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Abstract Vaccination is still the most promising strategy for combating influenza virus pandemics. However, the highly variable characteristics of influenza virus make it difficult to develop antibody-based universal vaccines, until now. Lung tissue-resident memory T cells (TRM), which actively survey tissues for signs of infection and react rapidly to eliminate infected cells without the need for a systemic immune reaction, have recently drawn increasing attention towards the development of a universal influenza vaccine. We previously designed a sequential immunization strategy based on orally administered Salmonella vectored vaccine candidates. To further improve our vaccine design, in this study, we used two different dendritic cell (DC)-targeting strategies, including a single chain variable fragment (scFv) targeting the surface marker DC-CD11c and DC targeting peptide 3 (DCpep3). Oral immunization with Salmonella harboring plasmid pYL230 (S230), which displayed scFv-CD11c on the bacterial surface, induced dramatic production of spleen effector memory T cells (TEM). On the other hand, intranasal boost immunization using purified DCpep3-decorated 3M2e-ferritin nanoparticles in mice orally immunized twice with S230 (S230inDC) significantly stimulated the differentiation of lung CD11b+ DCs, increased intracellular IL-17 production in lung CD4+ T cells and elevated chemokine production in lung sections, such as CXCL13 and CXCL15, as determined by RNAseq and qRT‒PCR assays, resulting in significantly increased percentages of lung TRMs, which could provide efficient protection against influenza virus challenge. The dual DC targeting strategy, together with the sequential immunization approach described in this study, provides us with a novel “prime and pull” strategy for addressing the production of protective TRM cells in vaccine design.

ABSTRACT The influenza virus continues to pose a great threat to public health due to the frequent variations in RNA viruses. Vaccines targeting conserved epitopes, such as the extracellular domain of the transmembrane protein M2 (M2e), a nucleoprotein, and the stem region of hemagglutinin proteins, have been developed, but more efficient strategies, such as nanoparticle-based vaccines, are still urgently needed. However, the labor-intensive in vitro purification of nanoparticles is still necessary, which could hinder the application of nanoparticles in the veterinary field in the future. To overcome this limitation, we used regulated lysis Salmonella as an oral vector with which to deliver three copies of M2e (3M2e-H1N1)-ferritin nanoparticles in situ and evaluated the immune response. Then, sequential immunization using Salmonella-delivered nanoparticles followed by an intranasal boost with purified nanoparticles was performed to further improve the efficiency. Compared with 3M2e monomer administration, Salmonella-delivered in situ nanoparticles significantly increased the cellular immune response. Additionally, the results of sequential immunization showed that the intranasal boost with purified nanoparticles dramatically stimulated the activation of lung CD11b dendritic cells (DCs) and elevated the levels of effector memory T (TEM) cells in both spleen and lung tissues as well as those of CD4 and CD8 tissue-resident memory T (TRM) cells in the lungs. The increased production of mucosal IgG and IgA antibody titers was also observed, resulting in further improvements to protection against a virus challenge, compared with the pure oral immunization group. Salmonella-delivered in situ nanoparticles efficiently increased the cellular immune response, compared with the monomer, and sequential immunization further improved the systemic immune response, as shown by the activation of DCs, the production of TEM cells and TRM cells, and the mucosal immune response, thereby providing us with a novel strategy by which to apply nanoparticle-based vaccines in the future. IMPORTANCE Salmonella-delivered in situ nanoparticle platforms may provide novel nanoparticle vaccines for oral administration, which would be beneficial for veterinary applications. The combination of administering Salmonella-vectored, self-assembled nanoparticles and an intranasal boost with purified nanoparticles significantly increased the production of effector memory T cells and lung resident memory T cells, thereby providing partial protection against an influenza virus challenge. This novel strategy could open a novel avenue for the application of nanoparticle vaccines for veterinary purposes.

BackgroundFollicular mucinosis (FM) is generally divided into a primary benign idiopathic form and a secondary form associated with mycosis fungoides.ObjectiveTo analyze the clinical and pathological features of FM and explore the pathological significance of CD103 expression.MethodsIn this case series, we retrospective analysis the clinical, pathological, treatment and follow-up treatment of 15 cases of FM. The expression of CD103 in all cases was detected by immunohistochemistry.ResultA total of 15 patients were enrolled, 7 were primary follicular mucinosis (P-FM) and 8 were mycosis fungoides-associated follicular mucinosis (MF-FM). Lesions of both P-FM and MF-FM are difficult to distinguish, present with red or dark red plaques and follicular papules. Pathologically, MF-FM showed more significant infiltrates of folliculotropic lymphoid cells, and the amount and proportion of CD103+ cells were significantly higher than that in P-FM. Follow-up data were available for 13 patients. Three cases were resolved after surgical resection, two patients were marked improved after oral administration of hydroxychloroquine and three times ALA photodynamic therapy respectively. The rest patients showed only modest efficacy.ConclusionFM should be differentiated based on pathological characteristics and treatment response, CD103 is helpful in differential diagnosis of FM.

The influenza virus continues to pose a great threat to public health due to the frequent variations in RNA viruses. Vaccines targeting conserved epitopes, such as the extracellular domain of the transmembrane protein M2 (M2e), a nucleoprotein, and the stem region of hemagglutinin proteins, have been developed, but more efficient strategies, such as nanoparticle-based vaccines, are still urgently needed. However, the labor-intensive in vitro purification of nanoparticles is still necessary, which could hinder the application of nanoparticles in the veterinary field in the future. To overcome this limitation, we used regulated lysis Salmonella as an oral vector with which to deliver three copies of M2e (3M2e-H1N1)-ferritin nanoparticles in situ and evaluated the immune response. Then, sequential immunization using Salmonella-delivered nanoparticles followed by an intranasal boost with purified nanoparticles was performed to further improve the efficiency. Compared with 3M2e monomer administration, Salmonella-delivered in situ nanoparticles significantly increased the cellular immune response. Additionally, the results of sequential immunization showed that the intranasal boost with purified nanoparticles dramatically stimulated the activation of lung CD11b dendritic cells (DCs) and elevated the levels of effector memory T (T EM ) cells in both spleen and lung tissues as well as those of CD4 and CD8 tissue-resident memory T (T RM ) cells in the lungs. The increased production of mucosal IgG and IgA antibody titers was also observed, resulting in further improvements to protection against a virus challenge, compared with the pure oral immunization group. Salmonella-delivered in situ nanoparticles efficiently increased the cellular immune response, compared with the monomer, and sequential immunization further improved the systemic immune response, as shown by the activation of DCs, the production of T EM cells and T RM cells, and the mucosal immune response, thereby providing us with a novel strategy by which to apply nanoparticle-based vaccines in the future. IMPORTANCE Salmonella-delivered in situ nanoparticle platforms may provide novel nanoparticle vaccines for oral administration, which would be beneficial for veterinary applications. The combination of administering Salmonella-vectored, self-assembled nanoparticles and an intranasal boost with purified nanoparticles significantly increased the production of effector memory T cells and lung resident memory T cells, thereby providing partial protection against an influenza virus challenge. This novel strategy could open a novel avenue for the application of nanoparticle vaccines for veterinary purposes., Competing Interests: The authors declare no conflict of interest.

Background: Follicular mucinosis (FM) is generally divided into a primary benign idiopathic form and a secondary form associated with mycosis fungoides., Objective: To analyze the clinical and pathological features of FM and explore the pathological significance of CD103 expression., Methods: In this case series, we retrospective analysis the clinical, pathological, treatment and follow-up treatment of 15 cases of FM. The expression of CD103 in all cases was detected by immunohistochemistry., Result: A total of 15 patients were enrolled, 7 were primary follicular mucinosis (P-FM) and 8 were mycosis fungoides-associated follicular mucinosis (MF-FM). Lesions of both P-FM and MF-FM are difficult to distinguish, present with red or dark red plaques and follicular papules. Pathologically, MF-FM showed more significant infiltrates of folliculotropic lymphoid cells, and the amount and proportion of CD103+ cells were significantly higher than that in P-FM. Follow-up data were available for 13 patients. Three cases were resolved after surgical resection, two patients were marked improved after oral administration of hydroxychloroquine and three times ALA photodynamic therapy respectively. The rest patients showed only modest efficacy., Conclusion: FM should be differentiated based on pathological characteristics and treatment response, CD103 is helpful in differential diagnosis of FM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Wang, Zhou, Wang, Zhu, Li and Shen.)