RESUMO
INTRODUCTION: Nucleic acids represent a promising platform for creating vaccines. One disadvantage of this approach is its relatively low immunogenicity. Electroporation (EP) is an effective way to increase the DNA vaccines immunogenicity. However, due to the different configurations of devices used for EP, EP protocols optimization is required not only to enhance immunogenicity, but also to ensure greater safety and tolerability of the EP procedure. AREA COVERED: An data analysis for recent years on the DNA vaccines delivery against viral and parasitic infections using EP was carried out. The study of various EP physical characteristics, such as frequency, pulse duration, pulse interval, should be considered along with the immunogenic construct design and the site of delivery of the vaccine, through the study of the immunogenic and protective characteristics of the latter. EXPERT OPINION: Future research should focus on regulating the humoral and cellular response required for protection against infectious agents by modifying the EP protocol. Significant efforts will be directed to establishing the possibility of redirecting the immune response toward the Th1 or Th2 response by changing the EP physical parameters. It will allow for an individual selective approach during EP, depending on the pathogen type of an infectious disease.
Assuntos
Vacinas de DNA , Humanos , Eletroporação/métodos , DNARESUMO
While the nasal mucosa is a potentially useful site for human immunization, toxin-based nasal adjuvants are generally unsafe and less effective in humans. Safe mucosal adjuvants that activate protective immunity via mucosal administration are highly dependent on barrier antigen sampling by epithelial and DCs. Here, we demonstrate that protein antigens formulated in unique oil-in-water nanoemulsions (NEs) result in distinctive transcellular antigen uptake in ciliated nasal epithelial cells, leading to delivery into nasal associated lymphoid tissue. NE formulation also enhances MHC class II expression in epithelial cells and DC activation/trafficking to regional lymphoid tissues in mice. These materials appear to induce local epithelial cell apoptosis and heterogeneous cytokine production by mucosal epithelial cells and mixed nasal tissues, including G-CSF, GM-CSF, IL-1a, IL-1b, IL-5, IL-6, IL-12, IP-10, KC, MIP-1a, TGF-ß, and TSLP. This is the first observation of a nasal adjuvant that activates calreticulin-associated apoptosis of ciliated nasal epithelial cells to generate broad cytokine/chemokine responses in mucosal tissue.
Assuntos
Adjuvantes Imunológicos , Apoptose , Citocinas/biossíntese , Células Dendríticas/imunologia , Mucosa Nasal/imunologia , Animais , Transporte Biológico/imunologia , Calreticulina , Movimento Celular , Células Cultivadas , Células Dendríticas/metabolismo , Emulsões , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Genes MHC da Classe II , Interleucina-6/imunologia , Interleucina-6/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Nasal/metabolismoRESUMO
Natural transmission of human immunodeficiency virus type 1 (HIV-1) occurs through gastrointestinal and vaginal mucosa. These mucosal tissues are major reservoirs for initial HIV replication and amplification, and the sites of rapid CD4(+) T cell depletion. In both HIV-infected humans and SIV-infected macaques, massive loss of CD4(+) CCR5(+) memory T cells occurs in the gut and vaginal mucosa within the first 10-14 days of infection. Induction of local HIV-specific immune responses by vaccines may facilitate effective control of HIV or SIV replication at these sites. Vaccines that induce mucosal responses, in particular CD8(+) cytotoxic T lymphocytes (CTL), have controlled viral replication at mucosal sites and curtailed systemic dissemination. Thus, there is strong justification for development of next generation vaccines that induce mucosal immune effectors against HIV-1 including CD8(+) CTL, CD4(+) T helper cells and secretory IgA. In addition, further understanding of local innate mechanisms that impact early viral replication will greatly inform future vaccine development. In this review, we examine the current knowledge concerning mucosal AIDS vaccine development. Moreover, we propose immunization strategies that may be able to elicit an effective immune response that can protect against AIDS as well as other mucosal infections.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Mucosa/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Imunidade nas Mucosas , Mucosa/virologiaRESUMO
CD8(+) cytotoxic T lymphocyte (CTL) responses are crucial in establishing the control of persistent virus infections. Population studies of HIV-1-infected individuals suggest that CD8(+) CTL responses targeting epitopes that take the greatest toll on virus replication are instrumental in immune control. A major question for vaccine design is whether incorporating epitopes responsible for controlling a persistent virus will translate into protection from natural infection or serve solely as a fail-safe mechanism to prevent overt disease in infected individuals. Here, we discuss qualitative parameters of the CD8(+) CTL response and mechanisms operative in the control of persistent virus infections and suggest new strategies for design and delivery of HIV vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Humanos , Imunidade nas Mucosas/imunologiaRESUMO
Adjuvant effects on innate as well as adaptive immunity may be critical for inducing protection against mucosal HIV and simian immunodeficiency virus (SIV) exposure. We therefore studied effects of Toll-like receptor agonists and IL-15 as mucosal adjuvants on both innate and adaptive immunity in a peptide/poxvirus HIV/SIV mucosal vaccine in macaques, and made three critical observations regarding both innate and adaptive correlates of protection: (i) adjuvant-alone without vaccine antigen impacted the intrarectal SIVmac251 challenge outcome, correlating with surprisingly long-lived APOBEC3G (A3G)-mediated innate immunity; in addition, even among animals receiving vaccine with adjuvants, viral load correlated inversely with A3G levels; (ii) a surprising threshold-like effect existed for vaccine-induced adaptive immunity control of viral load, and only antigen-specific polyfunctional CD8(+) T cells correlated with protection, not tetramer(+) T cells, demonstrating the importance of T-cell quality; (iii) synergy was observed between Toll-like receptor agonists and IL-15 for driving adaptive responses through the up-regulation of IL-15Ralpha, which can present IL-15 in trans, as well as for driving the innate A3G response. Thus, strategic use of molecular adjuvants can provide better mucosal protection through induction of both innate and adaptive immunity.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Regulação da Expressão Gênica , Interleucina-15/imunologia , Interleucina-15/farmacologia , Macaca mulatta , Mucosa/imunologia , Mucosa/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Linfócitos T/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologiaRESUMO
Survival of antigen-specific CD8(+) T cells in peripheral lymphoid organs during viral infection is known to be dependent predominantly on IL-7 and IL-15. However, little is known about a possible influence of tissue environmental factors on this process. To address this question, we studied survival of memory antigen-specific CD8(+) T cells in the small intestine. Here, we show that 2 months after vaccinia virus infection, B8R(20-27) /H2-K(b) tetramer(+) CD8(+) T cells in the small intestinal intraepithelial (SI-IEL) layer are found in mice deficient in IL-15 expression. Moreover, SI-IEL and lamina propria lymphocytes do not express the receptor for IL-7 (IL-7Rα/CD127). In addition, after in vitro stimulation with B8R(20-27) peptide, SI-IEL cells do not produce high amounts of IFN-γ neither at 5 days nor at 2 months postinfection (p.i.). Importantly, the lack of IL-15 was found to shape the functional activity of antigen-specific CD8(+) T cells, by narrowing the CTL avidity repertoire. Taken together, these results reveal that survival factors, as well as the functional activity, of antigen-specific CD8(+) T cells in the SI-IEL compartments may markedly differ from their counterparts in peripheral lymphoid tissues.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , Interleucina-15/imunologia , Interleucina-7/imunologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Feminino , Imunidade nas Mucosas/imunologia , Memória Imunológica , Interferon gama/biossíntese , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Subunidade alfa de Receptor de Interleucina-7/imunologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Interleucina-7/imunologia , Receptores de Interleucina-7/metabolismo , Vacínia/imunologiaRESUMO
For acute self-limiting infections a vaccine is successful if it elicits memory at least as good as the natural experience; however, for persistent and chronic infections such as HIV, hepatitis C virus (HCV), human papillomavirus (HPV), and human herpes viruses, this paradigm is not applicable. At best, during persistent virus infection the person must be able to maintain the integrity of the immune system in equilibrium with controlling replicating virus. New vaccine strategies are required that elicit both potent high-avidity CD8(+) T-cell effector/memory and central memory responses that can clear the nidus of initial virus-infected cells at mucosal surfaces to prevent mucosal transmission or significantly curtail development of disease. The objective of an HIV-1 T-cell vaccine is to generate functional CD8(+) effector memory cells at mucosal portals of virus entry to prevent viral transmission. In addition, long-lived CD8(+) and CD4(+) central memory cells circulating through secondary lymphoid organs and resident in bone marrow, respectively, are needed to provide a concerted second wave of defense that can contain virus at mucosal surfaces and prevent systemic dissemination. Further understanding of factors which can influence long-lived effector and central memory cell differentiation will significantly contribute to development of effective T-cell vaccines. In this review we will focus on discussing mechanisms involved in T-cell memory and provide promising new approaches toward expanding current vaccine strategies to enhance antiviral memory.
