exposure to the laser under the specific parameters disclosed herein causes enough stress on cells of the epidermal and dermal layer of the skin sufficient to result in the secretion of a significant amount heat shock protein 70 molecules into the extracellular matrix, but not enough to result in irreversible cellular damage. The end result of this irradiation is the enhanced response of the epidermal immune mechanisms to administered vaccines, leading to a better immune response in the immunized subject. As an example, FIG. 1 shows the ideal ranges of pulse duration, frequency and power for an Nd:YAG laser emitting at 578 nm for use in the methods of the invention.
  • a wavelength or combination of wavelengths of light must be selected that are significantly absorbed by one or more chromophores, which are chemical compounds capable of selective light absorbance, located in the epidermal or dermal layers of the skin.
  • chromophores are melanin, hemoglobin, riboflavin, and cytochrome. It is suggested that this preferential absorption is the basis for the subsequent photothermal, photokinetic and photochemical effects that leads to enhanced immune response in immunized subjects.
  • the skin must be exposed to the laser light at a site at or near the site of injection.
  • the site of injection is exposed to the laser.
  • the skin can be exposed to the laser at a site near the injection site provided that the skin exposed to the laser is within the area in which the vaccine would diffuse between the time of the injection and the time of exposure to the laser.
  • the non-damaging laser-based methods of the invention are distinct from prior medical uses of lasers.
  • the approved, routine medical applications of lasers have largely been methods that intentionally damage tissue including:
  • the methods provided herein are not based on creating irreversible damage or destruction of cells. Instead the methods are based on exposing cells to a laser of relatively high power without causing significant or irreversible damage to the cells. Irreversible damage does not include a cell stress response in which the cell may enter a period of senescence prior to cell division. Reversible damage can be induced and detected, for example, by the activation of one or more cell cycle checkpoints which can result in a pause in the cell cycle or by the detection of a transient increase in membrane permeability.
  • Irreversible damage to cells can be caused by the destructive application of heat on proteins or lipids in the cell (denaturation or coagulation), by thermal expansion or vaporization of water in and around the cell (ablation), or by triggering the release of destructive chemical compounds within the cell such as radical oxygen or nitrogen species (photochemical destruction).
  • Denaturation, coagulation and ablation are principally the result of the conversion of laser light energy into heat energy. This conversion is mediated by chromophores, which are chemical compounds capable of selective light absorbance. Once the laser energy is converted into heat via the chromophore, heat begins to dissipate into the surrounding tissue.
  • thermal containment When light energy is absorbed by the chromophore faster than it can be dissipated to the surrounding tissue, the result is an increase in the temperature within a local region. This condition is called thermal containment.
  • a local region which can be an organelle, a cell, or a microstructure
  • damage to or destruction of the tissue results either by alteration of the biological material of the cell or by significant and rapid heating of intra- or extracellular water.
  • the denaturation or coagulation of tissues occurs when a rise in temperature alters the structures within the tissue, causing a loss of function.
  • Denaturation of proteins occurs when the kinetic energy caused by heat overcomes the weak hydrogen bonds and van der Waals interactions that help maintain the three-dimensional structure of proteins.
  • FIG. 2 shows the threshold temperature for tissue damage in the skin at different time intervals (Welch 1984).
  • An alternative method of causing irreversible tissue destruction is through the ablation of the tissue using thermal expansion or vaporization of water.
  • Ablation refers to the explosive removal of tissue from the site of irradiation.
  • water itself is the chromophore for the laser.
  • laser ablation a small volume of water is very rapidly heated to the point where either its expansion within a confined area causes a loss of the structural integrity of the tissue or cell, or to the point where the water experiences a rapid phase change into a gas, referred to as a phase explosion.
  • water exists in a superheated metastable liquid state and then rapidly moves into an equilibrium state as a gas.
  • Irreversible damage to the cells can also result from the photochemical effects of lasers, which occur when laser energy catalyzes a chemical process that is toxic to the cell.
  • lasers One of the major ways lasers can cause cell and tissue destruction through photochemical effects is the generation of reactive oxygen or nitrogen species. The precise mechanisms of action are not understood, but it is hypothesized that when the energy of a photon from a laser exceeds the energy needed to remove an electron from a molecule, a collision with that molecule might then lead to generation of free radicals. Therefore, the initial cellular damage may be due to the local formation of hydroxyl or other reactive radicals that may then generate longer lived organic radicals including peroxy- or alkoxy-radicals (Kim 2002). Typical targets for this kind of laser damage are cell membranes, mitochondria, nitric acid complexes, proteins and lipids.
  • Another photochemical effect is the direct damage of cellular DNA by laser light. In some cases absorption of energetic photons can cause breaks in the DNA structure, leading in some cases to irreversible cell damage and subsequent apoptosis.
  • Irreversible damage to cells from photothermal and photochemical effects result in either the apoptosis or necrosis of the cell, depending on the site and extent of the damage, or the disruption of the entire tissue (in the case of laser ablation).
  • Cells need not be killed immediately upon exposure to the laser, but instead, they may die over a longer period of time as a direct result of laser treatment (e.g., initiate apoptosis, become necrotic, somatic fusion and subsequent mitotic catastrophe).
  • the exact cause or causes of irreversible damage is not a limitation of the invention. Irreversible damage can result from a combination of mechanisms.
  • Thermal damage to a culture of cells or living tissue can be expressed by the parameter ⁇ , which is the logarithm of the ratio of the concentration of the intact protein in a sample before damage and the concentration of intact protein in a sample after exposure.
  • ⁇ is dimensionless, exponentially dependent on temperature, and linearly dependent on time of exposure (Diller and Pearce 1999).
  • Calculation of ⁇ is a function of the ratio of the concentration of viable cells at time point 0 and at time point (t). A value of 1 for ⁇ at the time point (t) means that 63.2% of cells in a given sample have been damaged at that point, while a value of 10 means that virtually all cells in the sample have been destroyed.
  • Measurement of the concentration of viable cells in a particular tissue or culture is routine in the art. Methods and kits are known in the art to detect apoptosis and necrosis. The number of cells undergoing apoptosis or necrosis can be readily scored and expressed as a percentage of cells exposed to the laser.
  • Apoptosis can be detected using flow cytometry techniques. For example, apoptotic cells show an increased uptake of the vital dye HO342 compared to live cells due to a changes in membrane permeability. Apoptosis can be measured using a number of other assay-based approaches including measurement of DNA fragmentation, membrane phospholipid changes, interleukin-1beta converting enzyme-like protease activation, or nucleosomal fragmentation by DNA agarose gel electrophoresis. Finally, apoptosis can be detected through visual means such as changes in cell morphology.
  • Necrotic cells can be detected by flow cytometry techniques, such as the addition of the nucleic acid stain PI, which binds to DNA or RNA but cannot permeate cell membranes and therefore is visible under fluorescence only if the cell membrane has been compromised, or stains such as propidium iodide or 7-AAD that discriminate cells which have lost membrane integrity. In addition, they can be visualized optically using standard staining and microscopy techniques.
  • the degree of irreversible damage to tissue samples may also be determined by several methods known in the art, such as the measurement of changes in optical characteristics of the tissue. For example, concentration of viable cells in muscles and collagen can be measured by the relative birefringence of tissue when viewed through a polarized filter (Diller and Pearce 1999), Undamaged tissue is birefringent and appears bright while damaged, nonbirefringent tissue is dark. The proportion of damaged to undamaged tissue in a particular sample can be calculated by measuring optical intensity. Other methods of optical measurement are also possible (see for example Roggan and Müller 1995). Additional methods exist in the art to quantify damage in tissue includes enzyme deactivation (Bhowmick and Bischof 1998) and extravasation of fluorescent-tagged proteins (Green and Diller 1978).
  • the method of this invention utilizes high power laser irradiation of tissue, but is designed to avoid significant and irreversible damage to cells. Preferably less than 1% of cells are irreversibly damaged upon laser exposure using the methods of the invention, which would correspond to an ⁇ value of 0.01 or less. In an embodiment, significant and irreversible damage of the cells is limited or prevented by limiting the increase in temperature in the cells and/or tissues exposed to the laser in the methods of the invention to below the levels below the critical thresholds illustrated in FIG. 2 . In addition, the method does not result in the heating of intracellular or extracellular water to supercritical temperatures required for vaporization.
  • the method is distinguished from the use of relatively low power laser irradiation to modify the metabolic activity of cells in order to accelerate burn or wound healing.
  • Such methods which are sometimes referred to as low-level laser therapy (LLLT) use very low power irradiation (in the range of 10 milliwatts to 10 watts) with a wide variety of wavelengths, pulse durations and frequencies with the intention of stimulating the production of factors such as proangiogenic or other growth factors that can lead to accelerated healing processes.
  • LLLT low-level laser therapy
  • the wavelength of laser light determines the depth of its penetration into the skin.
  • Laser light of wavelengths in the near infrared spectrum penetrate furthest into the skin.
  • Absorption of laser light by surrounding tissues also depends on the wavelength of the laser used.
  • visible light laser radiation is absorbed by pigments in the skin (chromophores).
  • Absorption by a particular pigment is dependent on the laser's wavelength.
  • Near infrared energy such as that produced by the neodymium:yttrium aluminum garnet (Nd:YAG) laser at 1.06 ⁇ m, has little pigment specificity, while the 10.6 ⁇ m wavelength light emitted by the CO 2 laser is effectively absorbed by water.
  • laser pulses at an appropriate frequency (Hz, or KHz).
  • the methods herein rely predominantly on the use of a combination of the desired pulse length, preferably in the nanosecond range; frequency, preferably in the kilohertz range; and power, preferably in the range of about 1-10 watts.
  • HSP 70 increased local concentration of HSP 70 at or near the site of laser exposure, particularly extracellular HSP 70, increased local concentration or number of Langerhans cells at or near the site of exposure, and/or increased response to antigen as compared to a subject not exposed to laser, without causing significant or irreversible damage to the cells or tissue exposed to the laser.
  • the method is practiced to minimize the exposure time required to yield these effects to seconds or minutes. Variation in wavelength, laser type, beam size, fluence, and frequency of laser administration on a macro time scale (e.g., minutes, hours, days, rather than fractions of seconds) is well within the ability of those in the art. Methods of testing specific parameters using the methods provided herein as well as those known to those of skill in the art is routine.
  • tissue can be heated to a supraphysiologic level, eventually leading to significant and irreversible cell injury, death or destruction of tissue through superheating of water within and around the tissues (Venugopalan et al. 1996, incorporated herein by reference). Because cellular injury or death is a property of the heat exposure duration and not solely of the temperature (Moritz and Henrique 1947, incorporated herein by reference), the heating effect of a laser can be controlled to keep it below a level that causes significant cell damage or death. This heating effect leads to two biological processes in the skin which together create the conditions for effective immunostimulation suitable for enhancing vaccination: production and secretion of heat shock proteins and concentration stimulation of Langerhans cells.
  • HSPs heat shock proteins
  • skin cells heat shock response
  • HSPs heat shock proteins
  • cells produce HSPs in response to stress caused by heat, poisons or signals from nerves or hormones.
  • heat shock proteins play an important protective role against these stresses, specifically assisting in protein maintenance, folding, chaperoning and degredation, and, when expressed on the surface of the cell, assist with the stabilization of the cell membrane.
  • HSP70 heat shock proteins
  • HSP70 is involved in protein folding and chaperoning activities inside the cell, outside the cell it acts as a chaperone of protein antigens to dendritic cells and stimulates their maturation (Bendz et al. 2007).
  • Dendritic cells are key actors in the immune response against both infectious pathogens and cancerous tumors (Banchereau and Steinman 1998). In an immature state, dendritic cells capture and process antigens with high efficiency. After maturation, they migrate to lymphoid organs to present these antigens, as well as co-stimulatory molecules, to T cells.
  • antigen-presenting dendritic cells Approximately 40% of the body's antigen-presenting dendritic cells are located in the skin. These antigen presenting cells, called Langerhans cells, are concentrated in the in the basal, spinous, and granular layers of the epidermis, showing a preference for the suprabasal portion of the epidermis.
  • HSP70 can also be elicited by use of a laser capable of creating thermal confinement sufficient to raise temperatures of cells above normal but below the critical temperature where cells begin to die (Souil et al. 2001).
  • cells vary in their ability to produce HSPs. For example, keratinocytes in the skin highly express HSP70 relative to other cell types (Trautinger et al. 1993)
  • This method utilizes a laser to rapidly and non-destructively (i.e., without significant or irreversible damage) cause the secretion of significant quantities of HSP70 in the skin and yield enhancements in the response to vaccination.
  • lasers of different types can enhance the expression of heat shock protein in living tissues without causing significant damage to the living cells (Ferrando et al. 1993; Destura et al. 2001; Souil et al. 2001; Emohare et al. 2004). To date, no research has demonstrated that a non-destructive exposure to laser irradiation leads to enhanced secretion of HSP70 from cells.
  • HSP70 The secretion of HSP70 introduces a significant additional number of chaperone proteins into the surrounding epidermal tissues that are capable of enhancing presentation the protein antigens from a vaccine to Langerhans cells. HSP70 is capable of inducing maturation of immature Langerhans cells, rapidly multiplying the number that migrates to the lymph system, where they prime T-cells to mount an immune response against a cell or virus expressing the vaccine antigen.
  • this invention leads to the concentration of immature Langerhans cells in the local area of the skin subject to laser irradiation.
  • FIG. 5 shows a specific example of the increase in number of Langerhans cells in the epidermal tissues of a mouse subject to laser irradiation. This concentration also serves to increase the effectiveness of vaccination by increasing the number of Langerhans cells available to receive vaccine antigens and migrate to the lymph system after maturation.
  • Fibroblasts cells derived from mouse embryo were cultured in the Petri dish using the Eagle or No. 199 medium for 24 h. Cell culture was exposed to the light beam of the Cu laser. The levels of HSP70 have been determined before and after irradiation using antibodies to Hsp70 (Russian Patent No. 2242764 7, Aug. 2003. After a 1 minute exposure, the separated supernatant solution displayed 350% increase of the extracellular HSP70, from 3.2 to 11.2 ng/L. The culture cells remained viable after laser irradiation as established by staining with the toluidine blue 0.2%
  • Healthy white mice (CBA, Rappolovo, 50 animals) were treated using Cu laser by exposing a section of an ear to the laser light with a diameter of 5 mm over a period of 1-3 min. After the experiments, the animals were sacrificed at different time points. Immunohistochemical staining using antibodies to HSP70 (Russian Patent No. 2242764, 7, Aug. 2003) of the ventricular epidermal sheets of the ear ( FIG. 6 ) provided a qualitative estimate of the increased levels of the secreted HSP70 as compared to the tissue from untreated ear from the same animal. These data were supported by the results of immunoblotting analysis of the processed ventricular epidermal sheets tissues though these results did not differentiate between secreted and intracellular HSP70.
  • mice received 50 ⁇ L of a commercial H3N2 influenza vaccine (VAXIGRIP®, Aventis Pasteur) by subcutaneous injection to the left ear and served as a control group.
  • VAXIGRIP® commercial H3N2 influenza vaccine
  • 50 mice were administered the same vaccine together with laser treatment (Cu laser at 578/511 nm, 1-3 ns pulses at 10 MHz) of the left ear.
  • laser treatment Cu laser at 578/511 nm, 1-3 ns pulses at 10 MHz
  • a subgroup of 25 animals was a subject of a one minute exposure whereas another subgroup of 25 mice was a subject of a two-minute exposure.
  • mice received the vaccine and exogenous bovine HSP70 (10 ⁇ g per injection, dissolved in 50 ⁇ L saline solution).
  • the animals were sacrificed and antibody titers were measured 28 days after vaccination following a standard literature protocol [“The immune drugs for grippe prophylactic and diagnostic effectiveness testing methods” Russian Federal Methodologic Standarts—MY 3.3.2.1758-03′′ (28 Sep. 2003)].
  • Results of the experiment are summarized in the Table 1.
  • the most optimal treatment consisted in using vaccine and laser treatment for 2 min that resulted in 86% increase of the antibody titers as compared to vaccine alone. 1 min exposure was also effective providing a 54% increase in titers. Consistent with our hypothesis about the role of HSP in improved presentation of the vaccine antigens, combination of the influenza vaccine with exogenous HSP70 produced as much as 68% increase in the antibody titers.
  • mice received a commercial H3N2 influenza vaccine (VAXIGRIP®, Aventis Pasteur) by subcutaneous injection to the left ear (50 ⁇ L).
  • VAXIGRIP® Aventis Pasteur
  • All mice received a lethal dose of the H3N2 influenza virus (strain A/Aichi/2/68) by inhalation using a “Mussson” type ultrasound inhalator coupled with special murine inhalation mask 14 days post-vaccination.
  • H3N2 influenza virus strain A/Aichi/2/68
  • the first group of 22 immunocompromised patients received a standard influenza vaccination (VAXIGRIP®, Aventis Pasteur), the second group of 22 immunocompromised patients received vaccination after a one-minute laser exposure of the skin.
  • 22 healthy control subjects received standard vaccination. Seven different measurements of immune function and activity were assessed 30 days after immunization. Results were normalized to the set of vaccinated healthy adults. Based on an overall assessment of all measurements, 75% of the healthy patients had a significant response to the vaccine whereas only 25% of the vaccinated immunocompromised patients had a significant response. In comparison, over 50% of the immunocompromised patients receiving vaccination with laser showed significant response: double that of the vaccine only group.
  • hepatitis B vaccine Recombinant Yeast Hepatitis B vaccine, Combiotech, Russia
  • hepatitis B vaccination at 0, 1 and 3 months
  • IL-2 2,500,000 IE SQ
  • nine patients received the same vaccination schedule coupled with the copper laser treatment of the skin at the time of injection (power-1.5 watts, exposure time 2 minutes, vaccine injection 5 minutes after laser skin treatment).
  • Responders were determined by the level of protective antibodies titer (>10 IE/ml).
  • Arm 1 subjects receive the standard of care—intramuscular vaccination with the influenza vaccine manufactured to provide prophylaxis against the current season's influenza strain.
  • Arm 2 subjects receive the same vaccine in an intradermal delivery (e.g., intradermal microinjection system, Becton, Dickinson & Company). Intradermal dosage is adjusted appropriately to site of delivery.
  • Arm 3 subjects receive the same vaccine in an intradermal dosage coupled with a short-duration exposure to a laser vaccine adjuvant device immediately before administration of the vaccine.
  • the time of laser exposure is about one minute, but may be adjusted to match skin color, and patients with darker skin receive shorter duration of irradiation. Sufficient numbers of patients are enrolled in each arm to ensure sufficient statistical powering at the end of the study, and enrollees in each arm are case-matched demographically. Enrollment is conducted at the start of the influenza season (November to May).
  • the study shows that combination of laser irradiation and epidermal vaccination is statistically superior to both standard of therapy vaccination and epidermal vaccination alone in the improvement of antibody titers and the concentration of Granzyme-secreting cells, and that patients receiving both the epidermal vaccination and laser treatment have a statistically significant decrease in clinical events related to influenza, particularly with respect to hospitalization.
  • mice in group 1 receive the standard intramuscular avian influenza vaccine.
  • Mice in group 2 receive the standard avian influenza vaccine administered epidermally in the ear.
  • Groups 1 and 2 act as control groups for the experiment. Preliminary experiments are used to establish the doses of the vaccine for each group that correspond to protective levels of H5N1 antibody.
  • mice in group 3 receive the standard avian influenza vaccine by intramuscular injection coupled with a short-duration laser exposure immediately before administration of the vaccine.
  • Mice in group 4 receive the standard avian influenza vaccine administered using epidermal vaccination to the ear coupled with a short-duration exposure to a laser vaccine adjuvant device immediately before administration of the vaccine.
  • the duration of laser exposure in groups 3 and 4 will be approximately one minute. Mice in group 3 are shaved in the area of intramuscular injection to maximum effect of the laser.
  • mice in each group and subgroup are adequate to sufficiently power the study for statistical analysis.
  • a population of healthy individuals is assessed for the difference in response to a novel DNA-based Hepatitis B vaccine compared with an existing, approved hepatitis B vaccine (RECOMBIVAX HB®, Merck and Co.).
  • Subjects are randomized to receive either the DNA-based hepatitis B vaccine, the hepatitis B vaccine combined with laser vaccine adjuvant, or the standard hepatitis B vaccine.
  • Administration of each type of vaccine, including dosage, frequency of vaccination, and methods of administration, follows FDA-approved approaches.
  • the time of exposure to the laser is about one minute, but may be adjusted to match skin color, and patients with darker skin receive shorter duration of irradiation.
  • blood samples are drawn from each patient and assessed for hepatitis B surface antigen levels.
  • a population of healthy individuals is assessed for the difference in response to a novel subunit-based MMR (measles, mumps, rubella) vaccine compared with an existing, approved MMR vaccine that is composed of attenuated live viruses (MMR II®, Merck and Co.).
  • Subjects are randomized to receive either the subunit-based MMR vaccine, the subunit MMR vaccine combined with laser vaccine adjuvant, or the standard MMR vaccine.
  • Administration of each type of vaccine, including dosage, frequency of vaccination, and methods of administration follows FDA-approved approaches.
  • the time of exposure to the laser is about one minute, but may be adjusted to match skin color, and patients with darker skin receive shorter duration of irradiation.
  • Sufficient numbers of patients are enrolled in each arm to ensure sufficient statistical powering at the end of the study, and enrollees in each arm are case-matched demographically.
  • Exclusion criteria for the study includes previous vaccination against or exposure to measles, mumps, or rubella.
  • a population of healthy individuals is assessed for the difference in response to a novel synthetic peptide vaccine against bacterial meningitis compared with an existing, approved bacterial meningitis vaccine that is composed of meningococcal polysaccharides conjugated to the diphtheria toxoid (MENACTRA®, Sanofi Pasteur).
  • Subjects are randomized to receive either the synthetic peptide vaccine, the synthetic peptide vaccine combined with laser vaccine adjuvant, or the standard conjugated vaccine.
  • Administration of each type of vaccine, including dosage, frequency of vaccination, and methods of administration follows FDA-approved approaches.
  • the time of exposure to the laser is about one minute, but may be adjusted to match skin color, and patients with darker skin receive shorter duration of irradiation.
  • Sufficient numbers of patients are enrolled in each arm to ensure sufficient statistical powering at the end of the study, and enrollees in each arm are case-matched demographically. Exclusion criteria for the study includes previous vaccination against or exposure to bacterial meningitis.

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AU2012301730A1 (en) * 2011-09-01 2014-04-17 The General Hospital Corporation Laser adjuvants for enhancing immune response
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US11622943B2 (en) 2019-05-10 2023-04-11 Chamkurkishtiah Panduranga Rao System and method for allergen-specific epicutaneous immunotherapy

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