RESUMEN
CMV, a ubiquitous herpesvirus, elicits an extraordinarily large T cell response that is sustained or increases over time, a phenomenon termed 'memory inflation.' Remarkably, even latent, non-productive infection can drive memory inflation. Despite intense research on this phenomenon, the infected cell type(s) involved are unknown. To identify the responsible cell type(s), we designed a Cre-lox murine CMV (MCMV) system, where a spread-deficient (ΔgL) virus expresses recombinant SIINFEKL only in Cre+ host cells. We found that latent infection of endothelial cells (ECs), but not dendritic cells (DCs) or hepatocytes, was sufficient to drive CD8 T cell memory inflation. Infection of Lyve-1-Cre and Prox1-CreERT2 mice revealed that amongst EC subsets, infection of lymphatic ECs was sufficient. Genetic ablation of ß2m on lymphatic ECs did not prevent inflation, suggesting another unidentified cell type can also present antigen to CD8 T cells during latency. This novel system definitively shows that antigen presentation by lymphatic ECs drives robust CD8 T cell memory inflation.
Asunto(s)
Infecciones por Citomegalovirus , Infección Latente , Muromegalovirus , Animales , Ratones , Células Endoteliales , Linfocitos T CD8-positivos , Antígenos , Memoria InmunológicaRESUMEN
Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Muromegalovirus/fisiología , Sarcoma/inmunología , Neoplasias Cutáneas/inmunología , Animales , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/trasplante , Muerte Celular , Línea Celular Tumoral , Cricetinae , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Infecciones por Herpesviridae/terapia , Humanos , Inmunoglobulina G/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Metilcolantreno , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Ratas , Sarcoma/terapia , Neoplasias Cutáneas/terapiaRESUMEN
Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alum's functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNA's adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell-DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC-T-cell interactions.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Presentación de Antígeno/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , ADN/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Antígenos/inmunología , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Ratones DesnudosRESUMEN
NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Muromegalovirus/inmunología , Animales , Relación CD4-CD8 , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/patología , Memoria Inmunológica , Células Asesinas Naturales/virología , Hígado/inmunología , Hígado/patología , Hígado/virología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Bazo/inmunología , Bazo/patología , Bazo/virología , Células Madre/inmunología , Células Madre/patología , Células Madre/virologíaRESUMEN
Pancreatic cancer remains a formidable challenge due to limited treatment options and its aggressive nature. In recent years, the naturally occurring anticancer compound juglone has emerged as a potential therapeutic candidate, showing promising results in inhibiting tumor growth and inducing cancer cell apoptosis. However, concerns over its toxicity have hampered juglone's clinical application. To address this issue, we have explored the use of polymeric micelles as a delivery system for juglone in pancreatic cancer treatment. These micelles, formulated using Poloxamer 407 and D-α-Tocopherol polyethylene glycol 1000 succinate, offer an innovative solution to enhance juglone's therapeutic potential while minimizing toxicity. In-vitro studies have demonstrated that micelle-formulated juglone (JM) effectively decreases proliferation and migration and increases apoptosis in pancreatic cancer cell lines. Importantly, in-vivo, JM exhibited no toxicity, allowing for increased dosing frequency compared to free drug administration. In mice, JM significantly reduced tumor growth in subcutaneous xenograft and orthotopic pancreatic cancer models. Beyond its direct antitumor effects, JM treatment also influenced the tumor microenvironment. In immunocompetent mice, JM increased immune cell infiltration and decreased stromal deposition and activation markers, suggesting an immunomodulatory role. To understand JM's mechanism of action, we conducted RNA sequencing and subsequent differential expression analysis on tumors that were treated with JM. The administration of JM treatment reduced the expression levels of the oncogenic protein MYC, thereby emphasizing its potential as a focused, therapeutic intervention. In conclusion, the polymeric micelles-mediated delivery of juglone holds excellent promise in pancreatic cancer therapy. This approach offers improved drug delivery, reduced toxicity, and enhanced therapeutic efficacy.
RESUMEN
It has been recognized for nearly 80 years that insoluble aluminum salts are good immunologic adjuvants and that they form long-lived nodules in vivo. Nodule formation has long been presumed to be central for adjuvant activity by providing an antigen depot, but the composition and function of these nodules is poorly understood. We show here that aluminum salt nodules formed within hours of injection and contained the clotting protein fibrinogen. Fibrinogen was critical for nodule formation and required processing to insoluble fibrin by thrombin. DNase treatment partially disrupted the nodules, and the nodules contained histone H3 and citrullinated H3, features consistent with extracellular traps. Although neutrophils were not essential for nodule formation, CD11b(+) cells were implicated. Vaccination of fibrinogen-deficient mice resulted in normal CD4 T-cell and antibody responses and enhanced CD8 T-cell responses, indicating that nodules are not required for aluminum's adjuvant effect. Moreover, the ability of aluminum salts to retain antigen in the body, the well-known depot effect, was unaffected by the absence of nodules. We conclude that aluminum adjuvants form fibrin-dependent nodules in vivo, that these nodules have properties of extracellular traps, and the nodules are not required for aluminum salts to act as adjuvants.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Aluminio/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Aluminio/farmacología , Animales , Fibrina/metabolismo , Fibrinógeno/metabolismo , Histonas/metabolismo , Ratones , Ratones Noqueados , Sales (Química) , Linfocitos T/inmunología , Trombina/metabolismo , VacunaciónRESUMEN
As a transcription factor that promotes cell growth, proliferation, and apoptosis, c-MYC (MYC) expression in the cell is tightly controlled. Disruption of oncogenic signaling pathways in human cancers can increase MYC protein stability, due to altered phosphorylation ratios at two highly conserved sites, Threonine 58 (T58) and Serine 62 (S62). The T58 to Alanine mutant (T58A) of MYC mimics the stabilized, S62 phosphorylated, and highly oncogenic form of MYC. The S62A mutant is also stabilized, lacks phosphorylation at both Serine 62 and Threonine 58, and has been shown to be nontransforming in vitro. However, several regulatory proteins are reported to associate with MYC lacking phosphorylation at S62 and T58, and the role this form of MYC plays in MYC transcriptional output and in vivo oncogenic function is understudied. We generated conditional c-Myc knock-in mice in which the expression of wild-type MYC (MYCWT), the T58A mutant (MYCT58A), or the S62A mutant (MYCS62A) with or without expression of endogenous Myc is controlled by the T-cell-specific Lck-Cre recombinase. MYCT58A expressing mice developed clonal T-cell lymphomas with 100% penetrance and conditional knock-out of endogenous Myc accelerated this lymphomagenesis. In contrast, MYCS62A mice developed clonal T-cell lymphomas at a much lower penetrance, and the loss of endogenous MYC reduced the penetrance while increasing the appearance of a non-transgene driven B-cell lymphoma with splenomegaly. Together, our study highlights the importance of regulated phosphorylation of MYC at T58 and S62 for T-cell transformation. IMPLICATIONS: Dysregulation of phosphorylation at conserved T58 and S62 residues of MYC differentially affects T-cell development and lymphomagenesis.
Asunto(s)
Linfoma de Células T , Proteínas Proto-Oncogénicas c-myc , Treonina , Animales , Carcinogénesis , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Serina/metabolismo , Linfocitos T/metabolismo , Treonina/genética , Factores de Transcripción/metabolismoRESUMEN
To understand more about how the body recognizes alum we characterized the early innate and adaptive responses in mice injected with the adjuvant. Within hours of exposure, alum induces a type 2 innate response characterized by an influx of eosinophils, monocytes, neutrophils, DCs, NK cells and NKT cells. In addition, at least 13 cytokines and chemokines are produced within 4 h of injection including IL-1beta and IL-5. Optimal production of some of these, including IL-1beta, depends upon both macrophages and mast cells, whereas production of others, such as IL-5, depends on mast cells only, suggesting that both of these cell types can detect alum. Alum induces eosinophil accumulation partly through the production of mast cell derived IL-5 and histamine. Alum greatly enhances priming of endogenous CD4 and CD8 T cells independently of mast cells, macrophages, and of eosinophils. In addition, Ab levels and Th2 bias was similar in the absence of these cells. We found that the inflammation induced by alum was unchanged in caspase-1-deficient mice, which cannot produce IL-1beta. Furthermore, endogenous CD4 and CD8 T cell responses, Ab responses and the Th2 bias were also not impacted by the absence of caspase-1 or NLRP3. These data suggest that activation of the inflammasome and the type 2 innate response orchestrated by macrophages and mast cells in vivo are not required for adjuvant effect of alum on endogenous T and B cell responses.