Assuntos
Memória Imunológica , Linfócitos T/imunologia , Vacinas/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular , Diferenciação Celular , Citocinas/imunologia , Humanos , Interferons/imunologia , Ativação Linfocitária , Modelos Imunológicos , Linfócitos T/citologia , Viroses/imunologia , Viroses/prevenção & controleRESUMO
The route of vaccination is important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. Immune responses required for mucosal protection can differ vastly depending on the individual pathogen. For some mucosal pathogens, including acute self-limiting infections, high-titer neutralizing Abs that enter tissue parenchyma or transude into the mucosal lumen are sufficient for clearing cell-free virus. However, for pathogens causing chronic infections such as HIV, hepatitis C virus, herpes viruses, mycobacteria, and fungal and parasitic infections, a single arm of the immune response generated by systemic vaccination may be insufficient for protection. Induction of the mucosal innate and adaptive immune systems, including CD4+ T help, Th17, high avidity CD8+ CTL, and secretory IgA and IgG1 neutralizing Abs, at the site of pathogen entry may be required for effective protection against highly invasive pathogens that lead to chronic infection and may be generated predominantly by mucosal vaccination.
Assuntos
Vias de Administração de Medicamentos , Imunidade nas Mucosas , Infecções/imunologia , Vacinas/administração & dosagem , Animais , HumanosRESUMO
Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Vacinas/imunologia , Animais , Células Cultivadas , Feminino , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/imunologiaRESUMO
The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T-cell and B-cell epitopes as the constituents of a single immunogen. Here we report on the design, chemical synthesis, and immunogenicity of three Plasmodium falciparum MAP vaccines that incorporated antigenic epitopes from the sporozoite, liver, and blood stages of the life cycle. Antibody and cellular responses were determined in three inbred (C57BL/6, BALB/c, and A/J) strains, one congenic (HLA-A2 on the C57BL/6 background) strain, and one outbred strain (CD1) of mice. All three MAPs were immunogenic and induced both antibody and cellular responses, albeit in a somewhat genetically restricted manner. Antibodies against MAP-1, MAP-2, and MAP-3 had an antiparasite effect that was also dependent on the mouse major histocompatibility complex background. Anti-MAP-1 (CSP-based) antibodies blocked the invasion of HepG2 liver cells by P. falciparum sporozoites (highest, 95.16% in HLA-A2 C57BL/6; lowest, 11.21% in BALB/c). Furthermore, antibodies generated following immunizations with the MAP-2 (PfCSP, PfLSA-1, PfMSP-1(42), and PfMSP-3b) and MAP-3 (PfRAP-1, PfRAP-2, PfSERA, and PfMSP-1(42)) vaccines were able to reduce the growth of blood stage parasites in erythrocyte cultures to various degrees. Thus, MAP-based vaccines remain a viable option to induce effective antibody and cellular responses. These results warrant further development and preclinical and clinical testing of the next generation of candidate MAP vaccines that are based on the conserved protective epitopes from Plasmodium antigens that are widely recognized by populations of divergent HLA types from around the world.
