Scientific and Regulatory Policy Committee Points to Consider: Biological Sample Retention From Nonclinical Toxicity Studies.

Samples of biologic specimens and their derivatives (eg, wet tissues, paraffin-embedded tissue blocks, histology slides, frozen tissues, whole blood, serum/plasma, and urine) are routinely collected during the course of nonclinical toxicity studies. Good Laboratory Practice regulations and/or guidance specify minimum requirements for specimen retention duration, with the caveat that retention of biologic specimens need not extend beyond the duration of sample stability. However, limited availability of published data regarding stability for various purposes following storage of each specimen type has resulted in confusion, uncertainty, and inconsistency as to the appropriate duration for storage of these specimens. To address these issues, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to review published information, regulations, and guidance pertinent to this topic and to summarize the current practices and rationales for retention duration through a survey-based approach. Information regarding experiences reaccessing biologic specimens and performing sample stability investigations was also collected. Based on this combined information, the working group developed several points to consider that may be referenced when developing or revising sample retention practices. [Box: see text].

The Nonclinical Safety Profile of GalNAc-conjugated RNAi Therapeutics in Subacute Studies.

Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.

Evolutionary reconstructions of the transferrin receptor of Caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs.

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.

Limited transferrin receptor clustering allows rapid diffusion of canine parvovirus into clathrin endocytic structures.

Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here we used fluorescence microscopy to directly visualize the association of single canine parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that most capsids associated with fewer than five TfRs and that ∼25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissociated from the cell surface with a half-life of ∼30 s. Together, our results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.

Role of multiple hosts in the cross-species transmission and emergence of a pandemic parvovirus.

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Spontaneous cervicovaginal lesions and immune cell infiltrates in nonhuman primates.

Nonhuman primates, particularly rhesus macaques (Macaca mulatta), provide important model systems for studying human reproductive infectious diseases such as human immunodeficiency virus, human papillomavirus, and Chlamydia spp. An understanding of the spectrum of spontaneous cervical disease provides essential context for interpreting experimental disease outcomes in the female reproductive tract. This retrospective study characterizes the incidence of inflammatory and/or proliferative cervicovaginal lesions seen over a 14-year period in a multispecies nonhuman primate colony, focusing on rhesus macaques. The most common observations included a spectrum of lymphocytic accumulation from within normal limits to lymphoplasmacytic cervicitis, and suppurative inflammation with occasional squamous metaplasia or polyp formation. These inflammatory spectra frequently occurred in the context of immunosuppression following experimental simian immunodeficiency virus (SIV) infection. Cervical neoplasias were uncommon and included leiomyomas and carcinomas. Cervical sections from 13 representative cases, with an emphasis on proliferative and dysplastic lesions, were surveyed for leukocyte infiltration, abnormal epithelial proliferation, and the presence of papillomavirus antigens. Proliferative lesions showed sporadic evidence of spontaneous papillomavirus infection and variable immune cell responses. These results underscore the importance of pre screening potential experimental animals for the presence of preexisting reproductive tract disease, and the consideration of normal variability within cycling reproductive tracts in interpretation of cervical lesions.

Examining the cross-reactivity and neutralization mechanisms of a panel of mAbs against adeno-associated virus serotypes 1 and 5.

Neutralizing antibodies play a central role in the prevention and clearance of viral infections, but can be detrimental to the use of viral capsids for gene delivery. Antibodies present a major hurdle for ongoing clinical trials using adeno-associated viruses (AAVs); however, relatively little is known about the antigenic epitopes of most AAV serotypes or the mechanism(s) of antibody-mediated neutralization. We developed panels of AAV mAbs by repeatedly immunizing mice with AAV serotype 1 (AAV1) capsids, or by sequentially immunizing with AAV1 followed by AAV5 capsids, in order to examine the efficiency and mechanisms of antibody-mediated neutralization. The antibodies were not cross-reactive between heterologous AAV serotypes except for a low level of recognition of AAV1 capsids by the AAV5 antibodies, probably due to the initial immunization with AAV1. The neutralization efficiency of different IgGs varied and Fab fragments derived from these antibodies were generally poorly neutralizing. The antibodies appeared to display various alternative mechanisms of neutralization, which included inhibition of receptor-binding and interference with a post-attachment step.

Early steps in cell infection by parvoviruses: host-specific differences in cell receptor binding but similar endosomal trafficking.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are closely related parvoviruses that differ in their host ranges for cats and dogs. Both viruses bind their host transferrin receptor (TfR), enter cells by clathrin-mediated endocytosis, and traffic with that receptor through endosomal pathways. Infection by these viruses appears to be inefficient and slow, with low numbers of virions infecting the cell after a number of hours. Species-specific binding to TfR controls viral host range, and in this study FPV and strains of CPV differed in the levels of cell attachment, uptake, and infection in canine and feline cells. During infection, CPV particles initially bound and trafficked passively on the filopodia of canine cells while they bound to the cell body of feline cells. That binding was associated with the TfR as it was disrupted by anti-TfR antibodies. Capsids were taken up from the cell surface with different kinetics in canine and feline cells but, unlike transferrin, most did not recycle. Capsids labeled with fluorescent markers were seen in Rab5-, Rab7-, or Rab11-positive endosomal compartments within minutes of uptake, but reached the nucleus. Constitutively active or dominant negative Rab mutants changed the intracellular distribution of capsids and affected the infectivity of virus in cells.

The parvovirus capsid odyssey: from the cell surface to the nucleus.

During cellular entry and infection, the parvovirus capsid follows a complex path from the cell surface to the nucleus, where the DNA is replicated. Various receptors have been characterized that bind to different parvoviruses and mediate their entry into cells. However, the subsequent trafficking pathways within the endosomal system, cytoplasm and into the nucleus are still not well defined. Studies of viruses entering various cell types under different conditions show particles located in many different endosomal compartments, within the cytoplasm and in the nucleus with significant variations in timing and distribution. Here, we define the previously unresolved issues that are now better understood for the infection pathways of these viruses, and outline some of the areas that remain to be clarified in future studies.

Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.

Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus.

BACKGROUND: Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047-2056. RESULTS: Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression. CONCLUSION: We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

Effects of canine parvovirus strain variations on diagnostic test results and clinical management of enteritis in dogs.

OBJECTIVE: To estimate the prevalence of canine parvovirus (CPV) strains among dogs with enteritis admitted to a referral hospital in the southwestern United States during an 11-month period and to compare diagnostic test results, disease severity, and patient outcome among CPV strains. DESIGN: Prospective observational study. ANIMALS: 72 dogs with histories and clinical signs of parvoviral enteritis. PROCEDURES: For each dog, a fecal sample or rectal swab specimen was evaluated for CPV antigen via an ELISA. Subsequently, fecal samples (n = 42 dogs) and pharyngeal swab specimens (16) were obtained and tested for CPV antigen via an ELISA and CPV DNA via a PCR assay. For specimens with CPV-positive results via PCR assay, genetic sequencing was performed to identify the CPV strain. RESULTS: 56 dogs tested positive for CPV via ELISA or PCR assay. For 42 fecal samples tested via both ELISA and PCR assay, 27 had positive results via both assays, whereas 6 had positive PCR assay results only. Ten pharyngeal swab specimens yielded positive PCR assay results. Genetic sequencing was performed on 34 fecal or pharyngeal swab specimens that had CPV-positive PCR assay results; 25 (73.5%) were identified as containing CPV type-2c, and 9 (26.5%) were identified as containing CPV type-2b. No association was found between CPV strain and disease severity or clinical outcome. CONCLUSIONS AND CLINICAL RELEVANCE: CPV type-2b and CPV type-2c posed similar health risks for dogs; therefore, genetic sequencing of CPV does not appear necessary for clinical management of infected patients. The diagnostic tests used could detect CPV type-2c.