Brief Exposure of Skin to Near-Infrared Laser Modulates Mast Cell Function and Augments the Immune Response.

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.

Near-Infrared 1064 nm Laser Modulates Migratory Dendritic Cells To Augment the Immune Response to Intradermal Influenza Vaccine.

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.

Passive collection of ticks in New Hampshire reveals species-specific patterns of distribution and activity.

Ticks and tick-borne diseases are increasing in the United States, including New Hampshire (NH). We report on the findings of an ongoing free crowdsourcing program spanning four years within NH. The date of tick's submission was recorded along with species, sex, stage, location they were collected (translated into latitude and longitude), the activity the individual was doing when the tick was found, and host species. A total of 14,252 ticks belonging to subclass Acari, family Ixodidae and genera Ixodes, Dermacentor, Amblyomma, and Haemaphysalis was recorded from the period 2018-2021 throughout NH. A total of 2,787 Ixodes scapularis and 1,041 Dermacentor variabilis, were tested for the presence of Borrelia sp. (Spirochaetales: Spirochaetaceae), B. burgdorferi sensu lato, B. miyamotoi, B. mayonii, Babesia microti (Piroplasmida: Babesiidae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), Francisella tularensis (Thiotrichales: Francisellaceae), and Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by PCR. For the I. scapularis ticks tested, the pathogen prevalence was 37% B. burgdorferi s.l. 1% B. miyamotoi, 6% A. phagocytophilum, and 5% Ba. microti. Only one D. variabilis resulted positive to F. tularensis. We created state-wide maps informing the differences of ticks as detailed by administrative divisions. Dermacentor variabilis peaked in June and I. scapularis peaked in May and October. The most reported activity by people with tick encounters was while walking/hiking, and the least was biking. Using the reported distribution of both species of ticks, we modeled their climate suitability in the target territory. In NH, I. scapularis and D. variabilis have distinct patterns of emergence, abundance, and distribution. Tick prevention is important especially during April-August when both tick species are abundant and active.

Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale.

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale.

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements.

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.

Anaplasma marginale type IV secretion system proteins VirB2, VirB7, VirB11, and VirD4 are immunogenic components of a protective bacterial membrane vaccine.

Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serological responses of OM vaccinees to the OM proteome previously identified over 20 antigenic proteins, including three type IV secretion system (T4SS) proteins, VirB9-1, VirB9-2, and VirB10. Subsequent studies showed that these three proteins also stimulated CD4(+) T-cell responses in OM vaccinees. The T4SS, composed of a complex of proteins spanning the inner and outer membranes of certain bacteria, is an important virulence factor but is relatively unexplored as a vaccine target. The goal of this study was to determine if additional T4SS proteins are immunogenic for animals immunized with the protective OM fraction of A. marginale. T4SS proteins expressed by in vitro transcription and translation were screened for stimulating proliferation of T cells from OM vaccinees, and immunogenic proteins were expressed as recombinant proteins in Escherichia coli and their immunogenicity was verified. VirB2, a putative VirB7, VirB11, and VirD4 were immunogenic for OM vaccinees expressing several common major histocompatibility complex (MHC) class II haplotypes. VirB2 is encoded by multiple genes that share a conserved central region, and epitope mapping revealed T-cell epitopes in this region. The discovery of novel immunogenic T4SS proteins recognized by outbred individuals with common MHC haplotypes further justifies evaluating the T4SS as a potential vaccine candidate for pathogenic bacteria.

Semiconductor diode laser device adjuvanting intradermal 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.