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Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8+ or %CD4+ cytokine-positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty-three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry.
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Citocinas , Neoplasias , Citometría de Flujo , Humanos , Inmunoterapia , Neoplasias/terapia , Reproducibilidad de los Resultados , Coloración y EtiquetadoRESUMEN
Background: GEN-003 is a candidate therapeutic vaccine for genital herpes simplex virus type 2 (HSV-2). We compared virologic and clinical impact of varying GEN-003 doses. Methods: Adults with symptomatic HSV-2 received placebo or GEN-003 (30 or 60 µg antigen with 25, 50, or 75 µg adjuvant). Viral shedding and lesion rates before vaccination were compared with those measured immediately after vaccination, then at weeks 29-33 and 53-57 after last dose. Results: Compared with baseline shedding rates, the rate ratios for viral shedding immediately after treatment were as follows: 0.82 (95% confidence interval [CI], 0.49-1.36), 30 µg antigen/25 µg adjuvant (30/25) dose; 0.64 (95% CI, 0.45-0.92), 30/50 dose; 0.63 (95% CI, 0.37-1.10), 30/75 dose; 0.56 (95% CI, 0.36-0.88), 60/25 dose; 0.58 (95% CI, 0.38-0.89), 60/50 dose; 0.45 (95% CI, 0.16-0.79), 60/75 dose; and 0.98 (95% CI, 0.76-1.26), placebo. Lesion rate reductions by GEN-003 ranged from 31% to 69%, but lesion rates also decreased among placebo recipients (62%). Reductions in shedding and lesion rate were durable for 12 months for the 60 µg antigen plus 50 or 75 µg adjuvant groups. No serious adverse events occurred with vaccination. Conclusions: The most efficacious vaccine combinations for GEN-003 were the 60 µg/50 µg and 60 µg/75 µg doses.
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Herpes Genital/terapia , Herpesvirus Humano 2/inmunología , Inmunoterapia , Vacunas Virales/uso terapéutico , Adyuvantes Inmunológicos , Adolescente , Adulto , Femenino , Herpes Genital/virología , Humanos , Masculino , Persona de Mediana Edad , Vacunación , Vacunas Virales/administración & dosificación , Esparcimiento de Virus , Adulto JovenRESUMEN
Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitis; however, MnB is most commonly associated with asymptomatic carriage in the nasopharyngeal cavity, as opposed to the disease state. Two vaccines are now licensed for the prevention of MnB disease; a possible additional benefit of these vaccines could be to protect against disease indirectly by disrupting nasopharyngeal carriage (e.g., herd protection). To investigate this possibility, accurate diagnostic approaches to characterize MnB carriage isolates are required. In contrast to invasive meningococcal disease (IMD) isolates, which can be readily serogrouped, carriage isolates often lack capsule expression, making standard phenotypic assays unsuitable for strain characterization. Several antibody-based methods were evaluated for their abilities to serogroup isolates and were compared with two genotyping methods (real-time PCR [rt-PCR] and whole-genome sequencing [WGS]) to identify which approach would most accurately ascertain the polysaccharide groups associated with carriage isolates. WGS and rt-PCR were in agreement for 99% of IMD isolates, including those with coding sequences for MnB, MnC, MnW, and MnY, and the phenotypic methods correctly identified serogroups for 69 to 98% of IMD isolates. In contrast, only 47% of carriage isolates were groupable by genotypic methods, due to mutations within the capsule operon; of the isolates identified by genotypic methods, ≤43% were serogroupable with any of the phenotypic methods tested. These observations highlight the difficulties in the serogrouping and capsular genogrouping of meningococcal carriage isolates. Based on our findings, WGS is the most suitable approach for the characterization of meningococcal carriage isolates.
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Cápsulas Bacterianas/clasificación , Portador Sano/microbiología , Técnicas de Genotipaje/métodos , Neisseria meningitidis/clasificación , Infecciones por Neisseriaceae/microbiología , Serotipificación/métodos , Adolescente , Adulto , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/inmunología , Estudios Epidemiológicos , Femenino , Humanos , Masculino , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Adulto JovenRESUMEN
Pancreatic and colorectal cancers are often KRAS mutated and are incurable when tumor DNA or protein persists or recurs after curative intent therapy. Cancer vaccine ELI-002 2P enhances lymph node delivery and immune response using amphiphile (Amph) modification of G12D and G12R mutant KRAS (mKRAS) peptides (Amph-Peptides-2P) together with CpG oligonucleotide adjuvant (Amph-CpG-7909). We treated 25 patients (20 pancreatic and five colorectal) who were positive for minimal residual mKRAS disease (ctDNA and/or serum tumor antigen) after locoregional treatment in a phase 1 study of fixed-dose Amph-Peptides-2P and ascending-dose Amph-CpG-7909; study enrollment is complete with patient follow-up ongoing. Primary endpoints included safety and recommended phase 2 dose (RP2D). The secondary endpoint was tumor biomarker response (longitudinal ctDNA or tumor antigen), with exploratory endpoints including immunogenicity and relapse-free survival (RFS). No dose-limiting toxicities were observed, and the RP2D was 10.0 mg of Amph-CpG-7909. Direct ex vivo mKRAS-specific T cell responses were observed in 21 of 25 patients (84%; 59% both CD4+ and CD8+); tumor biomarker responses were observed in 21 of 25 patients (84%); biomarker clearance was observed in six of 25 patients (24%; three pancreatic and three colorectal); and the median RFS was 16.33 months. Efficacy correlated with T cell responses above or below the median fold increase over baseline (12.75-fold): median tumor biomarker reduction was -76.0% versus -10.2% (P < 0.0014), and the median RFS was not reached versus 4.01 months (hazard ratio = 0.14; P = 0.0167). ELI-002 2P was safe and induced considerable T cell responses in patients with immunotherapy-recalcitrant KRAS-mutated tumors. ClinicalTrials.gov identifier: NCT04853017 .
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Neoplasias Colorrectales , Vacunas , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Recurrencia Local de Neoplasia/patología , Biomarcadores de Tumor/genética , Vacunas/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Péptidos , Antígenos de Neoplasias/uso terapéuticoRESUMEN
Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.
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Citocinas/metabolismo , Citometría de Flujo/normas , Neoplasias/terapia , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citometría de Flujo/métodos , Humanos , Inmunoterapia , Cooperación Internacional , Laboratorios/normas , Ensayos de Aptitud de Laboratorios , Neoplasias/inmunología , Neoplasias/patología , Reproducibilidad de los Resultados , Coloración y EtiquetadoRESUMEN
Staphylococcus aureus and other staphylococci cause severe human disease, and there are currently no vaccines available. We evaluated whether manganese transport protein C (MntC), which is conserved across the staphylococcal species group, could confer protection against S. aureus and Staphylococcus epidermidis. In vivo analysis of S. aureus MntC expression revealed that expression occurs very early during the infectious cycle. Active immunization with MntC was effective at reducing the bacterial load associated with S. aureus and S. epidermidis infection in an acute murine bacteremia model. Anti-MntC monoclonal antibodies have been identified that can bind S. aureus and S. epidermidis cells and are protective in an infant rat passive protection model and induce neutrophil respiratory burst activity. This is the first description of a protein that has the potential to provide protection across the staphylococcal species group.
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Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Proteínas de la Membrana/inmunología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Bacteriemia/inmunología , Bacteriemia/prevención & control , Bacteriemia/terapia , Carga Bacteriana , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Proteínas de la Membrana/genética , Ratones , Conejos , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/terapia , Vacunas Estafilocócicas/administración & dosificación , Vacunas Estafilocócicas/genética , Resultado del TratamientoRESUMEN
The recent emergence of a causal link between Epstein-Barr virus (EBV) and multiple sclerosis has generated considerable interest in the development of an effective vaccine against EBV. Here we describe a vaccine formulation based on a lymph node targeting Amphiphile vaccine adjuvant, Amphiphile-CpG, admixed with EBV gp350 glycoprotein and an engineered EBV polyepitope protein that includes 20 CD8+ T cell epitopes from EBV latent and lytic antigens. Potent gp350-specific IgG responses are induced in mice with titers >100,000 in Amphiphile-CpG vaccinated mice. Immunization including Amphiphile-CpG also induces high frequencies of polyfunctional gp350-specific CD4+ T cells and EBV-specific CD8+ T cells that are 2-fold greater than soluble CpG and are maintained for >7 months post immunization. This combination of broad humoral and cellular immunity against multiple viral determinants is likely to provide better protection against primary infection and control of latently infected B cells leading to protection against the development of EBV-associated diseases.
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Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Ratones , Animales , Infecciones por Virus de Epstein-Barr/prevención & control , Linfocitos T CD8-positivos , Epítopos de Linfocito T , Ganglios Linfáticos , Vacunas de SubunidadRESUMEN
Despite the success of currently authorized vaccines for the reduction of severe COVID-19 disease risk, rapidly emerging viral variants continue to drive pandemic waves of infection, resulting in numerous global public health challenges. Progress will depend on future advances in prophylactic vaccine activity, including advancement of candidates capable of generating more potent induction of cross-reactive T cells and durable cross-reactive antibody responses. Here we evaluated an Amphiphile (AMP) adjuvant, AMP-CpG, admixed with SARS-CoV-2 Spike receptor binding domain (RBD) immunogen, as a lymph node-targeted protein subunit vaccine (ELI-005) in mice and non-human primates (NHPs). AMP-mediated targeting of CpG DNA to draining lymph nodes resulted in comprehensive local immune activation characterized by extensive transcriptional reprogramming, inflammatory proteomic milieu, and activation of innate immune cells as key orchestrators of antigen-directed adaptive immunity. Prime-boost immunization with AMP-CpG in mice induced potent and durable T cell responses in multiple anatomical sites critical for prophylactic efficacy and prevention of severe disease. Long-lived memory responses were rapidly expanded upon re-exposure to antigen. In parallel, RBD-specific antibodies were long-lived, and exhibited cross-reactive recognition of variant RBD. AMP-CpG-adjuvanted prime-boost immunization in NHPs was safe and well tolerated, while promoting multi-cytokine-producing circulating T cell responses cross-reactive across variants of concern (VOC). Expansion of RBD-specific germinal center (GC) B cells in lymph nodes correlated to rapid seroconversion with variant-specific neutralizing antibody responses exceeding those measured in convalescent human plasma. These results demonstrate the promise of lymph-node adjuvant-targeting to coordinate innate immunity and generate robust adaptive responses critical for vaccine efficacy.
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Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.
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Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Lipopolisacáridos/química , Modelos Moleculares , Neisseria meningitidis/química , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Humanos , Lipopolisacáridos/inmunología , Ratones , Micelas , Neisseria meningitidis/inmunología , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína/fisiologíaRESUMEN
A bivalent factor H binding protein (fHBP) vaccine for the prevention of disease caused by Neisseria meningitidis serogroup B is currently in clinical development. Since fHBP is also expressed by other meningococcal serogroups, anti-fHBP antibodies may have bactericidal activity against meningococci independent of serogroup. To begin examining the susceptibility of other meningococcal serogroups to anti-fHBP antibodies, meningococcal serogroup C invasive isolates (n = 116) were collected from the Centers for Disease Control and Prevention's Active Bacterial Core surveillance (ABCs) sites during 2000-2001. These isolates were analyzed for the presence of the fhbp gene. All serogroup C isolates contained the gene, and sequence analysis grouped the proteins into two subfamilies, A and B. Flow cytometry analysis demonstrated that fHBP was expressed on the surface of ~70% of isolates in vitro with varying levels of expression. fHBP was accessible to antibodies on the cell surface even in the presence of the polysaccharide capsule. Nine isolates from different geographic regions were identified which harboured an identical single nucleotide deletion that could result in a truncated subfamily B fHBP. Analysis by flow cytometry using a polyclonal fHBP antibody preparation revealed that a subpopulation of each of these isolates expressed fHBP. Rabbit and non-human primate immune sera generated with bivalent fHBP vaccine were tested for bactericidal activity against a panel of diverse serogroup C clinical isolates using human complement. Sera from both species demonstrated serum bactericidal antibody activity against the serogroup C isolates tested. These promising findings suggest that a bivalent fHBP vaccine may be capable of providing protection against meningococcal disease caused by both serogroup C and B.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo C/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/análisis , Antígenos Bacterianos/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Secuencia de Bases , Actividad Bactericida de la Sangre , Análisis por Conglomerados , Proteínas del Sistema Complemento/inmunología , ADN Bacteriano/química , ADN Bacteriano/genética , Femenino , Citometría de Flujo , Genotipo , Humanos , Macaca fascicularis , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Infecciones Meningocócicas/microbiología , Vacunas Meningococicas/administración & dosificación , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria meningitidis Serogrupo C/química , Neisseria meningitidis Serogrupo C/genética , Conejos , Análisis de Secuencia de ADNRESUMEN
The profound consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mandate urgent development of effective vaccines. Here, we evaluated an Amphiphile (AMP) vaccine adjuvant, AMP-CpG, composed of diacyl lipid-modified CpG, admixed with the SARS-CoV-2 Spike-2 receptor binding domain protein as a candidate vaccine (ELI-005) in mice. AMP modification efficiently delivers CpG to lymph nodes, where innate and adaptive immune responses are generated. Compared to alum, immunization with AMP-CpG induced >25-fold higher antigen-specific T cells that produced multiple T helper 1 (TH1) cytokines and trafficked into lung parenchyma. Antibody responses favored TH1 isotypes (IgG2c and IgG3) and potently neutralized Spike-2-ACE2 receptor binding, with titers 265-fold higher than natural convalescent patient COVID-19 responses; T cell and antibody responses were maintained despite 10-fold dose reduction in Spike antigen. Both cellular and humoral immune responses were preserved in aged mice. These advantages merit clinical translation to SARS-CoV-2 and other protein subunit vaccines.
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Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , Inmunidad Celular , Inmunidad Humoral , Ganglios Linfáticos/inmunología , SARS-CoV-2/inmunología , Tensoactivos/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/virología , Vacunas contra la COVID-19/inmunología , Femenino , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Neutralización , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Resultado del Tratamiento , Vacunación/métodos , Vacunas de Subunidad/inmunologíaRESUMEN
Neoantigens are critical targets of antitumor T-cell responses. The ATLAS bioassay was developed to identify neoantigens empirically by expressing each unique patient-specific tumor mutation individually in Escherichia coli, pulsing autologous dendritic cells in an ordered array, and testing the patient's T cells for recognition in an overnight assay. Profiling of T cells from patients with lung cancer revealed both stimulatory and inhibitory responses to individual neoantigens. In the murine B16F10 melanoma model, therapeutic immunization with ATLAS-identified stimulatory neoantigens protected animals, whereas immunization with peptides associated with inhibitory ATLAS responses resulted in accelerated tumor growth and abolished efficacy of an otherwise protective vaccine. A planned interim analysis of a clinical study testing a poly-ICLC adjuvanted personalized vaccine containing ATLAS-identified stimulatory neoantigens showed that it is well tolerated. In an adjuvant setting, immunized patients generated both CD4+ and CD8+ T-cell responses, with immune responses to 99% of the vaccinated peptide antigens. SIGNIFICANCE: Predicting neoantigens in silico has progressed, but empirical testing shows that T-cell responses are more nuanced than straightforward MHC antigen recognition. The ATLAS bioassay screens tumor mutations to uncover preexisting, patient-relevant neoantigen T-cell responses and reveals a new class of putatively deleterious responses that could affect cancer immunotherapy design.This article is highlighted in the In This Issue feature, p. 521.
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Antígenos de Neoplasias/inmunología , Inmunidad Celular , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Genómica/métodos , Humanos , Inmunogenicidad Vacunal , Melanoma Experimental , Ratones , Mutación , Neoplasias/genética , Neoplasias/terapia , Linfocitos T/metabolismo , Linfocitos T/patología , Resultado del Tratamiento , VacunaciónRESUMEN
BACKGROUND: Genital herpes simplex virus (HSV) type 2 is a common persistent infection that frequently reactivates to cause recurrent lesions and recurrent viral shedding which is incompletely controlled by antiviral therapy. GEN-003 is a candidate therapeutic vaccine containing 2 HSV-2 proteins, gD2 and ICP4, and Matrix-M2 adjuvant (M2). METHODS: HSV-2 seropositive persons with genital herpes were randomized into three dose cohorts of Gen-003 (60⯵g antigen/50⯵gâ¯M2, 60⯵g/75⯵gâ¯M2 or Placebo). Three intramuscular doses 21â¯days apart of GEN-003 or placebo were administered. Participants obtained genital area swabs twice-daily for HSV-2 detection and monitored genital lesions for 12â¯months. The rates of virus shedding and lesion rates before vaccination were compared to 3 defined periods after vaccination; Days 43-71, Month 6 and Month 12. RESULTS: GEN-003 at a dose of 60⯵g each antigen/50⯵gâ¯M2 reduced HSV shedding immediately after dosing with a rate ratio of 0.58, compared to 0.75 for the GEN-003 60⯵g/75⯵gâ¯M2 and 1.06 for placebo. Lesion rates, recurrence rates, and duration of recurrences were also reduced. Reactogenicity was higher with the 75⯵gâ¯M2 dose than the 50⯵gâ¯M2 dose, specifically for pain, tenderness, malaise and fatigue. Antibody and cellular immune responses were stimulated by both doses and persisted to 12â¯months. CONCLUSIONS: GEN-003 vaccine manufactured with a scalable process gave results similar to those observed in prior clinical trials. GEN-003 had an acceptable safety profile and stimulated both humoral and cellular immune responses. The 60⯵g antigen/50⯵gâ¯M2 provided the maximal effect on virologic and clinical measures and warrants further development. (Funded by Genocea; ClinicalTrials.gov number NCT02515175).
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Herpes Genital/inmunología , Herpesvirus Humano 2/inmunología , Vacunas Virales/inmunología , Esparcimiento de Virus/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adolescente , Adulto , Femenino , Humanos , Inmunidad Celular/inmunología , Inmunoterapia/métodos , Masculino , Persona de Mediana Edad , Vacunación/métodos , Adulto JovenRESUMEN
Bivalent rLP2086 (Trumenba), a vaccine for prevention of Neisseria meningitidis serogroup B (NmB) disease, was licensed for use in adolescents and young adults after it was demonstrated that it elicits antibodies that initiate complement-mediated killing of invasive NmB isolates in a serum bactericidal assay with human complement (hSBA). The vaccine consists of two factor H binding proteins (fHBPs) representing divergent subfamilies to ensure broad coverage. Although it is the surrogate of efficacy, an hSBA is not suitable for testing large numbers of strains in local laboratories. Previously, an association between the in vitro fHBP surface expression level and the susceptibility of NmB isolates to killing was observed. Therefore, a flow cytometric meningococcal antigen surface expression (MEASURE) assay was developed and validated by using an antibody that binds to all fHBP variants from both fHBP subfamilies and accurately quantitates the level of fHBP expressed on the cell surface of NmB isolates with mean fluorescence intensity as the readout. Two collections of invasive NmB isolates (n = 1,814, n = 109) were evaluated in the assay, with the smaller set also tested in hSBAs using individual and pooled human serum samples from young adults vaccinated with bivalent rLP2086. From these data, an analysis based on fHBP variant prevalence in the larger 1,814-isolate set showed that >91% of all meningococcal serogroup B isolates expressed sufficient levels of fHBP to be susceptible to bactericidal killing by vaccine-induced antibodies.IMPORTANCE Bivalent rLP2086 (Trumenba) vaccine, composed of two factor H binding proteins (fHBPs), was recently licensed for the prevention of N. meningitidis serogroup B (NmB) disease in individuals 10 to 25 years old in the United States. This study evaluated a large collection of NmB isolates from the United States and Europe by using a flow cytometric MEASURE assay to quantitate the surface expression of the vaccine antigen fHBP. We find that expression levels and the proportion of strains above the level associated with susceptibility in an hSBA are generally consistent across these geographic regions. Thus, the assay can be used to predict which NmB isolates are susceptible to killing in the hSBA and therefore is able to demonstrate an fHBP vaccine-induced bactericidal response. This work significantly advances our understanding of the potential for bivalent rLP2086 to provide broad coverage against diverse invasive-disease-causing NmB isolates.
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Antibacterianos/farmacología , Anticuerpos Antibacterianos/farmacología , Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Vacunas Meningococicas/inmunología , Viabilidad Microbiana/efectos de los fármacos , Neisseria meningitidis Serogrupo B/efectos de los fármacos , Neisseria meningitidis Serogrupo B/fisiología , Actividad Bactericida de la Sangre , Citometría de Flujo/métodos , Humanos , Neisseria meningitidis Serogrupo B/química , Neisseria meningitidis Serogrupo B/aislamiento & purificaciónRESUMEN
BACKGROUND: Bivalent rLP2086 (Trumenba), 1 of 2 meningococcal serogroup B (MnB) vaccines recently approved in the United States for the prevention of MnB disease in individuals 10-25 years of age, is composed of 2 lipidated factor H binding proteins from subfamilies A and B. This study evaluated the breadth of MnB strain coverage elicited by bivalent rLP2086 measured with serum bactericidal assays using human complement (hSBAs). METHODS: hSBA responses to diverse MnB clinical strains circulating in the United States and Europe (n = 23), as well as recent US university outbreak strains (n = 4), were evaluated. Individual prevaccination and postvaccination sera from adolescents and young adults previously enrolled in phase 2 clinical studies of bivalent rLP2086 were assessed. Responders were defined by an hSBA titer ≥1:8, which is more stringent than the accepted correlate of protection (hSBA titer ≥1:4). RESULTS: Baseline hSBA response rates were generally low; robust increases were observed after 2 and 3 doses of bivalent rLP2086, with hSBA responses to all test strains ranging from 31.8% to 100% and 55.6% to 100%, respectively. hSBA responses to strains expressing prevalent subfamily A and B factor H binding protein variants in the United States and Europe, A22 and B24, ranged from 88.0% to 95.0% and 81.0% to 100.0%, respectively, after dose 3. Substantial responses were also observed for recent US outbreak strains. CONCLUSIONS: Bivalent rLP2086 elicits robust hSBA responses to MnB strains expressing 14 factor H binding protein variants representing approximately 80% of MnB invasive isolates and different from vaccine antigens, suggesting that bivalent rLP2086 confers broad protection against diverse MnB disease-causing strains.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Brotes de Enfermedades/prevención & control , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/inmunología , Niño , Ensayos Clínicos Fase II como Asunto , Estudios de Cohortes , Brotes de Enfermedades/estadística & datos numéricos , Humanos , Infecciones Meningocócicas/epidemiología , Infecciones Meningocócicas/microbiología , Vacunas Meningococicas/administración & dosificación , Vacunas Meningococicas/química , Adulto JovenRESUMEN
OBJECTIVES: Bivalent rLP2086 (Trumenba®; MenB-FHbp), composed of two factor H binding proteins (FHbps), is a vaccine approved in the United States for prevention of Neisseria meningitidis serogroup B (MnB) invasive meningococcal disease (IMD). Bactericidal activity of sera from subjects vaccinated with bivalent rLP2086 was assessed against MnB isolates from recent disease outbreaks in France. METHODS: MnB isolates from IMD cases were characterized by whole genome sequencing and FHbp expression was assessed using a flow cytometry-based assay. Sera from subjects (11-<19years old) vaccinated with bivalent rLP2086 at 0, 2, and 6months were evaluated. Bactericidal activity was measured in serum bactericidal assays using human complement (hSBAs). The response rate (RR) represents the percentage of subjects with an hSBA titer ⩾1:4. RESULTS: The six MnB outbreak isolates expressed diverse FHbp variants: A22, B03, B24 (two isolates), B44, and B228. FHbp expression levels ranged from 1309 to 8305 (mean fluorescence intensity units). The RR of preimmune sera from subjects was 7% to 27%. RRs increased for all isolates after each vaccine dose. After two doses, RRs ranged from 40% to 93%. After dose 3, RRs were ⩾73% for all isolates (range, 73%-100%). CONCLUSIONS: Each of the representative French outbreak isolates was killed by sera from subjects vaccinated with bivalent rLP2086. Vaccination elicited an immune response with bactericidal activity against these diverse isolates in a large proportion of subjects at risk. These results provide additional support for the licensure strategy of testing MnB strains expressing vaccine-heterologous FHbp variants in hSBAs and further illustrate the breadth of efficacy of this protein-based MnB vaccine.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Actividad Bactericida de la Sangre , Vacunas Meningococicas/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Adolescente , Antígenos Bacterianos/análisis , Antígenos Bacterianos/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Niño , Proteínas del Sistema Complemento/inmunología , Brotes de Enfermedades , Femenino , Francia/epidemiología , Perfilación de la Expresión Génica , Humanos , Masculino , Vacunas Meningococicas/administración & dosificación , Viabilidad Microbiana , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/aislamiento & purificaciónRESUMEN
BACKGROUND: The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to Streptococcus pneumoniae. METHODS: We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC) or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS) using microengraving (a single-cell analysis method) and single-cell RT-PCR. RESULTS: Seven days after boosting, the mean frequency of antigen-specific memory B cells was significantly increased in macaques vaccinated with 13vPnC compared to those receiving 23vPS. The 13vPnC-vaccinated macaques also exhibited a more even distribution of antibody specificities to four polysaccharides in the vaccine (PS4, 6B, 14, 23F) that were examined. However, single-cell analysis of the antibody variable region sequences from antigen-specific B cells elicited by unconjugated and conjugated vaccines indicated that both the germline gene segments forming the heavy chains and the average lengths of the Complementary Determining Region 3 (CDR3) were similar. CONCLUSIONS: Our results confirm that distinctive differences can manifest between antigen-specific memory B cell repertoires in nonhuman primates immunized with conjugated and unconjugated pneumococcal polysaccharide vaccines. The study also supports the notion that the conjugated vaccines have a favorable profile in terms of both the frequency and breadth of the anamnestic response among antigen-specific memory B cells.
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Linfocitos B/metabolismo , Vacuna Neumocócica Conjugada Heptavalente/administración & dosificación , Macaca/inmunología , Vacunas Neumococicas/administración & dosificación , Animales , Anticuerpos Antibacterianos/inmunología , Vacuna Neumocócica Conjugada Heptavalente/inmunología , Inmunización Secundaria , Memoria Inmunológica , Vacunas Neumococicas/inmunología , Análisis de la Célula Individual , Streptococcus pneumoniae/inmunologíaRESUMEN
BACKGROUND: The decline in immune function with age is a challenge to vaccine development. Following an initial study in adults aged 18-64years, this study evaluated the safety and immunogenicity of Staphylococcus aureus (S. aureus) 4-antigen (SA4Ag) and 3-antigen (SA3Ag) vaccine in older adults. SA3Ag included capsular polysaccharide serotypes 5 and 8 (CP5 and CP8) conjugated to the nontoxic mutant form of diphtheria toxin (CRM197) and a recombinant version of clumping factor A (ClfA). SA4Ag included these antigens, with the addition of a recombinant manganese transporter C (rP305A or MntC). Both vaccines were unadjuvanted. METHODS: In this double-blind, sponsor-unblinded, placebo-controlled, phase 1/2 study, 284 healthy adults (aged 65-85years) were randomised to receive a single dose of one of three formulations of SA4Ag with escalating dose levels of rP305A, SA3Ag, or placebo. Functional immune responses were measured using opsonophagocytic activity (OPA) killing and fibrinogen-binding inhibition (FBI) assays; immunogenicity was also assessed using a competitive Luminex® immunoassay (cLIA). T-cell responses were measured in a small subgroup of subjects using intracellular cytokine staining (ICS) assays. RESULTS: The results demonstrated rapid and robust functional immune responses to all antigens in healthy older adults. A high proportion of active vaccine recipients met the pre-defined antibody thresholds for each antigen at Day 29. SA4Ag elicited a dose-level response to rP305A with up to a 13-fold rise in cLIA titres at Day 29. Opsonophagocytic activity (OPA) assays showed >50- and >20-fold rises in functional titres using S. aureus strains expressing CP5 and CP8, respectively, at Day 29. T-cell cytokine responses were not substantially above background levels. There were no safety concerns in this study population and no increases in adverse events with higher rP305A dose levels. CONCLUSIONS: Single-dose vaccination of SA4Ag and SA3Ag in healthy adults aged 65-85years safely induced rapid and robust functional immune responses, supporting further development of SA4Ag for the prevention of S. aureus disease in adults up to age 85years. TRIAL REGISTRATION NUMBER: NCT01643941.
