RESUMO
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.
Assuntos
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
BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Chlamydia trachomatis/genética , Escherichia coli/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Células Cultivadas , Infecções por Chlamydia/prevenção & controle , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/metabolismo , Feminino , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease but in turn have also resulted in replacement with non-vaccine serotypes. One such serotype, 35B, a multidrug resistant type, has been associated with an increase in disease. Mice were immunized intramuscularly with monovalent pneumococcal polysaccharide 35B conjugated to CRM197 containing aluminum phosphate adjuvant on days 0, 14, and 28. Pneumococcal enzyme-linked immunosorbent assay, opsonophagocytic killing assays, and competition OPA were performed for STs 35B and 29 to measure serotype-specific binding and functional antibodies. On day 52, mice were intratracheally challenged with S. pneumoniae ST29 to evaluate cross-protection. 35B-CRM197 immunized mice had binding and functional antibodies to both PnPs 35B and 29. 35B-CRM197 immunized mice were 100% protected from IT challenge with S. pneumoniae ST29 as compared to 30% survival in the naïve group. Future vaccines containing polysaccharide 35B, such as the investigational 21-valent PCV, V116, may provide cross protection against the non-vaccine serotype 29 due to structural similarity.
Assuntos
Infecções Pneumocócicas , Pneumonia , Animais , Camundongos , Sorogrupo , Proteção Cruzada , Streptococcus pneumoniae , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas , Vacinas Conjugadas , Anticorpos AntibacterianosRESUMO
Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Coelhos , Camundongos , Animais , Vacinas Pneumocócicas , Vacinas Conjugadas , Macaca mulatta , Anticorpos Antibacterianos , Infecções Pneumocócicas/prevenção & controle , Sorogrupo , Modelos Animais de DoençasRESUMO
IMPORTANCE: We show that simultaneous study of stool and nasopharyngeal microbiome reveals divergent timing and patterns of maturation, suggesting that local mucosal factors may influence microbiome composition in the gut and respiratory system. Antibiotic exposure in early life as occurs commonly, may have an adverse effect on vaccine responsiveness. Abundance of gut and/or nasopharyngeal bacteria with the machinery to produce lipopolysaccharide-a toll-like receptor 4 agonist-may positively affect future vaccine protection, potentially by acting as a natural adjuvant. The increased levels of serum phenylpyruvic acid in infants with lower vaccine-induced antibody levels suggest an increased abundance of hydrogen peroxide, leading to more oxidative stress in low vaccine-responding infants.
Assuntos
Microbioma Gastrointestinal , Microbiota , Vacinas , Lactente , Criança , Humanos , Metaboloma , VacinaçãoRESUMO
We describe here a novel platform technology for the discovery of small molecule mimetics of conformational epitopes on protein antigens. As a model system, we selected mimetics of a conserved hydrophobic pocket within the N-heptad repeat region of the HIV-1 envelope protein, gp41. The human monoclonal antibody, D5, binds to this target and exhibits broadly neutralizing activity against HIV-1. We exploited the antigen-binding property of D5 to select complementary small molecules using a high throughput screen of a diverse chemical collection. The resulting small molecule leads were rendered immunogenic by linking them to a carrier protein and were shown to elicit N-heptad repeat-binding antibodies in a fraction of immunized mice. Plasma from HIV-1-infected subjects shown previously to contain broadly neutralizing antibodies was found to contain antibodies capable of binding to haptens represented in the benzylpiperidine leads identified as a result of the high throughput screen, further validating these molecules as vaccine leads. Our results suggest a new paradigm for vaccine discovery using a medicinal chemistry approach to identify lead molecules that, when optimized, could become vaccine candidates for infectious diseases that have been refractory to conventional vaccine development.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Peptidomiméticos/imunologia , Vacinas contra a AIDS/farmacologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Feminino , Infecções por HIV/sangue , Infecções por HIV/prevenção & controle , Haptenos/imunologia , Haptenos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peptidomiméticos/farmacologiaRESUMO
Invasive pneumococcal disease (IPD) is responsible for serious illnesses such as bacteremia, sepsis, meningitis, and pneumonia in young children, older adults, and persons with immunocompromising conditions and often leads to death. Although the most recent pneumococcal conjugate vaccines (PCVs) have been designed to target serotypes identified as the primary causative agents of IPD, the epidemiological landscape continues to change stressing the need to develop new PCVs. We have developed an investigational 24-valent PCV (PCV24) including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F all conjugated to CRM197 and evaluated this vaccine in adult monkeys. PCV24 was shown to be immunogenic and induced functional antibody for all vaccine serotypes. Of the serotypes common to PCV13 and V114 (PCV15), PCV24 had a similar immunogenic response with the exceptions of 23F which had higher IgG GMCs for PCV13 and V114, and 7F which had higher GMCs for PCV13. Functional antibody responses were similar for the serotypes in common between PCV24, PCV13 and V114 vaccines, with the exception of serotype 7F which was greater for PCV13. Overall, this study shows that PCV24 provided similar immunogenicity as the lower valent vaccines in adult monkeys with no apparent serotype interference. In addition, PCV24 also provided protection against pneumococcal infection in a mouse challenge model.
Assuntos
Infecções Pneumocócicas , Vacinas Pneumocócicas , Idoso , Animais , Anticorpos Antibacterianos , Pré-Escolar , Haplorrinos , Humanos , Lactente , Camundongos , Infecções Pneumocócicas/prevenção & controle , Vacinas ConjugadasRESUMO
Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo-trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR-FTIR) reduction in ß-sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α-helix content increased by 7% (CD) to 13% (ATIR-FTIR). Maintenance of a native-like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Animais , Linhagem Celular , Chlamydia muridarum/química , Chlamydia trachomatis/química , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Cricetulus , Humanos , Peso Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sorogrupo , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Vacinas de Subunidades Antigênicas/químicaRESUMO
Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for approximately 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti-C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.
Assuntos
Chlamydia trachomatis/citologia , Chlamydia trachomatis/crescimento & desenvolvimento , Corpos de Inclusão/microbiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Contagem de Colônia Microbiana/instrumentação , Contagem de Colônia Microbiana/métodos , Cricetinae , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Pneumococcal disease continues to be a medical need even with very effective vaccines on the market. Globally, there are extensive research efforts to improve serotype coverage with novel vaccines; therefore, conducting preclinical studies in different animal models becomes essential. The work presented herein focuses on evaluating a 15-valent pneumococcal conjugate vaccine (PCV15) in mice. Initially we evaluated several doses of PCV15 in Balb/c mice. The optimal vaccine dose was determined to be 0.4µg per pneumococcal polysaccharide (PS) (0.8µg of 6B) for subsequent studies. This PS dose was chosen for PCV evaluation in mice based on antibody levels determined by multiplexed electrochemiluminescent (ECL) assays, T-cell responses following in vitro stimulation with CRM197 peptides and protection from pneumococcal challenge. We then selected four mouse strains for evaluation: Balb/c, C3H/HeN, CD1 and Swiss Webster (SW), immunized with PCV15 by either intraperitoneal (IP) or intramuscular (IM) routes. We assessed IgG responses by ECL assays and functional antibody activity by multiplexed opsonophagocytic assays (MOPA). Every mouse strain evaluated responded to all 15 serotypes contained in the vaccine. Mice tended to have lower responses to serotypes 6B, 23F and 33F. The IP route of immunization resulted in higher antibody titers for most serotypes in Balb/c, C3H and SW. CD1 mice tended to respond similarly for most serotypes, regardless of route of immunization. Similar trends were observed with the four mouse strains when evaluating functional antibody activity. Given the differences in antibody responses based on mouse strain and route of immunization, it is critical to evaluate pneumococcal vaccines in multiple animal models to determine the optimal formulation before moving to clinical trials.
Assuntos
Anticorpos Antibacterianos/biossíntese , Imunoglobulina G/biossíntese , Vacinas Pneumocócicas/administração & dosagem , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/efeitos dos fármacos , Vacinação , Animais , Proteínas de Bactérias/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Injeções Intramusculares , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Vacinas Pneumocócicas/síntese química , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/microbiologia , Polissacarídeos Bacterianos/administração & dosagem , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Sorogrupo , Especificidade da Espécie , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas ConjugadasRESUMO
Chlamydia trachomatis is a human pathogen that infects genital tracts in women. Disease control may be achieved through development of an efficacious vaccine. A mouse genital tract model serves as a tool for evaluation of vaccine candidates. Currently, assessment of infection in mice is performed by enumeration of inclusion-forming units (IFUs) through microscopic counting of fluorescently stained bacteria. We have developed a highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR) assay for enumeration of Chlamydia from mouse genital tracts to increase assay sensitivity, remove subjectivity, and improve sample throughput. The qPCR assay uses a 16S ribosomal gene sequence that is conserved across Chlamydia species and serovars, resulting in detection of multiple serovars of C. trachomatis, as well as Chlamydia muridarum and Chlamydia pneumoniae. The PCR assay provided results similar to IFU enumeration (94% agreement between the 2 assays) and is highly sensitive and specific with less inherent subjectivity than traditional enumeration methods.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Infecções por Chlamydia/microbiologia , Chlamydia/isolamento & purificação , Genitália Feminina/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , Linhagem Celular , Chlamydia/genética , Primers do DNA/genética , DNA Bacteriano/genética , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vagina/microbiologiaRESUMO
The enzyme-linked immunospot (ELISPOT) assay is a powerful tool for measuring antigen-specific cellular immune responses. The ability to use frozen peripheral blood mononuclear cells (PBMC) facilitates testing samples in multicenter clinical trials; however, unreliable ELISPOT responses may result if samples are not handled properly. Exposure of frozen PBMC to suboptimal storage temperature (-20 degrees C) or repeated cycling between more optimal storage temperatures (less than -130 degrees C and -70 degrees C) reduced the quality of frozen PBMC, as assessed by cell viability and functional ELISPOT response measures. Cell viability as assessed by trypan blue dye exclusion was reduced, and the percentage of apoptotic cells, as determined by the Guava Nexin assay, was significantly increased after these events. The functional gamma interferon ELISPOT responses to phytohemagglutinin (PHA) mitogen, a CD4 T-cell-specific antigen (varicella-zoster virus), and a CD8 T-cell-specific antigen (pool containing known cytomegalovirus, Epstein-Barr virus, and influenza virus peptides) were all significantly reduced after suboptimal storage events. However, for a given suboptimal storage event, the magnitude of the reduction varied between individuals and even among aliquots within an individual bleed, indicating the need for sample-specific acceptance criteria (AC). The percent viable or percent apoptotic cells after thaw, as well as the functional ELISPOT response to PHA, were all effective when applied with limits as AC for separating samples damaged during storage from valid control samples. Although all three AC measures could be effectively applied, the apoptosis AC limit applied was best for separating samples that could respond to antigenic stimulation from samples that could not effectively respond.
Assuntos
Criopreservação , Ensaio de Imunoadsorção Enzimática/métodos , Imunidade Celular , Leucócitos Mononucleares/imunologia , Temperatura , Criopreservação/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Interferon gama/análiseRESUMO
Measurements of serum-neutralizing antibody and anti-rotavirus immunoglobulin A (IgA) are the current standard for assessing immune responses following rotavirus vaccination. However, there is ongoing debate as to whether antibody titers correlate with protection against rotavirus gastroenteritis. Children recovering from rotavirus gastroenteritis have increased gamma interferon release from cultured peripheral blood mononuclear cells (PBMCs), suggesting that cell-mediated immunity (CMI) may play a role in viral clearance and protection from subsequent gastroenteritis. We have developed a gamma interferon enzyme-linked immunospot (ELISPOT) assay for evaluation of CMI responses to rotavirus using frozen PBMCs obtained from healthy adults. Responses to three different rotavirus antigen types were analyzed-a peptide pool based on the human VP6 sequence; reassortant human:bovine vaccine strains; and cell culture-adapted (CCA) human G1, G2, G3, G4, and bovine (WC3) G6 strains. The reassortant strains consist of a bovine WC3 genome background expressing the human rotavirus surface proteins VP7 (G1, G2, G3, or G4) or VP4 (P1). Responses to titrations of the peptide pool as well as CCA and reassortant strains were assessed. Gamma interferon ELISPOT responses were similar for CCA and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled. For most subjects, responses to the VP6 peptide pool positively correlated with responses to CCA and reassortant strains. Cell depletion studies indicate the memory responses detected with these frozen adult PBMCs were primarily due to the CD4+ T-cell population. This gamma interferon ELISPOT assay provides a new tool to apply in clinical studies for the characterization of natural or vaccine-induced CMI to rotavirus.
Assuntos
Técnicas Imunoenzimáticas/métodos , Memória Imunológica , Interferon gama , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Antígenos Virais/imunologia , Células Cultivadas , Humanos , Imunidade Celular , Interferon gama/biossíntese , Leucócitos Mononucleares , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
An interferon-gamma ELISPOT assay has been developed for assessment of cellular immune responses to Varicella-Zoster Virus (VZV) in large, multi-center clinical vaccine trials. We show that the assay performed best when testing peripheral blood mononuclear cells (PBMCs) that had been isolated and then frozen on the same day as blood was drawn, and that freezing PBMCs from blood that was stored overnight before processing resulted in dramatically reduced responses. This assay was used to monitor cell-mediated immunity (CMI) in response to a booster immunization with an investigational live, attenuated VZV vaccine in an elderly population that had been vaccinated 8-10 years previously. The booster vaccine elicited a 1.6- to 1.7-fold rise in the VZV-specific cellular immune response as measured by the ELISPOT assay. The increase from pre to post booster vaccination response was more pronounced (approximately 2.2-fold rise) in a subset of subjects who had received two prior immunizations with a live, attenuated vaccine.
Assuntos
Vacina contra Varicela/administração & dosagem , Ensaio de Imunoadsorção Enzimática/métodos , Herpesvirus Humano 3/imunologia , Imunidade Celular , Interferon gama/análise , Idoso , Humanos , Imunização Secundária , Técnicas In Vitro , Interferon gama/biossíntese , Linfócitos T/imunologiaRESUMO
The safety and immunogenecity of a booster dose of live attenuated varicella-zoster virus (VZV) vaccine was evaluated in 196 healthy subjects, >or=60 years old, who had already received a VZV vaccine >5 years before. This repeat booster dose was well tolerated. Cell-mediated immunity (CMI) to VZV was measured by an interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell (ELISPOT) assay and a limiting dilution responder cell frequency (RCF) assay. Prevaccination responses decreased as a function of increasing age but were detectable in all subjects by use of the IFN-gamma ELISPOT assay. In most subjects, VZV-specific CMI was increased at 6 weeks postvaccination. The magnitude of the vaccine-induced IFN-gamma ELISPOT response was inversely related to prevaccination values. Although there was a significant correlation between the IFN-gamma ELISPOT and RCF assays, the ELISPOT assay had greater sensitivity and a wider dynamic range. A live attenuated VZV vaccine is safe and immunogenic in an elderly population, and the vaccine-induced immunity may be monitored by the IFN-gamma ELISPOT assay.