Sharks in Captivity: The Role of Husbandry, Breeding, Education, and Citizen Science in Shark Conservation.

The role of public aquariums in promoting conservation has changed substantially over the decades, evolving from entertainment attractions to educational and research centres. In many facilities, larger sharks are an essential part of the collection and represent one of the biggest draws for the public. Displaying healthy elasmobranchs comes with many challenges, but improvements in husbandry techniques have enabled aquariums to have success with a variety of species. The establishment of organisations such as the Association of Zoos and Aquariums, and the completion of texts like the Elasmobranch Husbandry Manual, has helped set high standards of care for sharks in captivity and promoted international conservation efforts. Aquariums keeping sharks are in a unique position to influence local, regional, and international attitudes and policies by acting as both educational and research facilities. Interactions with multiple stakeholders of diverse educational and demographic backgrounds through the use of in-house advocacy, public outreach, media interviews, and partnerships with academic and government institutions enable these facilities to engage and share information with a broad audience. Although the data collected on sharks in captivity often cannot be directly translated to animals in the wild, it offers better insight into a number of life history traits and poorly understood behaviours, and has been the foundation for many captive breeding programs. Several Northeast Pacific (NEP) shark species are commonly displayed for long durations or bred in aquariums, while other less studied species have been held for short periods to collect valuable data that can be applied towards ongoing studies and conservation measures. Here, we discuss past and current tangible benefits of holding NEP sharks in captivity, as well as noting several ways in which future research and education activities will continue to inform and shape public opinions on shark management and conservation.

Detection of avian retroviruses in vaccines by amplification on DF-1 cells with immunostaining and fluorescent product-enhanced reverse transcriptase endpoint methods.

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Feeding Behavior of Subadult Sixgill Sharks (Hexanchus griseus) at a Bait Station.

This is the first in-situ study of feeding behaviors exhibited by bluntnose sixgill sharks. Bait was placed beneath the Seattle Aquarium pier situated on the waterfront in Elliott Bay, Puget Sound, Washington at 20m of water depth. Cameras and lights were placed around the bait box to record sixgill shark presence and behavior while feeding. Analysis of feeding behavior revealed that sixgills utilize a bite comparable to many other elasmobranchs and aquatic vertebrates, have the ability to protrude their upper jaw, change their feeding behavior based on the situation, and employ sawing and lateral tearing during manipulation. The versatility of their feeding mechanism and the ability of sixgills to change their capture and food manipulation behaviors may have contributed to the species' worldwide distribution and evolutionary success.

Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode.

Respiratory syncytial virus (RSV) is an important human pathogen, and is the most frequent viral cause of severe respiratory disease in infants. In addition, it is increasingly being recognized as an important cause of respiratory disease in the elderly and immunocompromised. Although a passive prophylactic treatment does exist for high-risk neonates and children, the overall disease burden warrants the development of a safe and effective prophylactic vaccine for use in otherwise healthy newborns and children. RSV is known to be an extremely labile virus, prone to aggregation and loss of infectious titer during virus handling and preparation procedures. To date infective RSV virions have been prepared by methods which are not readily scalable, such as density gradient ultracentrifugation. In this study we describe a scalable, chromatography-based purification procedure for preparation of highly pure, infectious RSV. The purification scheme is based on core bead technology and hollow fiber tangential flow filtration (TFF) and results in a ∼60% recovery of infectious virus titer. This method can be used to prepare highly purified wild type or live-attenuated vaccine strain viruses with titers as high as 1×10(8) plaque forming units per mL. A live-attenuated RSV vaccine prepared by this method was found to be immunogenic and protective in vivo, and its purity was 50-200-fold greater with respect to host cell dsDNA and Vero host cell proteins, than the raw feed stream. The results presented here can be considered a starting point for downstream process development of a live-attenuated vaccine approach for prevention of disease by RSV.

Determination of HSV-1 UL5 and UL29 gene copy numbers in an HSV complementing Vero cell line.

The genetic stability of transgenes is a critical characteristic used to assess constructed cell lines used for vaccine production. The evaluation of gene copy numbers by a qPCR method, is one of the most common approaches used to assess the consistency of transgenes in a constructed cell line. The cell line AV529-19 is a Vero-based cell line specifically engineered to express the HSV-1 UL5 and UL29 open reading frames. AV529-19 is used to support the replication of a defective HSV-2 viral candidate vaccine called HSV529. To assess the genetic stability of the UL5 and UL29 transgenes in AV529-19 cells, a digital PCR-based approach was developed. During characterization of the test method, the specificity, accuracy, and intermediate precision of the assay was investigated based on regulatory guidelines. The developed assay was used to monitor the stability of the transgenes in the manufactured AV529-19 cell lines by comparison of transgene copy numbers in the master cell bank (MCB) with their copy numbers in the extended cell bank (ECB). Results showed that the UL29 and UL5 transgenes are stable in that there are one and three copies of the UL29 and UL5 genes, respectively, per cell in both the AV529-19 MCB and ECB.

Murine responses to recombinant MVA versus ALVAC vaccines against tumor-associated antigens, gp100 and 5T4.

Virally vectored cancer vaccines comprise a new form of immunotherapy that aim to generate anti-tumor immune responses with potential for tumor clearance and enhanced patient survival. Here, we compared 2 replication-deficient poxviruses modified vaccinia Ankara (MVA) and ALVAC(2) in their ability to induce antigen expression and immunogenicity of the tumor-associated antigens (TAAs) 5T4 and gp100. To facilitate the comparison, recombinant MVA-gp100M and ALVAC(2)-5T4 were constructed to complement existing ALVAC(2)-gp100M and MVA-5T4 vectors. Recombinant TAA expression in chicken embryo fibroblast cells was confirmed by Western blot analysis. 5T4 expression was approximately equal for both viruses, whereas ALVAC-derived gp100 was quickly degraded, at a time point when MVA-derived gp100 was still stable and expressed at high levels. Human leukocyte antigen-A2 transgenic mice were vaccinated with recombinant viruses and the CD8 T-cell responses elicited against each TAA were monitored by interferon-γ enzyme-linked immunospot. No 5T4 peptide responses were detected using splenocytes from mice vaccinated with either vector, whereas vaccination with MVA elicited a significantly higher gp100-specific response than ALVAC(2) at 10 PFU (P<0.001). In CD-1 mice, each vector elicited similar 5T4 antibody responses, whereas MVA was more potent and induced gp100 antibody responses at a lower immunization dose than ALVAC (P<0.001). In this study, immunogenicity varied depending on the viral vector used and reflected vector-associated differences in in vitro TAA expression and stability. These findings suggest that novel vector-transgene combinations must be assessed individually when designing vaccines, and that stability of vector-encoded proteins produced in vitro may be useful as a predictor for in vitro immunogenicity.

Preclinical qualification of a new multi-antigen candidate vaccine for metastatic melanoma.

New therapies are urgently required for the treatment of patients with melanoma. Here we describe the generation and preclinical evaluation of 3 new recombinant ALVAC(2) poxviruses vCP2264, vCP2291, and vCP2292 for their ability to induce the desired cellular immune responses against the encoded melanoma-associated antigens. This was done either in HLA-A2/K transgenic mice or using in vitro antigen-presentation studies. These studies demonstrated that the vaccine was able to induce HLA-A*0201-restricted T-cell responses against gp100 and NY-ESO-1, detectable directly ex vivo, in HLA-A2/K-transgenic mice. The in vitro antigen presentation studies, in the absence of appropriate animal models, demonstrated that target cells infected with the vaccine construct were lysed by MAGE-1, MAGE-3 or MART-1 peptide-specific T cells. These data indicate that ALVAC(2)-encoded melanoma-associated antigens can be properly processed and presented to induce antigen-specific cytotoxic T-cell responses. To enhance the immunogenicity of the melanoma antigens, a TRIad of COstimulatory Molecules (TRICOM) were also cloned into all 3 vectors. Increased in vitro proliferation and IFN-γ production was observed with all ALVAC(2) poxviruses encoding TRICOM, confirming the immune-enhancing effect of the ALVAC-encoded TRICOM. These studies demonstrated that all components of the vaccine were functionally active and provide a rationale for moving this candidate vaccine to the clinic.

Characterization of antigen-specific immune responses induced by canarypox virus vaccines.

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against cancer and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host immune system. We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based vaccine against carcinoembryonic Ag (ALVAC-CEA), a tumor-associated autoantigen commonly overexpressed in colorectal cancers. The protective CEA-specific immunity induced by this vaccine consisted of CD4(+) T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by potent humoral responses, but not by CEA-specific CD8(+) CTL immunity. In contrast, protective immunity induced by a CEA-specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine.