Near-Infrared Laser Adjuvant for Influenza Vaccine

Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants

Effect of the laser adjuvant on cell-mediated anti-influenza immune responses.

<p>Systemic CD4+ helper T-cell responses were measured 4 days after challenge by re-stimulating 2×10⁶ splenocytes with a nucleoprotein (NP) major histocompatibility class-II complex (MHC) or class-I influenza-specific peptide. Percentages of (A) CD4+IFN-γ+ (B) CD4+IL-5+ (C) CD4+IL-17+ and (D) CD8+IFN-γ+ T cells are shown. Also shown are representative FACS plots. Error bars show means ± s.e.m. Experimental and control groups: (A–D) <i>n</i> = 16, 18, 13, 20, 10 for no vaccine, vaccine i.d., vaccine i.d. + PW 532 nm, vaccine i.d. + CW 1064 nm, and vaccine + Alum i.d. vaccine groups, respectively.</p

Effect of the near-infrared (NIR) laser adjuvant on the function of dendritic cells (DCs).

<p>A–E, Quantitation of DC activation markers (A) MHC class-II, (B) CD40, (C) CD86 and (D) CD80, and (E) the number of CD11c+OVA₆₄₇+ DCs in skin-draining lymph nodes 24 hours after vaccination with 40 µg Alexa Fluor-647-labeled OVA (OVA₆₄₇) with or without the 1-minute CW 1064 nm NIR laser treatment. Data are the ratio of median fluorescence intensity (MFI) of each marker normalized to no OVA controls. <i>n</i> = 7, 9, 15 for no OVA, OVA₆₄₇ i.d., and OVA₆₄₇ i.d. + CW 1064 nm, respectively; ANOVA with Bonferroni correction. AU, arbitorary units. A–E, Data are derived from at least three independent experiments.</p

List of enrolled subjects and tolerated doses.

<p>Study subjects were screened for inclusion and exclusion criteria detailed in Materials and Methods. Subjects were selected with either skin phototype V or VI because, at 1064 nm, levels of laser power and exposure time that proved to be non-painful and non-damaging in subjects with the darkest skin types would be predicted to be non-painful and non-damaging for all other skin types.</p

Effect of laser on the humoral immune response to a model vaccine.

<p>A, Serum ovalbumin- (OVA)-specific IgG titers 3, 6, and 12 weeks following vaccination with 40 µg OVA with or without laser illumination. Endpoint titer of OVA-specific serum IgG was determined by ELISA. Plates were coated with OVA. <i>n</i> = 33, 19, 15, 12 and 11, 5 and 6, 6 and 6 for no OVA, OVA i.d., OVA +Alum i.d., OVA i.d. + PW 532 nm 1 and 4 minutes, OVA i.d. + PW 1064 nm 1 and 4 minutes, OVA i.d. + CW 1064 nm 1 and 4 minutes, respectively. Error bars show means ± s.e.m. *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 as compared to OVA i.d. B, The relationships between anti-OVA antibody titers following 1–4 minutes PW 532 nm, PW 1064 nm and CW 1064 nm laser-treated groups at 6 weeks (logIgG) and maximal skin surface temperature (Tm) was not statistically significant; a Pearson's correlation coefficient <i>r</i> = −0.237 (<i>P</i> = 0.08), where log(IgG) = −0.193Tm+10.75 (linear regression, R² = 0.056). <i>n</i> = 20, 17, 18, for OVA i.d. + PW 532 nm 1–4 minutes, OVA i.d. + PW 1064 nm 1–4 minutes, OVA i.d. + CW 1064 nm 1–4 minutes.</p

Reported sensations and signs of skin damage on each NIR laser dose.

<p>Consenting human subjects were exposed to a range of laser doses from 0.5 to 3.7 W/cm² (16 doses) each up to 120 seconds. Sensations felt by subjects were classified as mild (warmth, tingling, itching, pinprick/needle sensations), moderate (hotness, dull pain), or severe (burning, sharp pain), and recorded. The operator also recorded any signs of skin damage.</p>1<p>Warmth</p>2<p>Hotness</p>3<p>Burning</p>4<p>Pinprick/needle sensations</p>5<p>Dull pain</p>6<p>Sharp pain</p>7<p>Tingling</p>8<p>Itching</p>9<p>Skin appearance change; No changes in skin appearance or damage were noted on all exposures, except transient skin darkening (transient hyperpigmentation) occurred in some subjects, which was due to changes in capillary blood flow in the treated area. These changes were not observed during follow-up examination after 2 hours.</p>10<p>Skin damage</p

NIR laser safety study in humans.

<p>A, Schedule of laser treatment and follow-up skin appearance documentation. Five healthy adults aged 20 to 46 years old with either skin phototype V or VI were enrolled. B, A plastic grid was used to separate the laser exposure sites. An aqueous gel was applied in each section of the grid to enhance the dissipation of heat from the skin's surface. The bar indicates 1 inch. C and D, Representative images of the laser-exposed skin are shown at (C) 1 hour and (D) 2 days after completion of the treatment. No detectable skin damage on visual inspection was observed following laser exposure at any irradiance used.</p

Effect of laser on skin tissue.

<p>A–C, Dose-temperature responses of the PW 532 nm laser and the PW and CW 1064 nm laser in mouse skin. <i>n</i> = 1–4 (4–16 exposures in total) for each group. PW, pulse wave; CW, continuous wave; Tm, maximal skin surface temperature. Error bars show means ± s.e.m. D, Images of the back of mice for visual inspection at 0 and 24 hours after the CW 1064 nm NIR laser treatment. Representative images for each group are presented. A–D, <i>n</i> = 1–4 (4–16 exposures in total) for each group. E, Microscopic assessment of skin damage and inflammatory infiltration after laser treatment. Representative time-course images of hematoxylin-eosin-stained skin tissue are presented. The bar indicates 50 µm. F, Quantification of polymorphonuclear leukocytes (PMN) after the NIR laser treatment. E–F, <i>n</i> = 3 for each group.</p

Effect of the laser adjuvant on protective immunity.

<p>A, EID₅₀ was determined by serial titration of lung homogenate in eggs at 4 days after challenge. EID₅₀, the 50% egg infectious dose. B, Kaplan-Meier survival plots of influenza-vaccinated mice for 15 days following lethal challenge; Gehan-Breslow-Wilcoxon test. C, HAI titers (C) in pre-challenge (4 weeks after vaccination) and (D) post-challenge (4 days after challenge) serum. Experimental and control groups: (A) <i>n</i> = 9, 10, 6, 9, 6 (B) <i>n</i> = 16, 20, 13, 21, 11 (C) <i>n</i> = 31, 38, 24, 41, 6 (D) <i>n</i> = 23, 31, 13, 34, 12 for no vaccine, vaccine i.d., vaccine i.d. + PW 532 nm, vaccine i.d. + CW 1064 nm, and vaccine + Alum i.d. vaccine groups, respectively.</p