RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.
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Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/genética , Descoberta de Drogas/métodos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Animais , COVID-19/virologia , Células Cultivadas , Proteases 3C de Coronavírus/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Engenharia Genética , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , SARS-CoV-2/metabolismo , Relação Estrutura-Atividade , Replicação Viral , Tratamento Farmacológico da COVID-19RESUMO
SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.
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Vacinas contra COVID-19 , Expressão Gênica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Vacinas contra COVID-19/química , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/farmacologia , Humanos , Camundongos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , SARS-CoV-2/química , SARS-CoV-2/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/farmacologiaRESUMO
Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm's pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.
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Imunidade , Proteínas do Leite/imunologia , Tricuríase/prevenção & controle , Trichuris/imunologia , Animais , Anticorpos Anti-Helmínticos/metabolismo , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/farmacologia , Imunidade/efeitos dos fármacos , Imunidade/genética , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Knockout , Camundongos SCID , Proteínas do Leite/genética , Proteínas do Leite/farmacologia , Tricuríase/imunologia , Trichuris/genética , Vacinação/métodosRESUMO
E6020 is a synthetic agonist of Toll-like receptor-4 (TLR4). The purpose of this study was to evaluate the effect of different doses of E6020-SE on Trypanosoma cruzi-specific immune responses and its ability to confer protection against acute lethal infection in mice. Forty female BALB/c were infected with 500 trypomastigotes of T cruzi H1 strain, divided into four groups (n = 10) and treated at 7- and 14-day post-infection (dpi) with different doses of E6020-SE or PBS (control). Survival was followed for 51 days, mice were euthanized and hearts were collected to evaluate parasite burden, inflammation and fibrosis. We found significantly higher survival and lower parasite burdens in mice injected with E6020-SE at all doses compared to the control group. However, E6020-SE treatment did not significantly reduce cardiac inflammation or fibrosis. On the other hand, E6020-SE modulated Th1 and Th2 cytokines, decreasing IFN-γ and IL-4 in a dose-dependent manner after stimulation with parasite antigens. We conclude that E6020-SE alone increased survival by decreasing cardiac parasite burdens in BALB/c mice acutely infected with T cruzi but failed to prevent cardiac damage. Our results suggest that for optimal protection, a vaccine antigen is necessary to balance and orient a protective immune response.
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Doença de Chagas/tratamento farmacológico , Fosfolipídeos/uso terapêutico , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Doença de Chagas/imunologia , Citocinas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma cruzi/imunologiaRESUMO
Proteins primarily absorb UV light due to the presence of tryptophan, tyrosine, and phenylalanine residues, with absorbance maxima at 280, 275, and 258 nm, respectively. We now demonstrate that a simple value obtained by relating the absorbance at all three wavelengths, [A280/A275 + A280/A258], is a generally useful, robust, and sensitive probe of protein 'foldedness', and thus can be used to investigate unfolding, refolding, disulfide bonds, stability, buffer excipients, and even protein-protein and protein-ligand interactions.
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Ácido Aspártico Proteases/química , Pepsina A/química , Raios Ultravioleta , Ácido Aspártico Proteases/metabolismo , Concentração de Íons de Hidrogênio , Pepsina A/metabolismo , Conformação Proteica , Dobramento de Proteína , Espectrofotometria UltravioletaRESUMO
Chagas disease affects 6 to 7 million people worldwide, resulting in significant disease burdens and health care costs in countries of endemicity. Chemotherapeutic treatment is restricted to two parasiticidal drugs, benznidazole and nifurtimox. Both drugs are highly effective during acute disease but are only minimally effective during chronic disease and fraught with significant adverse clinical effects. In experimental models, vaccines can be used to induce parasite-specific balanced TH1/TH2 immune responses that effectively reduce parasite burdens and associated inflammation while minimizing adverse effects. The objective of this study was to determine the feasibility of vaccine-linked chemotherapy for reducing the amount of benznidazole required to significantly reduce blood and tissue parasite burdens. In this study, we were able to achieve a 4-fold reduction in the amount of benznidazole required to significantly reduce blood and tissue parasite burdens by combining the low-dose benznidazole with a recombinant vaccine candidate, Tc24 C4, formulated with a synthetic Toll-like 4 receptor agonist, E6020, in a squalene oil-in-water emulsion. Additionally, vaccination induced a robust parasite-specific balanced TH1/TH2 immune response. We concluded that vaccine-linked chemotherapy is a feasible option for advancement to clinical use for improving the tolerability and efficacy of benznidazole.
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Doença de Chagas/tratamento farmacológico , Doença de Chagas/imunologia , Nitroimidazóis/uso terapêutico , Tripanossomicidas/uso terapêutico , Doença Aguda , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Doença de Chagas/parasitologia , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imuno-Histoquímica , Nitroimidazóis/farmacologia , Carga Parasitária , Vacinas Protozoárias/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/imunologia , VacinaçãoRESUMO
INTRODUCTION: Pseudoviruses are recombinant, replication-incompetent, viral particles designed to mimic the surface characteristics of native enveloped viruses. They are a safer, and cost-effective research alternative to live viruses. With the potential emergence of the next major infectious disease, more vaccine scientists must become familiar with the pseudovirus platform as a vaccine development tool to mitigate future outbreaks. AREAS COVERED: This review aims at vaccine developers to provide a basic understanding of pseudoviruses, list their production methods, and discuss their utility to assess vaccine efficacy against enveloped viral pathogens. We further illustrate their usefulness as wet-lab simulators for emerging mutant variants, and new viruses to help prepare for current and future viral outbreaks, minimizing the need for gain-of-function experiments with highly infectious or lethal enveloped viruses. EXPERT OPINION: With this platform, researchers can better understand the role of virus-receptor interactions and entry in infections, prepare for dangerous mutations, and develop effective vaccines.
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Vacinas , Vírus , Humanos , Desenvolvimento de Vacinas , Anticorpos AntiviraisRESUMO
INTRODUCTION: Zebrafishes represent a proven model for human diseases and systems biology, exhibiting physiological and genetic similarities and having innate and adaptive immune systems. However, they are underexplored for human vaccinology, vaccine development, and testing. Here we summarize gaps and challenges. AREAS COVERED: Zebrafish models have four potential applications: 1) Vaccine safety: The past successes in using zebrafishes to test xenobiotics could extend to vaccine and adjuvant formulations for general safety or target organs due to the zebrafish embryos' optical transparency. 2) Innate immunity: The zebrafish offers refined ways to examine vaccine effects through signaling via Toll-like or NOD-like receptors in zebrafish myeloid cells. 3) Adaptive immunity: Zebrafishes produce IgM, IgD,and two IgZ immunoglobulins, but these are understudied, due to a lack of immunological reagents for challenge studies. 4) Systems vaccinology: Due to the availability of a well-referenced zebrafish genome, transcriptome, proteome, and epigenome, this model offers potential here. EXPERT OPINION: It remains unproven whether zebrafishes can be employed for testing and developing human vaccines. We are still at the hypothesis-generating stage, although it is possible to begin outlining experiments for this purpose. Through transgenic manipulation, zebrafish models could offer new paths for shaping animal models and systems vaccinology.
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Imunidade Adaptativa , Adjuvantes Imunológicos , Imunidade Inata , Modelos Animais , Desenvolvimento de Vacinas , Vacinas , Peixe-Zebra , Peixe-Zebra/imunologia , Animais , Adjuvantes Imunológicos/administração & dosagem , Humanos , Vacinas/imunologia , Vacinas/administração & dosagem , Vacinologia/métodosRESUMO
Chagas disease (CD) (American trypanosomiasis caused by Trypanosoma cruzi) is a parasitic disease endemic in 21 countries in South America, with increasing global spread. When administered late in the infection, the current antiparasitic drugs do not prevent the onset of cardiac illness leading to chronic Chagasic cardiomyopathy. Therefore, new therapeutic vaccines or immunotherapies are under development using multiple platforms. In this study, we assessed the feasibility of developing an mRNA-based therapeutic CD vaccine targeting two known T. cruzi vaccine antigens (Tc24âa flagellar antigen and ASP-2âan amastigote antigen). We present the mRNA engineering steps, preparation, and stability of the lipid nanoparticles and evaluation of their uptake by dendritic cells, as well as their biodistribution in c57BL/J mice. Furthermore, we assessed the immunogenicity and efficacy of two mRNA-based candidates as monovalent and bivalent vaccine strategies using an in vivo chronic mouse model of CD. Our results show several therapeutic benefits, including reductions in parasite burdens and cardiac inflammation, with each mRNA antigen, especially with the mRNA encoding Tc24, and Tc24 in combination with ASP-2. Therefore, our findings demonstrate the potential of mRNA-based vaccines as a therapeutic option for CD and highlight the opportunities for developing multivalent vaccines using this approach.
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Doença de Chagas , Vacinas Protozoárias , Camundongos , Animais , RNA , Distribuição Tecidual , Doença de Chagas/prevenção & controle , Antígenos de Protozoários/genética , RNA Mensageiro , TecnologiaRESUMO
OBJECTIVE: Design and evaluate immune responses of neonatal foals to a mRNA vaccine expressing the virulence-associated protein A (VapA) of Rhodococcus equi. ANIMALS: Cultured primary equine respiratory tract cells; Serum, bronchoalveolar lavage fluid (BALF), and peripheral blood mononuclear cells (PBMCs) from 30 healthy Quarter Horse foals. METHODS: VapA expression was evaluated by western immunoblot in cultured equine bronchial cells transfected with 4 mRNA constructs encoding VapA. The mRNA construct with greatest expression was used to immunize foals at ages 2 and 21 days in 5 groups: (1) 300 µg nebulized mRNA (n = 6); (2) 600 µg nebulized mRNA (n = 4); (3) 300 µg mRNA administered intramuscularly (IM) (n = 5); (4) 300 µg VapA IM (positive controls; n = 6); or (5) nebulized water (negative controls; n = 6). Serum, BALF, and PBMCs were collected at ages 3, 22, and 35 days and tested for relative anti-VapA IgG1, IgG4/7, and IgA activities using ELISA and cell-mediated immunity by ELISpot. RESULTS: As formulated, nebulized mRNA was not immunogenic. However, a significant increase in anti-VapA IgG4/7 activity (P < .05) was noted exclusively in foals immunized IM with VapA mRNA by age 35 days. The proportion of foals with anti-VapA IgG1 activity > 30% of positive control differed significantly (P = .0441) between negative controls (50%; 3/6), IM mRNA foals (100%; 5/5), and IM VapA (100%; 6/6) groups. Natural exposure to virulent R equi was immunogenic in some negative control foals. CLINICAL RELEVANCE: Further evaluation of the immunogenicity and efficacy of IM mRNA encoding VapA in foals is warranted.
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Infecções por Actinomycetales , Doenças dos Cavalos , Rhodococcus equi , Animais , Cavalos , Animais Recém-Nascidos , Imunidade Humoral , Vacinas de mRNA , Proteínas de Bactérias/genética , Rhodococcus equi/genética , Leucócitos Mononucleares , Imunoglobulina G , RNA Mensageiro/genética , Infecções por Actinomycetales/prevenção & controle , Infecções por Actinomycetales/veterinária , Doenças dos Cavalos/prevenção & controle , Fatores de Virulência/genéticaRESUMO
The development of broad-spectrum coronavirus vaccines is essential to prepare for future respiratory virus pandemics. We demonstrated broad neutralization by a trivalent subunit vaccine, formulating the receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 XBB.1.5 with Alum and CpG55.2. Vaccinated mice produced cross-neutralizing antibodies against all three human Betacoronaviruses and others currently exclusive to bats, indicating the epitope preservation of the individual antigens during co-formulation and the potential for epitope broadening.
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BACKGROUND: Chagas disease, chronic infection with Trypanosoma cruzi, mainly manifests as cardiac disease. However, the liver is important for both controlling parasite burdens and metabolizing drugs. Notably, high doses of anti-parasitic drug benznidazole (BNZ) causes liver damage. We previously showed that combining low dose BNZ with a prototype therapeutic vaccine is a dose sparing strategy that effectively reduced T. cruzi induced cardiac damage. However, the impact of this treatment on liver health is unknown. Therefore, we evaluated several markers of liver health after treatment with low dose BNZ plus the vaccine therapy in comparison to a curative dose of BNZ. METHODOLOGY: Female BALB/c mice were infected with a bioluminescent T. cruzi H1 clone for approximately 70 days, then randomly divided into groups of 15 mice each. Mice were treated with a 25mg/kg BNZ, 25µg Tc24-C4 protein/ 5µg E6020-SE (Vaccine), 25mg/kg BNZ followed by vaccine, or 100mg/kg BNZ (curative dose). At study endpoints we evaluated hepatomegaly, parasite burden by quantitative PCR, cellular infiltration by histology, and expression of B-cell translocation gene 2(BTG2) and Peroxisome proliferator-activated receptor alpha (PPARα) by RT-PCR. Levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were quantified from serum. RESULTS: Curative BNZ treatment significantly reduced hepatomegaly, liver parasite burdens, and the quantity of cellular infiltrate, but significantly elevated serum levels of ALT, AST, and LDH. Low BNZ plus vaccine did not significantly affect hepatomegaly, parasite burdens or the quantity of cellular infiltrate, but only elevated ALT and AST. Low dose BNZ significantly decreased expression of both BTG2 and PPARα, and curative BNZ reduced expression of BTG2 while low BNZ plus vaccine had no impact. CONCLUSIONS: These data confirm toxicity associated with curative doses of BNZ and suggest that while dose sparing low BNZ plus vaccine treatment does not reduce parasite burdens, it better preserves liver health.
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Doença de Chagas , Nitroimidazóis , Tripanossomicidas , Trypanosoma cruzi , Vacinas , Feminino , Animais , Camundongos , Hepatomegalia/tratamento farmacológico , Infecção Persistente , PPAR alfa/farmacologia , PPAR alfa/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/prevenção & controle , Doença de Chagas/parasitologia , Tripanossomicidas/farmacologiaRESUMO
Background: Chagas disease, chronic infection with Trypanosoma cruzi, mainly manifests as cardiac disease. However, the liver is important for both controlling parasite burdens and metabolizing drugs. Notably, high doses of anti-parasitic drug benznidazole (BNZ) causes liver damage. We previously showed that combining low dose BNZ with a prototype therapeutic vaccine is a dose sparing strategy that effectively reduced T. cruzi induced cardiac damage. However, the impact of this treatment on liver health is unknown. Therefore, we evaluated several markers of liver health after treatment with low dose BNZ plus the vaccine therapy in comparison to a curative dose of BNZ. Methodology: Female BALB/c mice were infected with a bioluminescent T. cruzi H1 clone for approximately 70 days, then randomly divided into groups of 15 mice each. Mice were treated with a 25mg/kg BNZ, 25µg Tc24-C4 protein/5µg E6020-SE (Vaccine), 25mg/kg BNZ followed by vaccine, or 100mg/kg BNZ (curative dose). At study endpoints we evaluated hepatomegaly, parasite burden by quantitative PCR, cellular infiltration by histology, and expression of B-cell translocation gene 2(BTG2) and Peroxisome proliferator-activated receptor alpha (PPARα) by RT-PCR. Levels of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were quantified from serum. Results: Curative BNZ treatment significantly reduced hepatomegaly, liver parasite burdens, and the quantity of cellular infiltrate, but significantly elevated serum levels of ALT, AST, and LDH. Low BNZ plus vaccine did not significantly affect hepatomegaly, parasite burdens or the quantity of cellular infiltrate, but only elevated ALT and AST. Low dose BNZ significantly decreased expression of both BTG2 and PPARα, and curative BNZ reduced expression of BTG2 while low BNZ plus vaccine had no impact. Conclusions: These data confirm toxicity associated with curative doses of BNZ and suggest that the dose sparing low BNZ plus vaccine treatment better preserves liver health.
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Post-infectious conditions, where clinical symptoms fail to resolve even after pathogen clearance, present major health burdens. However, the mechanisms involved remain poorly understood. In Chagas disease (CD), caused by the parasite Trypanosoma cruzi, antiparasitic agents can clear T. cruzi but late-stage treatment does not improve clinical cardiac outcomes. In this study, we revealed differential metabolic trajectories of cardiac regions during T. cruzi infection, matching sites of clinical symptoms. Incomplete, region-specific, cardiac metabolic restoration was observed in animals treated with the antiparasitic benznidazole, even though parasites were successfully cleared. In contrast, superior metabolic restoration was observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy (Tc24-C4 T. cruzi flagellar protein and TLR4 agonist adjuvant), even though parasite burden reduction was lower. Overall, these results provide a mechanism to explain prior clinical treatment failures in CD and to test novel candidate treatment regimens. More broadly, our results demonstrate a link between persistent metabolic perturbation and post-infectious conditions, with broad implications for our understanding of post-infectious disease sequelae.
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Introduction: Chagas disease, caused by chronic infection with the protozoan parasite Trypanosoma cruzi, affects 6-7 million people worldwide. The major clinical manifestation of Chagas disease is chronic Chagasic cardiomyopathy (CCC), which encompasses a spectrum of symptoms including arrhythmias, hypertrophy, dilated cardiomyopathy, heart failure, and sudden death. Current treatment is limited to two antiparasitic drugs, benznidazole (BNZ) and nifurtimox, but both have limited efficacy to halt the progression of CCC. We developed a vaccine-linked chemotherapy strategy using our vaccine consisting of recombinant Tc24-C4 protein and a TLR-4 agonist adjuvant in a stable squalene emulsion, in combination with low dose benznidazole treatment. We previously demonstrated in acute infection models that this strategy parasite specific immune responses, and reduced parasite burdens and cardiac pathology. Here, we tested our vaccine-linked chemotherapy strategy in a mouse model of chronic T. cruzi infection to evaluate the effect on cardiac function. Methods: Female BALB/c mice infected with 500 blood form T. cruzi H1 strain trypomastigotes were treated beginning 70 days after infection with a low dose of BNZ and either low or high dose of vaccine, in both sequential and concurrent treatments streams. Control mice were untreated, or administered only one treatment. Cardiac health was monitored throughout the course of treatment by echocardiography and electrocardiograms. Approximately 8 months after infection, endpoint histopathology was performed to measure cardiac fibrosis and cellular infiltration. Results: Vaccine-linked chemotherapy improved cardiac function as evidenced by amelioration of altered left ventricular wall thickness, left ventricular diameter, as well as ejection fraction and fractional shortening by approximately 4 months of infection, corresponding to two months after treatment was initiated. At study endpoint, vaccine-linked chemotherapy reduced cardiac cellular infiltration, and induced significantly increased antigen specific IFN-γ and IL-10 release from splenocytes, as well as a trend toward increased IL-17A. Discussion: These data suggest that vaccine-linked chemotherapy ameliorates changes in cardiac structure and function induced by infection with T. cruzi. Importantly, similar to our acute model, the vaccine-linked chemotherapy strategy induced durable antigen specific immune responses, suggesting the potential for a long lasting protective effect. Future studies will evaluate additional treatments that can further improve cardiac function during chronic infection.
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Doença de Chagas , Trypanosoma cruzi , Vacinas , Feminino , Animais , Camundongos , Infecção Persistente , Doença de Chagas/parasitologia , Coração , Proteínas RecombinantesRESUMO
Post-infectious conditions present major health burdens but remain poorly understood. In Chagas disease (CD), caused by Trypanosoma cruzi parasites, antiparasitic agents that successfully clear T. cruzi do not always improve clinical outcomes. In this study, we reveal differential small molecule trajectories between cardiac regions during chronic T. cruzi infection, matching with characteristic CD apical aneurysm sites. Incomplete, region-specific, cardiac small molecule restoration is observed in animals treated with the antiparasitic benznidazole. In contrast, superior restoration of the cardiac small molecule profile is observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy, even with less parasite burden reduction. Overall, these results reveal molecular mechanisms of CD treatment based on simultaneous effects on the pathogen and on host small molecule responses, and expand our understanding of clinical treatment failure in CD. This link between infection and subsequent persistent small molecule perturbation broadens our understanding of infectious disease sequelae.
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Doença de Chagas , Nitroimidazóis , Tripanossomicidas , Trypanosoma cruzi , Animais , Tripanossomicidas/farmacologia , Tripanossomicidas/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Nitroimidazóis/farmacologia , Nitroimidazóis/uso terapêutico , Coração , Progressão da DoençaRESUMO
Tc24 is a Trypanosoma cruzi-derived flagellar protein that, when formulated with a TLR-4 agonist adjuvant, induces a balanced immune response in mice, elevating IgG2a antibody titers and IFN-γ levels. Furthermore, vaccination with the recombinant Tc24 protein can reduce parasite levels and improve survival during acute infection. Although some mRNA vaccines have been proven to elicit a stronger immune response than some protein vaccines, they have not been used against T. cruzi. This work evaluates the immunogenicity of a heterologous prime/boost vaccination regimen using protein and mRNA-based Tc24 vaccines. Mice (C57BL/6) were vaccinated twice subcutaneously, three weeks apart, with either the Tc24-C4 protein + glucopyranosyl A (GLA)-squalene emulsion, Tc24 mRNA Lipid Nanoparticles, or with heterologous protein/mRNA or mRNA/protein combinations, respectively. Two weeks after the last vaccination, mice were euthanized, spleens were collected to measure antigen-specific T-cell responses, and sera were collected to evaluate IgG titers and isotypes. Heterologous presentation of the Tc24 antigen generated antigen-specific polyfunctional CD8+ T cells, a balanced Th1/Th2/Th17 cytokine profile, and a balanced humoral response with increased serum IgG, IgG1 and IgG2c antibody responses. We conclude that heterologous vaccination using Tc24 mRNA to prime and Tc24-C4 protein to boost induces a broad and robust antigen-specific immune response that was equivalent or superior to two doses of a homologous protein vaccine, the homologous mRNA vaccine and the heterologous Tc24-C4 Protein/mRNA vaccine.
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OBJECTIVE: To examine the susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus relative to human bronchial epithelial cells (HBECs). SAMPLE: Primary EBEC cultures established from healthy adult horses and commercially sourced human bronchial epithelial cells (HBECs) were used as a positive control. METHODS: Angiotensin-converting enzyme 2 (ACE2) expression by EBECs was demonstrated using immunofluorescence, western immunoblot, and flow cytometry. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were transduced with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media for 96 hours. After 96 hours, eGFP expression in EBECs was assessed by fluorescence microscopy of cell cultures and quantitative PCR. RESULTS: ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean ΔCt values from quantitative PCR were significantly (P < .0001) higher in EBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. CLINICAL RELEVANCE: Equine respiratory tract cells were susceptible to cell entry with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infective to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted.
Assuntos
COVID-19 , Doenças dos Cavalos , Cavalos , Animais , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Internalização do Vírus , COVID-19/veterinária , Células EpiteliaisRESUMO
(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.
RESUMO
The development of SARS-CoV-2 main protease (Mpro) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate Mpro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of Mpro. An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using Mpro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of Mpro with low nanomolar Ki values. Furthermore, these compounds demonstrate efficacy against mutant forms of Mpro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.