RESUMEN
Nipah virus (NiV) disease is a bat-borne zoonosis responsible for outbreaks with high lethality and is a priority for vaccine development. With funding from the Coalition of Epidemic Preparedness Innovations (CEPI), we are developing a chimeric vaccine (PHV02) composed of recombinant vesicular stomatitis virus (VSV) expressing the envelope glycoproteins of both Ebola virus (EBOV) and NiV. The EBOV glycoprotein (GP) mediates fusion and viral entry and the NiV attachment glycoprotein (G) is a ligand for cell receptors, and stimulates neutralizing antibody, the putative mediator of protection against NiV. PHV02 is identical in construction to the registered Ebola vaccine (Ervebo) with the addition of the NiV G gene. NiV ephrin B2 and B3 receptors are expressed on neural cells and the wild-type NiV is neurotropic and causes encephalitis in affected patients. It was therefore important to assess whether the NiV G alters tropism of the rVSV vector and serves as a virulence factor. PHV02 was fully attenuated in adult hamsters inoculated by the intramuscular (IM) route, whereas parental wild-type VSV was 100% lethal. Two rodent models (mice, hamsters) were infected by the intracerebral (IC) route with graded doses of PHV02. Comparator active controls in various experiments included rVSV-EBOV (representative of Ebola vaccine) and yellow fever (YF) 17DD commercial vaccine. These studies showed PHV02 to be more neurovirulent than both rVSV-EBOV and YF 17DD in infant animals. PHV02 was lethal for adult hamsters inoculated IC but not for adult mice. In contrast YF 17DD retained virulence for adult mice inoculated IC but was not virulent for adult hamsters. Because of the inconsistency of neurovirulence patterns in the rodent models, a monkey neurovirulence test (MNVT) was performed, using YF 17DD as the active comparator because it has a well-established profile of quantifiable microscopic changes in brain centers and a known reporting rate of neurotropic adverse events in humans. In the MNVT PHV02 was significantly less neurovirulent than the YF 17DD vaccine reference control, indicating that the vaccine will have an acceptable safety profile for humans. The findings are important because they illustrate the complexities of phenotypic assessment of novel viral vectors with tissue tropisms determined by transgenic proteins, and because it is unprecedented to use a heterologous comparator virus (YF vaccine) in a regulatory-enabling study. This approach may have value in future studies of other novel viral vectors.
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Infecciones por Henipavirus , Estomatitis Vesicular , Vacunas Virales , Animales , Modelos Animales de Enfermedad , Vacunas contra el Virus del Ébola , Glicoproteínas/genética , Fiebre Hemorrágica Ebola/prevención & control , Infecciones por Henipavirus/prevención & control , Humanos , Ratones , Virus Nipah/genética , Vacunas Atenuadas/efectos adversos , Vacunas Sintéticas/efectos adversos , Estomatitis Vesicular/prevención & control , Vacunas Virales/efectos adversosRESUMEN
Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.
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Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Pandemias , Receptores Virales/inmunología , Anticuerpos Antivirales/metabolismo , Afinidad de Anticuerpos , Antígenos Virales/metabolismo , Sitios de Unión de Anticuerpos , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Modelos Moleculares , Conformación Proteica , Receptores Virales/metabolismo , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: The MIMIX platform is a novel microneedle array patch (MAP) characterized by slowly dissolving microneedle tips that deploy into the dermis following patch application. We describe safety, reactogenicity, tolerability and immunogenicity for MIMIX MAP vaccination against influenza. METHODOLOGY: The trial was a Phase 1, exploratory, first-in-human, parallel randomized, rater, participant, study analyst-blinded, placebo-controlled study in Canada. Forty-five healthy participants (18 to 39 years of age, inclusive) were randomized in a 1:1:1 ratio to receive either 15 µg or 7.5 µg of an H1N1 influenza vaccine, or placebo delivered via MIMIX MAP to the volar forearm. A statistician used a computer program to create a randomization scheme with a block size of 3. Post-treatment follow-up was approximately 180 days. Primary safety outcomes included the incidence of study product related serious adverse events and unsolicited events within 180 days, solicited application site and systemic reactogenicity through 7 days after administration and solicited application site erythema and/or pigmentation 14, 28, 56 and 180 days after administration. Immunogenicity outcomes included antibody titers and percentage of seroconversion (SCR) and seroprotection (SPR) rates determined by the hemagglutination inhibition (HAI) assay. Exploratory outcomes included virus microneutralization (MN) titers, durability and breadth of the immune response. The trial was registered with ClinicalTrials.gov, number NCT06125717. FINDINGS: Between July 7, 2022 and March 13, 2023 45 participants were randomized to a treatment group. One participant was lost to follow up in the 15 µg group and 1 participant withdrew from the 7.5 µg dose group. Safety analyses included n = 15 per group, immunogenicity analyses included n = 14 for the 15 µg and 7.5 µg treatment groups and n = 15 for the placebo group. No SAEs were reported in any of the treatment groups. All treatment groups reported solicited local events within 7 days after vaccination, with mild (Grade 1) erythema being the most frequent symptom reported. Other local symptoms reported included mostly mild (Grade 1) induration/swelling, itching, pigmentation, skin flaking, and tenderness. Within 7 days after vaccination, 2 participants (4.4%) reported moderate (Grade 2) erythema, 1 participant (2.2%) reported moderate (Grade 2) induration/swelling, and 1 participant (2.2%) reported moderate (Grade 2) itching. There was an overall reduction in erythema and pigmentation reported on Days 15, 29, 57, and 180 among all treatment groups. Systemic symptoms reported within 7 days after vaccination, included mild (Grade 1) fatigue reported among all treatment groups, and mild (Grade 1) headache reported by 1 participant in the 7.5 µg treatment group. No study drug related severe symptoms were reported in the study. Group mean fold rises in HAI titers ranged between 8.7 and 12-fold, SCRs were >76% and SPRs were >92% for both VX-103 dose groups thereby fulfilling serological criteria established by the EMA and FDA for seasonal influenza vaccines. Longitudinal assessments demonstrate persistence of the immune response through at least Day 180. CONCLUSIONS: The MIMIX MAP platform is safe, well tolerated and elicits robust antibody responses.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Humanos , Adulto , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/administración & dosificación , Masculino , Femenino , Subtipo H1N1 del Virus de la Influenza A/inmunología , Adulto Joven , Adolescente , Gripe Humana/prevención & control , Gripe Humana/inmunología , Agujas , Voluntarios Sanos , Vacunación/métodos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Método Doble Ciego , Inmunogenicidad VacunalRESUMEN
Background. We evaluated the safety and immunogenicity of VAX2012Q, a quadrivalent influenza vaccine comprising 4 hemagglutinin subunits fused to flagellin. Methods. In this dose-ranging, open-label study, healthy adults (18-40 years) were divided into 7 cohorts for evaluation of 5 dose levels and 3 component ratios. Dose levels were as follows: (1) 1 mcg per component of VAX128C (H1N1), VAX181 (H3N2), VAX173 (B-YAM), and VAX172 (B-VIC), respectively; (2) 2 mcg per component, respectively; (3) 2, 4, 4, and 4 mcg of each component, respectively; (4) 2, 4, 6, and 6 mcg of each component, respectively; and (5) 3 mcg per component, respectively. Tolerability and immunogenicity data were analyzed. Results. Three hundred sixteen subjects received VAX2012Q (309 per protocol). At all dose levels, 54% to 65% of subjects reported mild injection site pain, the most common local reaction. Moderate injection site pain increased at dose levels 2 through 5 (22%-42%, compared with 20% at dose level 1). Systemic symptoms were mostly mild to moderate with moderate symptoms increasing in dose levels 3 and 4. Three dose level 3 subjects (6%) reported severe, transient chills and or fever. Mean fold rises in hemagglutination inhibition titers ranged from 2.5 to 6.9 despite high baseline titers. Mean seroprotection rates were ≥90% and mean seroconversion rates were ≥40% for all strains in all groups postvaccination. Conclusions. VAX2012Q elicited immune responses at all dose levels with no significant safety concerns. Doses of 2 or 3 mcg per component provided a favorable balance of tolerability and immunogenicity.
