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An in vitro relative potency (IVRP) assay has been developed as an alternative to the mouse potency assay used to release Merck's human papillomavirus (HPV) vaccine, Gardasil ® , for early phase clinical trials. The mouse potency assay is a classical, in vivo assay, which requires 4-6 weeks to complete and exhibits variability on the order of 40% relative standard deviation (RSD). The IVRP assay is a sandwich-type immunoassay that is used to measure relative antigenicity of the vaccine product. The IVRP assay can be completed in three days, has a variability of approximately 10% RSD and does not require the sacrifice of live animals. Because antigen detection is achieved using H16.V5, a neutralizing monoclonal antibody, which binds to a clinically-relevant epitope, the relative antigenicity measured by the IVRP assay is believed to be a good predictor of in vivo potency.In this study, the relationship between immunogenicity, as measured by the mouse potency assay and antigenicity as measured by the IVRP assay, is demonstrated. Freshly manufactured and aged samples produced using two different manufacturing processes were tested using both methods. The results demonstrate that there is an inverse correlation between the IVRP and mouse potency assays. Additionally, clinical results indicate IVRP is predictive of human immunogenicity. Thus, antigenicity, as defined by the H16.V5 epitope, can be used as a surrogate for immunogenicity and the IVRP assay is suitable for use as the sole potency test for Gardasil samples.

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The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.The life cycle of human papillomaviruses (HPV) is intimately connected to the differentiation program of host epithelial tissues (14,20,22,36). The use of an organotypic (raft) epithelial culture system has allowed for the development of an in vitro culture system capable of reproducing the complete HPV life cycle, including the propagation of infectious viral particles (20,22). The raft culture system has been used to describe in detail the steps in the HPV life cycle (2,8,11,18,(25)(26)(27)(28)(29)(30), including the kinetics and spatial patterns of HPV gene expression (18,(25)(26)(27)(28)(29). Flores et al. used the raft culture system to begin a genetic analysis of the HPV life cycle by using an E7-deficient HPV type 16 (HPV16) genome (7). They found that this genome, while being maintained episomally, failed to amplify its DNA and expressed reduced levels of the L1 capsid protein. That study was done by using a spontaneously immortalized keratinocyte cell line (1). Attempts have been made to use a genetic approach to study the HPV life cycle by using primary keratinocytes (15, 39). These studies found that the majority of mutations examined, both in noncoding and in coding regions, were unstable in their ability to maintain the viral DNA (vDNA) in an episomal state.It has been reported that the interaction of the HPV nonstructural proteins, in particular, E2, with the structural capsid proteins, L1 and L...

BackgroundEscherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax™, or probiotic, Dairyman's Choice™ paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.FindingsDairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillusflavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax™ removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice™ paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax™ removed the cytotoxicity in vitro. Celmanax™ also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice™ paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.ConclusionThe current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrha...