Recommendations for Clinical Pathology Data Generation, Interpretation, and Reporting in Target Animal Safety Studies for Veterinary Drug Development.

Clinical pathology testing is routinely performed in target animal safety studies in order to identify potential toxicity associated with administration of an investigational veterinary pharmaceutical product. Regulatory and other testing guidelines that address such studies provide recommendations for clinical pathology testing but occasionally contain outdated analytes and do not take into account interspecies physiologic differences that affect the practical selection of appropriate clinical pathology tests. Additionally, strong emphasis is often placed on statistical analysis and use of reference intervals for interpretation of test article-related clinical pathology changes, with limited attention given to the critical scientific review of clinically, toxicologically, or biologically relevant changes. The purpose of this communication from the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology is to provide current recommendations for clinical pathology testing and data interpretation in target animal safety studies and thereby enhance the value of clinical pathology testing in these studies.

Best practices for veterinary toxicologic clinical pathology, with emphasis on the pharmaceutical and biotechnology industries.

The purpose of this paper by the Regulatory Affairs Committee (RAC) of the American Society for Veterinary Clinical Pathology (ASVCP) is to review the current regulatory guidances (eg, guidelines) and published recommendations for best practices in veterinary toxicologic clinical pathology, particularly in the pharmaceutical and biotechnology industries, and to utilize the combined experience of ASVCP RAC to provide updated recommendations. Discussion points include (1) instrumentation, validation, and sample collection, (2) routine laboratory variables, (3) cytologic laboratory variables, (4) data interpretation and reporting (including peer review, reference intervals and statistics), and (5) roles and responsibilities of clinical pathologists and laboratory personnel. Revision and improvement of current practices should be in alignment with evolving regulatory guidance documents, new technology, and expanding understanding and utility of clinical pathology. These recommendations provide a contemporary guide for the refinement of veterinary toxicologic clinical pathology best practices.

Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes.

Novel approaches for the prevention of allergy are required, because of the inevitably increasing prevalence of allergic diseases during the last 30 years. Here, a recombinant chimeric protein, which comprises the whole amino acid sequences of three bee venom major allergens has been engineered and used in prevention of bee venom sensitization in mice. Phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 3) fragments with overlapping amino acids were assembled in a different order in the Api m (1/2/3) chimeric protein, which preserved entire T cell epitopes, whereas B cell epitopes of all three allergens were abrogated. Accordingly, IgE cross-linking leading to mast cell and basophil mediator release was profoundly reduced in humans. Supporting these findings, the Api m (1/2/3) induced 100 to 1000 times less type-1 skin test reactivity in allergic patients. Treatment of mice with Api m (1/2/3) led to a significant reduction of specific IgE development towards native allergen, representing a protective vaccine effect in vivo. These results demonstrate a novel prototype of a preventive allergy vaccine, which preserves the entire T cell epitope repertoire, but bypasses induction of IgE against native allergen, and side effects related to mast cell/basophil IgE FcepsilonRI cross-linking in sensitized individuals.

Improving the therapeutic index of CpG oligodeoxynucleotides by intralymphatic administration.

Signal transduction initiated by TLR such as TLR9, a natural receptor for unmethylated cytosine-guanine-rich motifs (CpG), results in activation of transcription factors, including NF-kappaB, with substantial impact on the innate and adaptive immunity. However, practical application of new adjuvants such as CpG oligodeoxynucleotides (ODN) remains a challenge, since prominent systemic activation of NF-kappaB may result in severe side effects reminiscent of septic shock, thus limiting their therapeutic index (TI). Low-dose administration of CpG ODN into lymph nodes has been evaluated as a means to reduce systemic side effects while retaining strong adjuvant properties. To this aim, a prototype immune-stimulating CpG ODN was used to enhance the antibody production against the antigen phospholipase A(2) and the CD8(+) T cell responses to ovalbumin in mice. When administered subcutaneously, high CpG ODN doses (>10 nmol) were required to enhance antibody and CD8(+) T cell responses. In contrast, when administered directly into a lymph node, much lower amounts of CpG (<0.1 nmol) were sufficient for a similar immune-enhancing effect. Systemic adverse reactions induced by CpG ODN were only detected at higher doses (1-10 nmol), independently of the route of administration. Finally, low-dose CpG ODN, administered in a targeted fashion to HLA-A2.1(+) transgenic mice, greatly elevated anti-tumor CD8(+) T cell immunity. Thus, intralymphatic administration of CpG ODN considerably improves the TI and may greatly enable a safe and effective use in the clinic.

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy.

BACKGROUND: Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. OBJECTIVE: The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. METHODS: By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). RESULTS: The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. CONCLUSION: The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.

CpG oligodeoxynucleotides block human immunodeficiency virus type 1 replication in human lymphoid tissue infected ex vivo.

Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4(+) T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4(+) T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4(+) T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.