RESUMEN
BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.
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Citocinas/sangre , Citometría de Flujo/normas , Linfocitos T/química , Conservación de la Sangre , Criopreservación , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo/métodos , Liofilización , Humanos , Indicadores y Reactivos , Laboratorios , Linfocitos/química , Fosfoproteínas/sangre , Reproducibilidad de los Resultados , Manejo de Especímenes , Proteínas de la Matriz Viral/sangreRESUMEN
There is considerable interest in developing immunotherapeutic approaches to elicit tumor-specific CTL responses in cancer patients. Epitope-based approaches aim to deliver the antigenic peptides or epitopes recognized by CTLs rather than the intact tumor antigen. Many tumor-associated proteins are nonmutated self proteins for which the dominant peptide epitopes are usually poorly immunogenic. The subdominant epitopes, however, can elicit robust T cell responses if optimized for their ability to bind to class I MHC molecules. Only the epitopes for a few tumor antigens expressed in human cancers have been defined to this level, mainly for technical reasons. The means to rapidly screen and characterize the binding of epitopes derived from complex tumor-associated antigens is an important enabling technology. Here, we have used the high-throughput technology iTopia to identify those peptides derived from the tumor-associated antigen survivin that bind 8 class I alleles. A library of overlapping nonamers spanning the length of the survivin protein was initially screened for peptides capable of binding each allele. Nineteen HLA-A*0201, zero HLA-A*0101, seven HLA-A*0301, twelve HLA-A*1101, twenty-four HLA-A*2402, six HLA-B*0702, six HLA-B*0801, and eight HLA-B*1501 binding peptides were identified based on an arbitrary cutoff. Peptides capable of binding a given allele were further characterized by their affinity for MHC class I molecules and by the rate of dissociation of the complex. This information should help guide functional studies and future epitope-based immunotherapies.
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Antígenos HLA/inmunología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Alelos , Antígenos de Neoplasias , Antígenos HLA/genética , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/inmunología , Proteínas de Neoplasias/inmunología , Mapeo Peptídico , Péptidos/inmunología , Unión Proteica , Survivin , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The envelope (E) protein of flaviviruses includes three domains, EI, EII, and EIII, and is the major protective antigen. Because EIII is rich in type-specific and subcomplex-specific neutralizing epitopes and is easy to express, it is particularly attractive as a recombinant vaccine antigen. VaxInnate has developed a vaccine platform that genetically links vaccine antigens to bacterial flagellin, a Toll-like receptor 5 ligand. Here we report that tetravalent dengue vaccines (TDVs) consisting of four constructs, each containing two copies of EIII fused to flagellin (R3.2x format), elicited robust and long-lived neutralizing antibodies (geometric mean titers of 200 to 3,000), as measured with a 50% focus reduction neutralization test (FRNT50). In an immunogenicity study, rhesus macaques (n = 2) immunized subcutaneously with 10 µg or 90 µg of TDV three or four times, at 4- to 6-week intervals, developed neutralizing antibodies to four dengue virus (DENV) serotypes (mean post-dose 3 FRNT50 titers of 102 to 601). In an efficacy study, rhesus macaques (n = 4) were immunized intramuscularly with 16 µg or 48 µg of TDV or a placebo control three times, at 1-month intervals. The animals that received 48-µg doses of TDV developed neutralizing antibodies against the four serotypes (geometric mean titers of 49 to 258) and exhibited reduced viremia after DENV-2 challenge, with a group mean viremia duration of 1.25 days and 2 of 4 animals being completely protected, compared to the placebo-treated animals, which all developed viremia, with a mean duration of 4 days. In conclusion, flagellin-EIII fusion vaccines are immunogenic and partially protective in a nonhuman primate model.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Vacunas de Subunidad/inmunología , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Citocinas/sangre , Citocinas/inmunología , Vacunas contra el Dengue/administración & dosificación , Virus del Dengue/genética , Modelos Animales de Enfermedad , Flagelina/administración & dosificación , Flagelina/inmunología , Inyecciones Subcutáneas , Macaca mulatta/inmunología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Receptor Toll-Like 5/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas de Subunidad/administración & dosificación , Viremia/prevención & controlRESUMEN
The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.
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VIH/metabolismo , Interferón gamma/biosíntesis , Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Separación Celular , Ensayos Clínicos como Asunto , Criopreservación , Citometría de Flujo , Seropositividad para VIH , Interferón gamma/metabolismo , Leucocitos Mononucleares/virología , Péptidos/química , Factores de TiempoRESUMEN
Previously, we demonstrated that for H1N1 and H5N1 influenza strains, the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2) is highly immunogenic in preclinical models and man (Song et al. (2008) [13]; Song et al. (2009) [14]; Taylor et al. (2012) [12]). Further we showed that the vaccine format, or point of attachment of the vaccine antigen to flagellin, can dramatically affect the immunogenicity and safety profile of the vaccine. However, Influenza B vaccines based on these formats are poor triggers of TLR5 and consequently are poorly immunogenic. Through rational design, here we show that we have identified a fusion position within domain 3 of flagellin that improves TLR5 signaling and consequently, immunogenicity of multiple influenza B vaccines. Our results demonstrate that, similar to influenza A strains, the protective subunit of the influenza B HA can be fused to flagellin and produced in a standard prokaryotic expression system thereby allowing for cost and time efficient production of multivalent seasonal influenza vaccines.
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Flagelina/inmunología , Virus de la Influenza B/inmunología , Vacunas contra la Influenza/inmunología , Receptor Toll-Like 5/agonistas , Secuencia de Aminoácidos , Animales , Femenino , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/prevención & control , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/inmunología , Receptor Toll-Like 5/inmunologíaRESUMEN
In order to meet the global demand for rapid production of pandemic influenza vaccines, we have developed a recombinant fusion vaccine platform in which the globular head of hemagglutinin (HA) antigen is genetically fused to bacterial flagellin (a TLR5 ligand). These flagellin-HA fusion vaccine candidates elicit highly protective immunity against a lethal challenge with 2009 pandemic H1N1 (Liu, et al. PLoS ONE 2011; 6:e20928) or H5N1 influenza A/Vietnam/1203/04 (A/VN) infections in mice (Song, et al. Vaccine 2009;27:5875-88). Here we provide the first evidence showing that two A/VN vaccine candidates elicited HA-specific IgG, reduced nasal virus shedding, and conferred full protection against a lethal A/VN infection in ferrets. Furthermore, we show that similar flagellin-HA vaccine candidates of two other H5N1 HPAIV are immunogenic and/or efficacious in mice. Vaccines of A/Indonesia/5/05 (A/IN) induced significant HAI titers to homologous and heterologous A/Anhui/1/05 (A/AN) H5N1 viruses. Two subcutaneous immunizations with doses of either 0.3 µg or 3 µg of A/IN candidates resulted in ≥ 2.5 log(10) unit reduction in day 5 lung virus titer and 90-100% protection against a lethal A/IN challenge in mice. Both R3.HA5 IN and R3.2xHA5 IN vaccines elicited robust neutralizing antibody responses that last for at least 9 months and demonstrated a significant anamnestic antibody response upon further booster immunization. Finally, we found that two vaccine candidates of A/AN induced significant HAI titers in mice. Taken together, our recombinant flagellin-HA platform has been successfully used to generate potent H5N1 HPAIV vaccine candidates. These promising preclinical results justify the advancement of these candidates into the clinic.
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Adyuvantes Inmunológicos/administración & dosificación , Flagelina/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Adyuvantes Inmunológicos/genética , Animales , Anticuerpos Antivirales/sangre , Modelos Animales de Enfermedad , Hurones , Flagelina/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Inmunoglobulina G/sangre , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Inyecciones Subcutáneas , Masculino , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/virología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Análisis de Supervivencia , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Esparcimiento de VirusRESUMEN
BACKGROUND: We evaluated the safety and immunogenicity profiles of 3 novel influenza vaccine constructs consisting of the globular head of the HA1 domain of the Novel H1N1 genetically fused to the TLR5 ligand, flagellin. HA1 was fused to the C-terminus of flagellin in VAX128A, replaced the D3 domain of flagellin in VAX128B and was fused in both positions in VAX128C. METHODS: In a dose escalation trial, 112 healthy subjects 18-49 and 100 adults ≥65 years old were enrolled in a double blind, placebo controlled clinical trial at two centers. Vaccines were administered IM at doses ranging from 0.5 to 20 µg. VAX128C was selected for second study performed in 100 subjects 18-64 years old comparing 1.25 and 2.5 µg doses. All subjects were followed for safety and sera collected pre- and post-vaccination were tested by hemagglutination-inhibition (HAI). Serum C-reactive protein and cytokine levels were also measured. CONCLUSIONS: In the first study high HAI titers and high seroconversion and seroprotection rates were observed at doses ≥2.5 µg in adults 18-49. In adults ≥65 years, the vaccines doses of ≥4 µg were required to induce a ≥4-fold rise in HAI titer, 50% seroconversion and 70% seroprotection. Based on safety, VAX128A was tested up to 8 µg, VAX128B to 16 µg and VAX128C to 20 µg. Dose escalation for VAX128A was stopped at 8 µg because one subject had temperature 101.6°F associated with a high CRP response, VAX128B was stopped at 16 µg because of a severe AE associated with a high CRP and IL-6 response. VAX128C was not stopped before reaching the 20 µg dose. In the second study VAX128C was well tolerated among 100 subjects who received 1.25 or 2.5 µg. The peak GMT was 385 (95%CI 272-546), 79% (71-87%) seroconversion and 92% (84-96%) seroprotection. DISCUSSION: Flagellin adjuvanted vaccines can be designed to minimize reactogenicity and retain immunogenicity, thereby representing a promising next generation vaccine technology.
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Flagelina/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antivirales/sangre , Proteína C-Reactiva/inmunología , Método Doble Ciego , Femenino , Pruebas de Inhibición de Hemaglutinación , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Interleucina-6/inmunología , Masculino , Persona de Mediana Edad , Conejos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Adulto JovenRESUMEN
BACKGROUND: The ectodomain of matrix protein 2 (M2e) is a promising candidate for a broadly protective influenza A vaccine because it is highly conserved and antibodies to M2e are protective in animal models. STF2.4xM2e (VAX102) is a recombinant fusion protein that links four tandem copies of the M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand used as an adjuvant. The objectives of this first-in-human study were to assess the safety and immunogenicity of VAX102 given as a prime-boost regimen to healthy adults. METHODS: Sixty subjects 18-49 years old were enrolled in a multicenter, double-blind, randomized, placebo-controlled trial (Study 1). Based on pre-clincial data, initial design included doses starting at 10 µg, with an escalation plan. After reactogenicity was noted at the 10 µg dose, the trial was redesigned to evaluate 0.3, 1.0, and 3 µg doses. Following this study, 16 subjects were enrolled in Study 2, an open label, low dose study, to evaluate doses of 0.03 and 0.1 µg. In both trials, vaccine or placebo was given intramuscularly (i.m.) at 0 and 28 days. Clinical and laboratory safety assessments took place 1 and 7 days after immunization. Immune responses to M2e and flagellin were assessed by ELISA at 7, 14 and 28 days after each dose. Seroconversion was defined as a serum IgG anti-M2e antibody value ≥0.174 µg/ml and a fourfold rise in concentration. RESULTS: Doses of 0.03-1 µg were safe and well tolerated in all subjects. Doses of 0.03 and 0.1 µg produced limited immunogenicity (38% and 75% respectively), after the second dose of vaccine. Doses of 0.3 and 1.0 µg were immunogenic in 18 (75%) of 24 vaccinees after the first dose and 23 (96%) after the second dose. In the 1.0 µg group, the geometric mean M2e antibody concentration was 0.4 µg/ml after the first dose and 1.7 µg/ml after the booster dose. M2e antibody concentrations and seroconversion rates were not significantly different at higher doses (p>0.05). Immune response to flagellin was robust but did not appear to interfere with M2e antibody responses after the booster dose. Following the first injection of VAX102 at higher doses (3 and 10 µg), self-limited but severe symptoms were noted in some subjects and were associated with elevated levels of C-reactive protein. Although not directly measured, this reaction was believed to be mediated by cytokine release. CONCLUSIONS: VAX102 was safe and induced high antibody levels to M2e at 0.3 and 1.0 µg doses. The TLR5 ligand, S. typhimurium flagellin, is a novel approach to adjuvant-like activity through activation of innate immunity, and when fused to multiple copies of the M2e protein, the vaccine was able to induce a fourfold rise in antibody in humans, to a previously non-immunogenic, highly-conserved portion of the influenza virus. Clinical correlates of protection that may be afforded by M2e antibody in humans are a future focus of investigation.
