Patent Grant
11510983
The present invention is directed to a method and apparatus for increasing the efficacy of various vaccines. Such enhancement can be achieved using a method and apparatus that include generating a plurality of small (for example, sub-millimeter) regions of tissue preparation in the vaccination site to increase the local population of antigen-presenting cells prior to introduction of the vaccine.
Flu viral infection continues to be a significant public health problem, and vaccination is one of the most cost-effective means to prevent the spread of the virus and decrease flu-caused morbidity and mortality. However, annual flu vaccination presents a huge burden, economically, clinically and in production efforts, for the public health system. In particular, when an outbreak of a new strain or pandemic of flu virus occurs, manufacturers face an enormous challenge to double or triple the production of flu vaccines in order to cope with both seasonal and pandemics of flu virus. A method and system that provides dose-sparing of existing and stockpiled vaccines can greatly enhance a capacity to cope with flu vaccine shortages in the event of a flu pandemic, and could result in huge cost savings for seasonal flu vaccines. In addition, vaccine enhancement can provide further benefits such as improving the efficacy of vaccines in the elderly and immunocompromised patients.
Most vaccines may benefit from adjuvants to induce sufficient immune responses in the human body. An “adjuvant” can refer to any aid or contributor that can increase the efficacy or potency of a vaccine for the prevention, amelioration, or cure of a disease as compared to a vaccine or agent administered without the adjuvant. For example, 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, a reduction in the likelihood and/or severity of undesirable side effects of the vaccine, and/or a more rapid or robust response to the vaccine (e.g., a higher antibody titer).
Currently, very few chemical or biological vaccine adjuvants are approved for human use in the Western Hemisphere. Aluminum salts (aluminum hydroxide, aluminum phosphate or alum) have been used in vaccine preparations for a long time. These salts can create more antigenic precipitates with some vaccines, or increase the local concentration of antigen at the injection site to enhance vaccine uptake by antigen-presenting cells, among other possible effects. However, aluminum salts can also cause side effects in a percentage of the population that receives such vaccinations and may be undesirable for skin vaccination due to severe skin irritation it can cause. Certain other adjuvants are undergoing stringent approval testing in various countries, but have yet to gain wide acceptance.
In general, safety of vaccines (and associated adjuvants) is considered to be critical because vaccines are used to prevent illness, not to treat a disease. Any potential risks or unwanted side effects of a vaccine must be weighed against the reduction in likelihood of contracting the actual disease they are designed to treat, and the health risks of such disease if contracted. Accordingly, finding safe and effective vaccine adjuvants is becoming increasingly important.
One approach to produce an adjuvant by exposing the injection site to laser light having certain characteristics is described, e.g., in U.S. Patent Application Publication No. 2011/0059116 of Onikienko et al. The described method appears to stress the cells to release immunostimulatory factors such as heat shock proteins that can improve vaccine efficacy. However, the method requires certain narrow combinations of wavelengths, pulse power, and pulse durations to evoke the desired effects while avoiding significant irreversible damage (e.g., necrosis or aptosis) to skin cells exposed to the laser energy (for example, limiting irreversible damage to 1% or less than of the exposed cells). Such limitations on the parameters of the laser energy that can produce the desired effects may include limitations based on skin color or pigmentation, and thus may limit the application of such methods based on clinical skill and/or within certain populations.
Fractional resurfacing, or fractional photothermolysis, is a dermatological technique in which small regions of skin tissue (epidermis and/or dermis) are thermally damaged or ablated using electromagnetic energy (generally optical energy from a laser, flashlamp, laser diode, etc.) to stimulate a healing response in the skin. An example of a “fractional” approach to skin resurfacing is described, e.g., in U.S. Patent Publication No. 2006/0155266 of Manstein et al. In fractional resurfacing, the width of the skin regions exposed to energy is typically less than 1 millimeter, or even less than 0.5 millimeters. Such small damage zones, which can extend to the dermal layer, are well-tolerated by the skin, partially because the small regions of damage remain surrounded by healthy tissue. Fractional photothermolysis can produce effects such as skin tightening, wrinkle reduction, and improvement in pigmentation appearance. Fractional photothermolysis can be ablative (where a portion of the skin tissue is vaporized in the exposed regions), or non-ablative (where skin tissue is thermally damaged but not vaporized). For example, non-ablative fractional photothermolysis (NAFP) maintains a substantially intact stratum corneum and reduces a risk of infection as compared to other skin resurfacing techniques.
Other dermatological treatments have been developed based on the fractional damage concept. Various modalities for heating, ablating, or otherwise disrupting small regions of tissue (e.g., less than 1 millimeter wide or less than 0.5 millimeters wide) can be used to achieve various cosmetic effects. Generally, formation of small regions of thermal damage in tissue that are surrounded by healthy, unaffected tissue (e.g., fractional thermolysis) is generally well-tolerated.
Accordingly, it may be desirable to provide methods and apparatus for enhancing the efficacy of various vaccines that are not vaccine-specific and which are well-tolerated by the subject.
Embodiments of the present invention provide a method, system and apparatus for enhancing the efficacy of various vaccines by using, e.g., fractional thermolysis techniques and variations thereof, to generate a plurality of discrete microscopic regions of damaged tissue in a target zone of skin tissue, and then administering the vaccine into the target zone. For example, the damage can be generated thermally using, e.g., optical energy, radio frequency (rf) energy, other forms of electromagnetic energy, ultrasound energy, resistive heating, or the like to form microscopic thermal zones (MTZs) in the tissue. For example, a width of the MTZs can be less than about 1 millimeter, or less than 0.5 millimeters. A fractional areal coverage of the target zone by the MTZs is, for example, between about 5% and 40%. Certain topical adjuvants, such as Imiquimod (1-(2-Methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine, an immune response modifier) or the like, can be applied to the target zone to increase the vaccine efficacy further. As used herein and in the drawings, Imiquimod may be likewise referred to as IMIQ.
In one aspect, the invention provides a method of vaccinating a mammalian subject. The method includes the steps of: arranging a source of electromagnetic radiation proximate to a target zone of skin of the mammalian subject; controlling the source of electromagnetic radiation to deliver a dose of electromagnetic radiation to the target zone determined to create at least one thermally-denatured zone in the target zone; and intradermally injecting a vaccine within the target zone to vaccinate the mammalian subject. In one form, a width of one or more of the thermally denatured zones is less than about 1 millimeter. The thermally denatured zone may comprise columns having thermally denatured epidermal and dermal tissue and a stratum corneum that is not denatured, and wherein the columns are spatially separated from one another by an unirradiated area of skin. A fractional surface coverage of the target zone by the denatured zone(s) may be between 5% and 40%. The width of the columns may be about 0.1 millimeters. The source of electromagnetic radiation may includes at least one of a non-ablative laser, a diode laser, a fiber laser, a flashlamp, and a solid state laser. The electromagnetic radiation source can be a non-ablative laser. The method may further comprise applying an immune response modifier, such as Imiquimod, to the target zone.
In one version of the method, Hemagglutination inhibition (HAI) antibody titer in the mammalian subject is increased following vaccination compared to HAI antibody titer in a mammalian subject intradermally injected with the vaccine but not receiving the dose of electromagnetic radiation to produce the at least one thermally denatured zone.
In another version of the method, IgG antibody titer in the mammalian subject is increased following vaccination compared to IgG antibody titer in a mammalian subject intradermally injected with the vaccine but not receiving the dose of electromagnetic radiation to produce the at least one thermally denatured zone. The IgG antibody titer may be IgG2a antibody titer.
In another version of the method, the helper T cell immune response in the mammalian subject is increased following vaccination compared to the helper T cell immune response in a mammalian subject intradermally injected with the vaccine but not receiving the dose of electromagnetic radiation to produce the at least one thermally denatured zone. The helper T cell may be a Th1 helper T cell. The helper T cell may be a Th2 helper T cell.
In one version of the method, the vaccine is selected from the group consisting of a pneumococcal vaccine, a meningococcal vaccine, a hepatitis vaccine, a chickenpox vaccine, a diphtheria vaccine, a haemophilus influenza vaccine, an influenza vaccine, a measles vaccine, a mumps vaccine, a pertussis vaccine, a polio vaccine, a rotavirus vaccine, a rubella vaccine, a shingles vaccine, a respiratory syncytial virus vaccine, a human papillomavirus vaccine, and a tetanus vaccine. The method can provide at least a 5-fold dose-sparing of the vaccine. The method can overcome age-related immunosenescence.
In another aspect, the invention provides a system for enhancing the efficacy of a vaccine. The system includes a fractional arrangement configured to generate at least one region of thermal denaturation in a target zone of biological tissue; and a delivery arrangement configured to deliver a dose of the vaccine into the target zone. A width of the region(s) is less than about 1 millimeter, and each region is surrounded by substantially undamaged tissue. The fractional arrangement and the delivery arrangement can be provided in a single housing.
The fractional arrangement and the delivery arrangement can be provided in separate housings. The fractional arrangement can include a marking arrangement provided on a lower surface thereof, wherein the marking arrangement is configured to provide a visible marker on or proximal to the target zone to facilitate accurate placement of the delivery arrangement on the target zone. The system can further include a locating arrangement configured to be adhered to the target zone, wherein each of the fractional arrangement and the delivery arrangement are configured to be removably coupled to the locating arrangement. The fractional arrangement can include an immune response modifier delivery system provided on a lower surface thereof, wherein the delivery system is configured to provide an immune response modifier on or proximal to the target zone.
The fractional arrangement can include a source of optical energy. The fractional arrangement can include a plurality of needles configured to contact or penetrate into the target zone, and a source of radio frequency (rf) or thermal energy configured to be directed to the needles to generate the at least one region of thermal denaturation. The fractional arrangement can include a source of ultrasound energy configured to generate the at least one region of thermal denaturation. The delivery arrangement can be at least one of a needle, a fractional patch, a microneedle-array patch, or a microproject-array patch.
In the system, the vaccine can be selected from the group consisting of a pneumococcal vaccine, a meningococcal vaccine, a hepatitis vaccine, a chickenpox vaccine, a diphtheria vaccine, a haemophilus influenza vaccine, an influenza vaccine, a measles vaccine, a mumps vaccine, a pertussis vaccine, a polio vaccine, a rotavirus vaccine, a rubella vaccine, a shingles vaccine, a respiratory syncytial virus vaccine, a human papillomavirus vaccine, and a tetanus vaccine.
In another aspect, the invention provides a system for vaccinating a subject. The system can include an electromagnetic radiation source configured to be arranged proximate to a target zone on an exterior of the subject; a user control configured to selectively cause the electromagnetic radiation source to deliver a dose of electromagnetic radiation toward the target zone to create at least one thermally-denatured zone in the target zone; and a vaccine-delivery system configured to deliver a vaccine to the target zone. A width of the thermally-denatured zone(s) is less than about 1 millimeter, and each thermally-denatured zone can be surrounded by substantially undamaged tissue.
The electromagnetic radiation source, the user control, and the vaccine-delivery system can be provided in a single housing. The housing can include a masking arrangement provided on a lower surface thereof, wherein the masking arrangement includes at least one optically transparent opening. The masking arrangement can include a channel for receiving a needle of the vaccine-delivery system.
Alternatively, the electromagnetic radiation source can be provided in a first housing and the vaccine-delivery system can be provided in separate second housing. The first housing can include a marking arrangement provided on a lower surface thereof, wherein the marking arrangement is configured to provide a visible marker on or proximal to the target zone to facilitate accurate placement of the vaccine-delivery system on the target zone. The first housing can include a masking arrangement provided on a lower surface thereof, wherein the masking arrangement includes at least one optically transparent opening. The masking arrangement can include a channel for receiving a needle of the vaccine-delivery system. The system can further include a locating arrangement configured to be adhered to the target zone, wherein each of the first housing and the second housing are configured to be removably coupled to the locating arrangement. The locating arrangement can include a channel for receiving a needle of the vaccine-delivery system. The locating arrangement can include at least one optically transparent opening.
In the system, the electromagnetic radiation source can be a source of optical energy configured to generate the at least one thermally-denatured zone. The electromagnetic radiation source can be a source of ultrasound energy configured to generate the at least one thermally-denatured zone. The electromagnetic radiation source can be a non-ablative laser. The system can further include an immune response modifier delivery system configured to provide an immune response modifier on or proximal to the target zone. The immune response modifier can be Imiquimod.
In the system, the vaccine can be selected from the group consisting of a pneumococcal vaccine, a meningococcal vaccine, a hepatitis vaccine, a chickenpox vaccine, a diphtheria vaccine, a haemophilus influenza vaccine, an influenza vaccine, a measles vaccine, a mumps vaccine, a pertussis vaccine, a polio vaccine, a rotavirus vaccine, a rubella vaccine, a shingles vaccine, a respiratory syncytial virus vaccine, a human papillomavirus vaccine, and a tetanus vaccine.
These and other features of the present invention will become apparent upon reading the following detailed description of embodiments of the invention, when taken in conjunction with the appended claims.
Further features of the invention will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments, results and/or features of the exemplary embodiments of the present invention, in which:
Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components, or samples of the illustrated embodiments. Moreover, while the present invention will now be described in detail with reference to the figures, it is done so in connection with the illustrative embodiments and is not limited by the particular embodiments illustrated in the figures.
Recent public concerns about safety of adjuvantated influenza (flu) vaccine stress the urgent need of a safer adjuvant. We describe herein augmentation of flu vaccine in adult and old mice and in pigs by micro sterile inflammation, without involving injection of any adjuvant.
In one non-limiting illustrative example, the inoculation site was first illuminated for 1 second with an over-the-counter laser before flu vaccine was intradermally injected. Then, Imiquimod cream (IMIQ), an agonist for Toll-like receptor 7, was topically applied to the inoculation site. Non ablative fractional laser (NAFL) created an array of microthermal zones (MTZs) beneath the stratum corneum at the inoculation site, and the dying cells in each MTZ sent “danger” signals, provoking the production of various chemokines that attracted a large number of antigen presenting cells (APCs), in particular, plasmacytoid dendritic cells (pDCs) around each MTZ forming a micro-sterile inflammation array. The recruited pDCs were activated by IMIQ diffusing from the skin surface and secreted a high level of pro-inflammatory cytokines leading to a strong Th1 immune response. The Th1 immunity was abrogated significantly by systemic depletion of pDCs, a local TNF-α inhibitor, or in the absence of IFN regulatory factor 7 (IRF7), highlighting a pivotal role of this regulatory pathway in the adjuvanicity. In contrast to conventional adjuvants that stimulate persistent inflammation and severe skin lesion, NAFL/IMIQ caused little overt inflammation in the skin while sufficiently enhancing flu vaccine-mediated immune responses and hindering skin irritation evoked by flu vaccines. The micro-sterile inflammation strategy confers a means to improve the efficacy of flu vaccines, yet with diminished local and systemic adverse events.
Tremendous progress has been made in the past decade in understanding of sterile inflammation, which is a natural response to invaders or injury in protection of our body and induced by danger signals released from dying cells. The danger signals, named damage-associated molecular patterns (DAMPs), include uric acid, dsDNA and others, and can attract and activate antigen presenting cells (APCs). The sterile inflammation is one of the primary mechanisms behind alum and MF59 adjuvants, two licensed vaccine adjuvants and forms the basis for today's adjuvant development. The ability of sterile inflammation to boost immune responses against vaccines raises an intriguing possibility that skin injury can serve as safe “adjuvant” for cutaneous vaccination, provided that the injury is well under control. Non-ablative fractional laser (NAFL) can be such a great technology to controllably injure the skin at the site of vaccine inoculation. The laser treatment generated an array of micro-thermo zones (MTZs) in a desirable number, size, and depth without damaging skin's outer protective barrier, so that the skin barrier function is well preserved. Each MTZ is so small that it can be healed within one or two days by fast growing epithelial cells surrounding each MTZ, resulting in younger-looking skin. Indeed skin renewing by NAFL has been a cosmetic industry for decades. The micro-skin injury sharply contrasts the injury induced by intradermal (ID) injection of adjuvants that often evoke severe and persistent inflammation and severe skin lesion and is not acceptable for routine vaccination.
We hypothesized that laser-injured cells within the MTZs could send danger signals to induce sterile immunity and serve as a safe adjuvant for cutaneous vaccines. Skin is prone to adjuvant-induced inflammation and tends to cause more severe local reactogenicity including erythema, swelling, and ulceration that are persistent for weeks. Because of this unwanted adverse events, most current adjuvants such as Alum, oil-in-water emulsion adjuvants, and several TLR agonists are not suitable for skin vaccination. Adjuvant for cutaneous vaccination remains the urgent need for future needle-free vaccinations and for the ID immunization that is more potent than the conventional intramuscular (IM) immunization as shown by a number of studies.
We present herein that NAFL induces sterile inflammation at a micro-scale which causes no overt skin lesion while greatly augmenting flu vaccines ID administered. The danger signals released by dead cells in laser-generated MTZs preferably attracted plasmacytoid dendritic cells (pDCs) that were subsequently activated by topically applied Imiquimod (IMIQ) cream, leading to synergistic augmentation of the immune response against influenza (flu) vaccine in adult and old mice and in pigs, strikingly concomitant with improved local and systemic safety profiles.
The present invention relates to a method and apparatus for enhancing the efficacy of intradermal vaccines and increase subsequent immunological responses by generating a fractional pattern of thermal preparation regions, or micro-thermal zones (MTZs), at a target injection or administration site using optical energy, prior to introducing a vaccine into the target site. The MTZs can be generated using various arrangements, including ablative fractional photothermolysis (AFP) and non-ablative fractional photothermolysis (NAFP) methods and devices, ultrasound arrays, arrays of rf needle electrodes, or resistance-heated needles, etc. The vaccine is preferably administered after the MTZs have been formed in the injection site, which can avoid damage or modification to the vaccine components/compounds by any of the various forms of energy used to generate the MTZs. For example, the vaccine can be introduced immediately after formation of the MTZs, up to, for example, about 24 hours after their formation.
Without being bound by a particular theory, it has been observed that an increased number of antigen-presenting cells (APCs) tend to surrounding MTZs after they are generated in the skin. The MTZ-mediated enrichment of APCs can boost immune responses elicited by various vaccines administered intradermally. Fractional thermolysis treatment of a vaccine administration site can also enhance the motility of APCs and speed up their migration from the skin to draining lymph nodes. Further, fractional thermolysis pre-treatment of a vaccine administration site can augment immune responses against various vaccines independently of skin color while causing little skin damage visibly or histologically.
An exemplary vaccine apparatus 100 in accordance with exemplary embodiments of the present disclosure is illustrated in
In certain embodiments, the fractional arrangement 110 can include a source of energy 115 such as, e.g., a laser, a flashlamp, one or more lasers diodes, a patterned ultrasound generator, a plurality of needle electrodes, or the like. The apparatus 100 can include a source of power for the energy source 115 provided within a housing 105, or it can be connected to an external power source such as an electrical outlet. In further embodiments, the apparatus 100 can receive optical energy from an external source such as, e.g., laser energy generated by an external laser device (not shown) and delivered to the apparatus 100 via one or more optical fibers, waveguides, or the like (also not shown).
The fractional arrangement 110 can further include a control arrangement 118 configured to control aspects of the energy provided by the fractional arrangement 110 to form a plurality of MTZs on the target zone. In certain embodiments, the energy source 115 can provide optical energy, e.g., from a laser, flashlamp or the like, and the control arrangement 118 can include a plurality of lenses, a mechanical actuator, a movable mirror or prism, and/or a translator adapted to direct a beam or a series of pulses of the optical energy onto discrete locations in the target zone. The optical energy can be either ablative or nonablative. For example, the optical energy source can include a CO2 laser, a fiber laser, one or more diode lasers, an Er:YAG laser, or any other type of laser or optical energy source that can be used to generate fractional thermal damage in biological tissue. The control arrangement 118 can further include control circuitry to control an output of the energy source 115, e.g., to generate a plurality of timed pulses that coordinate with a translator, deflector, mirror, etc. to direct the pulses of energy onto particular locations on the target zone.
In further embodiments, the energy source 115 can be a flashlamp, a combustion lamp or the like, and the control arrangement 118 can work in conjunction with a plate 107, which can include a mask having a plurality of holes or other optically transparent openings therethrough provided between the energy source 115 and the target zone of tissue to be treated. Portions of the generated light can pass through the holes in the mask to irradiate portions of the target zone and generate MTZs, while the mask blocks light from irradiating tissue beneath the mask between the openings.
In still further embodiments, the energy source 115 can be a focused ultrasound device configured to direct a plurality of ultrasound pulses onto the target zone, and the control arrangement 118 can include circuitry, a translator, etc. configured to control the pattern of ultrasound energy applied to the tissue to form a plurality of MTZs.
In additional embodiments of the disclosure, the energy source 115 can include a plurality of small needles 180 configured to contact the surface of the target zone tissue or, alternatively, penetrate the tissue to a particular depth, when the apparatus 100 is placed on the target tissue. In further embodiments, the plurality or array of needles 180 can be configured to advance onto or into the tissue after the apparatus 100 has been placed on the tissue surface. In some embodiments, energy in the form of, e.g., rf energy (in monopolar or bipolar mode) can be directed into the needles 180 contacting or penetrating the target zone to form discrete MTZs therein. In still further embodiments, thermal energy provided by a resistance heater or the like can be conducted through the needles 180 to form MTZs in the target zone.
In general, the fractional arrangement 110 can be configured to direct energy, using any of the various modes described herein, onto or into a plurality of discrete locations on the target zone, to form MTZs that are, for example, less than about 1 millimeter wide, or less than 0.5 millimeters wide. The fractional areal coverage of the target zone by the MTZs can be, e.g., between about 5% and about 40%. This range of surface coverage can provide a sufficiently high volumetric amount of thermal preparation, such as denaturing or thermal damage in the target zone to achieve the desired effects described herein, while also preserving enough healthy tissue between the MTZs to facilitate rapid healing of the target zone and promote motility of the antigen-presenting cells and any other beneficial mechanisms that may operate to improve the vaccine efficacy. The target zone can be circular or square in shape, although other shapes can also be used. It can be between about 0.5 centimeters and 2 centimeters wide. This size range is generally large enough to provide a sufficient degree of local thermal damage in the skin tissue to achieve the beneficial results described herein, while not being so large as to create a target zone of thermally-treated tissue than is needed to enhance a dose of vaccine. In certain embodiments, the target zone can be somewhat larger or smaller than this exemplary range. For example, the target zone may be larger if a particular vaccine requires a relatively large volume of vaccine to be administered.
In embodiments where the fractional arrangement 110 provides optical energy to form the MTZs, each discrete location on the target zone can be irradiated with between, for example, about 1 mJ and 100 mJ of optical energy to form each of the MTZs. Such energy levels can be provided, e.g., by suitable configuration of the control arrangement 118 and/or control of the energy source 115.
The optical energy can have a wavelength greater than, for example, around 800 nanometers. For example, the optical energy can have a wavelength in any of the following examples of ranges: 1,200 to 1,600 nanometers, 1,850 to 2,100 nanometers, or 2,300 to 3,100 nanometers. Such wavelengths are often used in conventional NAFP devices and systems, as they are preferably absorbed by water as compared to pigment in the skin, such that the NAFP procedure will be relatively insensitive to pigmentation characteristics of the target zone. In some embodiments, the fractional arrangement 110 can deliver optical energy, for example, at 532 nanometers or 1410 nanometers to form MTZs in the target zone. In certain embodiments, optical energy containing a plurality of wavelengths can be used (e.g., the output of a flashlamp or combustion lamp).
In general, the control arrangement 118 can include control circuitry, switches, actuators, and/or other components configured to control an output of the energy source 115 for any of the energy modalities described herein, such that the energy source 115 will generate a plurality of MTZs having desired properties (e.g., percent of surface coverage, total energy per MTZ, etc.) in the target zone when activated.
The delivery arrangement 150 can include a reservoir 155 that contains the vaccine to be administered. For example, the reservoir 155 can be a removable and disposable vial that contains a single dose. In further embodiments, the reservoir 155 can be a container that holds a plurality of doses, where the delivery arrangement 150 is configured to deliver a single dose into the target zone when activated.
The delivery arrangement 150 further includes a needle 160 or other delivery conduit to direct the vaccine into the target zone. For example, the delivery arrangement 150 can include a fractional patch (such as a power-made patch), a microneedle-array patch, a microproject-array patch that contains vaccines printed in the same pattern as the pattern of MTZs on the target area, etc. to deliver the vaccine into the target zone. The microneedle or microproject-array can include biocompatible and/or dissolvable microneedles that can be formed using one or more vaccine compounds and various polymers. Other arrangements known in the art for delivering vaccines and similar substances into biological tissues can also be used in embodiments of the present disclosure. Certain components of the delivery arrangement 150, including but not limited to the reservoir 155, the needle 160, or the various patches can be disposable, e.g., they can be provided as a cartridge or other type of replaceable unit in the device.
The delivery arrangement 150 can be configured to deliver a dose of the vaccine into the target zone after the MTZs have been formed therein. For example, the needle 160 (if present) can be positioned to advance and penetrate a location within the target zone and inject the vaccine dose, as illustrated in
A lower portion of the housing 105 can be configured to be placed on or directly over the target zone, such that the delivery of energy by the fractional arrangement 110 and delivery of vaccine by the delivery arrangement 150 are each directed onto the target zone. In certain embodiments, the lower portion of the housing 105 can include a plate 107 configured to be placed over the target zone and allow passage of energy therethrough. For example, the plate 107, if present, can be optically transparent to the relevant wavelength(s) if the energy source 115 is configured to deliver optical energy onto the target zone to form MTZs. The plate 107 can include a plurality of holes 171 or openings therethrough that allow a plurality of needles to pass therethrough, if the energy source is configured to deliver rf or resistive heat energy through a needle array as described above. The plate 107 and energy source 115 can be configured to control the penetration depth of the plurality of needles into the target zone.
The plate 107, if present, can further include one or more channels 173 therethrough positioned to allow the needle 160 to pass therethrough, and to guide the needle 160 onto the target zone. In further embodiments, the lower portion of the housing 105 can include an opening configured to be placed over the target zone such that energy and the vaccine dose (from the fractional arrangement 110 and the delivery arrangement 150, respectively) are directed into the target zone. The plate 107 (or lower surface of the housing 105 if no plate 170 is present) and delivery arrangement 150 can be configured to control the penetration depth of the needle into the target zone. For example, these components can be configured such that the needle penetrates the target zone to a depth between about 0.5 and 2.5 millimeters to inject the vaccine. Microneedle arrays, if used, may penetrate to a similar or shallower depth. In this manner, the injection depth of the vaccine can be more precisely controlled than with an unsupported hypodermic needle.
Although a specific configuration of the components of the apparatus 100 are shown in
In certain embodiments, the fractional arrangement 110 and the delivery arrangement 150 can each be activated separately. In further embodiments, both the fractional arrangement 110 and the delivery arrangement 150 can be activated by a single switch, such that the fractional arrangement 110 forms MTZs in the target zone prior to administration of the vaccine dose by the delivery arrangement 150.
In a further embodiment, shown in
In a further embodiment, not illustrated in the figures, the fractional arrangement 110 and delivery arrangement 150 can also be provided as separate arrangements. A lower portion of the housing 205 of the fractional arrangement 110 can include a marking arrangement such as, e.g., an ink stamp or the like, that is configured to contact the surface of the target zone and outline the target zone. The delivery arrangement 150 can be a conventional syringe or the like. In typical use, this fractional arrangement 110 can be placed over the target zone and activated to form a plurality of MTZs within the target zone. The fractional arrangement 110 can then be removed from the target zone, where the marking arrangement 210 leaves an identifying marker on the tissue surface, e.g., to delineate the location of the treated target zone. The delivery arrangement 150 can then be directed onto the marked target zone and activated to deliver a vaccine does into the target zone.
In further embodiments, certain immune modifiers, such as agonists of toll-like receptors (TLRs), can be applied topically to the target zone and may synergistically augment the immune-enhancement mediated by the NAFP procedure. For example, it has been observed that NAFP can reduce skin irritation often caused by intradermal administration of influenza vaccines. NAFP followed by topical application of an immune response modifier, such as Imiquimod cream (5%), which is an agonist of toll-like receptor 7 (TLR-7), to the target zone did not worsen skin reaction induced by influenza vaccines but has been observed to further boost the associated immune responses. Such topical compounds can be applied to the target zone, e.g., manually, in a bandage or patch applied to the target zone, as a coating transferred from a portion of any exemplary apparatus or system described herein that contacts the target zone, etc. For example, a topical compound can be provided as a coating or layer on the lower portion of the housing 105 or plate 107 of apparatus 100, the lower portion of the housing 205 and/or the locating arrangement of system 200, etc.
Accordingly, systems and methods according to the present disclosure can both boost immune responses and reduce undesirable side effects. Further, in contrast to many vaccine adjuvants that alter a physical or chemical form of a given vaccine, NAFP is not vaccine-specific and can “prime” the body to generate improved responses to the vaccine. Accordingly, embodiments of the present disclosure can be used to boost any of a wide range of vaccine types.
In still further embodiments, the exemplary methods, apparatus and systems described herein can be used to deliver thermal energy to form MTZs and administer vaccines in target zones other than the surface of skin such as, e.g., mucosal areas to enhance efficacy of mucosal vaccinations or intramuscularly. For example, a catheter or fiber optic arrangement can be adapted to deliver optical energy to the nostril, the genital areas, the anus, muscle tissue, or other vaccination sites in the body in a pattern as described herein.
A number of exemplary studies of methods and observed results in accordance with embodiments of the present disclosure are presented below. Such examples, although not limited to the specific features described therein, can also be adapted to be used with the exemplary systems and devices described herein. In these Examples, “Laser” refers to treatment of the target zone with NAFP as described in the specific examples, although other energy modalities could also be used to form the MTZs in similar examples and applications.
The potency of Laser/Imiquimod creme (IMIQ, 3M Pharmaceuticals) adjuvant to enhance pandemic vaccine-induced humoral immune response was evaluated. Mice were intradermally immunized with 2009 H1N1 pandemic vaccine alone or in the presence of Laser, Imiquimod, or Laser/Imiquimod adjuvant. Four weeks later, serum HAI and specific antibody titer were quantified.
Young adult BALB/c mice (male, 6-8 weeks) were purchased from Charles River Laboratories. Old BALB/c mice (male, 18 months) were obtained from National Institute of Aging (NIA). Animals were housed in the animal facilities of Massachusetts General Hospital (MGH) and all procedures were in compliance with institutional, hospital, and NIH guidelines. A laser device was used that emits a 1410-nanometer laser light with 0.15 mrad beam divergence and 10 ms pulse duration, except as otherwise specified. One treatment using this device generates a 5×8 array of microthermal zones (MTZs) in a 7×10 mm2 area. In the current study, two treatments at the medium power were used to generate overlapped MTZs in the same target zone. Pandemic influenza virus A/California/7/2009 (H1N1) was obtained from American Type Culture Collection (ATCC, #FR-201). Influenza viruses were expanded in 10-day old embryonated chicken eggs at 35° C. for 3 days. Monovalent 2009 H1N1 pandemic vaccine was obtained from MGH pharmacy. 2009 H1N1 virus was adapted in mice for use in challenge studies after three cycles of intranasal instillation-lung homogenate preparation. The 50% tissue culture infectious dose (TCID50) of live virus was determined in Madin-Darby canine kidney (MDCK, ATCC, #CCL-34) cells. 50% lethal dose (LD50) was determined in young adult and old mice.
Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (20 mg/kg) for hair removal, immunization, and challenge. Mice were intradermally injected with 2009 H1N1 pandemic vaccine (0.06 μg HA antigen) by an insulin syringe equipped with a 29G hypodermic needle (ID) alone or with prior NAFP treatment of the target zone (Laser), post topical application of IMIQ, or both (Laser/IMIQ). Imiquimod-treated target zones were covered with a 3M Tegaderm patch to increase the adjuvant effects based on preliminary tests. Mice were challenged by intranasal instillation of 10×LD50 mouse-adapted 2009 H1N1 viruses. Body weight and survival were monitored for 14 days except otherwise specified. Mice with more than 25% body weight drop were euthanized. In some experiments, lung was harvested four days after challenge to measure lung viral titer by TCID50 assay.
The Hemagglutination inhibition (HAI) antibody titers assay was performed following published protocols (Koutsonanos D. G., PLoS One. 2009; 4(3)). Briefly, serum samples were incubated with receptor-destroying enzyme (Denka Seiken, Tokyo, Japan) at 37° C. overnight followed by heat inactivation at 56° C. for 30 minutes. Serum samples were then subjected to a 2-fold serial dilution, incubated with 4 HA units of pandemic viruses at 37° C. for 1 hour, and incubated with 0.5% chicken red blood cells (Charles River Laboratories) at room temperature for 30 minutes. The HAI titer was defined as the reciprocal of the highest dilution that inhibited hemagglutination. Vaccine-specific IgG, IgG1, and IgG2a antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). In brief, influenza vaccine containing 1 μg/ml HA was coated onto the ELISA plate. After incubation with serially diluted serum samples, HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a antibodies (Invitrogen, CA) were added to detect specific antibody subtypes.
Four weeks later, serum HAI (
As shown in
Due to the importance of cell-mediated immune response in clearance of virus-infected cells and the indication that Laser/IMIQ may induce cellular immunity based on the results above, the stimulation of IFNγ-secreting CD4+ and CD8+ cells in peripheral blood mononuclear cells (PBMCs) 7 days after immunization was measured. One week after mouse immunization as described above, 100-150 μl blood was collected in a heparinized tube by tail vein bleeding. Peripheral blood mononuclear cells were then isolated after red blood cell lysis. Peripheral blood mononuclear cells (106 cells/ml) were incubated in the presence of influenza vaccine (1 μg/ml HA content) and anti-CD28 antibodies (4 μg/ml) (BD Bioscience Pharmingen, San Diego, Calif.) overnight. Golgi-Plug was then added to prevent cytokine secretion. Five hours later, peripheral blood mononuclear cells were harvested, stained with fluorescence conjugated anti-CD4, -CD8, and -IFNγ antibodies and subjected to flow cytometry analysis. Non-immunized mice were used as control. Percentages of IFNγ-secreting CD4+ T cells and CD8+ T cells in peripheral blood mononuclear cells were significantly increased in Laser/IMIQ group as compared to Laser or IMIQ group (
BALB/c mice as described in Example 1 were intradermally immunized with pandemic vaccine (0.06 μg HA antigen) alone or in the presence of Laser, Imiquimod, or Laser/Imiquimod adjuvant. Five weeks later, immunized mice as well as non-immunized control mice were intranasally challenged with 10×LD50 of mouse-adapted 2009 H1N1 pandemic virus (0.06 μg HA antigen). Four days later, mice were euthanized and lung was isolated, homogenized and centrifuged.
Supernatants were used to infect MDCK cells to determine lung viral titer (
Percent survival (
Next, the dose-sparing effects of Laser/IMIQ were evaluated. Mice were intradermally immunized with high doses of pandemic vaccine (0.6 and 0.3 μg HA) without adjuvant or low doses of pandemic vaccine (0.06 and 0.03 μg HA) with or without Laser/IMIQ adjuvant. Four weeks later, serum HAI and IgG titer were measured. As shown in
Due to the importance of DCs to prime specific T and B cell responses, total and mature DCs were quantified in draining LNs. BALB/c mice were ID immunized with pandemic vaccine (0.06 μg HA antigen) alone or in the presence of Laser, Imiquimod, or Laser/Imiquimod adjuvant. Six and 18 hours later, draining LNs were harvested and total DCs and CD40- and CD80-expressing DCs were quantified. Single-cell suspensions were prepared, counted, stained with FITC-anti-CD11c (N418), APC-anti-CD40 (3/23) and PerCp-Cy5.5-anti-CD86 (GL-1) antibodies and analyzed by flow cytometry. Total CD11c+ cells and CD40- and CD86-expressed CD11c+ cells per LN were then calculated based on total LN cell number and percentage of each cell subsets.
Representative histograms of CD40+ and CD86+DCs 6 hours after immunization are shown in
Elderly populations often have a low response rate to seasonal or pandemic vaccine immunization due to age-induced immunosenescence. To find whether Laser/IMIQ adjuvant can enhance pandemic vaccine-induced immune response in the elderly, BALB/c mice (18 months old) were ID immunized with 0.6 μg pandemic vaccine without adjuvant or in the presence of Laser, IMIQ, or Laser/IMIQ adjuvant. Intramuscular (IM) immunization of old and young adult mice was used as controls. Four weeks later, serum HAI and IgG antibody titer were measured. Laser/IMIQ adjuvant significantly increased HAI antibody titer at levels comparable to or higher than IM immunization of the same antigen dose in young adult mice. No significant HAI antibody production in old mice IM immunized with the same amount of antigen was observed (
Given the importance of cell mediated immunity in immune protection against flu infection in the elderly, cell-mediated immune response was also evaluated. To measure cellular immune response, percentage of IFNγ-secreting CD4+ and CD8+ T cells was analyzed as in young adult mice, 1 week after immunization. In comparison with Laser or IMIQ alone, the percentages of both IFNγ-secreting CD4+ T cells and IFNγ-secreting CD8+ T cells were increased significantly in Laser/IMIQ group as compared to controls (
The potency of Laser/IMIQ adjuvant to enhance pandemic vaccine-induced immune response was further explored in larger mammals having skin properties similar to humans. Yorkshire pigs (˜4 months old) were obtained from Tufts University. Hair on the exterior hind leg skin of Yorkshire pigs was shaved. A 1 ml syringe equipped with a 26G hypodermic needle was used to inject 100 μl 2009 pandemic vaccine 2009 H1N1 vaccine (3 μg HA antigen) into the exterior hind leg skin alone or in the presence of Laser, Imiquimod, or Laser/Imiquimod adjuvant. Pigs were anesthetized by intramuscular injection of telazol (2.2 mg/kg)/xylazine (2.2 mg/kg)/Atropine (0.04 mg/kg) and maintained under inhalation isoflurane (2-3%) during hair removal and immunization.
Because the MTZs induced by the laser were limited within a depth of 200 μm from the skin surface, which was mainly within the epidermal tissue of the pig skin, a different clinical laser (Fraxel SR-1500) was used to generate MTZs to reach the dermal tissue of the pig skin. No significant difference was found in HAI antibody titer (
Whether a combination of the laser illumination, ID Flu vaccine, and Imiquimod caused any undesirable side effects was examined. No significant alteration in the skin appearance in mice was observed. As can be seen in
NAFP generates an array of MTZs that are micro-columns of thermally denatured skin with controlled width and depth, leaving up to 95% of the skin uninvolved. The healthy surrounding tissue allows fast epidermal repair via migration of the surrounding viable cells. The laser does not produce any holes in the skin as stratum corneum remains “intact,” protecting bacteria from entering the skin. However, the permeability of the MTZs to molecules may be increased, allowing better penetration of topically applied Imiquimod to the dermis and interaction with antigen-presenting cells around each MTZs (
These injured cells in MTZs can release “danger signals” that are recognized by the immune system to elicit inflammation aimed at clearance of the injured cells as the first step of a normal healing process, also called sterile inflammation. Such host-derived non-microbial stimuli released following tissue injury or cell death can activate the host immune system to induce sterile inflammation that either resolves the injury or leads to autoimmune disease. The “danger signals” released from the dead cells may include double strand (Ds) DNA, ATP, RNA, uric acid, hyaluronan, and so on. The “danger signals” can be sensed by Toll-like receptors (TLRs) on antigen presenting cells (APCs), which activates inflammasomes resulting in production of ILL IL-12, IFNβ1, etc. Indeed, non-ablative fractional laser (NAFL) significantly induced the production of these pro-inflammatory cytokines at the skin in the absence of antigen (
Antigen-presenting cell distribution following laser illumination in MHC II-EGFP mice was tracked by intravital confocal scanning microscopy. MHC-II molecule is expressed in antigen-presenting cells that include macrophages, dendritic cells, and B cells. As can be seen in
The over-the-counter, handheld, cosmetic NAFL generated a 6×9 array of MTZs in a 7×10 mm2 area of the skin. Each MTZ was similar in size as a hair, about 200 μm in diameter and 300 μm in depth and healed quickly, giving rise to new and younger skin. The sterile inflammation induced by laser-damage cells was restricted around each MTZ, leaving a majority of tissues unaffected, which ensured quick resolution of the sterile inflammation. To test whether this short life sterile inflammation could augment vaccination, a clinic H1N1 flu vaccine (A/California/7/2009) was ID inoculated into the site of laser illumination. After 4 weeks, hemagglutinin inhibition (HAI) antibody titers were found to be significantly higher in the presence of laser pre-treatment than in the absence of the treatment (
In humans, a majority of people are primed with influenza viral antigens either via vaccination or natural infection. To test if the adjuvant could boost the immune response efficiently in antigen-primed subjects, mice were primed with H1N1 influenza vaccine with or without NAFL or NAFL/IMIQ, followed by a booster immunization two weeks later. The adjuvant was found to boost immune responses in primed subjects as effectively as in naïve ones, regardless of whether or not the primary vaccination was given flu vaccine alone or the vaccine in combination with NAFL or NAFL/IMIQ adjuvant.
Measurement of serum IgG1 and IgG2a antibody titers revealed Th1-skewed immune responses evoked by the NAFL/IMIQ adjuvant. It profoundly elevated IgG2a production by 15-folds whereas only a 5-fold increase was seen with IMIQ or none with NAFL (
The superior humoral immune response was sustained at a ˜1:160 HAI titer for at least 9 months without any sign of decline (
Protection Against Viral Infection
The immunized mice were challenged with a mouse-adapted A/California/7/2009 H1N1 virus. The NAFL/IMIQ adjuvant greatly reduced lung viral titer to 2.2×101 PFU/mg tissue (p<0.001, ANOVA/Bonferroni) which was 1,000 times lower than that attained in mice receiving the vaccine alone (
A Favorable Safety Profile Compared to Squalene Based Adjuvant
A safety profile of the adjuvant was compared with that of AddaVax (Invivogen), which has a similar formulation and physical/chemical features as MF59 used in licensed flu vaccines in Europe. IM injection of flu vaccine emulsified with an equal amount of AddaVax provoked immune responses slightly inferior to that of the NAFL/IMIQ adjuvant, as measured by IgG and HAI titers (
Its safety and potency was further evaluated in swine because porcine skin resembles human skin in term of anatomy, immune responses, and pharmacokinetics. Because of a thicker skin of pigs than mice, we used another cosmetic, non-ablative fractional laser, named Fraxel SR-1500 (Solta Medical) for pig studies. This clinical device emitted a laser with 17% coverage, 93 MTZs/cm2/pass and 35 mJ/microbeam. After one pass of the laser treatment, the pigs were ID vaccinated with 3 μg HA of 2009 H1N1 vaccine, equivalent to a full dose of ID Flu vaccine in humans in the basis of body weight. The immunization enhanced HAI titers by ˜2-fold compared to ID vaccine alone (
To determine the underlying mechanism, we tracked MHC II+ cells at the inoculation site with or without laser treatment in mice expressing MHC II infused with green fluorescent protein (GFP). The MHC II+ cells in the skin are mainly DCs, Langerhans cells, and macrophages, broadly defined as APCs. An increase in the motility of APCs was noticed soon after laser treatment and these cells were gradually recruited to the vicinity of MTZs. Their accumulation around individual MTZs became apparent as early as 3 hours and peaked 24 hours after laser illumination (
CD11c+ cells only increased in ipsilateral lymph nodes but not in contralateral lymph nodes indicating that the adjuvant effect of NAFL/IMIQ impacts locally rather than systemically. Therefore we focused on local events to identify the underlying mechanism of synergistic adjuvant effect of NAFL and IMIQ. The active recruitment of APCs around each MTZ promoted us to study local chemokine production stimulated by NAFL treatment. Six out of nine chemokines examined were elevated after 6 hours of NAFL treatment, peaked in 24 hours, and dwindled down thereafter (
Among these cytokines induced at the inoculation site, TNF-α has been demonstrated to enhance emigration of dermal DCs into dLNs. We thus ID injected a TNF-α inhibitor, soluble TNF-Receptor Type I (TNF RI) into the inoculation site following the immunization. The inhibitor hampered adjuvant effects of NAFL/IMIQ, in particular, on IgG2a production (p<0.001, t-test), highlighting a critical role for TNF-α in NAFL/IMIQ-mediated adjuvanicity. Moreover, the adjuvanicity of NAFL/IMIQ in Th1 immunity was impaired in mice deficient in IFN regulatory factor? (IRF7−/−), suggesting contribution of IFN-α/β, two major activators of immature DCs to the immune-enhancement of NAFL/IMIQ as well. The Th1-predominant cytokine milieu at the inoculation site was pivotal for increasing percentages and numbers (
To directly address a pivotal role of pDCs in the synergistic adjuvant effect of NAFL and IMIQ, we depleted pDCs in Balb/c mice by injections of anti-mPDCA-1 antibodies prior to immunization as previously described. pDC depletion did not affect IgG1 levels, but profoundly impaired IgG2a production in the NAFL/IMIQ group (
Given the strong cell-mediated immune response evoked by the new adjuvant, we extended our investigation to old mice, because the elderly respond to current seasonal flu vaccine poorly. Yet, it is this population who suffers from a highest level of morbidity and mortality after flu infection and need the vaccine most. As can be seen in
Many vaccine adjuvants are being developed in preclinical studies or in various stages of clinical trials and the potency of these conventional adjuvants often comes at a great expense of safety. In markedly contrast, the current investigation explores a novel adjuvant that limits adverse effects locally and systemically while augmenting efficacy of flu vaccines over the vaccine alone or squalene-adjuvanted flu vaccines. Another advantage of this adjuvant is its standalone feature, like a topical adjuvant, which means neither pre-mixture nor re-formulation is required to extend it to any existing or new vaccines. Other TLR agonists may be employed in place of or in combination with IMIQ. The hand-held, small laser device is self-applicable and FDA approved for facial wrinkle removal at home, which requires a much higher stringent safety standard than laser illumination of a tiny spot on the upper arm for vaccination. The laser parameters in the pig study are well below those normally used in a clinical practice for skin resurfacing, and can be readily incorporated into the existing proprietary design of the small device. The 1410 nanometer light works on a stamp/scanning fashion and is equally effective regardless of skin colors, in contrast to the 532 nanometer laser adjuvant described previously. Its microprobe is tightly sealed by a thin plastic that prevents any skin materials from contaminating the probe, and the sealer is readily cleaned aseptically to prevent person-to-person contamination. The laser does not damage the stratum corneum or the most out layer of epidermis so that the integrity of skin barrier is preserved (
The mechanisms underlying the ability of NAFL/IMIQ adjuvant to augment flu vaccine while reducing adverse events are likely to be multifaceted. Laser-damaged cells in each MTZ release danger signals that stimulate the production of a number of chemokines and preferably attract pDCs from circulation leading to their increased presence around each MTZ. These pDCs are then activated by IMIQ penetrating from the skin surface because the cells express a high level of TLR7. Upon activation they secrete TNF-α, IFN-α/β and the like, and these cytokines direct maturation and differentiation of APCs in situ, and accelerate trafficking of these matured APCs into the dLNs via lymphatic vessels. NAFL-induced recruitment and IMIQ-mediated activation are likely to be the primary mechanisms for the observed synergy of these two adjuvants, resulting in a high level of TNF-α secretion at the inoculation site. An essential part of TNF-α in maturation and emigration of dermal DCs into dLNs was clearly demonstrated by the ability of TNF-α inhibitor to impede immune enhancement of NAFL/IMIQ (
Another major finding of this investigation is that the micro sterile inflammation induced by the adjuvant occurs only briefly, peaking at 24 hours and subsiding in 48 hours, but this short period of local sterile inflammation appears sufficiently enough to educate DCs in bridging an innate to adaptive immune responses. The fast resolution of the inflammation at the inoculation site warrants the super safety of the approach. On the contrary, injection of Alum or MF59 stimulates persistent local inflammation at the inoculation site, which is however not necessary for their adjuvant effects. An elegant experiment conducted by Hutchison et at corroborated that prolonged alum-induced local inflammation was dispense for alum adjuvant. They found that alum-mediated enhancement in antigen presentation and DC activation occurred primarily in the first 24-48 hours. A surgical removal of alum-containing site of inoculation 2 hours after vaccination did not affect the adjuvanticity of alum, an argument strongly supported by the current investigation. The controllable sterile inflammation at the inoculation site via a micro-sterile inflammation array could thus become a gold standard for future safe adjuvant development. Besides a shortened period of local inflammation, infiltration of pDCs may also contribute to the reduced skin irradiation, as Gregorio et at reported that pDCs could rapidly infiltrate into skin in response of skin injury, and improve the skin recovery. Finally, a combination of laser and IMIQ selectively reduces the production of the cytokines of the IL-1 family and TSLP that are well known to contribute to the local skin irritation, which may be another mechanism for reducing local and systemic adverse events of this topical adjuvant.
