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BACKGROUND: Cytomegalovirus (CMV) can cause congenital infection and is the leading cause of nongenetic newborn disabilities. V160, a conditionally replication-defective virus, is an investigational vaccine under evaluation for prevention of congenital CMV. The vaccine was well tolerated and induced both humoral and cellular immunity in CMV-seronegative trial participants. T-cell-mediated immunity is important for immune control of CMV. Here we describe efforts to understand the quality attributes of the T-cell responses induced by vaccination. METHODS: Using multicolor flow cytometry, we analyzed vaccine-induced T cells for memory phenotype, antigen specificity, cytokine profiles, and cytolytic potential. Moreover, antigen-specific T cells were sorted from 4 participants, and next-generation sequencing was used to trace clonal lineage development during the course of vaccination using T-cell receptor ß-chain sequences as identifiers. RESULTS: The results demonstrated that vaccination elicited polyfunctional CD4 and CD8 T cells to 2 dominant antigens, pp65 and IE1, with a predominantly effector phenotype. Analysis of T-cell receptor repertoires showed polyclonal expansion of pp65- and IE1-specific T cells after vaccination. CONCLUSION: V160 induced a genetically diverse and polyfunctional T-cell response and the data support further clinical development of V160 for prevention of CMV infection and congenital transmission. CLINICAL TRIALS REGISTRATION: NCT01986010.
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Linfócitos T CD8-Positivos , Infecções por Citomegalovirus , Vacinas contra Citomegalovirus , Imunidade Celular , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Humanos , VacinaçãoRESUMO
BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.
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Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Memória Imunológica , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Hipermutação Somática de Imunoglobulina , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Feminino , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Sorogrupo , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Streptococcus pneumoniae/imunologia , Vacinação , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêuticoRESUMO
Globally, Streptococcus pneumoniae is a leading cause of vaccine-preventable morbidity and mortality in infants and children. In recent decades, large-scale pediatric immunization programs have substantially reduced the incidence of invasive pneumococcal disease. Despite this, residual vaccine-type pneumococcal disease remains in the form of vaccine breakthrough and vaccine failure. This targeted literature review aims to discuss aspects of vaccine breakthrough and failure in infants and children, including disease epidemiology, clinical presentation, risk factors, vaccination schedules, vaccine serotypes, correlates of protection, comorbidities, disease surveillance, and potential implications for future vaccine development.
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Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among all infants worldwide and remains a significant cause of morbidity and mortality. To address this unmet medical need, MK-1654, a half-life extended RSV neutralizing monoclonal antibody, is in clinical development for the prevention of RSV disease in infants. This was a phase I, randomized, placebo-controlled, single-site, double-blind trial of MK-1654 in 44 healthy Japanese adults. The safety, tolerability, pharmacokinetics, antidrug antibodies (ADAs), and serum neutralizing antibody (SNA) titers against RSV were evaluated for 1 year after a single intramuscular (i.m.) or intravenous (i.v.) dose of MK-1654 or placebo in five groups (100 mg i.m., 300 mg i.m., 300 mg i.v., 1000 mg i.v., or placebo). MK-1654 was generally well-tolerated in Japanese adults. There were no serious drug-related adverse events (AEs) reported in any MK-1654 recipient and no discontinuations due to any AEs in the study. The half-life of MK-1654 ranged from 76 to 91 days across dosing groups. Estimated bioavailability was 86% for 100 mg i.m. and 77% for 300 mg i.m. One participant out of 33 (3.0%) developed detectable ADA with no apparent associated AEs. The RSV SNA titers increased in a dose-dependent manner among participants who received MK-1654. These data support the development of MK-1654 for use in Japanese infants.
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Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Humanos , Lactente , Japão , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/prevenção & controleRESUMO
In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.
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Imunidade Celular , Imunidade Humoral , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Vacinas de mRNA , Animais , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza/imunologia , Camundongos , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/prevenção & controle , Primatas , Vacinas Sintéticas , Vacinas de mRNA/imunologiaRESUMO
Dengue (DENV) is a mosquito-borne virus with four serotypes causing substantial morbidity in tropical and subtropical areas worldwide. V181 is an investigational, live, attenuated, quadrivalent dengue vaccine. In this phase 1 double-blind, placebo-controlled study, the safety, tolerability, and immunogenicity of V181 in baseline flavivirus-naïve (BFN) and flavivirus-experienced (BFE) healthy adults were evaluated in two formulations: TV003 and TV005. TV005 contains a 10-fold higher DENV2 level than TV003. Two-hundred adults were randomized 2:2:1 to receive TV003, TV005, or placebo on Days 1 and 180. Immunogenicity against the 4 DENV serotypes was measured using a Virus Reduction Neutralization Test (VRNT60) after each vaccination and out to 1 year after the second dose. There were no discontinuations due to adverse events (AE) or serious vaccine-related AEs in the study. Most common AEs after TV003 or TV005 were headache, rash, fatigue, and myalgia. Tri- or tetravalent vaccine-viremia was detected in 63.9% and 25.6% of BFN TV003 and TV005 participants, respectively, post-dose 1 (PD1). Tri- or tetravalent dengue VRNT60 seropositivity was demonstrated in 92.6% of BFN TV003, 74.2% of BFN TV005, and 100% of BFE TV003 and TV005 participants PD1. Increases in VRNT60 GMTs were observed after the first vaccination with TV003 and TV005 in both flavivirus subgroups for all dengue serotypes, and minimal increases were measured PD2. GMTs in the TV003 and TV005 BFE and BFN groups remained above the respective baselines and placebo through 1-year PD2. These data support further development of V181 as a single-dose vaccine for the prevention of dengue disease.