Asunto(s)
Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Adolescente , Adulto , Anticuerpos/sangre , Proteína C-Reactiva/análisis , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Flagelina/inmunología , Flagelina/uso terapéutico , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Masculino , Persona de Mediana Edad , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: Influenza vaccines perform poorly in the elderly with reduced serological response and vaccine efficacy. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in Escherichia coli. METHODS: 120 subjects ≥ 65 years old were enrolled at three clinical centers. VAX125 vaccine was administered at doses of 0.5, 1, 2, 3, 5 or 8 µg delivered i.m. as a single dose vaccination on Day 0 using a dose-escalation with 20 subjects in each dose level. Subjects were followed for adverse events and sera were tested by hemagglutination-inhibition (HAI) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP) and anti-flagellin antibody were also assessed. RESULTS: The mean age was 71 years. The vaccine was well tolerated at all dose levels, with no more than mild to moderate local or systemic symptoms. The geometric mean titers (GMT) increased in all dose groups. In the 5 µg group the day 14 post-vaccination HAI titer was 1:226 showing a 12-fold increase over baseline. The 8 µg group showed a similar post-vaccination GMT increase (â¼ 8-fold). In the combined 5 and 8 µg groups, the seroconversion rate was 75% and the seroprotection rate was 98%. CONCLUSIONS: A 5 µg dose of VAX125 was safe and able to induce a greater than 10-fold increase HAI antibody levels and nearly complete seroprotection in subjects over 65 years old. The use of flagellin to adjuvant influenza vaccines via the TLR5 innate immune pathway appears to be a useful approach to overcome poor immune responses in the elderly. VAX125 is a promising new candidate for prevention of influenza A disease in both young adults and the elderly.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Flagelina/administración & dosificación , Vacunas contra la Influenza/inmunología , Receptor Toll-Like 5/agonistas , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Escherichia coli/genética , Femenino , Flagelina/genética , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Masculino , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunologíaRESUMEN
Influenza vaccines are less effective in older people than younger people. This impaired ability to protect older people from influenza viral lung infection has important implications as older people suffer a higher morbidity and mortality from influenza viral lung infection than younger people. Therefore, the development of novel effective vaccines that induce protection from influenza viral infections in older people are urgently needed. We had previously shown that direct linking the TLR5 activator, flagellin, to viral peptides induces effective immunity to viral antigens in young mice and people, respectively. In this study, we tested the efficacy of this vaccine platform with the hemagglutinin peptide of the influenza A strain virus (vaccine denoted as STF2.HA1-2) in protecting aged mice from subsequent influenza viral lung infection. We found that a 3.0 µg dose of the vaccine was effective in reducing mortality and increasing clinical well-being during influenza viral lung infection in aged mice. However, this effect was inferior to the response induced in young mice. Defects in the adaptive immune system but not the innate immune system were associated with this reduced effectiveness of the vaccine with aging. Our results indicate that the STF2.HA1-2 vaccine is effective in protecting aged hosts from influenza lung infection, although defects in the adaptive immune system with aging may limit the effectiveness of this vaccine in older people.
Asunto(s)
Epítopos/inmunología , Flagelina/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Envejecimiento/inmunología , Animales , Epítopos/genética , Flagelina/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/genética , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/patología , Análisis de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
We have previously demonstrated that the globular head of the hemagglutinin (HA) antigen fused to flagellin of Salmonella typhimurium fljB (STF2, a TLR5 ligand) elicits protective immunity to H1N1 and H5N1 lethal influenza infections in mice (Song et al., 2008, PLoS ONE 3, e2257; Song et al., 2009, Vaccine 27, 5875-5888). These fusion proteins can be efficiently and economically manufactured in E. coli fermentation systems as next generation pandemic and seasonal influenza vaccines. Here we report immunogenicity and efficacy results of three vaccine candidates in which the HA globular head of A/California/07/2009 (H1N1) was fused to STF2 at the C-terminus (STF2.HA1), in replace of domain 3 (STF2R3.HA1), or in both positions (STF2R3.2xHA1). For all three vaccines, two subcutaneous immunizations of BALB/c mice with doses of either 0.3 or 3 µg elicit robust neutralizing (HAI) antibodies, that lead to >â=â2 Log(10) unit reduction in day 4 lung virus titer and full protection against a lethal A/California/04/2009 challenge. Vaccination with doses as low as 0.03 µg results in partial to full protection. Each candidate, particularly the STF2R3.HA1 and STF2R3.2xHA1 candidates, elicits robust neutralizing antibody responses that last for at least 8 months. The STF2R3.HA1 candidate, which was intermediately protective in the challenge models, is more immunogenic than the H1N1 components of two commercially available trivalent inactivated influenza vaccines (TIVs) in mice. Taken together, the results demonstrate that all three vaccine candidates are highly immunogenic and efficacious in mice, and that the STF2R3.2xHA1 format is the most effective candidate vaccine format.
Asunto(s)
Flagelina/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/epidemiología , Pandemias/prevención & control , Proteínas Recombinantes de Fusión/inmunología , Vacunas Virales/inmunología , Animales , Femenino , Flagelina/química , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Infecciones por Orthomyxoviridae/prevención & control , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Currently controversy exists about the immunogenicity of seasonal trivalent influenza vaccine in certain populations, especially the elderly. STF2.4×M2e (VAX102) is a recombinant fusion protein that links four copies of the ectodomain of influenza virus matrix protein 2 (M2e) antigen to Salmonella typhimurium flagellin, a TLR5 ligand. The objectives of this study were to assess the feasibility of giving VAX102 and TIV in combination in an effort to achieve greater immunogenicity and to provide cross-protection. METHODOLOGY/PRINCIPAL FINDINGS: Eighty healthy subjects, 18-49 years old, were enrolled in May and June 2009 in a double-blind, randomized, controlled trial at two clinical sites. Subjects were randomized to receive either TIV + VAX102 or TIV + placebo. Both arms tolerated the vaccines. Pain at the injection site was more severe with TIV + VAX102. Two weeks after immunization the HAI responses to the H1 and H3 antigens of TIV were higher in those that received TIV + VAX102 than in TIV + placebo (309 vs 200 and 269 vs 185, respectively), although statistically non-significant. There was no difference in the HAI of the B antigen. In the TIV + VAX102 arm, the geometric mean M2e antibody concentration was 0.5 µg/ml and 73% seroconverted. CONCLUSIONS/SIGNIFICANCE: The combination of TIV + VAX102 has the potential to increase the immune response to the influenza A components of TIV and to provide M2e immunity which may protect against influenza A strains not contained in seasonal TIV. TRIAL REGISTRATION: ClinicalTrials.gov NCT00921973.
