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1.
Methods Mol Biol ; 2197: 241-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827141

RESUMO

Human papillomavirus (HPV) is a contagious cause of anogenital and oropharyngeal cancers developing from persistently infected and subsequently transformed basal keratinocytes of mucosal epithelium. DNA-based immunotherapy offers great potential for the treatment of persisting HPV infections and associated cancers. Preclinical testing of therapeutic DNA-based HPV-targeted immunotherapy requires robust animal models which mimic HPV-associated cancer disease in humans. Here we describe a detailed protocol of intradermal delivery of a therapeutic DNA vaccine and a grafting model of neoantigen expressing skin to evaluate vaccine efficacy against HPV16 mediated hyperproliferative epithelium in mice.


Assuntos
Vacinas Anticâncer/imunologia , Papillomavirus Humano 16/imunologia , Neoplasias/etiologia , Neoplasias/terapia , Infecções por Papillomavirus/complicações , Vacinas contra Papillomavirus/imunologia , Vacinas de DNA/imunologia , Animais , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Imunização , Injeções Subcutâneas , Camundongos , Camundongos Transgênicos , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas de DNA/administração & dosagem
2.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
3.
PLoS One ; 15(5): e0233084, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32421735

RESUMO

BACKGROUND: Cervical cancer associated with high risk-human papillomavirus (HR-HPV) infection is becoming the one of the most common female cancer in many sub-Saharan African countries. First-generation immigrant African women living in Europe are at-risk for cervical cancer, in a context of social vulnerability, with frequent lack of cervical cancer screening and HPV vaccination. OBJECTIVE: Our objective was to address immunologically the issue of catch-up prophylactic HPV vaccination in first-generation African immigrant women living in France. METHODS: IgG immune responses and cross-reactivities to α7 (HPV-18, -45 and -68) and α9 (HPV-16, -31, -33, -35, -52 and -58) HPV types, including 7 HR-HPV targeted by the Gardasil-9® prophylactic vaccine, were evaluated in paired serum and cervicovaginal secretions (CVS) by HPV L1-virus-like particles-based ELISA. Genital HPV were detected by multiplex real time PCR (Seegene, Seoul, South Korea). RESULTS: Fifty-one immigrant women (mean age, 41.7 years; 72.5% HIV-infected) were prospectively included. More than two-third (68.6%) of them carried genital HPV (group I) while 31.4% were negative (group II). The majority (90.2%) exhibited serum IgG to at least one α7/α9 HR-HPV. Serum HPV-specific IgG were more frequently detected in group I than group II (100% versus 68.7%; P = 0.002). The distribution of serum and genital HPV-specific IgG was similar, but mean number of IgG reactivities to α7/α9 HR-HPV was higher in serum than CVS (5.6 IgG per woman in serum versus 3.2 in CVS; P<0.001). Rates of IgG cross-reactivities against HPV different from detected cervicovaginal HPV were higher in serum and CVS in group I than group II. Finally, the majority of groups I and II women (68.6% and 68.7%, respectively) exhibited serum or cervicovaginal IgG to Gardasil-9® HR-HPV, with higher mean rates in group I than group II (6.1 Gardasil-9® HR-HPV per woman versus 1.4; P<0.01). One-third (31.2%) of group II women did not show any serum and genital HPV-specific IgG. CONCLUSIONS: Around two-third of first-generation African immigrant women living in France showed frequent ongoing genital HPV infection and high rates of circulating and genital IgG to α7/α9 HPV, generally cross-reacting, avoiding the possibility of catch-up vaccination. Nevertheless, about one-third of women had no evidence of previous HPV infection, or showed only low levels of genital and circulating HR-HPV-specific IgG and could therefore be eligible for catch-up vaccination.


Assuntos
Anticorpos Antivirais/metabolismo , Vacina Quadrivalente Recombinante contra HPV tipos 6, 11, 16, 18/imunologia , Infecções por Papillomavirus/diagnóstico , Neoplasias do Colo do Útero/virologia , Adulto , África ao Sul do Saara/etnologia , Anticorpos Antivirais/sangue , Colo do Útero/imunologia , Detecção Precoce de Câncer , Emigrantes e Imigrantes/estatística & dados numéricos , Feminino , França/etnologia , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/imunologia , Vagina/imunologia
4.
PLoS One ; 15(4): e0227900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320400

RESUMO

OBJECTIVE: Identify the prevalence of HPV infections in the uterine cervix and oral cavity and HPV16 variants in HIV+ women. METHODS: A total of 174 HIV+ women attended an HIV+ specialized clinic in Mexico City. Cells were obtained from the oral cavity and cervix to extract DNA. Polymerase chain reaction (PCR) was used to amplify the HPV sequence with generic primers. We detected specific HPV types using the INNO-LiPA HPV Genotyping Extra II Kit (INNOGENETICS). The identification of variants was studied by sequencing the E6 gene with a Big Dye Terminator Kit and an Applied Biosystems 3500/3500xL genetic analyzer. RESULTS: HPV infection was very high in the uterine cervix (168/174, 96.6%) and oral cavity (161/174, 92.5%). The prevalence of HPV concurrent infections in the cervix and oral cavity was 155/174 (89.1%). We found hrHPVs to be more prevalent than low-risk HPVs (lrHPVs) in the oral cavity (90.2% versus 45.4%) and that infections simultaneously affected the cervix (94.3% versus 36.2%) and oral cavity (85.1% versus 20.1%). Surprisingly, only European variants of HPV type 16 were found in the uterine cervix of women and the oral cavity of all tested samples (52 oral cavity samples and 52 uterine cervix samples). CONCLUSIONS: The high prevalence of HPV, multiple infections and presence of the EP350G intravariant in both anatomical regions are strongly related to the persistence of the virus, which is fundamental for the development of cancer. Therefore, it is very important to control and monitor this high-risk population as well as implement programs for the early detection of HPV and vaccination.


Assuntos
Infecções por HIV/imunologia , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/epidemiologia , Neoplasias do Colo do Útero/prevenção & controle , Adulto , Fatores Etários , Colo do Útero/virologia , DNA Viral/isolamento & purificação , Feminino , Infecções por HIV/tratamento farmacológico , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , México/epidemiologia , Pessoa de Meia-Idade , Boca/virologia , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/transmissão , Infecções por Papillomavirus/virologia , Prevalência , Proteínas Repressoras/genética , Fatores de Risco , Comportamento Sexual/estatística & dados numéricos , Neoplasias do Colo do Útero/virologia
5.
Nat Commun ; 11(1): 1985, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332752

RESUMO

The unsatisfactory response rate of immune checkpoint blockade (ICB) immunotherapy severely limits its clinical application as a tumor therapy. Here, we generate a vaccine-based nanosystem by integrating siRNA for Cd274 into the commercial human papillomavirus (HPV) L1 (HPV16 L1) protein. This nanosystem has good biosafety and enhances the therapeutic response rate of anti-tumor immunotherapy. The HPV16 L1 protein activates innate immunity through the type I interferon pathway and exhibits an efficient anti-cancer effect when cooperating with ICB therapy. For both resectable and unresectable breast tumors, the nanosystem decreases 71% tumor recurrence and extends progression-free survival by 67%. Most importantly, the nanosystem successfully induces high response rates in various genetically modified breast cancer models with different antigen loads. The strong immune stimulation elicited by this vaccine-based nanosystem might constitute an approach to significantly improve current ICB immunotherapy.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/terapia , Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Nanopartículas/administração & dosagem , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Humanos , Imunidade Inata/genética , Camundongos , Recidiva Local de Neoplasia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Intervalo Livre de Progressão , RNA Interferente Pequeno/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
6.
PLoS One ; 15(3): e0229672, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214362

RESUMO

More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.


Assuntos
Anticorpos Antivirais/sangue , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Imunoensaio/métodos , Imunoglobulina G/sangue , Animais , Especificidade de Anticorpos , Reações Cruzadas , Proteínas de Ligação a DNA/imunologia , Técnicas Eletroquímicas , Ensaio de Imunoadsorção Enzimática , Feminino , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/estatística & dados numéricos , Humanos , Imunoensaio/estatística & dados numéricos , Limite de Detecção , Medições Luminescentes/métodos , Medições Luminescentes/estatística & dados numéricos , Macaca fascicularis , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologia
9.
Oral Oncol ; 100: 104488, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31835137

RESUMO

OBJECTIVE: To determine the influence of high-risk HPV genotype on outcomes in HNSCC patients. MATERIALS AND METHODS: This is a retrospective analysis of The Cancer Genome Atlas HNSCC cohort. RESULTS: Using multivariate Cox regression analysis, we revealed that HPV33+ HNSCC patients have inferior overall survival compared to HPV16+ HNSCC patients independent of anatomical site (HR 3.59, 95% CI 1.58-8.12; p = 0.002). A host anti-viral immune response, apolipoprotein B mRNA editing enzyme, and catalytic polypeptide-like mutational signature, was under represented and, aneuploidy and 3p loss were more frequent in HPV33+ tumors. A deconvolution RNA-Seq algorithm to infer immune cell fractions revealed that CD8+ cytotoxic T-cell infiltration was reduced in HPV33+ compared to HPV16+ tumors (1.3% vs. 2.7%, p = 0.007). TGFB1, a negative modulator of T-cell infiltration and function, showed expression and pathway enrichment in HPV33+ tumors. CONCLUSIONS: Our work reveals that HPV genotype, in particular HPV33, has a powerful impact on HNSCC patient survival. We argue that p16 immunohistochemistry as a surrogate biomarker for HPV+ status will lead to sub-optimal risk stratification and advocate HPV genotype testing as standard of care.


Assuntos
Neoplasias de Cabeça e Pescoço/virologia , Mutação , Papillomaviridae/classificação , Infecções por Papillomavirus/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica , Genótipo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/mortalidade , Papillomavirus Humano 16/classificação , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Humanos , Masculino , Análise Multivariada , Papillomaviridae/genética , Papillomaviridae/imunologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Prognóstico , Estudos Retrospectivos , Análise de Sequência de RNA , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Análise de Sobrevida , Sequenciamento Completo do Exoma
10.
Clin Exp Immunol ; 199(2): 131-142, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31628850

RESUMO

Recurrent respiratory papillomatosis (RRP) is characterized by benign exophytic lesions of the respiratory tract caused by the human papillomavirus (HPV), in particular low-risk HPV6 and HPV11. Aggressiveness varies greatly among patients. Surgical excision is the current standard of care for RRP, with adjuvant therapy used when surgery cannot control disease recurrence. Numerous adjuvant therapies have been used to control RRP with some success, but none are curative. Current literature supports a polarization of the adaptive immune response to a T helper type 2 (Th2)-like or T regulatory phenotype, driven by a complex interplay between innate immunity, adaptive immunity and HPV6/11 proteins. Additionally, certain immunogenetic polymorphisms can predispose individuals to an HPV6/11-tolerant microenvironment. As a result, immunomodulatory efforts are being made to restore the host immune system to a more balanced T cell phenotype and clear viral infection. Literature has shown exciting evidence for the role of HPV vaccination with Gardasil or Gardasil-9 as both primary prevention, by decreasing incidence through childhood vaccinations, and secondary prevention, by treating active RRP disease. Multi-institution randomized clinical trials are needed to better assess their efficacy as treatment for active disease. Interestingly, a DNA vaccine has recently shown in-vitro success in generating a more robust CD8+ T cell response. Furthermore, clinical trials for programmed death 1 (PD-1) inhibitors are under investigation for RRP management. Molecular insights into RRP, in particular the interplay between RRP and the immune system, are needed to advance our understanding of this disease and may lead to the identification of immunomodulatory agents to better manage RRP.


Assuntos
Predisposição Genética para Doença , Tolerância Imunológica , Infecções por Papillomavirus , Vacinas contra Papillomavirus/uso terapêutico , Polimorfismo Genético , Infecções Respiratórias , Vacinação , Criança , Papillomavirus Humano 11/imunologia , Papillomavirus Humano 16/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Imunidade Celular , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Infecções Respiratórias/genética , Infecções Respiratórias/imunologia , Infecções Respiratórias/prevenção & controle , Infecções Respiratórias/virologia , Linfócitos T/imunologia
11.
Sci China Life Sci ; 63(4): 582-591, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31231780

RESUMO

A new HPV-16/18 bivalent vaccine expressed by the Escherichia coli has been proven to be efficacious in adult women. A randomized, immunogenicity noninferiority study of this candidate vaccine was conducted in December 2015 in China. Girls aged 9-14 years were randomized to receive 2 doses at months 0 and 6 (n=301) or 3 doses at months 0, 1 and 6 (n=304). Girls aged 15-17 years (n=149) and women aged 18-26 years (n=225) received 3 doses. The objectives included noninferiority analysis of the IgG geometric mean concentration (GMC) ratio (95% CI, lower bound>0.5) to HPV-16 and HPV-18 at month 7 in girls compared with women. In the per-protocol set, the GMC ratio of IgG was noninferior for girls aged 9-17 years receiving 3 doses compared with women (1.76 (95% CI, 1.56, 1.99) for HPV-16 and 1.93 (95% CI, 1.69, 2.21) for HPV-18) and noninferior for girls aged 9-14 years receiving 2 doses compared with women (1.45 (95% CI, 1.25, 1.62) for HPV-16 and 1.17 (95% CI, 1.02, 1.33) for HPV-18). Noninferiority was also demonstrated for neutralizing antibodies. The immunogenicity of the HPV vaccine in girls receiving 3 or 2 doses was noninferior compared with that in young adult women.


Assuntos
Vacinas contra Escherichia coli/administração & dosagem , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Adolescente , Adulto , Anticorpos Neutralizantes/metabolismo , Criança , China , Relação Dose-Resposta à Radiação , Escherichia coli/metabolismo , Vacinas contra Escherichia coli/efeitos adversos , Feminino , Humanos , Imunogenicidade da Vacina , Vacinas contra Papillomavirus/efeitos adversos , Resultado do Tratamento
12.
J Natl Cancer Inst ; 112(2): 145-153, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31086947

RESUMO

BACKGROUND: The high cost and insufficient supply of human papillomavirus (HPV) vaccines have slowed the pace of controlling cervical cancer. A phase III clinical trial was conducted to evaluate the efficacy, safety, and immunogenicity of a novel Escherichia coli-produced bivalent HPV-16/18 vaccine. METHODS: A multicenter, randomized, double-blind trial started on November 22, 2012 in China. In total, 7372 eligible women aged 18-45 years were age-stratified and randomly assigned to receive three doses of the test or control (hepatitis E) vaccine at months 0, 1, and 6. Co-primary endpoints included high-grade genital lesions and persistent infection (over 6 months) associated with HPV-16/18. The primary analysis was performed on a per-protocol susceptible population of individuals who were negative for relevant HPV type-specific neutralizing antibodies (at day 0) and DNA (at day 0 through month 7) and who received three doses of the vaccine. This report presents data from a prespecified interim analysis used for regulatory submission. RESULTS: In the per-protocol cohort, the efficacies against high-grade genital lesions and persistent infection were 100.0% (95% confidence interval = 55.6% to 100.0%, 0 of 3306 in the vaccine group vs 10 of 3296 in the control group) and 97.8% (95% confidence interval = 87.1% to 99.9%, 1 of 3240 vs 45 of 3246), respectively. The side effects were mild. No vaccine-related serious adverse events were noted. Robust antibody responses for both types were induced and persisted for at least 42 months. CONCLUSIONS: The E coli-produced HPV-16/18 vaccine is well tolerated and highly efficacious against HPV-16/18-associated high-grade genital lesions and persistent infection in women.


Assuntos
Imunogenicidade da Vacina/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Adolescente , Adulto , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/efeitos adversos , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/patologia , Vacinação , Adulto Jovem
14.
Int J Nanomedicine ; 14: 8755-8768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31806970

RESUMO

Purpose: Single-chain variable fragments (scFvs) are one of the smallest antigen-binding units having the invaluable advantage to be expressed by a unique short open reading frame (ORF). Despite their reduced size, spontaneous cell entry of scFvs remains inefficient, hence precluding the possibility to target intracellular antigens. Here, we describe an original strategy to deliver scFvs inside target cells through engineered extracellular vesicles (EVs). This approach relies on the properties of a Human Immunodeficiency Virus (HIV)-1 Nef mutant protein referred to as Nefmut. It is a previously characterized Nef allele lacking basically all functions of wt Nef, yet strongly accumulating in the EV lumen also when fused at its C-terminus with a foreign protein. To gain the proof-of-principle for the efficacy of the proposed strategy, the tumor-promoting Human Papilloma Virus (HPV)16-E7 protein was considered as a scFv-specific intracellular target. The oncogenic effect of HPV16-E7 relies on its binding to the tumor suppressor pRb protein leading to a dysregulated cell duplication. Interfering with this interaction means impairing the HPV16-E7-induced cell proliferation. Methods: The Nefmut gene was fused in frame at its 3'-terminus with the ORF coding for a previously characterized anti-HPV16-E7 scFv. Interaction between the Nefmut-fused anti-HPV16-E7 scFv and the HPV16-E7 protein was tested by both confocal microscope and co-immunoprecipitation analyses on co-transfected cells. The in cis anti-proliferative effect of the Nefmut/anti-HPV16-E7 scFv was assayed by transfecting HPV16-infected cells. The anti-proliferative effect of EVs engineered with Nefmut/anti-HPV16-E7 scFv on HPV16-E7-expressing cells was evaluated in two ways: i) through challenge with purified EVs by a Real-Time Cell Analysis system and ii) in transwell co-cultures by an MTS-based assay. Results: The Nefmut/anti-HPV16-E7 scFv chimeric product is efficiently uploaded in EVs, binds HPV16-E7, and inhibits the proliferation of HPV16-E7-expressing cells. Most important, challenge with cell-free EVs incorporating the Nefmut/anti-HPV16-E7 scFv led to the inhibition of proliferation of HPV16-E7-expressing cells. The proliferation of these cells was hindered also when they were co-cultured in transwells with cells producing EVs uploading Nefmut/anti-HPV16-E7 scFv. Conclusion: Our data represent the proof-of-concept for the possibility to target intracellular antigens through EV-mediated delivery of scFvs. This finding could be relevant to design novel methods of intracellular therapeutic interventions.


Assuntos
Vesículas Extracelulares/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Anticorpos de Cadeia Única/administração & dosagem , Efeito Espectador , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Exossomos/imunologia , Exossomos/metabolismo , Vesículas Extracelulares/genética , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/patogenicidade , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/prevenção & controle , Anticorpos de Cadeia Única/genética , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
15.
Viral Immunol ; 32(10): 430-441, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31800372

RESUMO

Women with persistent human papillomavirus (HPV) infections have a high risk of developing cervical cancer (CaCx). HPV-16 alone accounts for more than 60% of CaCx worldwide. Most of the HPV infections are transient and only a subset of women develop persistent HPV-16 infection. Many studies have shown associations of different human leukocyte antigen (HLA) alleles with HPV-mediated CaCx, but there are only a few studies globally that relate to persistent HPV-16 infection. Furthermore, such studies from India are sparse. Hence, we investigated the association of HLA-A, B, DRB, and DQB alleles with persistent HPV-16 infection and HPV-16-positive CaCx in south India (Tamil Nadu). HPV-16 persistent infection was observed in 7% of normal women. A total of 50 women with HPV-16-positive CaCx, 21 women with HPV-16 persistent infection, and 74 HPV-16-negative normal women were recruited for this study. Low-resolution typing of HLA-A, B, DRB, and DQB alleles was performed. HLA-B*44 and DRB1*07 showed a significant association with persistent HPV-16 infection (odds ratio, p-value = 26.3, 0.03 and 4.7, 0.01, respectively). HLA-B*27 and DRB1*12 were significantly associated with both HPV-16+ CaCx and persistent HPV-16 infection (23.8, 0.03; 52.9, 0.01; 9.8, 0.0009; and 13.8, 0.009; respectively). HLA-B*15 showed a negative association with HPV-16-positive CaCx (0.1, 0.01), whereas DRB1*04 exhibited protection to both HPV-16-positive CaCx and persistent HPV-16 infection (0.3, 0.0001 and 0.1, 0.0002, respectively). Thus, we show HLA allelic association with HPV-16 infection in Tamil Nadu. Larger studies on high-resolution HLA typing coupled with HPV-16 genome diversity will offer further insights into host/pathogen genome coevolution.


Assuntos
Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe I/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/genética , Neoplasias do Colo do Útero/genética , Adulto , Alelos , Estudos de Casos e Controles , Colo do Útero/imunologia , Colo do Útero/virologia , DNA Viral/isolamento & purificação , Feminino , Predisposição Genética para Doença , Antígenos HLA-D/imunologia , Haplótipos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , Índia , Pessoa de Meia-Idade , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Polimorfismo Genético , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologia , Adulto Jovem
16.
Integr Cancer Ther ; 18: 1534735419893063, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31833799

RESUMO

Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.


Assuntos
Colo do Útero/microbiologia , Microbiota/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Lesões Intraepiteliais Escamosas/imunologia , Neoplasias do Colo do Útero/imunologia , Adulto , Colo do Útero/imunologia , Feminino , Papillomavirus Humano 16/imunologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/microbiologia , Lesões Intraepiteliais Escamosas/microbiologia , Neoplasias do Colo do Útero/microbiologia , Carga Viral/imunologia , Adulto Jovem
20.
Bull Cancer ; 106(11): 1008-1022, 2019 Nov.
Artigo em Francês | MEDLINE | ID: mdl-31606139

RESUMO

With more than 3300 new cases and almost 2500 deaths each year, cervical cancer (CC) ranks second among female cancers in Moroccan women. The majority of cases occurs in women aged 50 and over. In absence of a national cancer registry, data published in Morocco are limited to the number of cases recorded in some oncology centers, so the incidence of this cancer is likely much higher than estimated. A Moroccan national program against CC based on the practice of visual inspection after application of acetic acid was set up in 2010, allowing both screening and possibly immediate treatment of (pre)cancerous lesions. However, this program has not been implemented in all regions of the country. The CC develops slowly and most often without any symptoms, and so it is diagnosed at an advanced stage of the disease. Virtually, all CC are associated with persistent infection of high risk human papillomavirus (HPV), particularly HPV16 and 18. For more than ten years, two prophylactic vaccines targeting these two HPV genotypes have been marketed. They have proved their excellent immunogenicity and efficacy and they are well tolerated. However, HPV vaccine is not yet recommended by health authorities in Morocco. In this literature review, we focused on the current situation of CC, the prevalence of HPV infection and the prevention strategies against CC in Morocco.


Assuntos
Infecções por Papillomavirus/prevenção & controle , Lesões Pré-Cancerosas/prevenção & controle , Neoplasias do Colo do Útero/prevenção & controle , Ácido Acético/administração & dosagem , Adulto , Algoritmos , Coinfecção/epidemiologia , Detecção Precoce de Câncer/métodos , Feminino , Infecções por HIV/epidemiologia , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Incidência , Indicadores e Reagentes/administração & dosagem , Pessoa de Meia-Idade , Marrocos/epidemiologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Vacinas contra Papillomavirus/administração & dosagem , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/epidemiologia , Lesões Pré-Cancerosas/virologia , Prevenção Primária , Sistema de Registros/estatística & dados numéricos , Gestão de Riscos , Prevenção Secundária , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia
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