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1.
Mol Ther ; 31(8): 2360-2375, 2023 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-37403357

RESUMO

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.


Assuntos
Nanopartículas , RNA , Humanos , Camundongos , Animais , Distribuição Tecidual , RNA/genética , Antígenos , Imunidade Humoral , Inflamação
2.
Sci Immunol ; 6(56)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33579750

RESUMO

Microanatomical organization of innate immune cells within lymph nodes (LNs) is critical for the generation of adaptive responses. In particular, steady-state LN-resident dendritic cells (Res cDCs) are strategically localized to intercept lymph-draining antigens. Whether myeloid cell organization changes during inflammation and how that might affect the generation of immune responses are unknown. Here, we report that during type I, but not type II, inflammation after adjuvant immunization or viral infection, antigen-presenting Res cDCs undergo CCR7-dependent intranodal repositioning from the LN periphery into the T cell zone (TZ) to elicit T cell priming. Concurrently, inflammatory monocytes infiltrate the LNs via local blood vessels, enter the TZ, and cooperate with Res cDCs by providing polarizing cytokines to optimize T cell effector differentiation. Monocyte infiltration is nonuniform across LNs, generating distinct microenvironments with varied local innate cell composition. These spatial microdomains are associated with divergent early T cell effector programming, indicating that innate microenvironments within LNs play a critical role in regulating the quality and heterogeneity of T cell responses. Together, our findings reveal that dynamic modulation of innate cell microenvironments during type I inflammation leads to optimized generation of adaptive immune responses to vaccines and infections.


Assuntos
Comunicação Celular/imunologia , Microambiente Celular/imunologia , Linfonodos/imunologia , Infecções por Strongylida/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Movimento Celular/imunologia , Células Dendríticas/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Inflamação/imunologia , Inflamação/patologia , Linfonodos/citologia , Linfonodos/patologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Monócitos/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/parasitologia
3.
Cell Rep ; 31(3): 107523, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32320656

RESUMO

Recently developed approaches for highly multiplexed imaging have revealed complex patterns of cellular positioning and cell-cell interactions with important roles in both cellular- and tissue-level physiology. However, tools to quantitatively study cellular patterning and tissue architecture are currently lacking. Here, we develop a spatial analysis toolbox, the histo-cytometric multidimensional analysis pipeline (CytoMAP), which incorporates data clustering, positional correlation, dimensionality reduction, and 2D/3D region reconstruction to identify localized cellular networks and reveal features of tissue organization. We apply CytoMAP to study the microanatomy of innate immune subsets in murine lymph nodes (LNs) and reveal mutually exclusive segregation of migratory dendritic cells (DCs), regionalized compartmentalization of SIRPα- dermal DCs, and preferential association of resident DCs with select LN vasculature. The findings provide insights into the organization of myeloid cells in LNs and demonstrate that CytoMAP is a comprehensive analytics toolbox for revealing features of tissue organization in imaging datasets.


Assuntos
Tecido Linfoide/metabolismo , Células Mieloides/metabolismo , Animais , Camundongos , Análise Espacial
4.
Biomacromolecules ; 20(2): 854-870, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30608149

RESUMO

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (∼300 nm submicrometer particles, ∼10 nm micelles and ∼4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.


Assuntos
Adjuvantes Imunológicos/química , Linfócitos T CD8-Positivos/efeitos dos fármacos , Ativação Linfocitária , Micelas , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Hidrodinâmica , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica
5.
ACS Comb Sci ; 19(5): 286-298, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28383252

RESUMO

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.


Assuntos
Anticorpos/química , Haptenos/química , Nicotina/química , Animais , Anticorpos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linfócitos B/metabolismo , Simulação por Computador , Ensaio de Imunoadsorção Enzimática , Haptenos/imunologia , Humanos , Camundongos Endogâmicos BALB C , Nicotina/imunologia , Nicotina/farmacocinética , Estreptavidina/química , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia
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