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Vacinas contra Dengue , Vírus da Dengue , Dengue , Flavivirus , Adulto , Anticorpos Antivirais , Dengue/prevenção & controle , Método Duplo-Cego , Humanos , Imunogenicidade da Vacina , Vacinas Atenuadas , Vacinas CombinadasRESUMO
Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.
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Metapneumovirus , Vírus Sincicial Respiratório Humano , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Humanos , Células B de Memória , Proteínas Virais de FusãoRESUMO
Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.
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Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/fisiologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/fisiologia , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-H1 , Linhagem Celular , Doença Crônica , Humanos , Imunoglobulina G/fisiologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Morte Celular Programada 1 , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga ViralRESUMO
Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.
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Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Idoso , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Imunogenicidade da Vacina , Pessoa de Meia-Idade , RNA Mensageiro , Proteínas Virais de FusãoRESUMO
Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and related morbidity and mortality in infants. Passive immunization with an RSV-neutralizing antibody can provide rapid protection to this vulnerable population. Proof-of-concept for this approach has been demonstrated by palivizumab; however, the use of this antibody is generally restricted to the highest-risk infants due to monthly dosing requirements and its cost. To address the large unmet medical need for most infants, we are evaluating MK-1654, a fully human RSV-neutralizing antibody with half-life extending mutations targeting site IV of the fusion protein. In this 2-part, placebo-controlled, double-blind, first-in-human study, 152 healthy adults were randomized 3:1 to receive a single dose of MK-1654 or placebo in 5 cohorts (100 or 300 mg as an intramuscular dose or 300, 1000, or 3000 mg as an intravenous dose). Safety, pharmacokinetics, antidrug antibodies, and RSV serum-neutralizing antibody titers were evaluated through 1 year. MK-1654 serum concentrations increased proportionally with dose and resulted in corresponding elevations in RSV serum-neutralizing antibody titers. The antibody displayed a half-life of 73 to 88 days and an estimated bioavailability of 69% at the 300-mg dose. The overall safety profile of MK-1654 was similar to placebo, and treatment-emergent antidrug antibodies were low (2.6%) with no associated adverse events. These data support the continued development of MK-1654 for the prevention of RSV disease in infants.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Antivirais , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/efeitos adversos , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Antivirais/farmacocinética , Disponibilidade Biológica , Estudos de Coortes , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/imunologia , Adulto JovemRESUMO
The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.
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Results from Merck's phase II adenovirus type 5 (Ad5) gag/pol/nef test-of-concept trial showed that the vaccine lacked efficacy against human immunodeficiency virus (HIV) infection in a high-risk population. Among the many questions to be explored following this outcome are whether (i) the Ad5 vaccine induced the quality of T-cell responses necessary for efficacy and (ii) the lack of efficacy in the Ad5 vaccine can be generalized to other vector approaches intended to induce HIV type 1 (HIV-1)-specific T-cell responses. Here we present a comprehensive evaluation of the T-cell response profiles from cohorts of clinical trial subjects who received the HIV CAM-1 gag insert delivered by either a regimen with DNA priming followed by Ad5 boosting (n = 50) or a homologous Ad5/Ad5 prime-boost regimen (n = 70). The samples were tested using a statistically qualified nine-color intracellular cytokine staining assay measuring interleukin-2 (IL-2), tumor necrosis factor alpha, macrophage inflammatory protein 1beta, and gamma interferon production and expression of CD107a. Both vaccine regimens induced CD4(+) and CD8(+) HIV gag-specific T-cell responses which variably expressed several intracellular markers. Several trends were observed in which the frequencies of HIV-1-specific CD4(+) T cells and IL-2 production from antigen-specific CD8(+) T cells in the DNA/Ad5 cohort were more pronounced than in the Ad5/Ad5 cohort. Implications of these results for future vaccine development will be discussed.
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Adenoviridae/metabolismo , Genes gag/genética , Linfócitos T/virologia , Vacinas contra a AIDS/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Quimiocina CCL4/biossíntese , Estudos de Coortes , DNA/metabolismo , Humanos , Interleucina-2/biossíntese , Proteína 1 de Membrana Associada ao Lisossomo/biossíntese , Modelos Biológicos , Fenótipo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Linfócitos B/imunologia , Epitopos de Linfócito B/imunologia , Memória Imunológica , Vírus Sincicial Respiratório Humano/imunologia , Idoso , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Criança , Pré-Escolar , HumanosRESUMO
Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.
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Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Sequência Conservada , Glicoproteínas/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Humanos , Memória Imunológica , Modelos Moleculares , Ligação Proteica , SigmodontinaeRESUMO
The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection.
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Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Contagem de Linfócito CD4 , Doença Crônica , Estudos de Coortes , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Produtos do Gene gag/imunologia , Produtos do Gene nef/imunologia , Produtos do Gene pol/imunologia , Produtos do Gene tat/imunologia , Infecções por HIV/virologia , Hispânico ou Latino/estatística & dados numéricos , Humanos , Imunidade Celular , Interferon gama/imunologia , Masculino , RNA Viral/sangue , Carga Viral , População Branca/estatística & dados numéricos , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Respiratory syncytial virus (RSV) is a leading cause of serious lower respiratory tract disease in young children and older adults throughout the world. Prevention of severe RSV disease through active immunization is optimal but no RSV vaccine has been licensed so far. Immune mechanisms of protection against RSV infection in humans have not been fully established, thus a comprehensive characterization of virus-specific immune responses in a relevant animal model will be beneficial in defining correlates of protection. In this study, we infected juvenile naive AGMs with RSV A2 strain and longitudinally assessed virus-specific humoral and cellular immune responses in both peripheral blood and the respiratory tract. RSV viral loads at nasopharyngeal surfaces and in the lung peaked at around day 5 following infection, and then largely resolved by day 10. Low levels of neutralizing antibody titers were detected in serum, with similar kinetics as RSV fusion (F) protein-binding IgG antibodies. RSV infection induced CD8+, but very little CD4+, T lymphocyte responses in peripheral blood. Virus-specific CD8+ T cell frequencies were ~10 fold higher in bronchoaveolar lavage (BAL) compared to peripheral blood and exhibited effector memory (CD95+CD28-) / tissue resident memory (CD69+CD103+) T (TRM) cell phenotypes. The kinetics of virus-specific CD8+ T cells emerging in peripheral blood and BAL correlated with declining viral titers, suggesting that virus-specific cellular responses contribute to the clearance of RSV infection. RSV-experienced AGMs were protected from subsequent exposure to RSV infection. Additional studies are underway to understand protective correlates in these seropositive monkeys.
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Linfócitos T CD8-Positivos/imunologia , Imunidade Celular , Memória Imunológica , Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos CD/sangue , Antígenos CD/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Chlorocebus aethiops , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pulmão/metabolismo , Infecções por Vírus Respiratório Sincicial/sangue , Vírus Sinciciais Respiratórios/metabolismoRESUMO
Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.
Assuntos
Vacinas contra a AIDS/imunologia , Produtos do Gene gag/imunologia , Infecções por HIV/prevenção & controle , Linfócitos T/fisiologia , Adenoviridae , Adolescente , Adulto , Feminino , Antígenos HIV/imunologia , HIV-1 , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A vast body of evidence suggests that nanoparticles function as potent immune-modulatory agents. We have previously shown that Merck proprietary Lipid NanoParticles (LNPs) markedly boost B-cell and T-cell responses to sub-unit vaccine antigens in mice. To further evaluate the specifics of vaccine delivery and dosing regimens in vivo, we performed immunogenicity studies in BALB/c and C57BL/6 mice using two model antigens, Hepatitis B Surface Antigen (HBsAg) and Ovalbumin (OVA), respectively. To assess the requirement for co-administration of antigen and LNP for the elicitation of immune responses, we evaluated immune responses after administering antigen and LNP to separate limbs, or administering antigen and LNP to the same limb but separated by 24 h. We also evaluated formulations combining antigen, LNP, and aluminum-based adjuvant amorphous aluminum hydroxylphosphate sulfate (MAA) to look for synergistic adjuvant effects. Analyses of antigen-specific B-cell and T-cell responses from immunized mice revealed that the LNPs and antigens must be co-administered-both at the same time and in the same location-in order to boost antigen-specific immune responses. Mixing of antigen with MAA prior to formulation with LNP did not impact the generation of antigen-specific B-cell responses, but drastically reduced the ability of LNPs to boost antigen-specific T-cell responses. Overall, our data demonstrate that the administration of LNPs and vaccine antigen together enables their immune-stimulatory properties.
RESUMO
Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4(+) and CD8(+)T cell responses. Intracellular FACS analyses revealed that LNP and LNP+IMO formulated antigens led to higher frequency of antigen-specific IFNγ(+)TNFα(+)IL-2(+), multi-functional CD8(+)T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Linfócitos B/imunologia , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Animais , Feminino , Antígenos de Superfície da Hepatite B/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.