RESUMO
Vaccine hesitancy and the occurrence of elusive variants necessitate further treatment options for coronavirus disease 2019 (COVID-19). Accumulated evidence indicates that clinically used hypertensive drugs, angiotensin receptor blockers (ARBs), may benefit patients by mitigating disease severity and/or viral propagation. However, current clinical formulations administered orally pose systemic safety concerns and likely require a very high dose to achieve the desired therapeutic window in the lung. To address these limitations, we have developed a nanosuspension formulation of an ARB, entirely based on clinically approved materials, for inhaled treatment of COVID-19. We confirmed in vitro that our formulation exhibits physiological stability, inherent drug activity, and inhibitory effect against SARV-CoV-2 replication. Our formulation also demonstrates excellent lung pharmacokinetics and acceptable tolerability in rodents and/or nonhuman primates following direct administration into the lung. Thus, we are currently pursuing clinical development of our formulation for its uses in patients with COVID-19 or other respiratory infections.
Assuntos
COVID-19 , Infecções Respiratórias , Animais , Antagonistas de Receptores de Angiotensina/farmacologia , Antagonistas de Receptores de Angiotensina/uso terapêutico , Telmisartan , Sistema Renina-Angiotensina/fisiologia , SARS-CoV-2 , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Infecções Respiratórias/tratamento farmacológicoRESUMO
Although two vaccines for Zaire ebolavirus (EBOV) have been licensed and deployed successfully to combat recurring outbreaks of Ebolavirus Disease in West Africa, there are no vaccines for two other highly pathogenic members of the Filoviridae, Sudan ebolavirus (SUDV) and Marburg marburgvirus (MARV). The results described herein document the immunogenicity and protective efficacy in cynomolgus macaques of a single-vial, thermostabilized (lyophilized) monovalent (SUDV) and bivalent (SUDV & MARV) protein vaccines consisting of recombinant glycoproteins (GP) formulated with a clinical-grade oil-in-water nanoemulsion adjuvant (CoVaccine HT™). Lyophilized formulations of the vaccines were reconstituted with Water for Injection and used to immunize groups of cynomolgus macaques before challenge with a lethal dose of a human SUDV or MARV isolate. Sera collected after each of the three immunizations showed near maximal GP-binding IgG concentrations starting as early as the second dose. Most importantly, the vaccine candidates (monovalent or bivalent) provided 100% protection against severe and lethal filovirus disease after either SUDV or MARV infection. Although mild, subclinical infection was observed in a few macaques, all vaccinated animals remained healthy and survived the filovirus challenge. These results demonstrate the value that thermostabilized protein vaccines could provide for addressing an important gap in preparedness for future filovirus outbreaks.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Marburgvirus , Vacinas Virais , Animais , Humanos , Vacinas Combinadas , Sudão , Anticorpos Antivirais , Macaca fascicularis , ÁguaRESUMO
The issues of vaccine potency and stability constitute formidable challenges associated with the development and readiness of vaccines for biodefense. In most instances, the vaccines will be stockpiled (at considerable cost) for years and used only in the rare event of a public health emergency. It is therefore imperative that there be means to readily monitor overall stability of the stockpiled vaccines, preferably using reliable in vitro assays, without the need for expensive and labor-intensive animal studies. In this chapter, we describe an in vitro monoclonal antibody-based competition ELISA known as RiCoE for assessing the potency of a ricin toxin subunit vaccine. RiCoE can be applied to drug substance and drug products adsorbed to aluminum salts adjuvant. While RiCoE is specific for ricin toxin, the general methodologies and protocols described herein are amenable to virtually any subunit or even virus-like particle-based vaccine. Ultimately, RiCoE-like assays may replace or at least reduce the need for animal studies in vaccine potency determinations.
Assuntos
Potência de Vacina , Adjuvantes Farmacêuticos , Animais , Anticorpos Neutralizantes , Ricina , Vacinas de Subunidades Antigênicas , Vacinas de Partículas Semelhantes a VírusRESUMO
SQSTM1/p62 is an autophagic receptor that plays a major role in mediating stress and innate immune responses. Preclinical studies identified p62 as a target of the prototype innate defense regulator (IDR); however, the molecular mechanism of this process remains unclear. Here, we describe the structural basis and biological consequences of the interaction of p62 with the next generation of IDRs, dusquetide. Both electrostatic and hydrophobic contacts drive the formation of the complex between dusquetide and the ZZ domain of p62. We show that dusquetide penetrates the cell membrane and associates with p62 in vivo. Dusquetide binding modulates the p62-RIP1 complex, increases p38 phosphorylation, and enhances CEBP/B expression without activating autophagy. Our findings provide molecular details underlying the IDR action that may help in the development of new strategies to pharmacologically target p62.