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Antígenos Bacterianos/inmunología , Vacunas Estafilocócicas/efectos adversos , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Adyuvantes Inmunológicos/metabolismo , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Citocinas/análisis , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Femenino , Humanos , Masculino , Proteínas Opsoninas/sangre , Fagocitosis , Placebos/administración & dosificación , Polisacáridos Bacterianos/inmunología , Vacunas Estafilocócicas/administración & dosificación , Linfocitos T/inmunología , Resultado del Tratamiento , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunologíaRESUMEN
In this chapter, we describe a research and development pathway to identify and demonstrate the efficacy of a Neisseria meningitidis non-capsular vaccine, the recently licensed N. meningitidis serogroup B (MnB) vaccine, Trumenba(®). While other approaches have been followed in the identification of a MnB vaccine (Pizza et al. Science 287:1816-1820, 2000), the methods described here reflect the distinctive approach and experiences in discovering and developing Trumenba(®). In contrast to the development and licensure of polysaccharide-conjugate vaccines against meningococcal serotypes A, C, W, and Y, the development of a vaccine to produce broadly protective antibodies against meningococcal serogroup B has proved difficult, due to the antigenic mimicry of the serogroup B polysaccharide capsule, which is composed of polysialic acid structures similar to those expressed on human neuronal cells. Early development efforts for these vaccines failed because the MnB polysaccharide structures resemble autoantigens and thus were poorly immunogenic. The development of an MnB vaccine has therefore focused on non-polysaccharide approaches. It was critical to identify MnB cell surface-exposed antigens capable of inducing a protective response against diverse, circulating strains of invasive MnB to ensure global coverage. Once candidate antigens were identified, it was important to characterize antigenic variation and expression levels, and subsequently to assure that antigens were expressed broadly among diverse clinical isolates. Prior to the initiation of clinical trials in humans, candidate vaccine antigens were tested in functional immunogenicity assays and yielded responses that were correlated with protection from meningococcal disease. These functional immunogenicity assays (serum bactericidal assays using human complement, hSBAs) measure the titer of complement-dependent bactericidal antibodies in serum from immunized test animals using diverse clinical MnB isolates as targets. Following optimization of vaccine antigenic components based on hSBA responses in preclinical models, animal toxicology tests were performed. Initial clinical studies (Phase 1 and 2) subsequently provided data to support (1) safety and immunogenicity of the vaccine formulation, and (2) the dose and schedule. Phase 3 clinical trials were carried out in the target populations to provide the clinical confirmation of safety and efficacy required for vaccine licensure.
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Vacunas Bacterianas/inmunología , Descubrimiento de Drogas/métodos , Neisseria meningitidis Serogrupo B/inmunología , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/genética , Proteínas del Sistema Complemento/metabolismo , Genómica , Humanos , Inmunización , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/crecimiento & desarrollo , Seguridad , Coloración y EtiquetadoRESUMEN
Vaccines have had a major impact on the reduction of many diseases globally. Vaccines targeted against invasive meningococcal disease (IMD) due to serogroups A, C, W, and Y are used to prevent these diseases. Until recently no vaccine had been identified that could confer broad protection against Neisseria meningitidis serogroup B (MnB). MnB causes IMD in the very young, adolescents and young adults and thus represents a significant unmet medical need. In this brief review, we describe the discovery and development of a vaccine that has the potential for broad protection against this devastating disease.