Microneedle array has proven more efficient in stimulating humoral immunity than intramuscular vaccination. However, its effectiveness in inducing pulmonary CD8+ T cells remains elusive, which is essential to the frontline defense against pulmonary viral infections such as influenza and COVID-19 viruses. The current investigation reveals that superior CD8+ T-cell responses are elicited by immunization with a microneedle array over intradermal or intramuscular immunization using the model antigen ovalbumin, irrespective of whether or not the antigen is provided in the lung. Mechanistically, microneedle array-mediated immunization targeted the epidermal layer and stimulated predominantly Langerhans cells, resulting in increased expression of α4β1 adhesion molecules on the CD8+ T-cell surface, which may play a role in T-cell homing to the lung, whereas CD8+ T cells induced by intramuscular immunization did not express the adhesion molecule sufficiently. CD8+ T cells with a lung-homing propensity were also seen after intradermal vaccination, yet to a much lesser extent. Accordingly, microneedle array immunization provided stronger protection against influenza viral infection than intradermal or intramuscular immunization. The observations offer insights into a strong cross-talk between epidermal immunization and lung immunity and are valuable for designing and delivering vaccines against respiratory viral infections.

Background: More effective and safer immunotherapies to manage peanut allergy are in great demand despite extensive investigation of sublingual/oral immunotherapy and epicutaneous immunotherapy (EPIT) currently in the clinics. Objective: We sought to develop a powder-laden, dissolvable microneedle array (PLD-MNA) for epidermal delivery of powdered allergens and to evaluate the efficacy of this novel EPIT in peanut-sensitized mice. Methods: PLD-MNA was packaged with a mixture of powdered peanut allergen (PNA), 1,25-dihydroxyvitamin D-3 (VD3), and CpG. Its epidermal delivery and therapeutic efficacy were evaluated alongside PNA-specific forkhead box P3-positive regulatory T cells and IL-10(+) and TGF-beta 1(+) skin-resident macrophages. Results: PLD-MNAwas successfully laden with PNA/VD3/CpG powder and capable of epidermal delivery of most of its content 1 hour after application onto intact mouse skin concomitant with no significant leakage into the circulation or skin irritation. PLD-MNA-mediated EPIT substantially reduced clinical allergy scores to 1 from 3.5 in sham control mice (P <.001) after 6 treatments accompanied by lower levels of PNA-specific IgE and intestinal mucosal mast cells and eosinophils over sham treatments. Moreover, in comparison with allergens administered intradermally, powdered allergens delivered by means of PLD-MNA preferentially attracted immunoregulatory macrophages and stimulated the cells to produce IL-10, TGF-beta, or both at the immunization site, which might account for increased numbers of regulatory T-like cells in lymph tissues in association with systemic tolerance. PNA/VD3/CpG-laden PLD-MNA was safe and required only 6 treatments and one fifth of the PNA adjuvant dose, with improved outcomes when compared with 12 conventional intradermal immunotherapies. Conclusions: PLD-MNA holds great promise as a novel, safe, effective, and self-applicable modality to manage IgE-mediated allergies.

Current influenza vaccines only confer protection against homologous viruses. We synthesized pulmonary surfactant (PS)-biomimetic liposomes encapsulating 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an agonist of the interferon gene inducer STING (stimulator of interferon genes). The adjuvant (PS-GAMP) vigorously augmented influenza vaccine-induced humoral and CD8(+) T cell immune responses in mice by simulating the early phase of viral infection without concomitant excess inflammation. Two days after intranasal immunization with PS-GAMP-adjuvanted H1N1 vaccine, strong cross-protection was elicited against distant H1N1 and heterosubtypic H3N2, H5N1, and H7N9 viruses for at least 6 months while maintaining lung-resident memory CD8(+) T cells. Adjuvanticity was then validated in ferrets. When alveolar epithelial cells (AECs) lacked Sting or gap junctions were blocked, PS-GAMP-mediated adjuvanticity was substantially abrogated in vivo. Thus, AECs play a pivotal role in configuring heterosubtypic immunity.

More than 20% of all newborns admitted to the neonatal intensive care unit experience abnormally low platelet counts, called neonatal thrombocytopenia. Neonatal thrombocytopenia could increase a risk of hemorrhage, especially within the brain and adversely affect its development. No drugs or therapies are currently available for these sick newborns besides platelet transfusions. However, too many platelet transfusions could induce anti-platelet autoantibodies and other complications in some cases. One innovative idea for an alternative and non-invasive therapy for thrombocytopenia is low-level laser treatment (LLLT). LLLT is very safe, like blue-light-mediated treatment of newborn jaundice that has been used in clinics for decades with a long record of safety. LLLT stimulates the expression of a group of genes involved in mitochondrial generation in megakaryocytes (MKs) that are the precursor of platelets. This stimulation increases mitochondrial mass in MKs and enlarges the cells, which in turn elevates the rate of platelet production per MK. Our early investigation demonstrated that LLLT could quickly increase the number of circulating platelets and greatly reduce the risk of bleeding in thrombocytopenic adult animals. To explore a potential of this novel modality in management of neonatal thrombocytopenia, we investigated whether LLL could increase the platelet biogenesis in fetal liver and bone marrow MKs that intended to have low polyploidy compared to those MKs in adults. We found that LLL enhanced platelet production in MKs differentiated from fetal bone marrow and livers albeit at a slightly less efficiency. In addition to increased production of platelets from MKs, LLL appeared to increase the number of MKs differentiated from MK precursors in both bone marrow and liver of newborn mice in vivo as well, in contrast to little megakaryocytopoiesis induced by LLLT in adult mice. The ability of LLLT to increase not only platelet generation from MKs but also megakaryocytopoiesis in both livers and bone marrows in newborns suggests a potential for LLLT to manage neonatal thrombocytopenia and reduce the number of transfusions for thrombocytopenia-afflicted newborns.

Microbial biochemicals have been indicated as the primary stimulators of innate immunity, the first line of the body's defence against infections. However, the influence of topological features on a microbe's surface on immune responses remains largely unknown. Here we demonstrate the ability of TiO2 microparticles decorated with nanospikes (spiky particles) to activate and amplify the immune response in vitro and in vivo. The nanospikes exert mechanical stress on the cells, which results in potassium efflux and inflammasome activation in macrophages and dendritic cells during phagocytosis. The spiky particles augment antigen-specific humoral and cellular immune responses in the presence of monophosphoryl lipid A and elicit protective immunity against tumour growth and influenza viral infection. The study offers insights into how surface physical cues can tune the activation of innate immunity and provides a basis for engineering particles with increased immunogenicity and adjuvanticity.

Live attenuated Bacille Calmette-Guerin (BCG) bacillus is the only licensed vaccine for tuberculosis prevention worldwide to date. It must be delivered intradermally to be effective, which causes severe skin inflammation and sometimes, permanent scars. To minimize the side effects, we developed a novel microneedle array (MNA) that could deliver live attenuated freeze-dried BCG powder into the epidermis in a painless, lesion-free, and self-applicable fashion. The MNA was fabricated with biocompatible and dissolvable hyaluronic acid with a deep cave formed in the basal portion of each microneedle, into which BCG powder could be packaged directly. Viability of BCG vaccine packaged in the caves and the mechanical strength of the powder-laden MNA did not alter significantly before and after more than two months of storage at room temperature. Following insertion of the MNA into the skin, individual microneedle shafts melted away by interstitial fluid from the epidermis and upper dermis, exposing the powder to epidermal tissues. The powder sucked interstitial fluid, dissolved slowly, and diffused into the epidermis in a day against the interstitial fluid influx. Vaccination with BCG-MNA caused no overt skin irritation, in marked contrast to intradermal vaccination that provoked severe inflammation and bruise. While causing little skin irritation, vaccination efficacy of BCG-MNAs was comparable to that of intradermal immunization whether it was evaluated by humoral or cellular immunity. This powder-laden and dissolvable MNA represents a novel technology to sufficiently deliver live attenuated vaccine powders into the skin.

A previous amino acid sequence analyses from our laboratory reported nine potential sites in gp41 glycoprotein of HIV-1 that may contribute to virus escape from antibody neutralization. Besides four sites found outside the membrane of HIV-1 virus, five located in the C-terminal tail of gp41 specifically in the lentivirus lytic peptides motifs (LLPs). To further study the bioinformatical results, the virus infectivity assay and the standard neutralization assay were conducted on conservatively mutated virus. Two sites in the LLP3 domain stood out with the ability to alter the resistance of HIV-1 virus to certain broadly neutralizing antibodies (bNAbs). While the glycoprotein incorporation on the viral membrane and the interaction of the LLP3 domain with the lipid membrane remained unaltered, the increase in neutralization resistance of the mutant virus was associated with the changes on Env conformation. Our findings demonstrate different sensibility of bNAbs to mutations in the C-terminal tail and indicate an unrecognized potential role for even minor sequence variation in the C-terminal tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

Recent studies have demonstrated that the membrane-proximal external region (MPER) of human immunodeficiency virus 1 (HIV-1) glycoprotein 41 contains a series of epitopes for human monoclonal antibodies, including 2F5, Z13e1, 4E10, and 10E8, which were isolated from HIV-1-infected individuals and show broad neutralizing activities. This suggests that MPER is a good target for the development of effective HIV-1 vaccines. However, many studies have shown that it is difficult to induce antibodies with similar broad neutralizing activities using MPER-based peptide antigens. Here, we report that 10E8-like neutralizing antibodies with effective anti-HIV-1 activity were readily induced using a precisely designed conformational immunogenic peptide containing the 10E8-specific epitope. This immunogenic peptide (designated T10HE) contains a 15-mer MPER-derived 10E8-specific epitope fused to T-helper-cell epitopes from tetanus toxin (tt), which showed a significantly stabilized α-helix structure after a series of modifications, including substitution with an (S)-α-(2′-pentenyl) alanine containing an olefin-bearing tether and ruthenium-catalyzed olefin metathesis, compared with the unmodified T10E peptide. The stabilized α-helix structure of T10HE did not affect its capacity to bind the 10E8 antibody, as evaluated with an enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance binding assay (SPR assay). The efficacies of the T10HE and T10E epitope vaccines were evaluated after a standard vaccination procedure in which the experimental mice were primed with either the T10HE or T10E immunogen and boosted with HIV-1 JRFL pseudoviruses. Higher titers of 10E8-like antibodies were induced by T10HE than that by T10E. More importantly, the antibodies induced by T10HE showed enhanced antiviral potency against HIV-1 strains with both X4 and R5 tropism and a greater degree of broad neutralizing activity than the antibodies induced by T10E. These results indicate that a 10E8-epitope-based structure-specific peptide immunogen can elicit neutralizing antibodies when used as a vaccine prime.