The adjuvanticity of an ideal adjuvant should be localized and transient. Therefore, the finding that a strong immune response can be provoked by transient micro sterile inflammation at the inoculation site is of highly clinical significance for cutaneous vaccination. A growing body of evidence has shown that cutaneous vaccination is more effective than IM vaccination, as the skin is enriched in APCs and in networks of lymphatic vessels, in contrast to the muscle where few APCs reside. However, lack of a safe adjuvant for cutaneous vaccination hampers a broad application of this route of immunization, apart from inconvenience. Therefore, this micro sterile inflammation-based adjuvant merits clinic investigations for dose-sparing or augmenting flu vaccines in certain at risk groups like the elderly, with little concern of long term safety, because no any additive, apart from the vaccine itself is injected into the body.
Inbred BALB/c mice and outbred Swiss Webster mice at 6-8 weeks of age were purchased from Charles River Laboratories. Mice of both genders were used randomly with no notable difference. Eighteen-months-old BALB/c mice (old mice) were purchased from National Institute of Aging (NIA). Irf7−/− mice on C57BL/6J background were a kindly gift of Tokyo University and C57BL/6J control mice were obtained from Jackson Laboratories. MHC II-EGFP mice expressing MHC class II molecule infused into enhanced green fluorescent protein were a kindly gift of Harvard Medical School. Yorkshire pigs at 4 months of age were obtained from the Teaching and Research Resources at Tufts University. The animals were housed in the pathogen-free animal facilities of Massachusetts General Hospital (MGH) in compliance with institutional, hospital, and NIH guidelines. All studies were reviewed and approved by the MGH Institutional Animal Care and Use Committee.
A FDA-approved, home-use PaloVia laser was used in mice (PaloVia Skin Renewing Laser, Palomar Medical Technologies). The hand-held device emits a 1410-nanometer laser light and two passes at the medium power were used to generate overlapped MTZs at the inoculation site. Pigs were treated with one pass of Fraxel SR-1500 laser (Solta Medical) which produced a laser with 17% coverage, 93 MTZs/cm2/pass and 35 mJ/microbeam.
Pandemic A/California/7/2009 H1N1 flu virus was obtained from American Type Culture Collection (ATCC, #FR-201). The virus was expanded in 10-day-old embryonated chicken eggs at 35° C. for 3 days, harvested by ultracentrifugation, and frozen at −80° C. until use. Its quantity was determined with a 50% tissue culture infectious dose (TCID50) in Madin-Darby canine kidney (MDCK, ATCC, #CCL-34) cells. To challenge mice, the virus was adapted in mice for three cycles of intranasal instillation-lung homogenate preparation and infectivity of the resultant virus was assayed by a 50% lethal dose (LD50) in adult BALB/c mice following a standard protocol. Monovalent A/California/7/2009 H1N1 flu vaccine (Sanofi Pasteur, Inc.) was obtained from MGH pharmacy and BEI Resources, used at 0.06 μg/mouse or 3 μg/pig unless otherwise indicated.
Mice to be immunized were hair removed on the lower dorsal skin and ID inoculated next day with flu vaccine (ID) or illuminated with laser before flu or indicated vaccines were ID administered. After ID immunization, the inoculation site was either left alone or topically applied of Imiquimod cream (Aldara, 3M Pharmaceuticals). The inoculation site was then covered with a 3M Tegaderm film to protect it. For IM injection of adjuvanted flu vaccine, AddaVax®, a squalene based nano-emulsion adjuvant (Invivogen) with a formulation similar to commercial adjuvant MF59, was mixed at 1:1 ratio with flu vaccine (total volume 20 μl) and IM injected. Body temperature was monitored hourly after immunization in some of the mice by a temperature control device (FHC, ME). The immunized and control mice were challenged by intranasal instillation of 10×LD50 mouse-adapted 2009 H1N1 viruses. Body weight and survival were monitored daily for 14 days unless otherwise specified. In some infectious experiments, lungs were harvested 4 days after challenge to measure lung viral titer by TCID50 assay. To immunize pigs, the animals were anesthetized by IM injection of telazol (2.2 mg/kg)/xylazine (2.2 mg/kg)/atropine (0.04 mg/kg) and maintained under isoflurane (2-3%) inhalation during hair removal and immunization. Immunization procedure was similar as described in mice with 100 μl flu vaccine (3 μg HA content) inoculated into the exterior hind leg skin either alone or in the presence of NAFL, IMIQ, or NAFL/IMIQ adjuvant. To quantify local skin reactions, the inoculation sites were photographed and the erythema area at each inoculation site was circled and analyzed by Image Pro Premier software (Media Cybernetics, Inc.) for 3 times. Mean erythema area of each inoculation site was presented.
HAI titers were assayed according to a published protocol (PMID: 19274084). Briefly, serum samples were incubated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) at 37° C. overnight followed by heat inactivation at 56° C. for 30 minutes. The resultant serum samples were incubated with 4 hemagglutination (HA) units of flu virus at 37° C. for 1 hour after serial dilutions, and then with 0.5% chicken red blood cells (Charles River Laboratories) at room temperature for 30 minutes. The HAI titer was defined as the reciprocal of the highest dilution that inhibited hemagglutination.
Vaccine-specific IgG, IgG1, IgG2a and IgA antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). In brief, 1 μg/ml recombinant HA was coated onto ELISA plate in NaHCO3 buffer, pH 9.6. After incubation with serially diluted serum samples, HRP-conjugated goat anti-mouse IgG (NA931V), IgG1 (A90-105P), IgG2a (61-0220) or IgA (A90-103P) antibody was added to measure specific subtypes. For C57BL6 mice, anti-mouse IgG2c (1079-05) antibody was used in place of anti-IgG2a antibody.
One week after immunization, small blood samples were collected from immunized and control mice in a heparinized tube by tail vein bleeding. Peripheral blood mononuclear cells (PBMCs) were isolated after red blood cell lysis. PBMCs (106 cells/ml) were incubated with flu vaccine (1 μg/ml HA content) and anti-CD28 (37.51) antibody (4 μg/ml) overnight. Golgi-Plug was added to prevent cytokine secretion in the additional 5 hours of the incubation. The stimulated cells were stained with fluorescence conjugated anti- CD4 (RM4-5), -CD8 (53-6.7), and -IFNγ, (XMG1.2) antibodies and subjected to flow cytometric analysis as previously reported.
Mice were ID immunized with flu vaccine in the presence of NAFL/IMIQ adjuvant or IM with the vaccine mixed with AddaVax. One, three or seven days later, the tissues at the inoculation site were dissected, fixed and stained by a standard H&E procedure. Similar histological examination was carried out in pigs after 3 days of immunization. The slides were scanned and analyzed by NanoZoomer (Hamamatsu).
The ear of MHC II-EGFP transgenic mice was treated by NAFL or NAFL/IMIQ. GFP+ cells in epidermis and dermis were imaged by intravital two-photon confocal microscopy (Olympus FV-1000) as described. Three-D reconstruction was used to visualize accumulation of GFP+ cells around individual MTZs by Image J software.
The ear of MHC II-EGFP transgenic mice was immunized with flu vaccine in the presence of the indicated adjuvants. The outer ear flaps were prepared at indicated times post-immunization, fixed by 4% formaldehyde at 4° C. overnight, and blocked by 2% FBS/PBS for 2 hours at room temperature. The fixed samples were reacted with rat anti-LYVE-1 (223322) antibody to identify lymphatic vessels and rabbit anti-Collagen IV (ab6586) antibody at 4° C. overnight to label both lymphatic and blood vessels, after which the samples were stained with Cy3-conjugated anti-rat (A10522) and NL637-conjugated anti-rabbit (NL005) antibodies at 4° C. overnight. The stained samples were mounted and imaged by two-photon confocal microscopy (Olympus FV-1000).
The lower dorsal skin of mice was exposed to NAFL/IMIQ adjuvant. Twenty four hours later, full thickness of the skin at the site of laser illumination was excised, and frozen tissue sections were prepared and stained by anti-mouse Siglec H (440c) or isotype control antibody and DL594 goat anti-rat IgG (SA5-10081) antibody. The slides were mounted with DAPI contained mount medium, and imaged by confocal microscopy.
Skin at the inoculation site was excised, minced, and digested with Dispase II (Invitrogen, NY) and Collagenase D (Roche, Ind.) for 2 hours at 37° C. The digested skin tissues were passed through a 100 μm cell strainer to prepare single cell suspensions. The resultant cells were treated with anti-CD16/CD32 (93) for 20 minutes, followed by staining with the following fluorescence antibodies for 30 minutes on ice: APC-anti-PDCA-1(927), PE-anti-CD11c (N418), BV421-anti-CD11b (M1/70), FITC-anti-MHCII (M5/114.15.2), FITCanti-Ly6C (HK1.4), APC/Cy7-anti-B220 (RA3-6B2). The stained cells were acquired on a FACSAria (BD) and analyzed using FlowJo software (Tree Star).
Balb/c mice were i.p. injected with Anti-mPDCA-1 (JF05-1C2.4.1, Miltenyi Biotec Inc.) antibody (400 μg/mouse/time) twice at 24 and 0 hours before immunization as previously described. The depletion efficiency was confirmed by flow cytometry of pDCs in the blood samples. The cellular immune responses were analyzed one week later, and humoral immune responses were analyzed two weeks after immunization.
dLNs were collected and single-cell suspensions were prepared, counted, stained with FITC-anti-CD11c (N418), APC-anti-CD40 (3/23) and PerCp-Cy5.5-anti-CD86 (GL-1) antibodies and analyzed by flow cytometry as previously described. Total CD11c+ cells and CD40+ and CD86+ CD11c+ cells per LN were calculated in the basis of the total LN cell number and percentages of each cell subsets.
To analyze chemokine and inflammatory cytokine expression at the inoculation site, the full thickness of the skin area about 7×10 mm2 was excised, and total RNA was extracted, reverse-transcribed, and amplified by real-time qPCR using a SYBR Green PCR kit (Roche, Ind.). GAPDH was used as an internal control. All the primers were synthesized at MGH DNA core.
Two tailed t-test (t-test) was used to analyze a difference between two groups, and one way ANOVA was used among multiple groups. ANOVA followed by Bonferroni correction (ANOVA/Bonferroni) was used to compare selected pairs, and ANOVA followed by Dunnett's test (ANOVA/Dunnett's) was for comparing all groups with control group. The appropriate nonparametric tests were used if data failed to meet assumptions for parametric statistics. P value was calculated by PRISM software (GraphPad, CA) and a difference was regarded significant if p was less than 0.05. No animals were excluded from the analysis. The investigators were not blinded to the experiments which were carried out under highly standardized and predefined conditions, except for photo analysis and H&E slide examination.
The foregoing merely illustrates the principles of the invention. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous techniques which, although not explicitly described herein, embody the principles of the invention and are thus within the spirit and scope of the invention. All patents and publications cited herein are incorporated herein by reference in their entireties.
This application is a 371 application of PCT/US2014/025659 filed Mar. 13, 2014 which claims priority based on U.S. Provisional Patent Application No. 61/789,775 filed Mar. 15, 2013.
This invention was made with government support under grant numbers AI089779, AI070785, AI097696, and DA028378 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/025659 | 3/13/2014 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/151403 | 9/25/2014 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20010050083 | Marchitto | Dec 2001 | A1 |
| 20050049582 | DeBenedictis | Mar 2005 | A1 |
| 20050112135 | Cormier | May 2005 | A1 |
| 20060079947 | Tankovich | Apr 2006 | A1 |
| 20070142885 | Hantash | Jun 2007 | A1 |
| 20080208179 | Chan | Aug 2008 | A1 |
| 20080269735 | Vila Echague | Oct 2008 | A1 |
| 20090069741 | Altshuler | Mar 2009 | A1 |
| 20100082019 | Neev | Apr 2010 | A1 |
| 20110150924 | Della Rocca | Jun 2011 | A1 |
| 20110218464 | Iger | Sep 2011 | A1 |
| 20120143056 | Slayton | Jun 2012 | A1 |
| 20120150266 | Shalev | Jun 2012 | A1 |
| 20130066237 | Smotrich | Mar 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 2013033496 | Mar 2013 | WO |
| Entry |
|---|
| Schwan, H.P., Ultrasound and Electromagnetic Radiation in Hyperthermia—A Historical Perspective, Br. J. Cancer Suppl., Mar. 1982, vol. 5, pp. 84-92, p. 85, col. 2, paragraph 3, p. 91, col. 1, paragraphs 2, 4. |
| Manstein, D et al., Fractional Photothermolysis: A New Concept for Cutaneous Remodeling Using Microscopic Patterns of Thermal Injury, Laser Surg. Med. 2004, vol. 34, No. 5, pp. 426-438, abstract, p. 427, col. 1, paragraph 2, p. 427, col. 2, paragraph 5, figure 1c, Table 1, p. 428, col. 2, paragraph 1. |
| International Search Report and Written Opinion dated Jul. 24, 2014 for International Application No. PCT/US2014/025659. |
| Lee, WR et al. Laser-Assisted Topical Drug Delivery by Using a Low-Fluence Fractional Laser: Imiquimod and Macromolecules, Journal of Controlled Release, Aug. 10, 2011, vol. 153, No. 3, pp. 240-248, abstract, p. 240, col. 1, paragraph 2, p. 241, col. 1, paragraphs 1, 5. |
| Wilck, MB et al., Safety and Immunogenicity of Modified Vaccinia Ankara (ACAM3000): Effect of Dose and Route of Administration, J. Infect. Dis., May 1, 2010, vol. 201, No. 9, pp. 1361-1370, abstract, p. 1368, col. 1, paragraph 2. |
| Lee, B. et al., A Predictive Model of the Economic Effects of an Influenza Vaccine Adjuvant for the Older Adult (Age 65 and Over) Population, Vaccine, Apr. 6, 2009, vol. 27, No. 16, pp. 2251-2257, abstract, p. 2251, col. 1, paragraphs 3-4. |
| Number | Date | Country | |
|---|---|---|---|
| 20160038591 A1 | Feb 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61789775 | Mar 2013 | US |
