CD8+ T-cell exhaustion has been identified as a significant contributor to immunosuppression and immune escape in triple-negative breast cancer (TNBC). Dysfunction due to cell exhaustion is characterized by reduced effector capacity and sustained expression of inhibitory receptors (IRs). The factors contributing to CD8+ T-cell exhaustion are multifaceted, encompassing external influences such as the upregulation of IRs, reduction of effector cytokines, and internal changes within the immune cell, including transcriptomic alterations, epigenetic landscape remodeling, and metabolomic shifts. The impact of the altered TNBC tumor microenvironment (TME) on Tex is also a critical consideration. The production of exhausted CD8+ T-cells (CD8+ Tex) is positively correlated with poor prognosis and reduced response rates to immunotherapy in TNBC patients, underscoring the urgent need for the development of novel TNBC immunotherapeutic strategies that target the mechanisms of CD8+ T-cell exhaustion. This review delineates the dynamic trajectory of CD8+ T-cell exhaustion development in TNBC, provides an update on the latest research advancements in understanding its pathogenesis, and offers insights into potential immunotherapeutic strategies.

The majority of vaccines have been delivered into the muscular tissue. Skin contains large amounts of antigen-presenting cells and has been recognized as a more immunogenic site for vaccine delivery. Intradermal delivery has been approved to improve influenza vaccine efficacy and spare influenza vaccine doses. In response to the recent monkeypox outbreak, intradermal delivery has been also approved to stretch the limited monkeypox vaccine doses to immunize more people at risk. Incorporation of vaccine adjuvants is promising to further increase intradermal vaccine efficacy and spare more vaccine doses. Yet, intradermal vaccination is associated with more significant local reactions than intramuscular vaccination. Thus, adjuvants suitable to boost intradermal vaccination need to have a good local safety without inducing overt local reactions. This review introduces currently approved adjuvants in licensed human vaccines and their relative reactogenicity for intradermal delivery and then introduces emerging chemical and physical adjuvants with a good local safety to boost intradermal vaccination. The rational to develop physical adjuvants, the types of physical adjuvants, and the unique advantages of physical adjuvants to boost intradermal vaccination are also introduced in this review.

Nitric oxide (NO) is a well-known gaseous mediator that maintains vascular homeostasis. Extensive evidence supports that a hallmark of endothelial dysfunction, which leads to cardiovascular diseases, is endothelial NO deficiency. Thus, restoring endothelial NO represents a promising approach to treating cardiovascular complications. Despite many therapeutic agents having been shown to augment NO bioavailability under various pathological conditions, success in resulting clinical trials has remained elusive. There is solid evidence of diverse beneficial effects of the treatment with low-power near-infrared (NIR) light, defined as photobiomodulation (PBM). Although the precise mechanisms of action of PBM are still elusive, recent studies consistently report that PBM improves endothelial dysfunction via increasing bioavailable NO in a dose-dependent manner and open a feasible path to the use of PBM for treating cardiovascular diseases via augmenting NO bioavailability. In particular, the use of NIR light in the NIR-II window (1000-1700 nm) for PBM, which has reduced scattering and minimal tissue absorption with the largest penetration depth, is emerging as a promising therapy. In this review, we update recent findings on PBM and NO.