Asunto(s)
Flagelina/química , Virus de la Influenza A/metabolismo , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/metabolismo , Gripe Humana/prevención & control , Receptor Toll-Like 5/metabolismo , Adolescente , Adulto , Método Doble Ciego , Femenino , Humanos , Vacunas contra la Influenza/uso terapéutico , Ligandos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/metabolismoRESUMEN
BACKGROUND: The need for worldwide seasonal and pandemic vaccine production has increased interest in the development of innovative technologies for influenza vaccine production. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in E. coli. METHODS: 128 healthy adult subjects 18-49 years old were enrolled in a clinical trial conducted in three stages at a single center. Stage 1 was an open-label, dose escalation study in which the VAX125 vaccine was administered intramuscularly (im) at doses of 0.1 µg, 0.3 µg, 1 µg, 2 µg, 3 µg, 5 µg and 8 µg to groups of 8 subjects each. Stage 2 was a double-blind, placebo-controlled study in which subjects were randomized to receive 1.0 µg and 2.0 µg VAX125 vaccine doses or placebo, with 16 subjects per group. Finally, an additional 24 subjects received a 0.5 µg dose of VAX125 in stage 3, which was a non-randomized, open label study. In all parts subjects were followed for adverse events and sera was tested by hemagglutination-inhibition (HAI) and microneutralization (MN) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP), cytokine levels, and anti-flagellin antibody were also assessed. RESULTS: Vaccine was generally well tolerated and there were no serious adverse events. Pain at the injection site was the most common local adverse event, and was mild or moderate in intensity. Systemic symptoms after vaccination include fatigue and headache, and two subjects, who received either 3 or 8 µg, had moderately severe systemic symptoms accompanied by substantial increases in serum CRP. Serum antibody responses against SI were seen by HAI and MN in most study subjects, with the geometric mean titer of post vaccination antibody increasing in a dose-dependent fashion. Overall, four-fold or greater serum HAI responses were seen in 61 of 96 (64%) subjects who received doses of 0.5 µg or greater, including in 46 of 72 subjects who received doses from 0.5 µg to 2 µg. CONCLUSIONS: The globular head of the influenza HA expressed in a prokaryotic system was able to induce a functional antibody response against native virions. Vigorous responses were seen at relatively low doses of HA antigen suggesting that the addition of flagellin provided a substantial adjuvanting effect. The high levels of immune response at low doses of antigen and the relative ease of production associated with E. coli expression suggests that this approach may represent an effective strategy for enhancing the global influenza vaccine supply.
Asunto(s)
Flagelina/inmunología , Hemaglutininas Virales/inmunología , Vacunas contra la Influenza/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Anticuerpos Antivirales/sangre , Proteína C-Reactiva/análisis , Citocinas/sangre , Escherichia coli/genética , Femenino , Flagelina/genética , Expresión Génica , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas Virales/genética , Humanos , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/efectos adversos , Vacunas contra la Influenza/genética , Inyecciones Intramusculares , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Placebos/administración & dosificación , Vacunación/métodos , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Adulto JovenRESUMEN
Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.
Asunto(s)
Flagelina/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Línea Celular , Femenino , Flagelina/metabolismo , Pruebas de Inhibición de Hemaglutinación , Humanos , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes/inmunología , Receptor Toll-Like 5/metabolismoRESUMEN
The recognition of specific pathogen associated molecular patterns (PAMPs) by members of the Toll-like receptor (TLR) family is critical for the activation of the adaptive immune response. Thus, incorporation of PAMPs into vaccines should result in more potent, protective antigen-specific responses in the absence of adjuvants or complex formulations. Here we describe an influenza A vaccine that is refractory to the genetic instability of hemagglutinin and neuraminidase and includes a trigger of the innate immune response to enhance immunogenicity and efficacy. A recombinant protein comprising the TLR5 ligand flagellin fused to four tandem copies of the ectodomain of the conserved influenza matrix protein M2 (M2e) was expressed in Escherichia coli and purified to homogeneity. This protein, STF2.4xM2e, retained TLR5 activity and displayed the protective epitope of M2e defined by a monoclonal antibody, 14C2. Mice immunized with STF2.4xM2e in aqueous buffer, without adjuvants or other formulation additives, developed potent M2e-specific antibody responses that were quantitatively and qualitatively superior to those observed with M2e peptide delivered in alum. The antibody response was dependent on the physical linkage of the antigen to flagellin and recognized the epitope defined by monoclonal antibody 14C2, which has been shown to protect mice from challenge with influenza A virus. Moreover, immunization with STF2.4xM2e at a dose of 0.3 microg per mouse protected mice from a lethal challenge with influenza A virus, and significantly reduced weight loss and clinical symptoms. These data demonstrate that the linkage of specific TLR ligand with influenza M2e yields a vaccine candidate that offers significant promise for widespread protection against multiple influenza A virus strains.
Asunto(s)
Flagelina/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Escherichia coli/genética , Flagelina/genética , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/fisiopatología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Análisis de Supervivencia , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas de la Matriz Viral/genética , Pérdida de PesoRESUMEN
It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines.