Ligand binding assays in the 21st century laboratory: recommendations for characterization and supply of critical reagents.

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

Microsphere immunoassay for the detection of cytokines in domestic cat (Felis catus) plasma: elevated IL-12/23 in acute feline immunodeficiency virus infections.

We recently described the development and validation of a highly sensitive and specific microsphere immunoassay capable of simultaneously quantifying three domestic cat cytokines in tissue culture supernatant. Here we describe the modification of this assay to measure interferon gamma (IFNγ), interleukin (IL)-10 and IL-12/IL-23 p40 (IL-12/23) in domestic cat plasma, report values obtained from plasma collected after feline immunodeficiency virus (FIV) exposure, and compare plasma concentrations to blood cell mRNA expression. The validated quantitation limits of this assay are 31-1000 pg/ml for IFNγ, 63-2000 pg/ml for IL-10, and 20-625 pg/ml for IL-12/23. Plasma cytokine levels from domestic cats infected with pathogenic and/or apathogenic FIV were determined at 3-4 and 7-8 weeks post-infection. IL-12/23 was elevated (p<0.05) during acute infection with both FIV strains in two similar studies, conducted five years apart in different feline cohorts (n=44 total animals). IL-12/23 concentrations ranged from 377 to 1904 pg/ml in naïve cats and 552 to 3460 pg/ml in infected cats. In contrast, the majority of plasma samples had IFNγ and IL-10 concentrations below the lowest standard tested. The inability to consistently detect levels of IFNγ and IL-10 in plasma, despite the fact that mRNA changes were detected, suggests that these cytokines may be secreted and/or cleared in a more highly regulated manner than IL-12/23, or perhaps exert local effects under tighter peripheral constraints and/or at a lower effective concentration.

Prior virus exposure alters the long-term landscape of viral replication during feline lentiviral infection.

We developed a feline model of lentiviral cross-species transmission using a puma lentivirus (PLV or FIV(Pco)) which infects domestic cats but does not cause disease. Infection with PLV protects cats from CD4+ T-cell decline caused by subsequent infection with virulent feline immunodeficiency virus (FIV). Previous studies implicate innate immune and/or cellular restriction mechanisms for FIV disease attenuation in PLV-infected cats. In this study, we evaluated viral infection and cytokine mRNA transcription in 12 different tissue reservoirs approximately five months post infection. We quantitated tissue proviral load, viral mRNA load and relative transcription of IL-10, IL-12p40 and IFNγ from tissues of cats exposed to FIV, PLV or both viruses and analyzed these parameters using a multivariate statistical approach. The distribution and intensity of FIV infection and IFNγ transcription differed between single and co-infected cats, characterized by higher FIV proviral loads and IFNγ expression in co-infected cat tissues. Variability in FIV mRNA load and IFNγ was significantly more constrained in co-infected versus singly infected cat tissues. Single-infected:co-infected ratios of FIV mRNA load compared to FIV proviral load indicated that active viral transcription was apparently inhibited during co-infection. These results indicate that previous PLV infection increases activation of tissue innate immunity and constrains the ability of FIV to productively infect tissue reservoirs of infection for months, independent of FIV proviral load, supporting a model in which innate immunity and/or modulation of target cell susceptibility play a key role in PLV-induced protection from FIV disease.

Increased consistency and efficiency in routine potency testing by bioassay with direct use of cryopreserved (ready-to-plate) cells.

The use of cells as a cryopreserved, readily available reagent has facilitated high-throughput screening of new drug candidates by bioassay. This practice is considerably less labor intensive and allows more flexibility in laboratory testing than traditional continuous cell culture. We have shown that this practice can be adapted to cell proliferation and reporter gene assay formats used in routine sample testing for determination of relative potency of commercial product in a Quality Control Laboratory. The ability to use the same, optimized population of cells provides consistency in an assay over time. Measures of assay performance to indicate maintenance of the validated state of a method and to determine benefit on variation in potency results were compared between cultured and cryopreserved (frozen ready-to-plate) cells. Control of the cellular component, which is the most variable aspect of most cell based potency assays, allowed detection of more minor contributors to variability. In a cell proliferation assay format, the final result was a highly precise bioassay (repeatability CV of 2%). An improvement in process capability (Cp) was noted when ready-to-plate cells were used in the studies completed over long periods of time.

Profound differences in virus population genetics correspond to protection from CD4 decline resulting from feline lentivirus coinfection.

CD4 decline is a hallmark of disease onset in individuals infected with Feline Immunodeficiency Virus (FIV) or Human Immunodeficiency Virus type 1 (HIV-1). Cats that are infected with a poorly replicating, apathogenic FIV (PLV) prior to exposure to a virulent FIV strain (FIVC) maintain CD4 numbers by mechanisms that are not correlated with a measurable adaptive immune response or reduction in circulating viral load. We employed population genetic approaches based on the 3' portion of the viral genome to estimate the population structure of FIVC from single and dual infected cats. In dual infected cats, FIVC effective population size was decreased during the initial viral expansion phase, and after three weeks of infection, the population declined sharply. The FIVC population recovered to pre-bottleneck levels approximately seven weeks post-FIVC infection. However, the population emerging from the bottleneck in dual infected cats was distinct based on estimates of temporal population structure and substitution profiles. The transition to transversion rate ratio (κ) increased from early to late phases in dual infected cats due primarily to a decrease in transversions whereas in single infected cats, κ declined over time. Although one clone with extensive G to A substitutions, indicative of host cytidine deaminase editing, was recovered from a dual infected cat during the bottleneck, the post bottleneck population had an overall reduction in G to A substitutions. These data are consistent with a model of PLV-induced host restriction, putatively involving host DNA editing, that alters the dynamics of FIVC throughout the course of infection leading to disease attenuation.

Multivariate statistical analyses demonstrate unique host immune responses to single and dual lentiviral infection.

BACKGROUND: Feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the "immune profiles" that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNgamma, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells. CONCLUSIONS: Multivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline.

Ocelots on Barro Colorado Island are infected with feline immunodeficiency virus but not other common feline and canine viruses.

Transmission of pathogens from domestic animals to wildlife populations (spill-over) has precipitated local wildlife extinctions in multiple geographic locations. Identifying such events before they cause population declines requires differentiating spillover from endemic disease, a challenge complicated by a lack of baseline data from wildlife populations that are isolated from domestic animals. We tested sera collected from 12 ocelots (Leopardus pardalis) native to Barro Colorado Island, Panama, which is free of domestic animals, for antibodies to feline herpes virus, feline calicivirus, feline corona virus, feline panleukopenia virus, canine distemper virus, and feline immunodeficiency virus (FIV), typically a species-specific infection. Samples also were tested for feline leukemia virus antigens. Positive tests results were only observed for FIV; 50% of the ocelots were positive. We hypothesize that isolation of this population has prevented introduction of pathogens typically attributed to contact with domestic animals. The high density of ocelots on Barro Colorado Island may contribute to a high prevalence of FIV infection, as would be expected with increased contact rates among conspecifics in a geographically restricted population.

Prevention of immunodeficiency virus induced CD4+ T-cell depletion by prior infection with a non-pathogenic virus.

Immune dysregulation initiated by a profound loss of CD4+ T-cells is fundamental to HIV-induced pathogenesis. Infection of domestic cats with a non-pathogenic lentivirus prevalent in the puma (puma lentivirus, PLV or FIV(pco)) prevented peripheral blood CD4+ T-cell depletion caused by subsequent virulent FIV infection. Maintenance of this critical population was not associated with a significant decrease in FIV viremia, lending support to the hypothesis that direct viral cytopathic effect is not the primary cause of immunodeficiency. Although this approach was analogous to immunization with a modified live vaccine, correlates of immunity such as a serum-neutralizing antibody or virus-specific T-cell proliferative response were not found in protected animals. Differences in cytokine transcription profile, most notably in interferon gamma, were observed between the protected and unprotected groups. These data provide support for the importance of non-adaptive enhancement of the immune response in the prevention of CD4+ T-cell loss.

The molecular biology and evolution of feline immunodeficiency viruses of cougars.

Feline immunodeficiency virus (FIV) is a lentivirus that has been identified in many members of the family Felidae but domestic cats are the only FIV host in which infection results in disease. We studied FIVpco infection of cougars (Puma concolor) as a model for asymptomatic lentivirus infections to understand the mechanisms of host-virus coexistence. Several natural cougar populations were evaluated to determine if there are any consequences of FIVpco infection on cougar fecundity, survival, or susceptibility to other infections. We have sequenced full-length viral genomes and conducted a detailed analysis of viral molecular evolution on these sequences and on genome fragments of serially sampled animals to determine the evolutionary forces experienced by this virus in cougars. In addition, we have evaluated the molecular genetics of FIVpco in a new host, domestic cats, to determine the evolutionary consequences to a host-adapted virus associated with cross-species infection. Our results indicate that there are no significant differences in survival, fecundity or susceptibility to other infections between FIVpco-infected and uninfected cougars. The molecular evolution of FIVpco is characterized by a slower evolutionary rate and an absence of positive selection, but also by proviral and plasma viral loads comparable to those of epidemic lentiviruses such as HIV-1 or FIVfca. Evolutionary and recombination rates and selection profiles change significantly when FIVpco replicates in a new host.

Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis).

Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide an adequate alternative for screening of some species and is more easily adapted to field conditions.

Assessing flavivirus, lentivirus, and herpesvirus exposure in free-ranging ring-tailed lemurs in southwestern Madagascar.

The ring-tailed lemur (Lemur catta) is an endangered species found in southwestern Madagascar, and understanding infectious disease susceptibility is an essential step towards the preservation of wild and captive lemur populations. Lemurs are primates that are widely dispersed throughout the island of Madagascar and may serve as hosts or reservoirs for zoonotic infections. The aim of this study was to determine the prevalence of antibodies to West Nile virus (WNV), simian immunodeficiency virus (SIV), and herpes simplex virus type 1 (HSV-1) in a population of free-ranging ring-tailed lemur from the Beza Mahafaly Special Reserve, Madagascar. Samples were collected from 50 animals during field capture studies in June and July 2004 and assayed for presence of viral antibodies during the 12 mo following collection. Forty-seven of the 50 lemurs sampled had antibodies against WNV detectable by epitope-blocking enzyme-linked immunosorbent assay (ELISA). In addition, 50 of 50 samples had titers against WNV ranging from 80 to > or = 1,280 using plaque reduction neutralization test (PRNT(90)). Ten lemurs had antibodies against lentiviral antigens as determined by Western blot analysis. None of the lemurs had antibodies against HSV-1 using ELISA.

Recombination in feline lentiviral genomes during experimental cross-species infection.

Domestic cats develop an asymptomatic, productive infection with a feline immunodeficiency virus (PLV) derived from a naturally infected cougar (P. concolor). We previously demonstrated that there are extensive G to A substitutions, characteristic of host cytidine deaminase editing, and positive selection on reverse transcriptase in the PLV genome during this cross-species infection. In this study, we evaluated full-length viral genomes from each of four cats infected with PLV to determine if viral recombination occurred during this single source infection. Recombination rates were measurable in three of the four infected cats. In two of these animals, a single site in reverse transcriptase was under positive selection and there was significant topological incongruence among individual genes in the 3' half of the genomes. The break point was proximate to a splice site used for accessory gene expression. Our data indicate that recombination can facilitate lentivirus persistence in unfavorable environments such as a new host species.

Feline lentivirus evolution in cross-species infection reveals extensive G-to-A mutation and selection on key residues in the viral polymerase.

Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

Feline lentiviruses demonstrate differences in receptor repertoire and envelope structural elements.

Feline immunodeficiency virus (FIV) causes fatal disease in domestic cats via T cell depletion-mediated immunodeficiency. Pumas and lions are hosts for apparently apathogenic lentiviruses (PLV, LLV) distinct from FIV. We compared receptor use among these viruses by: (1) evaluating target cell susceptibility; (2) measuring viral replication following exposure to specific and non-specific receptor antagonists; and (3) comparing Env sequence and structural motifs. Most isolates of LLV and PLV productively infected domestic feline T cells, but differed from domestic cat FIV by infecting cells independent of CXCR4, demonstrating equivalent or enhanced replication following heparin exposure, and demonstrating substantial divergence in amino acid sequence and secondary structure in Env receptor binding domains. PLV infection was, however, inhibited by CD134/OX40 antibody. Thus, although PLV and LLV infection interfere with FIV superinfection, we conclude that LLV and PLV utilize novel, more promiscuous mechanisms for cell entry than FIV, underlying divergent tropism and biological properties of these viruses.

Puma lentivirus is controlled in domestic cats after mucosal exposure in the absence of conventional indicators of immunity.

A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.