Novel adjuvants are highly demanded to aid in development of improved or new vaccines against existing or emerging infectious diseases. Considering commonly used Alum and MF59 adjuvants induce tissue stress and release of endogenous danger signals to mediate their adjuvant effects, physical modalities may be used to induce tissue stress and endogenous danger signal release to enhance vaccine-induced immune responses. Furthermore, physical adjuvants are less likely to induce significant systemic adverse reactions due to their localized effects. Recently we found non-invasive radiofrequency (RF) pretreatment of the skin could significantly enhance intradermal vaccine-induced immune responses in murine models that included pandemic influenza vaccine, pre-pandemic vaccine, and influenza internal antigen vaccine. It remained to be explored whether the physical RF adjuvant (RFA) could be used to boost seasonal influenza vaccination, spare vaccine doses, and induce cross-protective immunity. This study found the physical RFA could significantly enhance seasonal influenza vaccine-induced immune responses against each viral strain and robustly enhance low-dose (nanograms) H3N2 vaccine-induced immune responses and protection in murine models. RFA also induced cross-protective immunity against heterologous and heterosubtypic influenza viruses. Further studies found heat shock protein 70 (inducible endogenous danger signal) and myeloid differentiation primary response 88 adaptor played a crucial role in dose-sparing effects of RFA. These data strongly support further development of the physical RFA to boost influenza vaccination.

With the development of laser technology in the 1960s, a technique was developed to inject intradermal vaccines immediately after irradiating the skin with laser light to elicit an adjuvant effect, referred to as "laser adjuvant." We have been investigating the mechanism of laser adjuvant in influenza mouse models using noninvasive continuous-wave (CW) near-infrared (NIR) light mainly at a wavelength of 1064 nm, and have shown that the production of reactive-oxygen-species (ROS) in the skin and mast cells in the skin tissue plays an important role in the laser adjuvant effect. The new wavelength of 1270 nm NIR light is characterized by its ability to elicit the same vaccine adjuvant effect as other wavelengths at a lower energy, and may be suitable for clinical applications. In this study, we investigated the physiological activity of CW1270 nm NIR light in mast cells, its biological activity on mouse skin, and the durability of the vaccine adjuvant effect in influenza vaccine mouse models. We show that irradiation of mast cells with 1270 nm NIR light produced ROS and ATP, and irradiation of isolated mitochondria also produced ATP. In mouse skin, the relative expression levels of chemokine mRNAs, such as Ccl2 and Ccl20, were increased by irradiation with 1270 and 1064 nm NIR light at minimum safe irradiance. However, the relative expression of Nfkb1 was increased at 1064 nm, but not at 1270 nm. Serum anti-influenza IgG antibody titers increased early after immunization with 1064 nm, whereas with 1270 nm, there was not only an early response of antibody production but also persistence of antibody titers over the medium- to long-term. Thus, to our knowledge, we show for the first time that 1270 nm NIR light induces ROS and ATP production in mitochondria as photoreceptors, initiating a cascade of laser adjuvant effects for intradermal vaccines. Additionally, we demonstrate that there are wavelength-specific variations in the mechanisms and effects of laser adjuvants. In conclusion, CW1270 nm NIR light is expected to be clinically applicable as a novel laser adjuvant that is equivalent or superior to 1064 nm NIR light, because it can be operated at low energy and has a wavelength-specific adjuvant effect with medium- to long-lasting antibody titer.

Immunotherapy, including immune checkpoint inhibitors, has revolutionized cancer treatment, but only a minor fraction of patients shows durable responses. A new approach to overcome this limitation is yet to be identified. Recently, we have shown that photobiomodulation (PBM) with near-infrared (NIR) light in the NIR-II window reduces oxidative stress and supports the proliferation of CD8+ T cells, suggesting that PBM with NIR-II light could augment anti-cancer immunity. Here, we report a novel approach to support tumor-infiltrating CD8+ T cells upon PBM with NIR-II laser with high tissue penetration depth. Brief treatments of a murine model of breast cancer with dual 1064 and 1270 nm lasers reduced the expression of the programmed cell death protein 1 (PD-1) in CD8+ T cells in a syngeneic mouse model of breast cancer. The direct effect of the NIR-II laser treatment on T cells was confirmed by the enhanced tumor growth delay by the adoptive transfer of laser-treated CD8+ T cells ex vivo against a model tumor antigen. We further demonstrated that specific NIR-II laser parameters augmented the effect of the immune checkpoint inhibitor on tumor growth. PBM with NIR-II light augments the efficacy of cancer immunotherapy by supporting CD8+ T cells. Unlike the current immunotherapy with risks of undesirable drug-drug interactions and severe adverse events, the laser is safe and low-cost. It can be broadly combined with other therapy without modification to achieve clinical significance. In addition, our study established a path to develop a novel laser-based therapy to treat cancer effectively.

There is solid evidence of the beneficial effect of photobiomodulation (PBM) with low-power near-infrared (NIR) light in the NIR-I window in increasing bioavailable nitric oxide (NO). However, it is not established whether this effect can be extended to NIR-II light, limiting broader applications of this therapeutic modality. Since we have demonstrated PBM with NIR laser in the NIR-II window, we determined the causal relationship between NIR-II irradiation and its specific biological effects on NO bioavailability. We analyzed the impact of NIR-II irradiation on NO release in cultured human endothelial cells using a NO-sensitive fluorescence probe and single-cell live imaging. Two distinct wavelengths of NIR-II laser (1064 and 1270 nm) and NIR-I (808 nm) at an irradiance of 10 mW/cm2 induced NO release from endothelial cells. These lasers also enhanced Akt phosphorylation at Ser 473, endothelial nitric oxide synthase (eNOS) phosphorylation at Ser 1177, and endothelial cell migration. Moreover, the NO release and phosphorylation of eNOS were abolished by inhibiting mitochondrial respiration, suggesting that Akt activation caused by NIR-II laser exposure involves mitochondrial retrograde signaling. Other inhibitors that inhibit known Akt activation pathways, including a specific inhibitor of PI3K, Src family PKC, did not affect this response. These two wavelengths of NIR-II laser induced no appreciable NO generation in cultured neuronal cells expressing neuronal NOS (nNOS). In short, NIR-II laser enhances bioavailable NO in endothelial cells. Since a hallmark of endothelial dysfunction is suppressed eNOS with concomitant NO deficiency, NIR-II laser technology could be broadly used to restore endothelial NO and treat or prevent cardiovascular diseases.

Pre-pandemic influenza H5N1 vaccine has relatively low immunogenicity and often requires high antigen amounts and two immunizations to induce protective immunity. Incorporation of vaccine adjuvants is promising to stretch vaccine doses during pandemic outbreaks. This study presents a physical radiofrequency (RF) adjuvant (RFA) to conveniently and effectively increase the immunogenicity and efficacy of H5N1 vaccine without modification of vaccine preparation. Physical RFA is based on a brief RF treatment of the skin to induce thermal stress to enhance intradermal vaccine-induced immune responses with minimal local or systemic adverse reactions. We found that physical RFA could significantly increase H5N1 vaccine-induced hemagglutination inhibition antibody titers in murine models. Intradermal H5N1 vaccine in the presence of RFA but not vaccine alone significantly lowered lung viral titers, reduced body weight loss, and improved survival rates after lethal viral challenges. The improved protection in the presence of RFA was correlated with enhanced humoral and cellular immune responses to H5N1 vaccination in both male and female mice, indicating no gender difference of RFA effects in murine models. Our data support further development of the physical RFA to conveniently enhance the efficacy of H5N1 vaccine.

Adjuvants are essential for ensuring the efficacy of modern vaccines. Considering frequent local and systemic adverse reactions, research into the development of safer and more effective adjuvants is being actively conducted. In recent years, the novel concept of laser vaccine adjuvants, which use the physical energy of light, has been developed. For long, light has been known to affect the physiological functions in living organisms. Since the development of lasers as stable light sources, laser adjuvants have evolved explosively in multiple ways over recent decades. Future laser adjuvants would have the potential not only to enhance the efficacy of conventional vaccine preparations but also to salvage candidate vaccines abandoned during development because of insufficient immunogenicity or owing to their inability to be combined with conventional adjuvants. Furthermore, the safety and efficacy of non-invasive laser adjuvants make them advantageous for vaccine dose sparing, which would be favorable for the timely and equitable global distribution of vaccines. In this review, we first describe the basics of light-tissue interactions, and then summarize the classification of lasers, the history of laser adjuvants, and the mechanisms by which different lasers elicit an immune response.

Rapid establishment of herd immunity with vaccination is effective to combat emerging infectious diseases. Although the incorporation of adjuvant and intradermal (ID) injection could augment early responses to the vaccine, the current chemical or biological adjuvants are inappropriate for this purpose with their side effects and high reactogenicity in the skin. Recently, a near-infrared (NIR) laser has been shown to augment the immune response to ID vaccination and could be alternatively used for mass vaccination programs. Here, we determined the effect of NIR laser as well as licensed chemical adjuvants on the immunogenicity 1, 2, and 4 weeks after ID influenza vaccination in mice. The NIR laser adjuvant augmented early antibody responses, while the widely used alum adjuvant induced significantly delayed responses. In addition, the oil-in-water and alum adjuvants, but not the NIR laser, elicited escalated T_H2 responses with allergenic immunoglobulin E (IgE) responses. The effect of the NIR laser was significantly suppressed in the basic leucine zipper transcription factor ATF-like 3 (Batf3) knockout mice, suggesting a critical role of the cluster of differentiation 103⁺ (CD103)⁺ dendritic cells. The current preliminary study suggests that NIR laser adjuvant is an alternative strategy to chemical and biological agents to timely combat emerging infectious diseases. Moreover, its immunomodulatory property could be used to enhance the efficacy of immunotherapy for allergy and cancer.

Influenza poses a huge threat to global public health. Influenza vaccines are the most effective and cost-effective means to control influenza. Current influenza vaccines mainly induce neutralizing antibodies against highly variable globular head of hemagglutinin and lack cross-protection. Vaccine adjuvants have been approved to enhance seasonal influenza vaccine efficacy in the elderly and spare influenza vaccine doses. Clinical studies found that MF59 and AS03-adjuvanted influenza vaccines could induce cross-protective immunity against non-vaccine viral strains. In addition to MF59 and AS03 adjuvants, experimental adjuvants, such as Toll-like receptor agonists, saponin-based adjuvants, cholera toxin and heat-labile enterotoxin-based mucosal adjuvants, and physical adjuvants, are also able to broaden influenza vaccine-induced immune responses against non-vaccine strains. This review focuses on introducing the various types of adjuvants capable of assisting current influenza vaccines to induce cross-protective immunity in preclinical and clinical studies. Mechanisms of licensed MF59 and AS03 adjuvants to induce cross-protective immunity are also introduced. Vaccine adjuvants hold a great promise to adjuvant influenza vaccines to induce cross-protective immunity.

COVID-19 is an evolving pandemic that has far reaching global effects, with a combination of factors that makes the virus difficult to contain. The symptoms of infection can be devastating or at the least very debilitating for vulnerable individuals. It is clear that the elderly are at most risk of the adverse impacts of the virus, including hospitalization and death. Others at risk are those with comorbidities such as cardiovascular disease and metabolic conditions and those with a hyper-excitable immune response. Treatment options for those with acute responses to the virus are limited and there is an urgent need for potential strategies that can mitigate these severe effects. One potential avenue for treatment that has not been explored is the microbiome gut/lung axis. In addition to those severely affected by their acute reaction to the virus, there is also a need for treatment options for those that are slow to recover from the effects of the infection and also those who have been adversely affected by the measures put in place to arrest the spread of the virus. One potential treatment option is photobiomodulation (PBM) therapy. PBM has been shown over many years to be a safe, effective, non-invasive and easily deployed adjunctive treatment option for inflammatory conditions, pain, tissue healing and cellular energy. We have also recently demonstrated the effectiveness of PBM to alter the gut microbiome. PBM therapy is worthy of consideration as a potential treatment for those most vulnerable to COVID-19, such as the elderly and those with comorbidities. The treatment may potentially be advantageous for those infected with the virus, those who have a slow recovery from the effects of the virus and those who have been denied their normal exercise/rehabilitation programs due to the isolation restrictions that have been imposed to control the COVID-19 pandemic.

Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology.

Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light.

In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature.

A specific combination of wavelengths at low irradiances (250 to 400  mW  /  cm2 for 1064 nm and 55 to 65  mW  /  cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes.

This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.

The use of an immunologic adjuvant to augment the immune response is essential for modern vaccines which are relatively ineffective on their own. In the past decade, researchers have been consistently reporting that skin treatment with a physical parameter, namely laser light, augments the immune response to vaccine and functions as an immunologic adjuvant. This "laser adjuvant" has numerous advantages over the conventional chemical or biological agents; it is free from cold chain storage, hypodermic needles, biohazardous sharp waste, irreversible formulation with vaccine antigen, undesirable biodistribution in vital organs, or unknown long-term toxicity. Since vaccine formulations are given to healthy populations, these characteristics render the "laser adjuvant" significant advantages for clinical use and open a new developmental path for a safe and effective vaccine. In addition, laser technology has been used in the clinic for more than three decades and is therefore technically matured and has been proved to be safe. Currently, four classes of laser adjuvant have been reported; ultrashort pulsed, non-pulsed, non-ablative fractional, and ablative fractional lasers. Since each class of the laser adjuvant shows a distinct mechanism of action, a proper choice is necessary to craft an effective vaccine formulation toward a desired clinical benefit for a clinical vaccine to maximize protection. In addition, data also suggest that further improvement in the efficacy is possible when a laser adjuvant is combined with chemical or biological adjuvant(s). To realize these goals, further efforts to uncover the molecular mechanisms of action of the laser adjuvants is warranted. This review provides a summary and comments of the recent updates in the laser adjuvant technology.

Refractory cutaneous warts are difficult to eliminate. In situ photo-immunotherapy (ISPI) is an innovative treatment concept combining local photothermal therapy (PTT) and topical immunotherapy using imiquimod. To compare the efficacy of ISPI vs topical imiquimod alone, a prospective randomized controlled trial was performed with patients suffering from refractory cutaneous warts. In both groups, approximately 50% of the skin surface containing warts was treated for 6 weeks. On the basis of topical imiquimod, ISPI includes an additional 808 nm laser irradiation. Treatment response, temperatures during irradiation and histopathologic examination were evaluated. The complete response rate in the ISPI-group (22/36, 61.1%) was significantly higher than in the imiquimod alone group (11/34, 32.4%). In the ISPI-group, the mean maximum temperature was 44.5 ± 5.1°C, and obvious lymphocytic infiltration was found in the perivasculature of the dermis. There was no recurrence or worsening in both groups during the 12-month follow-up. No obvious adverse reaction was observed. This study demonstrates that ISPI can be used as an effective and safe treatment modality for refractory cutaneous warts.

Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm2 to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a+ Langerhans cells in the dermis and CD11c+ dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

The treatment of skin with a low-power continuous-wave (CW) near-infrared (NIR) laser prior to vaccination is an emerging strategy to augment the immune response to intradermal vaccine, potentially substituting for chemical adjuvant, which has been linked to adverse effects of vaccines. This approach proved to be low cost, simple, small, and readily translatable compared with the previously explored pulsed-wave medical lasers. However, little is known on the mode of laser-tissue interaction eliciting the adjuvant effect. In this study, we sought to identify the pathways leading to the immunological events by examining the alteration of responses resulting from genetic ablation of innate subsets including mast cells and specific dendritic cell populations in an established model of intradermal vaccination and analyzing functional changes of skin microcirculation upon the CW NIR laser treatment in mice. We found that a CW NIR laser transiently stimulates mast cells via generation of reactive oxygen species, establishes an immunostimulatory milieu in the exposed tissue, and provides migration cues for dermal CD103+ dendritic cells without inducing prolonged inflammation, ultimately augmenting the adaptive immune response. These results indicate that use of an NIR laser with distinct wavelength and power is a safe and effective tool to reproducibly modulate innate programs in skin. These mechanistic findings would accelerate the clinical translation of this technology and warrant further explorations into the broader application of NIR lasers to the treatment of immune-related skin diseases.

