A Novel Laser Vaccine Adjuvant Increases the Motility of Antigen Presenting Cells

Background Development of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research. Methodology/Principal Findings We found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag+ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone. Conclusion/Significance The results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.National Institutes of Health (U.S.) (grant AI070785)National Institutes of Health (U.S.) (grant RC1 DA028378)Bill & Melinda Gates Foundation (Grand Challenges Explorations grant # 53273)Boston BioCom (Firm) (Sponsored Research agreement grant #2008A25652

Laser enhances Ag-uptake by DCs in the skin.

<p><b>A.</b> Representative flow cytometry histograms showing percentages of OVA⁺ CD11c⁺ cells. Single cell suspension was prepared from full thickness of the skin 6 hrs after 0.5 µg AF647-OVA in 20 µl or 20 µl PBS was <i>i.d.</i> administrated into the site of laser illumination or control. The cells were stained with anti-CD11c antibody, and analyzed for percentages of OVA⁺CD11c⁺ cells on the gated CD11c⁺ cell population. Percentages of OVA⁺ CD11c⁺ cells (<b>B</b> and <b>D</b>) and mean fluorescence intensity (MFI) of AF647-OVA (<b>C</b> and <b>E</b>) were analyzed at 6 hr (<b>B</b> and <b>C</b>) or 24 hr (<b>D</b> and <b>E</b>) after <i>i.d.</i> administration of AF647-OVA at the indicated doses. <i>n</i> = 6, blank bar, AF647-OVA alone; and black bar, laser + AF647-OVA. <b>F</b> and <b>G</b>. A light dose-dependent increase in Ag-uptake by DCs. The lower dorsal skin of mice was illuminated with laser at 0.3 W for the indicated time, corresponding to a light dose of 11.3, 22.5, 45, or 90 J/cm², respectively. AF674-OVA at 2 µg/ mouse was injected into the laser-illuminated site and 6 hrs later, percentages of OVA⁺ CD11c⁺ cells (<b>F</b>) and MFI of AF647-OVA (<b>G</b>) were analyzed and expressed as above.* and **, p<0.05 and 0.01, respectively.</p

Laser enhances cell-mediated immune responses induced by <i>i.m.</i> immunization.

<p>The posterior thigh muscle of BALB/c mice was illuminated by a 532-nm laser (KTP/532) delivered by a hair-like diffusing optical fiber followed by <i>i.m.</i> vaccination with 50 µg OVA (laser). Control groups received OVA immunization similarly without laser illumination (control). OVA-specific antibody (A) and IL4- and IFNγ-secreting CD4⁺ and CD8⁺ cells in the dLNs were analyzed three weeks later. OVA-specific Ab in the plasma was detected by ELISA as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013776#pone-0013776-g005" target="_blank">figure 5</a>. The numbers of IL4⁺-secreting CD4⁺ (B) and CD8+ cells (D) and IFNγ⁺-producing CD4⁺ (C) and CD8⁺ (E) cells per LN were identified by flow cytometric analyses after surface staining with anti-CD4 or anti-CD8 antibody followed by intracellular staining with anti-IL4 or anti-IFNγ Ab. n = 6, **, P<0.01; ***, P<0.001.</p

Laser increases Ag-uptake by DCs in the draining lymph node.

<p><b>A</b>. Representative flow cytometry profiles showing the number of OVA⁺CD11c⁺ cells. Single cell suspensions were prepared from the draining LN (inguinal) after 6 or 24 hrs of laser illumination and <i>i.d.</i> injection of 10 µg AF647-OVA. The cells were stained with anti-CD11c antibody and analyzed as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013776#pone-0013776-g003" target="_blank">Figure 3</a>. The absolute numbers of CD11c⁺OVA⁺ cells were given in the profile in one million cells counted. <b>B</b> and <b>C</b>. An increase in the number of Ag-captured DCs in the 1° draining LNs. The cells were prepared from the inguinal LN at 6 hr (<b>B</b>) or 24 hr (<b>C</b>) after laser illumination and AF647-OVA injection at the indicated dose and analyzed as above. Data are means ± SEM of absolute numbers of CD11c⁺OVA⁺ cells in 10⁴ LN cells from draining lymph nodes analyzed as <b>A</b>. <i>n</i> = 6; blank bar, AF647-OVA alone and black bar, laser + AF647-OVA. <b>D</b>. An increase in the number of Ag-captured DCs in the 2° draining LN at 24 hrs after 10 µg AF647-OVA was <i>i.d.</i> administered. The cells isolated from the ipsilateral axillary LN were analyzed and the data were expressed as above.</p

No significant alteration in skin histology after laser illumination for 2 min at 0.3 W.

<p>The lower dorsal skin of mice was exposed to laser at indicated output powers and times. Skin Tm was monitored during the exposure by an infrared camera (<b>A</b>). Histological examination was performed after 2 hrs (D0), 1 day (D1) or 3 days (D3) in the skin that was either illuminated with laser for 2 min at 0.3 W or <i>i.d.</i> injected with alum adjuvant (10 µl) or PBS as controls (<b>B</b>). Full thickness of skin tissues treated for 6 hrs as in <b>B</b> was evaluated for inflammatory cytokine expression by real-time RT-PCR (<b>C</b>). Scale bar in <b>B</b>, 100 µm. Data are representative of at least three experiments with similar results in <b>A</b> and <b>B</b>. Data in <b>C</b> are the means ± standard errors of the mean (SEM) of six samples. **, p<0.01.</p

Laser enhances flu vaccine-induced immune responses in both primary and booster immunizations.

<p>Laser augments not only primary (<b>A</b>) but also booster (<b>B</b>) immune responses. Flu-specific antibody was measured by ELISA at 3 weeks after the primary immunization with 0.3 µg flu vaccine at the site of laser illumination or control as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013776#pone-0013776-g005" target="_blank">Figure 5</a> (<b>A</b>). A booster immunization was carried out at the contralateral side similarly three weeks later after blood collection for assessing primary immune responses. Flu-specific antibody was detected in two weeks as above (<b>B</b>). Each symbol represents the data from individual mice. **, P<0.01; and ***, P<0.001.</p

Laser enhances humoral immune response induced by OVA.

<p><b>A</b>. Laser significantly augments and prolongs the production of serum OVA-specific antibody. Serum OVA-specific antibody was detected at indicated times after <i>i.d.</i> injection of 40 µg OVA with (filled) or without laser illumination (blank). <b>B</b>. A light-dose dependent augmentation of OVA-specific antibody production. The lower dorsal skin of mice received an increasing laser dose from 11.3 to 22.5, 45, 90, or 180 J/cm², which corresponded to irradiation at 0.3 W for 0.25, 0.5, 1, 2, and 4 min, respectively. <b>C</b>. Immune-enhancement effect of laser cannot be recapitulated with a Tm rise in the skin. Heat: the skin was warmed with a 42°C metal rod for 2 min mimicking the Tm rise caused by laser illumination as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013776#s2" target="_blank">Materials and Methods</a>. <b>D</b>. Laser-mediated immune enhancement is area-restricted. A significant increase in OVA-specific antibody production was attained only when OVA was injected into the illuminated site (direct) but not in a distal site (indirect). Serum Ab titers were measured 3 wks after immunization in <b>B</b>∼<b>D</b>. <b>E</b>. Laser increases the number of CD4⁺IL4-secreting T cells. T cells of OVA-immunized mice were activated <i>in vivo</i> for one week by <i>i.p.</i> injection of 40 µg OVA. CD4⁺IL4-secreting cells were analyzed by flow cytometry after surface staining with anti-CD4 antibody and then intracellular staining with anti-IL-4 antibody. Data are expressed as means ± SEM of absolute numbers of CD4⁺IL4⁺ cells in 10⁴ CD4⁺ splenocytes. <i>n</i> = 6 for each group except for <b>B</b> in which 3 mice were used in each time point. *, **, ***, <i>p</i><0.05, 0.01, and 0.001, respectively, using one-way analysis of variance (ANOVA) in <b>C</b> and <b>D</b>, and student <i>t</i>-test in <b>A</b> and <b>E</b>.</p