Assuntos
Antígenos de Protozoários , Vacinas Antimaláricas , Malária Falciparum/prevenção & controle , Vacinas de Subunidades Antigênicas , Vacinas Sintéticas , Sequência de Aminoácidos , Animais , Animais não Endogâmicos , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Desenho de Fármacos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Eritrócitos/parasitologia , Feminino , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Hepatócitos/parasitologia , Humanos , Imunização , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/síntese química , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologiaRESUMO
Effective frontline defenses against HIV-1 will require targeting vaccines to mucosal tissue in order to induce alphabeta CD8(+) lymphocytes in mucosal effector sites (lamina propria and intraepithelial compartment) as well as antibody secreting plasma cells that can neutralize and limit free virus. A concerted second wave of assault against the virus will require the activation and recruitment of antigen specific memory CD4(+) and CD8(+) T cells in mesenteric lymph nodes and distal secondary lymphoid organs. New delivery strategies targeting the "right" DC subsets in combination with delivery of mucosal adjuvants and innate signals for activating DC will be essential for mucosal vaccines in order to circumvent the naturally tolerogenic environment and the induction of Tregs. Mucosal delivery of antigen in combination with inflammatory signals has been shown to empower systemic immunization by directing responses to mucosal sites for imprinting optimum mucosal memory. Here, we discuss novel vaccine strategies and adjuvants for optimizing mucosal delivery of HIV vaccines.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Sistemas de Liberação de Medicamentos/métodos , Imunidade nas Mucosas/imunologia , Vacinas contra a AIDS/genética , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Células Dendríticas/imunologia , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Modelos Imunológicos , Vacinação/métodosRESUMO
IL-15 is critical for natural killer (NK)-cell development and function and for memory CD8(+) T-cell homeostasis. The IL-15 receptor consists of IL-15Ralpha, IL-2Rbeta, and the common cytokine receptor gamma chain (gamma(c)). IL-15Ralpha is known to "trans-present" IL-15 to an IL-2Rbeta/gamma(c) heterodimeric receptor on responding cells to initiate signaling. To investigate the importance of the IL-15Ralpha cytoplasmic domain, we generated a chimeric receptor consisting of the extracellular domain of IL-15Ralpha and intracellular domain of IL-2Ralpha (IL-15Ralpha(ext)/IL-2Ralpha(int)) and examined its function in 32D cells, in knock-in (KI) mice, and in adoptive-transfer experiments. The chimeric protein exhibited decreased cell-surface expression, and KI mice exhibited diminished NK, NKT, and CD8(+) T-cell development and defects in T-cell functional responses. However, 32D cells expressing the chimeric receptor had less IL-15-induced proliferation than wild-type (WT) transfectants with similar levels of IL-15Ralpha expression, indicating a signaling role for the IL-15Ralpha cytoplasmic domain beyond its effect on expression, and demonstrating that the IL-2Ralpha and IL-15Ralpha cytoplasmic domains are functionally distinct. Interestingly, adoptive-transfer experiments indicated that the chimeric IL-15Ralpha(ext)/IL-2Ralpha(int) receptor still supports trans-presentation. These experiments collectively indicate that IL-15Ralpha can act in cis in addition to acting in trans to present IL-15 to responding cells.