Assuntos
Imunidade Inata , Oligopeptídeos , Autofagia , Oligopeptídeos/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Inhalation of the biothreat agent, ricin toxin (RT), provokes a localized inflammatory response associated with pulmonary congestion, edema, neutrophil infiltration, and severe acute respiratory distress. The extreme toxicity of RT is the result of the toxin's B chain (RTB) promoting rapid uptake into alveolar macrophages and lung epithelial cells, coupled with the A chain's (RTA) potent ribosome-inactivating properties. We previously reported that intramuscular vaccination of rhesus macaques with a lyophilized, alum-adsorbed recombinant RTA subunit vaccine (RiVax®) was sufficient to confer protection against a lethal dose of aerosolized RT. That study implicated RT-specific serum IgG, toxin-neutralizing activity (TNA), and epitope-specific responses as being associated with immunity. However, it was not possible to define actual correlates of protection (COP) because all vaccinated animals survived the RT challenge. We addressed the issue of COP in the current study, by vaccinating groups of rhesus macaques with RiVax® following the previously determined protective regimen (100 µg on study days 0, 30 and 60) or one of two anticipated suboptimal regimens (100 µg on study days 30 and 60; 35 µg on study days 0, 30, and 60). Two unvaccinated animals served as controls. The animals were challenged with ~5 × LD50s of aerosolized RT on study day 110. We report that all vaccinated animals seroconverted prior to RT challenge, with the majority also having measurable TNA, although neither antibody levels nor TNA reached statistical significance with regard to a correlation with protection. By contrast, survival correlated with pre-challenge, epitope-specific serum IgG levels, derived from a competitive sandwich ELISA using a panel of toxin-neutralizing monoclonal antibodies directed against distinct epitopes on RiVax®. The identification of a species-neutral, competitive ELISA that correlates with vaccine-induced protection against RT in nonhuman represents an important advance in the development of medical countermeasures (MCM) against a persistent biothreat.
RESUMO
Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus are the filoviruses most commonly associated with human disease. Previously, we administered a three-dose regimen of trivalent vaccines comprising glycoprotein antigens from each virus in mice and non-human primates (NHPs). The vaccines, which contained a polysorbate 80-stabilized squalane-in-water emulsion adjuvant and were lyophilized from a solution containing trehalose, produced high antibody levels against all three filovirus antigens. Subsequently, single-vial formulations containing a higher concentration of adjuvant were generated for testing in NHPs, but these vaccines elicited lower neutralizing antibody titers in NHPs than previously tested formulations. In order to explain these results, in the current work we measured the size of adjuvant emulsion droplets and the peroxide levels present in the vaccines after lyophilization and reconstitution and tested the effects of these variables on the immune response in mice. Increases in squalane droplet sizes were observed when the ratio of adjuvant to trehalose was increased beyond a critical value, but antibody and neutralizing antibody titers in mice were independent of the droplet size. Higher levels of peroxides in the vaccines correlated with higher concentrations of adjuvant in the formulations, and higher peroxide levels were associated with increased levels of oxidative damage to glycoprotein antigens. Neutralizing titers in mice were inversely correlated with peroxide levels in the vaccines, but peroxide levels could be reduced by adding free methionine, resulting in retention of high neutralizing antibody titers. Overall, the results suggest that oxidation of glycoprotein antigens by peroxides in the polysorbate 80-stabilized squalane-in-water emulsion adjuvant, but not lyophilization-induced increases in adjuvant emulsion droplet size may have been responsible for the decreased neutralizing titers seen in formulations containing higher amounts of adjuvant.
Assuntos
Ebolavirus , Vacinas Virais , Camundongos , Animais , Anticorpos Neutralizantes , Polissorbatos , Trealose , Peróxidos , Emulsões , Anticorpos Antivirais , Adjuvantes Imunológicos/farmacologia , Glicoproteínas , Adjuvantes Farmacêuticos , Primatas , ÁguaRESUMO
FDA-approved and emergency use-authorized vaccines using new mRNA and viral-vector technology are highly effective in preventing moderate to severe disease; however, information on their long-term efficacy and protective breadth against severe acute respiratory syndrome coronavirus 2 variants of concern (VOCs) is currently scarce. Here, we describe the durability and broad-spectrum VOC immunity of a prefusion-stabilized spike (S) protein adjuvanted with liquid or lyophilized CoVaccine HT in cynomolgus macaques. This recombinant subunit vaccine is highly immunogenic and induces robust spike-specific and broadly neutralizing antibody responses effective against circulating VOCs (B.1.351 [Beta], P.1 [Gamma], and B.1.617 [Delta]) for at least three months after the final boost. Protective efficacy and postexposure immunity were evaluated using a heterologous P.1 challenge nearly three months after the last immunization. Our results indicate that while immunization with both high and low S doses shorten and reduce viral loads in the upper and lower respiratory tract, a higher antigen dose is required to provide durable protection against disease as vaccine immunity wanes. Histologically, P.1 infection causes similar COVID-19-like lung pathology as seen with early pandemic isolates. Postchallenge IgG concentrations were restored to peak immunity levels, and vaccine-matched and cross-variant neutralizing antibodies were significantly elevated in immunized macaques indicating an efficient anamnestic response. Only low levels of P.1-specific neutralizing antibodies with limited breadth were observed in control (nonvaccinated but challenged) macaques, suggesting that natural infection may not prevent reinfection by other VOCs. Overall, these results demonstrate that a properly dosed and adjuvanted recombinant subunit vaccine can provide protective immunity against circulating VOCs for at least three months.
Assuntos
COVID-19 , SARS-CoV-2 , Adjuvantes Imunológicos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Macaca , Vacinas de Subunidades AntigênicasRESUMO
Importance: Given that mycosis fungoides-cutaneous T-cell lymphoma (MF/CTCL) is chronic, there is a need for additional therapies with minimal short- and long-term adverse effects. Topical synthetic hypericin ointment, 0.25%, activated with visible light is a novel, nonmutagenic photodynamic therapy (PDT). Objectives: To determine the efficacy and safety of topical synthetic hypericin ointment, 0.25%, activated with visible light as a nonmutagenic PDT in early-stage MF/CTCL. Design, Settings, and Participants: This was a multicenter, placebo-controlled, double-blinded, phase 3 randomized clinical trial (FLASH study) conducted from December 2015 to November 2020 at 39 academic and community-based US medical centers. Participants were adults (≥18 years) with early-stage (IA-IIA) MF/CTCL. Interventions: In cycle 1, patients were randomized 2:1 to receive hypericin or placebo to 3 index lesions twice weekly for 6 weeks. In cycle 2, all patients received the active drug for 6 weeks to index lesions. In cycle 3 (optional), both index and additional lesions received active drug for 6 weeks. Main Outcomes and Measures: The primary end point was index lesion response rate (ILRR), defined as 50% or greater improvement in modified Composite Assessment of Index Lesion Severity (mCAILS) score from baseline after 6 weeks of therapy for cycle 1. For cycles 2 and 3, open label response rates were secondary end points. Adverse events (AEs) were assessed at each treatment visit, after each cycle, and then monthly for 6 months. Data analyses were performed on December 21, 2020. Results: The study population comprised 169 patients (mean [SD] age, 58.4 [16.0] years; 96 [57.8%] men; 120 [72.3%] White individuals) with early-stage MF/CTCL. After 6 weeks of treatment, hypericin PDT was more effective than placebo (cycle 1 ILRR, 16% vs 4%; P = .04). The ILRR increased to 40% in patients who received 2 cycles of hypericin PDT (P < .001 vs cycle 1 hypericin) and to 49% after 3 cycles (P < .001 vs cycle 1 hypericin). Significant clinical responses were observed in both patch and plaque type lesions and were similar regardless of age, sex, race, stage IA vs IB, time since diagnosis, and number of prior therapies. The most common treatment-related AEs were mild local skin (13.5%-17.3% across cycles 1-3 vs 10.5% for placebo in cycle 1) and application-site reactions (3.2%-6.9% across cycles 1-3 vs 4% for placebo in cycle 1). No drug-related serious AEs occurred. Conclusion and Relevance: The findings of this randomized clinical trial indicate that synthetic hypericin PDT is effective in early-stage patch and plaque MF/CTCL and has a favorable safety profile. Trial Registration: ClinicalTrials.gov Identifier: NCT02448381.
Assuntos
Linfoma Cutâneo de Células T , Micose Fungoide , Fotoquimioterapia , Neoplasias Cutâneas , Adulto , Antracenos , Feminino , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/patologia , Masculino , Pessoa de Meia-Idade , Micose Fungoide/patologia , Pomadas/uso terapêutico , Perileno/análogos & derivados , Fotoquimioterapia/efeitos adversos , Fármacos Fotossensibilizantes/efeitos adversos , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
The development of vaccines against biothreat toxins like ricin (RT) is considered an integral component of the U.S. national security efforts. RiVax is a thermostable, lyophilized RT subunit vaccine adsorbed to aluminum salt adjuvant intended for use by military personnel and first responders. Phase 1 studies indicated that RiVax is safe and immunogenic, while a three-dose intramuscular vaccination regimen in nonhuman primates elicited protection against lethal dose RT challenge by aerosol. Here, we investigated, in a mouse model, the durability of RiVax-induced antibody responses and corresponding immunity to lethal dose RT challenge. Groups of mice were subcutaneously administered 3 or 1 µg of RiVax on days 0 and 21 and challenged with 10× 50% lethal dose (LD50) RT by injection at six different intervals over the course of 12 months. Serum antibody titers and epitope-specific competition assays were determined prior to each challenge. We report that the two-dose, 3-µg regimen conferred near-complete protection against RT challenge on day 35 and complete protection thereafter (challenge days 65, 95, 125, 245, and 365). The two-dose, 3-µg regimen was superior to the 1-µg regimen as revealed by slight differences in survival and morbidity scores (e.g., hypoglycemia, weight loss) on challenge days 35 and 365. In separate experiments, a single 3-µg RiVax vaccination proved only marginally effective at eliciting protective immunity to RT, underscoring the necessity of a prime-boost regimen to achieve full and long-lasting protection against RT. IMPORTANCE Ricin toxin (RT) is a notorious biothreat, as exposure to even trace amounts via injection or inhalation can induce organ failure and death within a matter of hours. In this study, we advance the preclinical testing of a candidate RT vaccine known as RiVax. RiVax is a recombinant nontoxic derivative of RT's enzymatic subunit that has been evaluated for safety in phase I clinical trials and efficacy in a variety of animal models. We demonstrate that two doses of RiVax are sufficient to protect mice from lethal dose RT challenge for up to 1 year. We describe kinetics and other immune parameters of the antibody response to RiVax and discuss how these immune factors may translate to humans.
Assuntos
Epitopos/química , Ricina/química , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas/administração & dosagem , Aerossóis , Animais , Bioterrorismo , Feminino , Liofilização , Injeções Intramusculares , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The speed at which several COVID-19 vaccines went from conception to receiving FDA and EMA approval for emergency use is an achievement unrivaled in the history of vaccine development. Mass vaccination efforts using the highly effective vaccines are currently underway to generate sufficient herd immunity and reduce transmission of the SARS-CoV-2 virus. Despite the most advanced vaccine technology, global recipient coverage, especially in resource-poor areas remains a challenge as genetic drift in naïve population pockets threatens overall vaccine efficacy. In this study, we described the production of insect-cell expressed SARS-CoV-2 spike protein ectodomain constructs and examined their immunogenicity in mice. We demonstrated that, when formulated with CoVaccine HTTM adjuvant, an oil-in-water nanoemulsion compatible with lyophilization, our vaccine candidates elicit a broad-spectrum IgG response, high neutralizing antibody (NtAb) titers against SARS-CoV-2 prototype and variants of concern, specifically B.1.351 (Beta) and P.1. (Gamma), and an antigen-specific IFN-γ secreting response in outbred mice. Of note, different ectodomain constructs yielded variations in NtAb titers against the prototype strain and some VOC. Dose response experiments indicated that NtAb titers increased with antigen dose, but not adjuvant dose, and may be higher with a lower adjuvant dose. Our findings lay the immunological foundation for the development of a dry-thermostabilized vaccine that is deployable without refrigeration.
RESUMO
The speed at which several COVID-19 vaccines went from conception to receiving FDA and EMA approval for emergency use is an achievement unrivaled in the history of vaccine development. Mass vaccination efforts using the highly effective vaccines are currently underway to generate sufficient herd immunity and reduce transmission of the SARS-CoV-2 virus. Despite the most advanced vaccine technology, global recipient coverage, especially in resource-poor areas remains a challenge as genetic drift in naïve population pockets threatens overall vaccine efficacy. In this study, we described the production of insect-cell expressed SARS-CoV-2 spike protein ectodomain and examined its immunogenicity in mice. We demonstrated that, when formulated with CoVaccine HT™adjuvant, an oil-in-water nanoemulsion compatible with lyophilization, our vaccine candidates elicit a broad-spectrum IgG response, high neutralizing antibody titers, and a robust, antigen-specific IFN-γ secreting response from immune splenocytes in outbred mice. Our findings lay the foundation for the development of a dry-thermostabilized vaccine that is deployable without refrigeration.
RESUMO
Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filovirus. Previously, we showed that glycoprotein antigens from each virus could be lyophilized to create thermostable monovalent subunit vaccines. However, cross-protection is not expected from the monovalent vaccines and therefore developing a trivalent filovirus vaccine would be desirable. Subunit protein vaccines often require the addition of an adjuvant to sufficiently boost the immunogenicity. Typically, liquid suspensions or emulsions of adjuvants and lyophilized antigens are stored in separate vials to avoid destabilizing interactions and are only mixed immediately before administration. Herein, we describe the development and characterization of monovalent and trivalent filovirus vaccines that are co-lyophilized with a squalane-in-water emulsion adjuvant. We found that the single-vial presentation retained adjuvant particle diameter and zeta potential after lyophilization and reconstitution. Furthermore, the trivalent vaccines elicited high antibody levels against all three antigens in mice and non-human primates. These results advance the prospect of developing a single-vial trivalent filovirus vaccine, which would enable easier distribution and administration of the vaccine to resource-poor areas.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Vacinas Virais , Animais , Anticorpos Antivirais , Liofilização , Glicoproteínas , CamundongosRESUMO
Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas de Choque Térmico/imunologia , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Imunidade Inata/genética , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Proteína Sequestossoma-1 , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.
Assuntos
Anti-Infecciosos/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Peptídeos/farmacologia , Animais , Anti-Infecciosos/uso terapêutico , Anti-Infecciosos/toxicidade , Infecções Bacterianas/tratamento farmacológico , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Modelos Imunológicos , Peptídeos/toxicidade , Resultado do TratamentoRESUMO
Mouse challenge studies with death as an endpoint remain the gold standard in assessing the potency of ricin toxin, a Category B biothreat agent derived from the castor bean (Ricinus communis). However, animal studies are expensive, time consuming and ethically concerning. In an effort to reduce reliance on animals in vaccine development, we developed a monoclonal antibody (MAb)-based ricin competition ELISA (RiCoE) that indicates conformation integrity of ricin toxin. In forced degradation (heat-denaturation) experiments with native ricin holotoxin, we demonstrate a correlation between the decline in MAb reactivity in RiCoE and a corresponding loss of toxin potency in Vero cells (IC50) and mice (LD50). The RiCoE assay was applied to differentially sourced commercial lots of ricin toxin derived from R. communis blends and compared to toxin potency in mice. There was near perfect congruence between RiCoE values with two different MAbs (PB10, SyH7) and ricin potency in the mouse model using morbidity as an endpoint. In conclusion, we propose that RiCoE can serve as a rapid and sensitive substitute to mouse lethal dose challenge studies as a means to determine ricin toxin potency and will be valuable at various stages of vaccine development.
Assuntos
Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Ricina/toxicidade , Alternativas aos Testes com Animais , Animais , Especificidade de Anticorpos , Ligação Competitiva , Chlorocebus aethiops , Feminino , Epitopos Imunodominantes , Dose Letal Mediana , Camundongos Endogâmicos BALB C , Conformação Proteica , Desnaturação Proteica , Ricina/química , Ricina/imunologia , Relação Estrutura-Atividade , Células VeroRESUMO
The filoviruses Zaire ebolavirus (EBOV), Marburg marburgvirus (MARV), and Sudan ebolavirus (SUDV) are some of the most lethal infectious agents known. To date, the Zaire ebolavirus vaccine (ERVEBO®) is the only United States Food and Drug Administration (FDA) approved vaccine available for any species of filovirus. However, the ERVEBO® vaccine requires cold-chain storage not to exceed -60 °C. Such cold-chain requirements are difficult to maintain in low- and middle-income countries where filovirus outbreaks originate. To improve the thermostability of filovirus vaccines in order to potentially relax or eliminate these cold-chain requirements, monovalent subunit vaccines consisting of glycoproteins from EBOV, MARV, and SUDV were stabilized within amorphous disaccharide glasses through lyophilization. Lyophilized formulations and liquid controls were incubated for up to 12 weeks at 50 °C to accelerate degradation. To identify a stability-indicating assay appropriate for monitoring protein degradation and immunogenicity loss during these accelerated stability studies, filovirus glycoprotein secondary, tertiary, and quaternary structures and vaccine immunogenicity were measured. Size-exclusion chromatography was the most sensitive indicator of glycoprotein stability in the various formulations for all three filovirus immunogens. Degradation of the test vaccines during accelerated stability studies was reflected in changes in quaternary structure, which were discernible with size-exclusion chromatography. Filovirus glycoproteins in glassy lyophilized formulations retained secondary, tertiary, and quaternary protein structure over the incubation period, whereas the proteins within liquid controls both aggregated to form higher molecular weight species and dissociated from their native quaternary structure to form a variety of structurally-perturbed lower molecular weight species.
Assuntos
Ebolavirus , Glicoproteínas , Doença pelo Vírus Ebola , Marburgvirus , Vacinas , Ebolavirus/imunologia , Marburgvirus/imunologiaRESUMO
The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD50 of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration's "Animal Rule."
Assuntos
Ricina , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Formação de Anticorpos , Epitopos , Camundongos , Ricina/toxicidade , Vacinas de Subunidades AntigênicasRESUMO
Purpose: Characterization of a novel partial-body irradiation (PBI) shielding strategy in nonhuman primates (NHP; rhesus macaques), aimed at protecting the oral cavity, with respect to various gastrointestinal acute radiation syndrome (GI-ARS) syndrome parameters as well as buccal ulceration development.Materials and methods: NHPs were irradiated using a Cobalt-60 gamma source, in a single uniform dose, ranging from 9-13 Gy and delivered at 0.60-0.80 Gy min-1. Animals were either partially shielded via oral cavity shielding (PBIOS) or underwent total-body irradiation (TBI).Results: Clinical manifestations of GI-ARS, and also radiation-induced hematology and clinical chemistry changes, following PBIOS were comparable to the PBI NHP GI-ARS model utilizing shielding of the distal pelvic limbs and were significantly milder than TBI at similar radiation doses. Nadir citrulline levels were comparable between PBIOS and TBI but signs of recovery appeared earlier in PBIOS-treated animals. The PBIOS model prevented oral mucositis, whereas the TBI model presented buccal ulcerations at all tested radiation dose levels.Conclusions: Taken together, these results suggest that the PBIOS model is a suitable alternative to traditional PBI. For GI-ARS investigations requiring orally administered medical countermeasures, PBIOS confers added value due to the prevention of oral mucositis over traditional PBI.
Assuntos
Boca/efeitos da radiação , Proteção Radiológica/métodos , Síndrome Aguda da Radiação/sangue , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/patologia , Animais , Citrulina/sangue , Radioisótopos de Cobalto/efeitos adversos , Raios gama/efeitos adversos , Macaca mulatta , Masculino , Análise de Sobrevida , Úlcera/sangue , Úlcera/etiologia , Úlcera/patologiaRESUMO
Biodefense vaccine are destined to be stockpiled for periods of time and deployed in the event of a public health emergency. In this report, we compared the potency of liquid and lyophilized (thermostabilized) formulations of a candidate ricin toxin subunit vaccine, RiVax, adsorbed to aluminum salts adjuvant, over a 12-month period. The liquid and lyophilized formulations were stored at stressed (40⯰C) and unstressed (4⯰C) conditions and evaluated at 3, 6 and 12-month time points for potency in a mouse model of lethal dose ricin challenge. At the same time points, the vaccine formulations were interrogated in vitro by competition ELISA for conformational integrity using a panel of three monoclonal antibodies (mAbs), PB10, WECB2, and SyH7, directed against known immunodominant toxin-neutralizing epitopes on RiVax. We found that the liquid vaccine under stress conditions declined precipitously within the first three months, as evidenced by a reduction in in vivo potency and concomitant loss of mAb recognition in vitro. In contrast, the lyophilized RiVax vaccine retained in vivo potency and conformational integrity for up to one year at 4⯰C and 40⯰C. We discuss the utility of monitoring the integrity of one or more toxin-neutralizing epitopes on RiVax as a possible supplement to animal studies to assess vaccine potency.
Assuntos
Epitopos de Linfócito B/imunologia , Liofilização , Ricina/imunologia , Potência de Vacina , Vacinas de Subunidades Antigênicas/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Armas Biológicas , Mapeamento de Epitopos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Temperatura , Vacinas/química , Vacinas de Subunidades Antigênicas/químicaRESUMO
Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides.