RESUMO
Cholera, caused by Vibrio cholerae, persists in developing countries due to inadequate access to safe water, sanitation, and hygiene. There are approximately 4 million cases and 143,000 deaths each year due to cholera. The disease is transmitted fecally-orally via contaminated food or water. Severe dehydrating cholera can progress to hypovolemic shock due to the rapid loss of fluids and electrolytes, which requires a rapid infusion of intravenous (i.v.) fluids. The case fatality rate exceeds 50% without proper clinical management but can be less than 1% with prompt rehydration and antibiotics. Oral cholera vaccines (OCVs) serve as a major component of an integrated control package during outbreaks or within zones of endemicity. Water, sanitation, and hygiene (WaSH); health education; and prophylactic antibiotic treatment are additional components of the prevention and control of cholera. The World Health Organization (WHO) and the Global Task Force for Cholera Control (GTFCC) have set an ambitious goal of eliminating cholera by 2030 in high-risk areas.
Assuntos
Vacinas contra Cólera , Cólera , Antibacterianos , Cólera/diagnóstico , Cólera/epidemiologia , Cólera/prevenção & controle , Surtos de Doenças/prevenção & controle , Humanos , ÁguaRESUMO
BACKGROUND: Cholera remains a public health threat for low- and middle-income countries, particularly in Asia and Africa. Shanchol™, an inactivated oral cholera vaccine (OCV) is currently in use globally. OCV and oral poliovirus vaccines (OPV) could be administered concomitantly, but the immunogenicity and safety of coadministration among children aged 1-3 years is unknown. METHODS: We undertook an open-label, randomized, controlled, inequality trial in Dhaka city, Bangladesh. Healthy children aged 1-3 years were randomly assigned to 1 of 3 groups: bivalent OPV (bOPV)-alone, OCV-alone, or combined bOPV + OCV and received vaccines on the day of enrollment and 28 days later. Blood samples were collected on the day of enrollment, day 28, and day 56. Serum poliovirus neutralizing antibodies and vibriocidal antibodies against Vibrio cholerae O1 were assessed using microneutralization assays. RESULTS: A total of 579 children aged 1â3 years were recruited, 193 children per group. More than 90% of the children completed visits at day 56. Few adverse events following immunization were recorded and were equivalent among study arms. On day 28, 60% (90% confidence interval: 53%-67%) and 54% (46%-61%) of participants with co-administration of bOPV + OCV responded to polioviruses type 1 and 3, respectively, compared to 55% (47%-62%) and 46% (38%-53%) in the bOPV-only group. Additionally, >50% of participants showed a ≥4-fold increase in vibriocidal antibody titer responses on day 28, comparable to the responses observed in OCV-only arm. CONCLUSIONS: Co-administration of bOPV and OCV is safe and effective in children aged 1-3 years and can be cost-beneficial. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03581734).
Assuntos
Vacinas contra Cólera , Cólera , Poliomielite , Poliovirus , Humanos , Criança , Lactente , Pré-Escolar , Bangladesh , Cólera/prevenção & controle , Vacina Antipólio Oral , Vacinas de Produtos Inativados , Administração Oral , Poliomielite/prevenção & controleRESUMO
Several viruses are known to be associated with the development of certain cancers, including human papilloma virus (HPV), an established causative agent for a range of anogenital and head and neck cancers. However, the causality has been based on the presence of the virus, or its genetic material, in the sampled tumors. We have long wondered if viruses cause cancer via a "hit and run" mechanism such that they are no longer present in the resulting tumors. Here, we hypothesize that the absence of viral genes from the tumor does not necessarily exclude the viral aetiology. To test this, we used an HPV-driven oropharyngeal cancer (OPC) tumor model and CRISPR to delete the viral oncogene, E7. Indeed, the genetic removal of HPV E7 oncogene eliminates tumors in vivo. Remarkably, E7 deleted tumors recurred over time and develop new mutations not previously seen in HPV+ OPC tumors. Importantly, a number of these new mutations are found to be already present in HPV- OPC tumors.
Assuntos
Neoplasias de Cabeça e Pescoço , Proteínas Oncogênicas Virais , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Humanos , Papillomavirus Humano , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/patologia , Proteínas Repressoras/genética , Recidiva Local de Neoplasia , Neoplasias Orofaríngeas/complicações , Neoplasias Orofaríngeas/patologia , Proteínas E7 de Papillomavirus/genéticaRESUMO
Clinical and pre-clinical work for a number of cancer types has demonstrated relatively positive outcomes and effective tumour regression when the level and function of p53, a well-established tumour suppressor, is restored. Human papillomavirus (HPV)-driven cancers encode the E6 oncoprotein, which leads to p53 degradation, to allow the carcinogenic process to proceed. Indeed, there have been several attempts to revive p53 function in HPV-driven cancers by both pharmacological and genetic means to increase p53 bioavailability. Here, we employed a CRISPR activation (CRISPRa) approach to overcome HPV-mediated silencing of p53 by hyperexpressing the p53 gene promoter. Our data show that CRISPRa-mediated hyperexpression of p53 leads to HPV+ cervical cancer cell killing and the reduction of cell proliferation. This proof-of-concept data suggest that increasing p53 bioavailability may potentially be a promising therapeutic approach for the treatment of HPV-driven cancers.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Papillomavirus Humano , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismoRESUMO
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.
Assuntos
Tratamento Farmacológico da COVID-19 , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanopartículas/química , RNA de Cadeia Dupla/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , SARS-CoV-2/genética , Administração Intravenosa , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/metabolismo , COVID-19/virologia , Feminino , Inativação Gênica , Células HEK293 , Humanos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RNA de Cadeia Dupla/genética , RNA Viral/genética , Transcriptoma/efeitos dos fármacos , Resultado do TratamentoRESUMO
The recent advancements in CRISPR/Cas9 engineering have resulted in the development of more targeted and potentially safer gene therapies. The challenge in the cancer setting is knowing the driver oncogenes responsible, and the translation of these therapies is hindered by effective and safe delivery methods to target organs with minimal systemic toxicities, on-target specificity of gene editing, and demonstrated lack of long-term adverse events. Using a model system based on cervical cancer, which is driven by the ongoing expression of the human papillomavirus E6 and E7 proteins, we show that CRISPR/Cas9 delivered systemically in vivo using PEGylated liposomes results in tumor elimination and complete survival in treated animals. We compared treatment and editing efficiency of two Cas9 variants, wild-type (WT) Cas9 and the highly specific FokI-dCas9, and showed that the latter was not effective. We also explored high-fidelity repair but found that repair was inefficient, occurring in 6%-8% of cells, whereas non-homologous end joining (NHEJ) was highly efficient, occurring in â¼80% of the cells. Finally, we explored the post gene-editing events in tumors and showed that cell death is induced by apoptosis. Overall, our work demonstrates that in vivo CRISPR/Cas editing treatment of preexisting tumors is completely effective despite the large payloads.
Assuntos
Sistemas CRISPR-Cas , Sistemas de Liberação de Medicamentos , Terapia Genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/terapia , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Feminino , Edição de Genes , Humanos , Lipossomos/administração & dosagem , Camundongos , Proteínas Oncogênicas Virais/antagonistas & inibidores , Infecções por Papillomavirus/virologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologiaRESUMO
Piperacillin (Pip) is a broad spectrum ß-lactam against most Gram-positive and Gram-negative aerobic and anaerobic bacteria. However, bacterial resistance restricts its benefits for the treatment of infectious diseases. Recently, nanoliposomal systems have been investigated as encouraging strategies to address this issue owing to their immense potential. We aimed to encapsulate Pip in liposomal nanoparticles and study their antibacterial activities in vitro against Pseudomonas aeruginosa (P. aeruginosa). Different liposomes were prepared based on the freeze-drying of a monophase solution method. Then, they were characterized in terms of size, zeta potential, polydispersity-index, and morphology. For further analysis, spectra of ATR-FTIR and XRD were taken for liposomal Pip. Encapsulation efficiency (EE) was determined via agar diffusion assay. Also, minimum inhibitory concentrations (MICs) were investigated by the standard broth macro-dilution method. The liposomes were from 100.9 to 444.13 nm with z-potential of - 30.70 to - 10.57 mV. EE of the selected formulation was 53.1%. TEM results showed that the liposomes were nanosized and almost spherical. ATR-FTIR results confirmed the full encapsulation of Pip in nanoliposomes. The X-ray pattern indicated that the liposomal Pip was amorphous. The MIC (10.6 µg/ml) of the nanoliposomal Pip against P. aeruginosa was one-half of the MIC (21.25 µg/ml) of free Pip for the same organisms. Considering four aspects (nanosized liposomes, no need for sterilization, suitable EE and enhanced antibacterial effects), this preparation method seems promising and may be used to overcome the bacterial resistance relative to Pip.
Assuntos
Antibacterianos , Piperacilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Lipossomos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosaRESUMO
BACKGROUND: Considering the complex nature of gastrointestinal cancer, different methods including surgery, radiotherapy, and chemotherapy are considered for the treatment. Novel strategies including silencing of oncogenes using safe delivery systems could be considered as a novel approach in colorectal cancer treatment. The aim of this study was to investigate the silencing effect of high mobility group A2 (HMGA2) small interfering RNA (siRNA)-loaded nanoliposomes on gastrointestinal cancers. METHODS: The siRNA-lipoplexes were prepared using dioleoyl trimethylammonium propane (DOTAP)/cholesterol (Chol)/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) through the freeze-drying of a monophase solution method. The size, polydispersity index (PDI), and zeta-potential of nanoliposomes were determined using Zetasizer analyzer. The morphology of the nanoliposomes was determined by transmission electron microscopy (TEM). The agarose gel-retardation assay was carried out to confirm the loading of siRNAs into liposome. The silencing of the HMGA2 in cancer cells was evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of liposomes on cell cytotoxicity was studied by MTT assay. The inhibitory effect of siRNA-loaded liposomes was evaluated by a wound-healing assay. The apoptosis induction was investigated via the annexin V/propidium iodide assay. RESULTS: The size, PDI, and zeta-potential of the prepared liposomes were found to be 350 nm, 0.67, and 86.3 mV, respectively. They were spherical in shape and could efficiently associate with siRNA. The results of gene silencing showed that the optimum condition of HMGA2 silencing was 80 pmol HMGA2 and 24 hours after treatment in each cancer cell lines. MTT assays indicated that silencing of HMGA2 in optimal condition could reduce the viability of the cancer cells more than 60% in the three cell lines. The result of the apoptosis assay showed more than 50% of the cell deaths related to the apoptosis in all three cell lines. The gene expression evaluation confirmed that apoptosis was induced via the intrinsic pathway inducing both caspase-3 and -9 expressions. Also, the reduction in Bcl2 expression confirmed the activation apoptosis pathway in the treated cancer cells. The wound-healing assay showed the suppression of cancer cell migration after treatment with the prepared nanoliposomes. CONCLUSION: The results of this study showed the HMGA2 siRNA-loaded nanoliposomes could be effective in the treatment of gastrointestinal cancers.
Assuntos
Apoptose/fisiologia , Neoplasias Gastrointestinais/metabolismo , Proteína HMGA2/metabolismo , RNA Interferente Pequeno/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Neoplasias Gastrointestinais/genética , Inativação Gênica/fisiologia , Células HCT116 , Proteína HMGA2/genética , Humanos , Lipossomos/química , Microscopia Eletrônica de Transmissão , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
BACKGROUND/AIMS: Human metapneumovirus (hMPV) is an important human respiratory pathogen and is implicated in an array of respiratory illnesses, ranging from asymptomatic infection to severe bronchiolitis. Currently, there is no reliable vaccine or specific antiviral therapy for hMPV infection and treatment is supportive. The use of ribonucleic acid interference has the potential to change that with the targeting of essential viral genes via small interfering RNAs (siRNAs) offering the ability to directly and rapidly treat viral infections. METHOD: The human lung carcinoma epithelial cell line, A549, was transfected with siRNAs targeting the N and P genes before infecting with hMPV A2 CAN97-83. Viral growth inhibition was then measured by the viral plaque assay and nucleoprotein (N) and phosphoprotein (P) gene knockdown was determined by real-time PCR. RESULTS: In vitro prophylactic use of siRNAs targeting the 3'-abundantly expressed N and P genes of hMPV resulted in potent, sequence-specific viral inhibition. The viral inhibition was specific and not mediated by an anti-viral interferon-ß response or cell death. CONCLUSION: The findings presented here confirmed the highly potent, sequence-specific antiviral effect of siRNAs targeting the N and P gene of hMPV. These results may facilitate the development of a novel therapeutic agent for hMPV control.
Assuntos
Metapneumovirus/crescimento & desenvolvimento , Metapneumovirus/genética , Nucleoproteínas/genética , Fosfoproteínas/genética , Interferência de RNA , Células A549 , Técnicas de Silenciamento de Genes , Genes Virais , Humanos , RNA Interferente Pequeno/genética , RNA Viral/genética , Transfecção , Proteínas Virais/genéticaRESUMO
BACKGROUND INFORMATION: Interferons are a family of cytokines with growth inhibitory and antiviral functions, which exert their biological actions through the expression of interferon-stimulated genes (ISGs). The human ISG12 family of proteins comprises ISG12A, ISG12B, ISG12C and ISG6-16. Due to differential splicing and a gene variation, the human ISG12A protein exists as a full-length ISG12A form and three ISG12A variants. ISG12 genes have been found transcriptionally dysregulated in many disorders. High levels of ISG12A mRNA have been found in breast and ovarian cancers. Loss of heterozygosity at the position of the ISG12 genes often occurs in ovarian carcinomas and lymphoblastic leukemias. Both ISG12A and ISG6-16 are up-regulated in psoriasis. RESULTS: We demonstrate here that expression of the human full-length ISG12A protein sensitises cells for TNFα and the BH3 mimetic gossypol induced apoptosis, and the other ISG12A variants as well as ISG12B and ISG12C can induce apoptosis directly in HEK293 cells. Also ISG6-16 sensitises HEK293 cells for gossypol-induced apoptosis. In the ISG12 motif, two putative Bcl-2 homology (BH)3 like motifs were found, which may be decisive for the apoptotic properties of the ISG12 proteins. A series of BH3 mutants was made in ISG12AΔ-S, the smallest apoptosis-inducing ISG12A variant and our results indicate that ISG12AΔ-S indeed possesses features resembling those of BH3-only proteins. Supporting this notion are our findings that the full-length ISG12A co-immunoprecipitates with the Bcl-2 protein, and the apoptotic properties of the ISG12A variants are reduced in Bcl-2 expressing HEK293 cells. In addition, full-length ISG12A is able to form homodimers, which suggests a possible involvement in pore formation during apoptosis. The full-length ISG12A, the three ISG12A variants and the ISG12B proteins were found to be localised in the mitochondria. CONCLUSIONS: Our results suggest that the ISG12 family of proteins has an important role for the apoptotic properties induced by type 1 interferon. SIGNIFICANCE: The ISG12 family constitute small hydrophic proteins involved in apoptosis. This is the first comparison of the apoptotic potentials of the full-length ISG12A protein and the three ISG12A variants. The differential apoptotic potentials of these proteins might have an impact on the strategies to monitor and interpret their dysregulation associated with many disorders.
Assuntos
Apoptose , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Sequência Conservada , Gossipol/farmacologia , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Isoformas de Proteínas/fisiologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.
Assuntos
Células Epiteliais/metabolismo , Inativação Gênica , Proteínas E7 de Papillomavirus/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Colo do Útero/metabolismo , Colo do Útero/patologia , Colo do Útero/virologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Células HeLa , Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Camundongos , Proteínas E7 de Papillomavirus/genética , Polirribossomos/genética , Polirribossomos/metabolismo , Estabilidade de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: There is a dramatic rise in the incidence of Human papillomavirus (HPV) - associated head and neck squamous cell carcinoma (HNSCC) in the world, with considerable variation by geography, gender and ethnicity. Little is known about the situation in Bangladesh, where tobacco- and areca nut-related head and neck cancers (HNCs) are the most common cancers in men. We aimed to determine the prevalence of HPV in HNSCC in Bangladesh and to explore the possible value of cell cycle markers in clinical diagnostic settings. METHODS: One hundred and ninety six archival HNSCC tissue samples were analysed for the presence of HPV DNA. The DNA quality was assured, and then amplified using a nested PCR approach. The typing of HPV was performed by automated DNA sequencing. Cellular markers p53, Cyclin D1 and pRb were tested on all samples by immunohistochemistry (IHC), as well as p16 as a putative surrogate for the detection of HPV. RESULTS: HPV DNA was detected in 36/174 (~21%) samples: 36% of cancers from the oropharynx; 31% of oral cancers, and 22% from the larynx. HPV-16 was most common, being present in 33 samples, followed by HPV-33 (2 samples) and HPV-31 (1 sample). Twenty-eight out of 174 samples were positive for p16, predominantly in HPV-positive tissues (p < 0.001). No statistically significant association was observed between the cellular markers and HPV DNA positive cases. However, p16 positivity had excellent predictive value for the presence of HPV by PCR. CONCLUSION: There is a significant burden of HPV-associated HNSCC in Bangladesh, particularly in the oropharynx but also in oral and laryngeal cancers. Whilst a combination of PCR-based DNA detection and p16 IHC is useful, the latter has excellent specificity, acceptable sensitivity and good predictive value for carriage of HPV in this population and should be used for prognostic evaluation and treatment planning of all HNSCC patients in South Asia, as in the Western world.
Assuntos
Neoplasias de Cabeça e Pescoço/epidemiologia , Neoplasias de Cabeça e Pescoço/etiologia , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Adulto , Idoso , Bangladesh/epidemiologia , Biomarcadores Tumorais , DNA Viral , Feminino , Genótipo , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Vigilância da População , Prevalência , Adulto JovemRESUMO
Immunotherapy is one of the most promising strategies for the treatment of cancer. Human papillomavirus (HPV) is responsible for virtually all cases of cervical cancer. The main purpose of a therapeutic HPV vaccine is to stimulate CD8(+) cytotoxic T lymphocytes (CTLs) that can eradicate HPV infected cells. HPV oncoproteins E6 and E7 are continuously expressed and are essential for maintaining the growth of HPV-associated tumor cells. We designed polymer-based multi-antigenic formulations/constructs that were comprised of the E6 and E7 peptide epitopes. We developed an N-terminus-based epitope conjugation to conjugate two unprotected peptides to poly tert-butyl acrylate. This method allowed for the incorporation of the two antigens into a polymeric dendrimer in a strictly equimolar ratio. The most effective formulations eliminated tumors in up to 50% of treated mice. Tumor recurrence was not observed up to 3months post initial challenge.
Assuntos
Antígenos/química , Vacinas contra Papillomavirus/uso terapêutico , Peptídeos/química , Polímeros/química , Neoplasias do Colo do Útero/prevenção & controle , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Epitopos/química , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/imunologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in individuals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach; potentially ensuring that large numbers of immunized individuals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44-57) administered with Montanide ISA51 adjuvant.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Lipopeptídeos/química , Neoplasias/tratamento farmacológico , Proteínas E7 de Papillomavirus/efeitos dos fármacos , Infecções por Papillomavirus/terapia , Proteínas Recombinantes , Adjuvantes Imunológicos/síntese química , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/síntese química , Técnicas de Química Sintética , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Lipopeptídeos/síntese química , Lipopeptídeos/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies.
Assuntos
Adjuvantes Imunológicos/farmacologia , Alphapapillomavirus/imunologia , Neoplasias Experimentais/tratamento farmacológico , Infecções por Papillomavirus/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Infecções Tumorais por Vírus/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon-alfa/imunologia , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/patologiaRESUMO
INTRODUCTION: Respiratory syncytial virus (RSV) causes bronchiolitis and other respiratory issues in immunocompromised individuals, the elderly, and children. After six decades of research, we have only recently seen the approval of two RSV vaccines, Arexvy and Abrysvo. Direct-acting antivirals against RSV have been more difficult to develop with ribavirin and palivizumab giving very modest reductions in hospitalizations and no differences in mortality. Recently, nirsevimab was licensed and has proven to be much more effective when given prophylactically. These are delivered intravenously (IV) and intramuscularly (IM), but an intranasal (IN) antiviral has several advantages in terms of ease of use, lower resource need, and acting at the site of infection. AREAS COVERED: In this paper, we review the available literature on the current pre-clinical research landscape of anti-RSV therapeutics tested for IN delivery. EXPERT OPINION: As RSV is a respiratory virus that infects both the upper and lower respiratory tracts, efforts are focused on developing a therapeutic that can be delivered via the nasal route. The rationale is to directly target the replicating virus with an obvious respiratory tract tropism. This approach will not only pave the way for a nasal delivery approach aimed at reducing respiratory viral illness but also controlling aerosol virus transmission.
RESUMO
There remains a striking overall mortality burden of COVID-19 worldwide. Given the waning effectiveness of current SARS-CoV-2 antivirals due to the rapid emergence of new variants of concern (VOC), we employed a direct-acting molecular therapy approach using gene silencing RNA interference (RNAi) technology. In this study, we developed and screened several ultra-conserved small-interfering RNAs (siRNAs) before selecting one potent siRNA candidate for pre-clinical in vivo testing. This non-immunostimulatory, anti-SARS-CoV-2 siRNA candidate maintains its antiviral activity against all tested SARS-CoV-2 VOC and works effectively as a single agent. For the first time, significant antiviral effects in both the lungs and nasal cavities of SARS-CoV-2 infected mice were observed when this siRNA candidate was delivered intranasally (IN) as a prophylactic agent with the aid of lipid nanoparticles (LNPs). Importantly, a pre-exposure prophylactic IN-delivered anti-SARS-CoV-2 siRNA antiviral that can ameliorate viral replication in the nasal cavity could potentially prevent aerosol spread of respiratory viruses. An IN delivery approach would allow for the development of a direct-acting nasal spray approach that could be self-administered prophylactically.
Assuntos
COVID-19 , Animais , Camundongos , RNA Interferente Pequeno/genética , COVID-19/prevenção & controle , Cavidade Nasal , SARS-CoV-2/genética , Antivirais/uso terapêutico , PulmãoRESUMO
Long antisense RNAs (asRNAs) have been observed to repress HIV and other virus expression in a manner that is refractory to viral evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) disease, has a distinct ability to evolve resistance around antibody targeting, as was evident from the emergence of various SARS-CoV-2 spike antibody variants. Importantly, the effectiveness of current antivirals is waning due to the rapid emergence of new variants of concern, more recently the omicron variant. One means of avoiding the emergence of viral resistance is by using long asRNA to target SARS-CoV-2. Similar work has proven successful with HIV targeting by long asRNA. In this study, we describe a long asRNA targeting SARS-CoV-2 RNA-dependent RNA polymerase gene and the ability to deliver this RNA in extracellular vesicles (EVs) to repress virus expression. The observations presented in this study suggest that EV-delivered asRNAs are one means to targeting SARS-CoV-2 infection, which is both effective and broadly applicable as a means to control viral expression in the absence of mutation. This is the first demonstration of the use of engineered EVs to deliver long asRNA payloads for antiviral therapy.
Assuntos
COVID-19 , Vesículas Extracelulares , RNA Antissenso , SARS-CoV-2 , Vesículas Extracelulares/genética , Vesículas Extracelulares/virologia , Vesículas Extracelulares/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/efeitos dos fármacos , Humanos , RNA Antissenso/genética , RNA Antissenso/uso terapêutico , COVID-19/virologia , COVID-19/genética , COVID-19/terapia , Animais , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Células Vero , Chlorocebus aethiops , Antivirais/uso terapêutico , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19RESUMO
Dendrimers are structurally well-defined, synthetic polymers with sizes and physicochemical properties often resembling those of biomacromolecules (e.g., proteins). As a result, they are promising candidates for peptide-based vaccine delivery platforms. Herein, we established a synthetic pathway to conjugate a human papillomavirus (HPV) E7 protein-derived peptide antigen to a star-polymer to create a macromolecular vaccine candidate to treat HPV-related cancers. These conjugates were able to reduce tumor growth and eradicate E7-expressing TC-1 tumors in mice after a single immunization, without the help of any external adjuvant.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Proteínas E7 de Papillomavirus/uso terapêutico , Infecções por Papillomavirus/terapia , Fragmentos de Peptídeos/uso terapêutico , Neoplasias do Colo do Útero/terapia , Resinas Acrílicas/química , Adjuvantes Imunológicos/síntese química , Sequência de Aminoácidos , Animais , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Células Cultivadas , Química Click , Reação de Cicloadição , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Carga Tumoral , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) lung infection in vivo. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo. Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The study presented here demonstrates an attractive alternate delivery strategy for the prophylactic treatment of both future and emerging respiratory viral diseases.