Assuntos
Transferência Adotiva , Apresentação de Antígeno/fisiologia , Subunidade alfa de Receptor de Interleucina-15/química , Subunidade alfa de Receptor de Interleucina-15/fisiologia , Interleucina-15/metabolismo , Transferência Adotiva/veterinária , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Proliferação de Células , Células Cultivadas , Citoplasma/metabolismo , Feminino , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/fisiologia , Estrutura Terciária de Proteína/fisiologia , TransfecçãoRESUMO
Adequate presentation of self and foreign antigens is a key factor for efficient T-cell immunosurveillance against pathogens and tumors. Cells presenting foreign antigens usually comprise a rare population and are difficult to detect even at the peak of infection. Here we demonstrate a CD8(+) T-cell-based approach that allows detection of specific antigen-presenting cells (APC) at a frequency of less than 0.0005%. When T cells are in excess, they form rosettes with rare APCs, which appear as single spots in an IFN-gamma ELISPOT assay. Using this RELISPOT (Rosette ELISPOT) method we demonstrate the dynamic interplay between CD8 T cells and professional and non-professional APCs following virus challenge.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Vaccinia virus/imunologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/virologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/imunologia , Epitopos de Linfócito T , Feminino , Citometria de Fluxo , Corantes Fluorescentes/química , Interferon gama/biossíntese , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Ovário/imunologia , Ovário/virologia , Formação de Roseta , Vacínia/imunologiaRESUMO
Transcutaneous immunization (TCI), the application of vaccines on the skin, induces robust systemic and mucosal antibodies in animal models and in humans. The means by which mucosal immune responses to vaccine antigens are elicited by TCI has not been well characterized. We examined the effect of TCI with an HIV peptide vaccine on the induction of mucosal and systemic CTL responses and protective immunity against mucosal challenge with live virus in mice. Robust HIV-specific CTL responses in the spleen and in the gut mucosa were detected after TCI. The responses were dependent upon the addition of an adjuvant and resulted in protection against mucosal challenge with recombinant vaccinia virus encoding HIV gp160. Although it is clear that adjuvant-activated DCs migrated mainly to draining lymph nodes, coculture with specific T cells and flow cytometry studies with DCs isolated from Peyer's patches after TCI suggested that activated DCs carrying skin-derived antigen also migrated from the skin to immune-inductive sites in gut mucosa and presented antigen directly to resident lymphocytes. These results and previous clinical trial results support the observation that TCI is a safe and effective strategy for inducing strong mucosal antibody and CTL responses.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Pele/imunologia , Vacinas/imunologia , Administração Cutânea , Animais , Feminino , Camundongos , Linfócitos T Citotóxicos/imunologia , Vacinas/administração & dosagemRESUMO
In recent years, great strides in understanding and regulating the immune system have led to new hope for harnessing its exquisite specificity to destroy cancer cells without affecting normal tissues. This review examines the fundamental immunologic advances and the novel vaccine strategies arising from these advances, as well as the early clinical trials studying new approaches to treat or prevent cancer.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/farmacologia , Neoplasias/prevenção & controle , Vacinas Anticâncer/imunologia , Vetores Genéticos , Humanos , Imunoterapia , Neoplasias/imunologia , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologia , Vacinas de Subunidades Antigênicas/imunologia , VírusRESUMO
Among the most cost-effective strategies for preventing viral infections, vaccines have proven effective primarily against viruses causing acute, self-limited infections. For these it has been sufficient for the vaccine to mimic the natural virus. However, viruses causing chronic infection do not elicit an immune response sufficient to clear the infection and, as a result, vaccines for these viruses must elicit more effective responses--quantitative and qualitative--than does the natural virus. Here we examine the immunologic and virologic basis for vaccines against three such viruses, HIV, hepatitis C virus, and human papillomavirus, and review progress in clinical trials to date. We also explore novel strategies for increasing the immunogenicity and efficacy of vaccines.
Assuntos
Vacinas contra a AIDS/uso terapêutico , Infecções por HIV/terapia , Hepatite C Crônica/terapia , Infecções por Papillomavirus/terapia , Vacinas contra Hepatite Viral/uso terapêutico , Vacinas Virais/imunologia , Vacinas contra a AIDS/imunologia , Doença Crônica , Ensaios Clínicos como Assunto , Infecções por HIV/imunologia , Hepatite C Crônica/imunologia , Humanos , Imunidade Celular , Modelos Biológicos , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Hepatite Viral/imunologiaRESUMO
BACKGROUND: Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible. METHODS: Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining. RESULTS: Comparable lymphocyte subset percentages in WB or PBMCs were observed regardless of Optliyse method used (all Ps >/= 0.8). However, differences in fluorescence intensity for several markers were observed across procedures. Compared with the standard stain/fix procedures, fix/stain decreased the mean fluorescence intensities for CD4, CD8, CD19 and CD16/56 in WB and PBMCs (P = 0.03 for these markers P = 0.105 for CD8 in PBMCs). Further decreases in mean fluorescence intensity were seen with the fix/stain/freeze procedure. The stain/fix/freeze yielded intensities largely comparable to those seen with standard stain/fix procedure (P >/= 0.13), suggesting that, when the markers of interest are known at the time of field collection, implementation of this procedure might be desirable. Fix/freeze/stain resulted in diminution of intensity in general, but they tended to be more modest than those seen for fix/stain/freeze and therefore might be applicable to field studies in instances when the specific markers of interest cannot be defined upfront. CONCLUSIONS: Freezing of fixed WB and PBMCs before or after cell surface staining is a reliable method for preserving specimens in field sites for later determination of lymphocyte subset percentages, which are commonly assessed in immunodeficient and cancer patients.
Assuntos
Preservação de Sangue , Criopreservação/métodos , Citometria de Fluxo/métodos , Leucócitos Mononucleares/citologia , Subpopulações de Linfócitos/citologia , Fixação de Tecidos/métodos , Separação Celular , Fixadores , Humanos , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos/imunologiaRESUMO
Understanding key intervention points in developing immune responses may allow the rational inclusion of biological adjuvants into vaccines that could potentiate the immune response both quantitatively and qualitatively and enhance effective memory responses. Cytokine and chemokine combinations can potentially help target antigen to the appropriate antigen presenting cell and initiate maturation of these presenting cells, attract cells expressing different chemokine receptors, steer cellular immune responses toward Th1 and CD8 CTL, and enhance systemic and mucosal IgG and secretory IgA antibodies and determine their isotype balance. Animal protection studies suggest that synergistic combinations of cytokines and immunomodulating molecules may be required to protect from a viral challenge. For example, GM-CSF has been shown to be synergistic with IL-12 or CD40 ligand for induction of CTL and for antiviral protection, and the triple combination of GM-CSF, IL-12, and TNF alpha appears to induce the most effective protection in some mouse models. Chemokine-antigen fusions have also been shown to enhance immunogenicity of the antigen. Combinations of costimulatory molecules have been found to be synergistic when incorporated in a vaccine. Combined use of newer more potent vaccine constructs, containing codon optimized epitopes, relevant CpG motifs, cytokines, costimulatory molecules and chemokines, used in heterologous prime-boost strategies with viral vector vaccines or recombinant proteins, might afford the most potent vaccine approaches yet developed. In this review we will discuss the application and delivery of cytokines, costimulatory molecules, and chemokines toward improving current vaccine strategies.
Assuntos
Vacinas contra a AIDS , Adjuvantes Imunológicos , Quimiocinas/imunologia , Citocinas/imunologia , Animais , Quimiocinas/metabolismo , Ilhas de CpG , Citocinas/metabolismo , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Ativação Linfocitária , Modelos Imunológicos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The use of MHC multimers allows precise and direct detecting and analyzing of antigen-specific T-cell populations and provides new opportunities to characterize T-cell responses in humans and animals. MHC-multimers enable us to enumerate specific T-cells targeting to viral, tumor and vaccine antigens with exceptional sensitivity and specificity. In the field of HIV/SIV immunology, this technique provides valuable information about the frequencies of HIV- and SIV-specific CD8(+) cytotoxic T lymphocytes (CTLs) in different tissues and sites of infection, AIDS progression, and pathogenesis. Peptide-MHC multimer technology remains a very sensitive tool in detecting virus-specific T -cells for evaluation of the immunogenicity of vaccines against HIV-1 in preclinical trials. Moreover, it helps to understand how immune responses are formed following vaccination in the dynamics from priming point until T-cell memory is matured. Here we review a diversity of peptide-MHC class I multimer applications for fundamental immunological studies in different aspects of HIV/SIV infection and vaccine development.