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Inmunidad Celular/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Mediadores de Inflamación/farmacología , Macrófagos Peritoneales/inmunología , Mastocitos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Secuencia de Aminoácidos , Animales , Técnicas Biosensibles , Proteínas Portadoras/fisiología , Caspasa 1/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Mediadores de Inflamación/administración & dosificación , Mediadores de Inflamación/clasificación , Inyecciones Intraperitoneales , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
The dynamics of mouse cytomegalovirus (MCMV)-specific CD4 T cell responses and the mechanisms by which these cells contribute to viral control are not well understood, mainly due to lack of appropriate tools to characterize MCMV-specific CD4 T cells. We therefore generated MCMV-specific CD4 T cell hybridomas, then used an MCMV expression library and overlapping peptides to identify CD4 T cell epitopes. We used these novel tools to study the long-term kinetics and organ distribution of MCMV-specific CD4 T cells in comparison to MCMV-specific CD8 T cell responses. We demonstrate that the overall MCMV-specific CD4 T cell response stabilizes during the latent stage, which stands in contrast to subpopulations of MCMV-specific CD8 T cells and HCMV-specific CD4 T cells which accumulate over the course of CMV latency. Furthermore, MCMV-specific CD4 T cells displayed a Th1 phenotype, secreting high levels of IFN-gamma and TNF-alpha and to some extent IL-2, cytokines which are involved in protection from CMV disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Infecciones por Herpesviridae/inmunología , Muromegalovirus/inmunología , Latencia del Virus , Enfermedad Aguda , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Citocinas/inmunología , Epítopos de Linfocito T/metabolismo , Infecciones por Herpesviridae/virología , Humanos , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Péptidos/metabolismoRESUMEN
Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Melanoma Experimental/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.
Asunto(s)
Traslado Adoptivo , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Infecciones por Herpesviridae/inmunología , Muromegalovirus/inmunología , Animales , Linfocitos T CD8-positivos/virología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibroblastos/virología , Epítopos Inmunodominantes/genética , Cinética , Ratones , Ratones Endogámicos BALB C , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Herpesviruses establish lifelong infections, normally characterized by prolonged periods of latency with intermittent episodes of viral reactivation. Feline herpesvirus-1 (FHV-1) infects domestic cats, and epidemiological studies indicate that many or most domestic cats are exposed to FHV-1, but the strength and longevity of the antibody response to FHV-1 is not fully characterized. Here we describe development of an ELISA, using lysates of cat cells infected with FHV-1, that measure feline antibodies against FHV-1. The assay is sensitive, quantitative and has a large dynamic range. We found that serum anti-FHV-1 antibodies primarily recognize FHV-1 proteins of the Late (L) class and are primarily of the IgG isotype. We then analyzed serum from a cross-sectional cohort of 100 client-owned cats that differed in age, sex and vaccination history. While there was no difference in FHV-1 antibody responses between females and males, antibody levels were significantly increased in older cats in comparison with younger animals (p=0.01). Surprisingly, as the length of time since the most recent vaccination increased, there was no corresponding drop in serum anti-FHV-1 antibody. These data suggest that FHV-1 immunity is very long-lived and support the current recommendation that many cats do not require revaccination against FHV-1 annually.
Asunto(s)
Enfermedades de los Gatos/virología , Infecciones por Herpesviridae/veterinaria , Herpesviridae/inmunología , Factores de Edad , Animales , Anticuerpos Antivirales/inmunología , Enfermedades de los Gatos/inmunología , Gatos/inmunología , Gatos/virología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Inmunidad Humoral/inmunología , Inmunidad Humoral/fisiología , MasculinoRESUMEN
Trafficking of memory CD8+ T cells out of the circulation is essential to provide protective immunity against intracellular pathogens in nonlymphoid tissues. However, the molecular mechanisms that dictate the trafficking potential of diverse memory CD8+ T cell populations are not completely defined. We show that after infection or inflammatory challenge, central memory (TCM) CD8+ T cells rapidly traffic into nonlymphoid tissues, whereas most effector memory cells remain in the circulation. Furthermore, we demonstrate that cellular migration of memory CD8+ T cells into nonlymphoid tissues is driven by interleukin-15 (IL-15)-stimulated enzymatic synthesis of core 2 O-glycans, which generates functional ligands for E- and P-selectins. Given that IL-15-stimulated expression of glycosyltransferase enzymes is largely a feature of TCM CD8+ T cells, this allows TCM to selectively migrate out of the circulation and into nonlymphoid tissues. Collectively, our data indicate that entry of memory CD8+ T cells into inflamed, nonlymphoid tissues is primarily restricted to TCM cells that have the capacity to synthesize core 2 O-glycans.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Polisacáridos/inmunología , Animales , Linfocitos T CD8-positivos/enzimología , Movimiento Celular , Citoplasma/inmunología , Citoplasma/virología , Inflamación , Interleucina-15/genética , Interleucina-15/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Polisacáridos/biosíntesisRESUMEN
Cytomegalovirus (CMV)-based vaccines have shown remarkable efficacy in the rhesus macaque model of acquired immune deficiency syndrome, enabling 50% of vaccinated monkeys to clear a subsequent virulent simian immunodeficiency virus challenge. The protective vaccine elicited unconventional CD8 T cell responses that were entirely restricted by MHC II or the nonclassical MHC I molecule, MHC-E. These unconventional responses were only elicited by a fibroblast-adapted rhesus CMV vector with limited tissue tropism; a repaired vector with normal tropism elicited conventional responses. Testing whether these unusual protective CD8 T responses could be elicited in humans requires vaccinating human subjects with a fibroblast-adapted mutant of human CMV (HCMV). In this study, we describe the CD8 T cell responses of human subjects vaccinated with two fibroblast-adapted HCMV vaccines. Most responses were identified as conventional classically MHC I restricted, and we found no evidence for MHC II or HLA-E restriction. These results indicate that fibroblast adaptation alone is unlikely to explain the unconventional responses observed in macaques.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Vacunas contra Citomegalovirus/inmunología , Citomegalovirus/inmunología , Fibroblastos/inmunología , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Citomegalovirus/fisiología , Infecciones por Citomegalovirus/prevención & control , Infecciones por Citomegalovirus/virología , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/genética , Epítopos/inmunología , Fibroblastos/virología , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Células K562 , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , Microscopía Fluorescente , Mutación , VacunaciónRESUMEN
Plasmid DNA inoculations can induce both humoral and cellular immunity, and this technique is now being employed in developing vaccination regimens for a large number of applications. DNA vaccination studies require the preparation of large amounts of purified plasmid DNA with low endotoxin contamination, and the cost burden for multiple injections, multiple animal or large animal studies is significant. We recently reported that selective compaction with spermine can be used to purify large quantities of DNA. We wanted to determine whether this method would produce DNA suitable for vaccination. Endotoxin levels for spermine-compacted DNA were 0.3+/-0.01 endotoxin units (EU)/microg, well within the accepted range (less than 3 EU/microg) for in vivo use. When injected intramuscularly into mice, column-purified and spermine-compacted DNA induced an equivalent antigen-specific CD8+ T-cell response. The labor and time involved in purifying 5 mg of DNA by each method were similar, but the cost of spermine-compacted DNA was only 20% of the cost of column-purified DNA. We conclude that spermine compaction is an efficient and economical method for preparing vaccination-grade DNA.
Asunto(s)
ADN Recombinante/aislamiento & purificación , Espermina/química , Vacunas de ADN , Animales , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , ADN Recombinante/genética , Femenino , Interferones/metabolismo , Ratones , Ratones Endogámicos BALB C , Plásmidos , Linfocitos T Citotóxicos/inmunología , Factores de Tiempo , Vacunas de ADN/economía , Vacunas de ADN/inmunologíaRESUMEN
Preventing losses in vaccine potency due to accidental freezing has recently become a topic of interest for improving vaccines. All vaccines with aluminum-containing adjuvants are susceptible to such potency losses. Recent studies have described excipients that protect the antigen from freeze-induced inactivation, prevent adjuvant agglomeration and retain potency. Although these strategies have demonstrated success, they do not provide a mechanistic understanding of freeze-thaw (FT) induced potency losses. In the current study, we investigated how adjuvant frozen in the absence of antigen affects vaccine immunogenicity and whether preventing damage to the freeze-sensitive recombinant hepatitis B surface antigen (rHBsAg) was sufficient for maintaining vaccine potency. The final vaccine formulation or Alhydrogel(®) alone was subjected to three FT-cycles. The vaccines were characterized for antigen adsorption, rHBsAg tertiary structure, particle size and charge, adjuvant elemental content and in-vivo potency. Particle agglomeration of either vaccine particles or adjuvant was observed following FT-stress. In vivo studies demonstrated no statistical differences in IgG responses between vaccines with FT-stressed adjuvant and no adjuvant. Adsorption of rHBsAg was achieved; regardless of adjuvant treatment, suggesting that the similar responses were not due to soluble antigen in the frozen adjuvant-containing formulations. All vaccines with adjuvant, including the non-frozen controls, yielded similar, blue-shifted fluorescence emission spectra. Immune response differences could not be traced to differences in the tertiary structure of the antigen in the formulations. Zeta potential measurements and elemental content analyses suggest that FT-stress resulted in a significant chemical alteration of the adjuvant surface. This data provides evidence that protecting a freeze-labile antigen from subzero exposure is insufficient to maintain vaccine potency. Future studies should focus on adjuvant protection. To our knowledge, this is the first study to systematically investigate how FT-stress to adjuvant alone affects immunogenicity. It provides definitive evidence that this damage is sufficient to reduce vaccine potency.
Asunto(s)
Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Congelación , Vacunas contra Hepatitis B/inmunología , Potencia de la Vacuna , Animales , Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Femenino , Antígenos de Superficie de la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/inmunología , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Tamaño de la Partícula , Estructura Terciaria de ProteínaRESUMEN
The efficacy of vaccines depends on the presence of an adjuvant in conjunction with the antigen. Of these adjuvants, the ones that contain aluminium, which were first discovered empirically in 1926, are currently the most widely used. However, a detailed understanding of their mechanism of action has only started to be revealed. In this Timeline article, we briefly describe the initial discovery of aluminium adjuvants and discuss historically important advances. We also summarize recent progress in the field and discuss their implications and the remaining questions on how these adjuvants work.
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Adyuvantes Inmunológicos , Aluminio/inmunología , Adsorción/inmunología , Compuestos de Aluminio/química , Compuestos de Aluminio/inmunología , Compuestos de Aluminio/farmacología , Animales , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Antígenos/química , Antígenos/inmunología , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/fisiología , Caspasa 1/fisiología , Proteínas del Citoesqueleto/fisiología , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Ratones , Modelos Inmunológicos , Proteína con Dominio Pirina 3 de la Familia NLRRESUMEN
Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor.
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Antivirales/inmunología , Proteínas Portadoras/inmunología , Glicoproteínas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Muromegalovirus/inmunología , Receptores Inmunológicos/inmunología , Proteínas Virales/inmunología , Animales , Bioensayo , Infecciones por Herpesviridae/inmunología , Antígenos de Histocompatibilidad Clase I/química , Activación de Linfocitos , Ratones , Mutación/genética , Células 3T3 NIH , Estructura Terciaria de ProteínaRESUMEN
In this Commentary, McKee et al. highlight the properties of extrinsic vaccine adjuvants that must be considered to achieve the most protective immune response, as occurs naturally with many intrinsic pathogen-derived adjuvants.
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Adyuvantes Inmunológicos/farmacología , Sistema Inmunológico/fisiología , Infecciones/inmunología , Vacunas/inmunología , Animales , HumanosRESUMEN
Both human CMV and murine CMV (MCMV) elicit large CD8 T cell responses, despite the potent effects of viral genes that interfere with the MHC class I (MHC I) pathway of Ag presentation. To investigate the impact of immune evasion on CD8 T cell priming, we infected mice with wild-type (wt) MCMV or a mutant lacking its MHC I immune evasion genes, Deltam4+m6+m152 MCMV. In acute infection, the two viruses elicited a CD8 T cell response to 26 peptide epitopes that was virtually identical in total size, kinetics, and immunodominance hierarchy. This occurred despite results demonstrating that primary DCs are susceptible to the effects of MCMV's MHC I immune evasion genes. Eight months later, responses to both wt and mutant MCMV displayed the same CD8 T cell "memory inflation" and altered immunodominance that characterize the transition to chronic MCMV infection in C57BL/6 mice. Taken together, these findings suggest either that cross-priming dominates over direct CD8 T cell priming in both acute and chronic MCMV infection, or else that the MHC I immune evasion genes of MCMV are unable to alter direct CD8 T cell priming in vivo. At 2 years postinfection, differences in CD8 T cell immunodominance emerged between individual mice, but on average there were only slight differences between wt and mutant virus infections. Overall, the data indicate that the presence or absence of MHC I immune evasion genes has remarkably little impact on the size or specificity of the MCMV-specific CD8 T cell response over an entire lifetime of infection.