Добірка наукової літератури з теми "Influenza Vaccination RNA Immunology CD8 T cells"

Abstract Influenza remains a serious public health challenge as the current vaccination strategy cannot protect against new strains that emerge every season. Tissue resident memory T cells (TRM) are a non-circulating memory subset that are generated in the lung after influenza infection or intranasal live vaccination, and these lung TRM have been shown to confer broad cross-strain protection. However, the mechanisms by which both CD4+ and CD8+ TRM mediate their protective response are not well defined. We investigated the tissue specific events during an active murine influenza response, including TRM-mediated protection and effector T cell trafficking. To specifically determine the role of TRM, we treated mice with the drug Fingolimod (FTY720), which sequesters circulating lymphocytes into secondary lymphoid organs, enriching the lung for tissue resident memory cells. We found that TRM immediately confer protection, with reduced viral titers evident as early as day 3 after infection. At later time points beginning at days 4–5 post infection, we determined that CD4+ and CD8+ TRM enhance the recruitment of influenza-specific effector T cells into the lung resident niche as well as CD4+ and CD8+ lymphocyte in situ proliferation as measured by BrdU incorporation. Furthermore, total lung RNA sequencing during active infection and FTY720 treatment reveal candidate TRM-associated chemokine pathways for tissue lymphocyte trafficking. These findings indicate that TRM uniquely influence the cellular dynamics of the lung resident niche to mediate cross-strain protection against influenza.

Abstract A long-lived pool of potent memory cells is the defining feature of adaptive immunity. Memory CD8 T cells offer protection for the life of the host due to their unique capabilities to survive in the absence of antigen and respond rapidly to secondary challenge. Therefore, effective CD8 T cell memory is the goal of cell-mediated vaccination strategies. While it is well established that CD4 help is required for CD8 T cell memory formation, it is unclear when during CD8 differentiation this help is required. Further, the affect that CD4 help has on the transcriptional profiles of CD8 T cells and the molecular pathways they use during the generation and maintenance of memory CD8 T cells remains elusive. Using a mouse model of Influenza A virus infection, where priming occurs in the presence or absence of CD4 T cell help, we have pinpointed that help is required during the initial priming of CD8 T cells, and not during memory maintenance or recall. Genome wide RNA-sequencing analysis of the transcriptional signatures between resting “helped” and “ unhelped” memory CD8 T cells reveals surprisingly few differentially expressed genes. However, upon reactivation, “helped” memory CD8 T cells exhibited greater transcriptional up regulation than their “unhelped” counterparts, and utilization of alternate molecular pathways. Our analysis revealed that CD4 help during initial priming is essential for establishing a memory cell pool with enhanced transcriptional potential. Intriguing metabolism differences are currently being further dissected based on this RNA-sequencing data. Thus, CD4 T cell dependent programming likely underpins rapid responsiveness, a key characteristic of memory CD8 T cells.

BackgroundAntitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy.MethodsListeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61–80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing.ResultsCD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells.ConclusionCD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

Abstract Respiratory tract infections are among the leading causes of death. While current vaccines reduce severe disease, they provide suboptimal mucosal protection (e.g. against influenza virus and SARS-CoV-2). Nasal vaccines offer an advantage by quickly limiting viral shedding, in part by generating tissue-resident memory (Trm) CD8 T cells in the nasal mucosa. But little is known about the molecular requirements for this Trm formation. The common paradigm is that lymphocytes home to tissues after having been imprinted within draining lymph nodes to express a tissue-specific combination of trafficking molecules, and Trm cells form rapidly from this pool. To probe the paradigm of tissue homing to the nasal mucosa, we compared CD8 T cell trafficking following intranasal and intramuscular vaccination. To investigate the trafficking molecules expressed by CD8 T cells and nasal cells we generated a single-cell RNA sequencing profile of the nasal mucosa over the course of an acute influenza infection. We found significantly more CD8 Trm cells in the nasal mucosa following intranasal vaccination that rapidly cleared influenza infection. Notably, CD8 T cells could be “pulled” into the nasal mucosa with an inflammatory stimulus following intramuscular vaccination, suggesting a new strategy for generating nasal CD8 Trm cells. Unlike canonical T cell homing to the gut and skin, nasal CD8 T cell trafficking depended on a4/VCAM1 (not a4b7/MADCAM1 or P and E-Selectin), and CD8 T cells expressed high levels of CXCR3 and CXCR6. This combination of trafficking molecules suggests a distinct multi-step adhesion cascade for CD8 T cell recruitment to the nasal mucosa and provides critical insights to rationally design vaccines for respiratory tract protection. Supported by grants from NIH (R01 AR068383-01, P01 AI 112521) and HMS-AbbVie Alliance

Abstract Background: The use of in vitro transcribed RNA isolated from tumor cells or coding for defined tumor associated antigens was shown to be a very powerfull method to generate antigen specific T cells upon transfection into dendritic cells (DC). More recently it was demonstrated in a mouse model that application of RNA intra dermaly can elicit both, CD8 and CD4 mediated immune responses. Furthermore, in Her-2-neu transgenic mice vaccinations with naked RNA were able to elicit a specific immune response in vivo and delay the development of breast cancer in treated animals. Based on these results we analyzed the clinical and immunological responses in patients with metastatic renal cell carcinoma (RCC) vaccinated with mRNA encoding for tumor associated antigens (TAA) using two different treatment arms. Methods: In vitro transcribed RNA was generated using plasmids coding for the tumor antigens MUC1, CEA, Her-2/neu, telomerase, survivin and MAGE-1. RNA coding for HbsAg and Influenza matrix protein were included as controls. Vaccinations were performed intradermally on day 1,14, 28 and 42 in a first group of patients and on day 0–3, 7–10, 28 and 42 in a second group (intensified arm). Vaccinations were repeated afterwards monthly until tumor progression. One day after each RNA injection patients additionally received 1 injection of GM-CSF (250 μg) sc. The enhancement of T cell precursor was monitored using IFN-g ELISPOT and tetramer staining.. Results: 25 patients were included in this study. In 3 patients regression of metastases was induced and 6 patients had a stabilization of the disease. Specific CD8 and CD4 T cell responses in vivo were detected in the first analyzed patients. The treatment was well tolerated with no severe side effects. In most cases erythema and induration were observed after injections of GM-CSF. One patient developed an allergic exanthema after GM-CSF application. Conclusions: This study demonstrates that intradermal RNA-vaccination can be effective in the treatment of metastatic RCC and induces clinical and immunological responses.

ABSTRACT Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.

Abstract Vaccination remains the most cost-effective way to control Influenza outbreaks. However, current Influenza vaccines do not provide efficacious heterosubtypic immunity. Therefore, the development of a successful universal vaccination strategy is urgently needed. To achieve this goal, one approach is focused on the induction of a T cell based response versus more conserved flu antigens, such as the Nucleoprotein (NP) and the Matrix protein 1 (M1). The aim of this study was to evaluate the immunogenicity and protective efficacy conferred by conserved NP and M1 antigens expressed by a self-amplifying RNA-based vaccine (SAM(NP)® and SAM(M1)®). We demonstrate that SAM(NP)® and SAM(M1)® are immunogenic in Balb/c mice. SAM(NP)® formulation induced IFNγ+ and LAMP1+ NP-specific cytotoxic CD8+ T cells as well as multifunctional NP-specific CD4+ T cells. SAM(M1) ® elicited a robust polyfunctional CD4 T helper 1 response. Moreover, we show that SAM(NP)® and SAM(M1)® formulations, when administered separately or in combination, provided differential levels of protection against a lethal challenge with mouse-adapted A/PR/8/1934 (H1N1) Influenza virus. SAM(NP)® and SAM(M1)® formulation induced T cell immune responses, potent mediator of heterosubtypic immunity, therefore, they are promising antigens in the design of a cross-protective flu vaccine. Since the SAM® vaccine technology offers several advantages , it could potentially speed up the development of universal flu vaccine candidates.

Abstract Memory CD8+ T cells afford long-lived, durable protection against infection and malignancy. Despite the critical relevance of memory T cells to vaccination and immunotherapies, the transcriptional and epigenetic signals instructing memory T cell fate remain unclear. Here, we utilized a pooled RNA interference screen to evaluate the activity of transcription factors and chromatin modifiers governing memory T cell differentiation in vivo. The epigenetic ‘reader,’ Brd4 of the BET protein family, emerged as a top regulator of memory T cell differentiation. Through RNA interference, small molecule inhibition and inducible genetic deletion, we established a central role for Brd4 in mediating CD8+ T cell proliferation, differentiation and function during acute viral infection. Brd4 was required for optimal expression of fate-specifying transcription factors, and a deficiency of Brd4 activity resulted in diminished formation of effector memory and CD103+ tissue-resident memory CD8+ T cells. Given that BET inhibition has emerged as a powerful approach for suppressing tumor growth, we also evaluated how BET inhibition and Brd4-deficiency influence T cell activity during cancer. Brd4 was required for intratumoral accumulation and function of T cells in a mouse model of melanoma, and BET inhibition suppressed T cell-mediated control of tumor growth. However, epigenetic targeting of Brd4 constrained terminal differentiation of T cells within tumors, biasing tumor-resident T cells towards a reprogrammable TCF1-expressing phenotype. These studies establish a novel role for Brd4 in CD8+ T cell biology and provide insight for immunotherapy approaches designed to leverage the dynamic activity of CD8+ T cells in tumors.

Abstract Viral sensing by RIG-I and downstream activation of antiviral defenses along with the induction of innate immune cytokines is essential from protection against influenza A virus (IAV) infection. We have identified a novel, small-molecule RIG-I agonist, KIN1148, which binds and activates RIG-I to signal the activation of IRF3 and the innate immune response. We are developing this molecule as an adjuvant to enhance vaccination against pandemic H1N1 (pH1N1) IAV. Ex vivo treatment of dendritic cells with KIN1148 leads to their activation and maturation. We determined the ability of KIN1148 to enhance suboptimal IAV vaccine responses in vivo. Administration of KIN1148 leads enhanced protection during high dose pH1N1 infection following a single, intramuscular administration of KIN1148 with IAV vaccine. This increase in protection is accompanied by a significant reduction in virus titers, as well as lung pathology. Analysis of the immune response induced following vaccination with KIN1148 as well as challenge demonstrates an increase in chemoattractant cytokines, germinal center B cells, IAV-specific antibodies, and IAV-specific CD4 and CD8 T cells compared to vaccination alone, indicating the induction of a broad anti-IAV immune response. Together these results demonstrate that prophylactic drug targeting of the RIG-I pathway with a small molecule enhances vaccine protection and highlight the potential of KIN1148 to enhancing vaccines against RNA virus infection.

Little is known about the factors that govern the level of HIV-1 replication in infected individuals. Recent studies (using potent antiviral drugs) of the kinetics of HIV-1 replication in vivo have demonstrated that steady-state levels of viremia are sustained by continuous rounds of de novo infection and the associated rapid turnover of CD4+ T lymphocytes. However, no information is available concerning the biologic variables that determine the size of the pool of T cells that are susceptible to virus infection or the amount of virus produced from infected cells. Furthermore, it is not known whether all CD4+ T lymphocytes are equally susceptible to HIV-1 infection at a given time or whether the infection is focused on cells of a particular state of activation or antigenic specificity. Although HIV-1 replication in culture is known to be greatly facilitated by T cell activation, the ability of specific antigenic stimulation to augment HIV-1 replication in vivo has not been studied. We sought to determine whether vaccination of HIV-1-infected adults leads to activation of virus replication and the targeting of vaccine antigen-responsive T cells for virus infection and destruction. Should T cell activation resulting from exposure to environmental antigens prove to be an important determinant of the steady-state levels of HIV-1 replication in vivo and lead to the preferential loss of specific populations of CD4+ T lymphocytes, it would have significant implications for our understanding of and therapeutic strategies for HIV-1 disease. To begin to address these issues, HIV-1-infected individuals and uninfected controls were studied by measurement of immune responses to influenza antigens and quantitation of virion-associated plasma HIV-1 RNA levels at baseline and at intervals after immunization with the trivalent influenza vaccine. Influenza vaccination resulted in readily demonstrable but transient increases in plasma HIV-1 RNA levels, indicative of activation of viral replication, in HIV-1-infected individuals with preserved ability to immunologically respond to vaccine antigens. Activation of HIV-1 replication by vaccination was more often seen and of greater magnitude in individuals who displayed a T cell proliferative response to vaccine antigens at baseline and in those who mounted a significant serologic response after vaccination. The fold increase in viremia, as well as the rates of increase of HIV-1 in plasma after vaccination and rates of viral decline after peak viremia, were higher in individuals with higher CD4+ T cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)

Priming of antigen-specific CD8+ T cells by vaccination is key for subsequent viral clearance. However, to date, CD8+ T-cell characterization is performed under the assumption that cells of a particular type are identical, and not acknowledging their heterogeneous nature. It has recently been proven, in many different studies, that cells do differ quite significantly and can drive different responses. In order to characterize the heterogeneity of vaccine-induced CD8+ T cells, we performed gene expression profiling and protein characterization at the single-cell level, after delivery of influenza RNA-based vaccine (SAM (H1) (A/California/07/2009 (H1/N1)) or MF59TM -adjuvanted Monovalent Influenza Vaccine (aMIV (A/California/07/2009 (H1N1)). CD8+ T cells were isolated from mouse splenocytes, stained with MHC class-I HA533-554 pentamer and either single-cell sorted, lysed, and processed for reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR) analysis of 96 individual genes, or characterized by mass cytometry for the expression of 32 biomarkers. In parallel, the functionality of the CD8+ T cells was assessed in an in vivo cytotoxic assay. CD8+ T cells induced by the SAM(H1) vaccine showed increased antigen-specific lysis activity in vivo, compared to cells induced by aMIV. Ex vivo, higher frequencies of HA533-541-pentamer+ CD8+ T cells were induced by SAM(H1) compared to aMIV at all time points tested, and pentamer+ cells were single-cell sorted for gene expression analysis. Refined analysis of TEFF (il-7rα -- cd62l- ), TEM (il-7rα +- cd62l- ) and TCM (il-7rα +- cd62l+ )) highlighted significant differences in gene expression profiles between and within the vaccines. Overall, SAM(H1) induced mostly TEM cells, while aMIV induced equal number of TCM and TEM cells. Both vaccines induced few TEFF cells, and only CD8+ T cells from SAM(H1) showed TEFF terminally differentiated short-lived effector cells (SLEC) (klrg1+ il-7rα - cd62lcxcr3- tbet+ blimp-1 + ). Ten days after the second immunization, TEM cells, unlike TCM cells, showed a transcriptional difference between the vaccines. SAM(H1) and aMIV, with up regulation of members of the cytolytic/Fas Ligans (FasL) and tumor necrosis factor (TNF) Superfamily pathways, respectively. A specific molecular signature of 12 differentially expressed genes (DEG) was identified and further evaluated at the protein expression level using mass cytometry. Most of the proteins corresponding to the 12 DEG list were also expressed. The aim of this study was to apply state-of-the-art technology platforms to the study of gene and protein expression by immune cells in response to vaccination. With this approach, we identified distinct HA533-541- pentamer+ CD8+ T-cell subsets elicited by RNA- and adjuvanted protein-based vaccines, which revealed a particular biomarker signature associated to each vaccine. In conclusion, our results showed that these novel technology platforms might be extremely useful for the in-depth characterization of immune responses to vaccination and can be extended to other medical applications.

The severe disease associated with seasonal epidemics of influenza A virus (IAV), as well as pandemic outbreaks, have highlighted the necessity for novel, broadly cross-reactive vaccination and therapeutic strategies against IAV. Our studies have focused on the contribution of IAV-specific CD8 T cells to mediating protection following IAV vaccination and infection as IAV-specific CD8 T cells are required for clearance of IAV. Further, IAV-specific CD8 T cells are typically cross-protective as they are generally directed at highly conserved areas of IAV. Recently, influenza virus-like particles (VLPs) have been developed from recombinant baculoviruses containing influenza proteins hemagglutinin (HA) and/or neuraminidase (NA) on the surface and matrix (M1) in the VLP core. Influenza VLPs induce potent antibody responses and have been shown to provide protection from morbidity and mortality during lethal homo- and hetero-subtypic IAV challenge. This suggests that conserved, VLP-induced CD8 T cell responses may also contribute to the overall protective ability of VLPs. However, whether influenza VLPs can induce influenza-specific CD8 T cell responses and if these T cells are protective during IAV challenge remains unknown. Here, I demonstrate that a single, intranasal vaccination with VLPs containing HA and M1 leads to a significant increase in HA533-specific CD8 T cells in the lungs and lung-draining lymph nodes. Our results also indicate that HA533-specific CD8 T cells primed by influenza VLP vaccination are significantly increased in the lungs following lethal IAV challenge. These VLP-induced memory CD8 T cells are critical in providing protection from lethality following subsequent challenge infections, as depletion of CD8 T cells leads to increased mortality, even when total, but not VLP-induced memory, CD8 T cell numbers have been allowed to recover prior to lethal dose IAV challenge. In addition, my studies also importantly demonstrate that these VLP-induced, HA533-specific CD8 T cells aid in protection from high-dose, heterosubtypic IAV infections where CD8 T cell epitopes are conserved, but the targets of neutralizing antibodies have been destroyed. This dissertation further elucidates the requirements for the regulation of the IAV-specific CD8 T cell response in the periphery (i.e. lung) by pDC and CD8α+ DC. Our studies have previously demonstrated that pDC or CD8α+ DC must present viral antigen in the context of MHC class I along with trans-presentation of IL-15 to effector, IAV-specific CD8 T cells in the lungs to protect the T cells from apoptosis and allow generation of the full magnitude CD8 T cell response needed to clear IAV infection. Herein, I demonstrate that in addition to antigen presentation and IL-15, costimulatory molecules on the surface of pDC and CD8α+ DC are also required. However, the specific costimulatory molecules required depends upon both the mouse strain utilized for IAV infection as well as DC subset. In addition to costimulatory molecules, I also demonstrate that the requirement for pDC and CD8α+ DC to be infected differs in order for them to participate in this pulmonary rescue of the IAV-specific CD8 T cell response. While CD8α+ DC are able to efficiently cross-present exogenous antigen, pDC must be directly infected and utilize the endogenous, direct antigen presentation pathway to present viral antigen to IAV-specific CD8 T cells in the lungs during IAV infection. These data suggest there are distinct differences between pDC and CD8α+ DC in their mechanism of regulating the pulmonary IAV-specific CD8 T cell response, which had not been previously appreciated. Together, the results presented herein further detail the mechanism of regulation of effector IAV-specific CD8 T cells by DC as well as the contribution of IAV-specific CD8 T cells to a novel, IAV VLP vaccination strategy. These findings highlight the importance of IAV-specific CD8 T cells in mediating protection following IAV vaccination and infection.