Protein/subunit vaccines often require external adjuvants to induce protective immunity. Due to the safety concern of chemical adjuvants, physical adjuvants were recently explored to boost vaccination. Physical adjuvants use physical energies rather than chemicals to stimulate tissue stress and endogenous danger signal release to boost vaccination. Here we present the safety and potency of non-invasive radiofrequency treatment to boost intradermal vaccination in murine models. We show non-invasive radiofrequency can increase protein antigen-induced humoral and cellular immune responses with adjuvant effects comparable to widely used chemical adjuvants. Radiofrequency adjuvant can also safely boost pandemic 2009 H1N1 influenza vaccination with adjuvant effects comparable to MF59-like AddaVax adjuvant. We find radiofrequency adjuvant induces heat shock protein 70 (HSP70) release and activates MyD88 to mediate the adjuvant effects. Physical radiofrequency can potentially be a safe and potent adjuvant to augment protein/subunit vaccine-induced humoral and cellular immune responses.

Mass cytometry enables highly multiplexed profiling of cellular immune responses in limited-volume samples, advancing prospects of a new era of systems immunology. The capabilities of mass cytometry offer expanded potential for deciphering immune responses to infectious diseases and to vaccines. Several studies have used mass cytometry to profile protective immune responses, both postinfection and postvaccination, although no vaccine-development program has yet systematically employed the technology from the outset to inform both candidate design and clinical evaluation. In this article, we review published mass cytometry studies relevant to vaccine development, briefly compare immune profiling by mass cytometry to other systems-level technologies, and discuss some general considerations for deploying mass cytometry in the context of vaccine development.-Reeves, P. M., Sluder, A. E., Raju Paul, S., Scholzen, A., Kashiwagi, S., Poznansky, M. C. Application and utility of mass cytometry in vaccine development.

Brief exposure of skin to near-infrared (NIR) laser light has been shown to augment the immune response to intradermal vaccination and thus act as an immunologic adjuvant. Although evidence indicates that the NIR laser adjuvant has the capacity to activate innate subsets including dendritic cells (DCs) in skin as conventional adjuvants do, the precise immunological mechanism by which the NIR laser adjuvant acts is largely unknown. In this study we sought to identify the cellular target of the NIR laser adjuvant by using an established mouse model of intradermal influenza vaccination and examining the alteration of responses resulting from genetic ablation of specific DC populations. We found that a continuous wave (CW) NIR laser adjuvant broadly modulates migratory DC (migDC) populations, specifically increasing and activating the Lang⁺ and CD11b^-Lang^- subsets in skin, and that the Ab responses augmented by the CW NIR laser are dependent on DC subsets expressing CCR2 and Langerin. In comparison, a pulsed wave NIR laser adjuvant showed limited effects on the migDC subsets. Our vaccination study demonstrated that the efficacy of the CW NIR laser is significantly better than that of the pulsed wave laser, indicating that the CW NIR laser offers a desirable immunostimulatory microenvironment for migDCs. These results demonstrate the unique ability of the NIR laser adjuvant to selectively target specific migDC populations in skin depending on its parameters, and highlight the importance of optimization of laser parameters for desirable immune protection induced by an NIR laser-adjuvanted vaccine.

A brief exposure of skin to a low-power, non-tissue damaging laser light has been demonstrated to augment immune responses to intradermal vaccination. Both preclinical and clinical studies show that this approach is simple, effective, safe and well tolerated compared to standard chemical or biological adjuvants. Until now, these laser exposures have been performed using a diode-pumped solid-state laser (DPSSL) devices, which are expensive and require labor-intensive maintenance and special training. Development of an inexpensive, easy-to-use and small device would form an important step in translating this technology toward clinical application. Here we report that we have established a handheld, near-infrared (NIR) laser device using semiconductor diodes emitting either 1061, 1258, or 1301nm light that costs less than $4000, and that this device replicates the adjuvant effect of a DPSSL system in a mouse model of influenza vaccination. Our results also indicate that a broader range of NIR laser wavelengths possess the ability to enhance vaccine immune responses, allowing engineering options for the device design. This small, low-cost device establishes the feasibility of using a laser adjuvant approach for mass-vaccination programs in a clinical setting, opens the door for broader testing of this technology with a variety of vaccines and forms the foundation for development of devices ready for use in the clinic.

Vaccines are commonly administered by injection using needles. Although transdermal microneedles are less-invasive promising alternatives, needle-free topical vaccination without involving physical damage to the natural skin barrier is still sought after as it can further reduce needle-induced anxiety and simply administration. However, this long-standing goal has been elusive since the intact skin is impermeable to most macromolecules. Here, we show an efficient, non-invasive transdermal vaccination in mice by employing two key innovations: first, the use of hyaluronan (HA) as vaccine carriers and, second, non-ablative laser adjuvants. Conjugates of a model vaccine ovalbumin (OVA) and HA-HA-OVA conjugates-induced more effective maturation of dendritic cells in vitro, compared to OVA or HA alone, through synergistic HA receptor-mediated effects. Following topical administration in the back skin, HA-OVA conjugates penetrated into the epidermis and dermis in murine and porcine skins up to 30% of the total applied quantity, as revealed by intravital microscopy and quantitative fluorescence assay. Topical administration of HA-OVA conjugates significantly elevated both anti-OVA IgG antibody levels in serum and IgA antibody levels in bronchioalveolar lavage, with peak levels at 4 weeks, while OVA alone had a negligible effect. An OVA challenge at week 8 elicited strong immune-recall humoral responses. With pre-treatment of the skin using non-ablative fractional laser beams (1410 nm wavelength, 10 ms pulse duration, 0.2 mJ/pulse) as laser adjuvant, strong immunization was achieved with much reduced doses of HA-OVA (1 mg/kg OVA). Our results demonstrate the potential of the non-invasive patch-type transdermal vaccination platform.

The near-infrared radiation (NIR) window, also known as the "optical window" or "therapeutic window", is the range of wavelengths that has the maximum depth of penetration in tissue. Indeed, because NIR is minimally absorbed by water and hemoglobin, spectra readings can be easily collected from the body surface. Recent reports have shown the potential of NIR spectroscopy in various medical applications, including functional analysis of the brain and other tissues, as well as an analytical tool for diagnosing diseases. The broad applicability of NIR spectroscopy facilitates the diagnosis and therapy of diseases as well as elucidating their pathophysiology. This review introduces recent advances and describes new studies in NIR to demonstrate potential clinical applications of NIR spectroscopy.

Infrared irradiation (IR) is the most abundant fraction of sunlight reaching the earth's surface and provides heat. The fever response of an animal is known to regulate its immune responses. However, the non-thermal immune responses of IR were difficult to assess owing to its close association with heat. We hypothesized that IR irradiation induced differential immunological responses, independent of its associated heat. With an IR machine coupled with a delicate temperature control system, we investigated the non-thermal immunological effects of IR in vivo. With heating at 37 °C or 39 °C using an electric blanket or IR irradiation, we measured the skin's physiological parameters, including transepidermal water loss (TEWL), pH, skin hydration, elasticity, sebum production, and skin blood flow. We also measured the number of Langerhans cells in epidermal sheets and draining lymph nodes. Lymph node cells were activated by anti-CD3 antibody and their production of interleukin (IL)-5, 10, 13, 17, and interferon (IFN)-γ was measured by enzyme-linked immunosorbent assay (ELISA). The result showed that compared to heating alone, IR causes an enhanced activation of epidermal Langerhans cells, both in epidermal sheets and in draining lymph nodes. The activation of draining lymph node cells by anti-CD3 antibody in vitro induces both Th2 and Th1, but not Treg immune responses. Interestingly, IL-13, a Th2 cytokine, is induced the most. In contrast, physiological parameters and barrier functions of skin were not altered after IR irradiation. The study showed that IR alone without heat modulates immune responses in vivo, indicating that IR irradiation might regulate host immunity in a heat-independent manner.

Drug tolerance is the most common cause of treatment failure in neovascular age-related macular degeneration. "Low-intensity/high-density" subthreshold diode micropulse laser (SDM) has been reported effective for a number of retinal disorders without adverse effects. It has been proposed that SDM normalizes retinal pigment epithelial function. On this basis, it has been postulated that SDM treatment might restore responsiveness to anti-vascular endothelial growth factor drugs in drug-tolerant eyes.

Subthreshold diode micropulse laser treatment was performed in consecutive eyes unresponsive to all anti-vascular endothelial growth factor drugs, including at least three consecutive ineffective aflibercept injections. Monthly aflibercept was resumed 1 month after SDM treatment.

Thirteen eyes of 12 patients, aged 73 to 97 years (average, 84 years), receiving 16 to 67 (average, 34) anti-vascular endothelial growth factor injections before SDM treatment were included and followed for 3 months to 7 months (average, 5 months) after SDM treatment. After SDM treatment and resumption of aflibercept, 92% (12 of 13) of eyes improved, with complete resolution of macular exudation in 69% (9 of 13). Visual acuity remained unchanged. Central and maximum macular thicknesses significantly improved.

Subthreshold diode micropulse laser treatment restored drug response in drug-tolerant eyes with neovascular age-related macular degeneration. Based on these findings, a theory of SDM action is proposed, suggesting a wider role for SDM as retinal reparative/protective therapy.

Immunologic adjuvants are essential for current vaccines to maximize their efficacy. Unfortunately, few have been found to be sufficiently effective and safe for regulatory authorities to permit their use in vaccines for humans and none have been approved for use with intradermal vaccines. The development of new adjuvants with the potential to be both efficacious and safe constitutes a significant need in modern vaccine practice. The use of non-damaging laser light represents a markedly different approach to enhancing immune responses to a vaccine antigen, particularly with intradermal vaccination. This approach, which was initially explored in Russia and further developed in the US, appears to significantly improve responses to both prophylactic and therapeutic vaccines administered to the laser-exposed tissue, particularly the skin. Although different types of lasers have been used for this purpose and the precise molecular mechanism(s) of action remain unknown, several approaches appear to modulate dendritic cell trafficking and/or activation at the irradiation site via the release of specific signaling molecules from epithelial cells. The most recent study, performed by the authors of this review, utilized a continuous wave near-infrared laser that may open the path for the development of a safe, effective, low-cost, simple-to-use laser vaccine adjuvant that could be used in lieu of conventional adjuvants, particularly with intradermal vaccines. In this review, we summarize the initial Russian studies that have given rise to this approach and comment upon recent advances in the use of non-tissue damaging lasers as novel physical adjuvants for vaccines.

The downstream processing of virus particles for vaccination or gene therapy is becoming a critical bottleneck as upstream titers keep improving. Moreover, the growing pressure to develop cost-efficient processes has brought forward new downstream trains. This review aims at analyzing the state-of-the-art in viral downstream purification processes, encompassing the classical unit operations and their recent developments. Emphasis is given to novel strategies for process intensification, such as continuous or semi-continuous systems based on multicolumn technology, opening up process efficiency. Process understanding in the light of the pharmaceutical quality by design (QbD) initiative is also discussed. Finally, an outlook of the upcoming breakthrough technologies is presented.