RESUMEN
Capsomeres are considered to be an alternative to viruslike particle (VLP)-based vaccines as they can be produced in prokaryotic expression systems. So far, no detailed side-by-side comparison of VLPs and capsomeres has been performed. In the present study, we immunized mice with insect cell-derived human papillomavirus type 16 VLPs and capsomeres. VLPs induced consistently higher antibody titers than capsomeres but the two forms induced similar CD8 T-cell responses after subcutaneous, intranasal, and oral immunization, and at least 20 to 40 times more L1 in the form of capsomeres than in the form of VLPs was needed to achieve comparable antibody responses. These results were confirmed by DNA immunization. The lower immunogenicity of capsomeres was independent of the isotype switch, as it was also observed for the early immunoglobulin M responses. Although there were differences in the display of surface epitopes between the L1 particles, these did not contribute significantly to the differences in the immune responses. capsomeres were less immunogenic than VLPs in Toll-like receptor 4 (TLR4)-deficient mice, suggesting that the lower immunogenicity is not due to a failure of capsomeres to trigger TLR4. We observed better correlation between antibody results from enzyme-linked immunosorbent assays and neutralization assays for sera from VLP-immunized mice than for sera from capsomere-immunized mice, suggesting qualitative differences between VLPs and capsomeres. We also showed that the lower immunogenicity of capsomeres could be compensated by the use of an adjuvant system containing MPL. Taken together, these results suggest that, presumably because of the lower degree of complexity of the antigen organization, capsomeres are significantly less immunogenic than VLPs with respect to the humoral immune response and that this characteristic should be considered in the design of putative capsomere-based prophylactic vaccines.
Asunto(s)
Formación de Anticuerpos/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Cápside/inmunología , Cápside/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Proteínas de la Cápside/metabolismo , Línea Celular , ADN Viral/inmunología , Epítopos/inmunología , Papillomavirus Humano 16/metabolismo , Humanos , Inmunidad Mucosa/inmunología , Inmunogenética , Cambio de Clase de Inmunoglobulina/inmunología , Ratones , Proteínas Oncogénicas Virales/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Equine sarcoids are fibrosarcoma-like skin tumours with a prevalence of approximately 1-2 %. Strong evidence exists for a causative role of bovine papillomavirus (BPV) type 1 or type 2 in the development of sarcoids. No effective treatment of equine sarcoid is available and after surgical excision relapse of the tumours is very frequent. We developed chimeric virus-like particles (CVLPs) of BPV 1 L1-E7 for the immunotherapy of equine sarcoid. In a phase I clinical trial 12 horses suffering from equine sarcoid with an average number of more than 22 tumours per animal were vaccinated in a dose-escalation setting. The animals were followed-up for 63 days, eight of the twelve horses were followed-up for more than a year and side-effects, humoral immune responses and tumour appearance were recorded. BPV DNA was detected in tumours of 11 cases. CVLPs were well tolerated in all dose groups, a robust anti-L1 antibody response was induced in all but one of the horses. Anti-E7 antibodies were detected in five of the 12 animals at low titres. Two animals showed a clear improvement of the clinical status after treatment, i.e. the number of the tumours per horse was reduced. In another horse regression of five sarcoids was observed; three of them relapsed during the study. Two animals showed tumour regression as well as growth of new sarcoids. In two horses the clinical status remained unchanged, in another two horses growth of existing tumours or growth of additional tumours was observed. The remaining three animals showed simultaneously regression and growth of existing tumours. Neither the humoral immune responses nor the observed effects on the tumours was correlated with the dose group.
Asunto(s)
Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/aislamiento & purificación , Fibrosarcoma/virología , Enfermedades de los Caballos/virología , Infecciones por Papillomavirus/veterinaria , Sarcoidosis/veterinaria , Sarcoidosis/virología , Animales , Anticuerpos Antivirales/análisis , Formación de Anticuerpos , Biopsia , Quimera , ADN Viral/genética , ADN Viral/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosarcoma/inmunología , Fibrosarcoma/cirugía , Fibrosarcoma/veterinaria , Enfermedades de los Caballos/inmunología , Enfermedades de los Caballos/cirugía , Caballos , Inmunoterapia , Masculino , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/cirugía , Recurrencia , Sarcoidosis/inmunología , Sarcoidosis/cirugíaRESUMEN
In the present study we describe the isolation and characterization of putative equine granzyme B for which we propose the designation 'eqGrzmB'. Sequence analysis revealed characteristic features of a GrzmB protease such as the presence of a signal (leader-) peptide and an activation di-peptide. The isolated eqGrzmB is functionally active when expressed in human or in insect cells. Furthermore, exchange of any of three putative active site amino acids, which are highly conserved along granzyme B enzymes, led to a complete loss of enzymatic activity in the newly identified eqGrzmB. Phylogenetic analysis places eqGrzmB in the chymase-locus within the large family of granzymes in close proximity to putative equine mast cell protease and to granzyme B from mouse, rat, and human. eqGrzmB proteolytic activity has been kinetically characterized and can be specifically inhibited by granzyme B inhibitors. Taken together, we conclude that we have isolated a new member of the granzyme B family, the first granzyme identified in Equidae. The description of equine granzyme B might facilitate the development of immunological assays for the activity of equine lymphocytes.
Asunto(s)
Granzimas/genética , Granzimas/metabolismo , Caballos , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica , Granzimas/antagonistas & inhibidores , Granzimas/química , Caballos/genética , Humanos , Datos de Secuencia Molecular , Mutación , Filogenia , Especificidad por SustratoRESUMEN
Infections by human papillomaviruses (HPV) are the major cause of uterine cancer in women worldwide. Aiming to develop a combined prophylactic and therapeutic vaccine we have previously demonstrated immunogenicity of chimeric virus-like particles consisting of a C-terminally truncated HPV 16 L1 capsid protein fused to an E7 portion. Here we show that genetic vaccination with a corresponding DNA was inefficient in the induction of a L1-specific prophylactic immune response. DNA immunization with C-terminally truncated HPV 16 L1 genes of different lengths revealed that only short deletions (L1(1-498)) were tolerated for eliciting a humoral immune response against viral capsids. This correlates with the observation that the C-terminal sequences are critical for nuclear localization, capsomere and capsid assembly. However, only the ability of L1 protein to form capsomeres or capsids showed a direct influence on the outcome of the immune response. C-terminal insertion of 60 amino acids of E7 was tolerated in fusion constructs, whereas insertion of full-length E7(1-98) or shuffled E7 (149 aa) completely abolished the humoral immune response. The L1(1-498)/E7(1-60) fusion construct not only induced L1-specific antibodies but also L1- and E7-specific CTL responses after DNA vaccination.