- Open Access
Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3
- Sohela de Rozières†1,
- Christina H Swan†2,
- Dennis A Sheeter2,
- Karen J Clingerman3,
- Ying-Chuan Lin1,
- Salvador Huitron-Resendiz4,
- Steven Henriksen4,
- Bruce E Torbett2 and
- John H Elder1Email author
© de Rozières et al; licensee BioMed Central Ltd. 2004
- Received: 16 September 2004
- Accepted: 19 November 2004
- Published: 19 November 2004
The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates.
Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p ≤ 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p ≤ 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease.
The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection.
- Human Immunodeficiency Virus
- Viral Load
- Feline Immunodeficiency Virus
- High Viral Load
- Human Immunodeficiency Virus Protease
Feline immunodeficiency virus (FIV) is a lentivirus that infects domestic and feral cat populations worldwide. Infected cats exhibit similar disease patterns as human immunodeficiency virus (HIV) infected patients by developing multiple immuno-depletive symptoms collectively referred to as acquired immunodeficiency syndrome (AIDS). As with HIV, differences in virulence among the different FIV subgroups are evident [1–4]. Thus, the cat represents an amenable animal model for testing certain anti-HIV-1 drug modalities in vivo.
One of the major breakthroughs in HIV-1 treatment has been the use of specific inhibitors of the viral aspartic protease family as part of a drug cocktail, called highly active anti-retroviral therapy (HAART), with the ultimate goal of suppressing HIV-1 replication in patients to low or undetectable levels [5–8]. Effective HAART therapy continues to be dependent on the development of new drug modalities due to the rapid mutation rate of HIV-1, leading to drug resistance development . Therefore, an effective small animal model for evaluating new drugs and treatments for HIV is of paramount importance. Experimental testing of new protease inhibitors in cats has been of limited success due to the ineffectiveness of HIV-1 specific protease inhibitors against FIV [10, 11]. The promising development of the protease inhibitor TL-3, which inhibits FIV, HIV-1 and SIV (simian immunodeficiency virus) infections in vitro with similar effectiveness  led us to analyze its efficacy in the cat model. Initial in vivo studies using the predominantly neurotropic FIV-PPR strain, showed that TL-3 treatment lowered plasma viral loads and resulted in a significant protective effect against neurologic alterations in the CNS in FIV infected cats . In the present study, we employed the highly pathogenic FIV-C isolate (CABCpady00C), which causes a fulminant acute phase disease in the periphery, with high death rates from acute phase immunodeficiency disease .
In Vivo Infection
Twenty-two female specific pathogen-free (SPF) cats were randomly divided into five groups. Group 0 consisted of two cats, which received TL-3 treatment without viral infection and were considered controls. Group 1 (n = 5) received 0.1 ml (105 RNA copies/ml) of FIV-C-infected plasma I.V. with TL-3 drug treatment. Group 2 (n = 5) received 0.1 ml FIV-C-infected plasma without TL-3 treatment. Group 3 (n = 5) received 0.5 ml FIV-C-infected plasma with TL-3 and Group 4 (n = 5) received 0.5 ml FIV-C-infected plasma without TL-3 treatment. Blood (1 ml) was drawn from all cats prior to the start of the experiment, at weekly intervals for the first four weeks after infection, and at bi-monthly intervals from week 4 until the end of the study. Complete blood counts were assessed as a function of infection and TL-3 treatment. In addition, quantitative reverse transcription PCR (QRT-PCR) analyses were performed to assess plasma viral load. All animals were continuously observed for any changes in general health. No significant differences were noted between viral load or disease phenotype between the two plasma dosages used in infection and subsequent discussion will not distinguish between these two groups.
By week 6 post infection, seven animals (221, 222, 220, 234, 229, 219, 215) were showing clinical signs of debilitating acute phase disease. Four of the seven affected animals (215, 219, 220, 234) were from the untreated groups, and three animals (222, 221, 229) were from the TL-3 treated groups. Symptoms in all seven symptomatic cats varied from conjunctivitis, anorexia, corneal ulcerations, and gingivitis to increases in temperature, dermatitis and marked lethargy. Despite intensive antibiotic treatment, the general state of health of 6 of the cats did not improve (221, 220, 234, 229, 215, 219) and they received mandated euthanasia between six to seven weeks post-infection. Cat 222 (TL-3 treated) responded to antibiotic therapy and recovered from acute phase symptoms.
Brainstem auditory evoked potential changes (BAEPs)
Previous studies using FIV-PPR showed that the isolate induces marked and consistent delays in BAEPs of infected cats and that TL-3 could reverse this effect . We, therefore, analyzed the FIV-C infected animals for similar BAEP delays with or without TL-3 treatment. Animals were analyzed at two-week intervals for the first eight weeks of the experiment. Interestingly, no delays in BAEPs were noted with FIV-C infection (data not shown) in spite of high viral loads in the periphery (see below).
Viral load quantification
Consistent with previous reports [14–16], we also observed changes in the total neutrophil counts in the symptomatic FIV-C infected cats. Within one week post-infection, neutrophil numbers increased markedly in cats 220 (10, 962 cells/μl) and 234 (11, 926 cells/μl) as compared to other cats with identical TL-3 treatment (average = 6880 ± 2847 cells/μl). By week 4, neutrophil values fell drastically in the same two cats (220: 558 cells/μl; 234: 416 cells/μl) as well as in cats 215 (420 cells/μl) and 221 (400 cells/μl). By week 6, four of the symptomatic cats had neutrophil counts near zero. Only cat 220 slightly recovered its neutrophil cell count (3675 cells/μl) prior to mandated euthanasia.
Protease escape variants
FIV Protease Escape Mutants (HIV equivalent residue)
Ki vs TL-3 (nM)
The protease inhibitor, TL-3, demonstrated broad efficacy against FIV, SIV and HIV in tissue culture , as well as against drug-resistant HIV isolates . Furthermore, TL-3 treatment had a very strong protective effect on early neurologic alterations in the CNS of FIV-PPR infected cats . However, molecularly cloned FIV-PPR causes little acute phase disease in the periphery. We, therefore, sought to test TL-3 efficacy in vivo in the context of the highly pathogenic, uncloned CABCpady00C species (FIV-C) , which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. Although only partial protection was afforded by TL-3 in our studies, the results are promising in that average peak viral loads in some cats were lower in the presence of drug, even in the face of a highly aggressive infection (Figure 2B).
Of 20 cats infected with uncloned FIV-C, seven animals showed signs of immunodepletive disease early on (Figure 4) and developed full-fledged acute phase AIDS symptoms with anorexia, conjunctivitis, corneal ulcerations, gingivitis and marked lethargy by week 6, mandating euthanasia of six animals. The symptomatic cats had viral RNA loads significantly higher (>108 RNA copies/ml, p ≤ 0.05) than asymptomatic infected animals independent of drug treatment. This finding suggests that the intense viral infection severely compromised the immune system leading to immunodeficiency and the development of concomitant AIDS, as evidenced by the rapid loss of CD4+ T cells as well as neutrophils in the affected cats during the first few weeks (Figure 4). Cats receiving TL-3 treatment had lower peak viral loads compared to cats not receiving TL-3 at weeks 4 and 8, indicating that the protease inhibitor reduced systemic expansion of viral infection. Previous studies have correlated disease progression with high initial peak viral loads . Of the five out of eight cats treated with TL-3 and having higher viral loads at weeks 4 and 8 compared to non-TL-3-treated cats, three (222, 228, 231) were evaluated for FIV resistance to TL-3. None of the protease genes recovered from cat 228, whose viral levels fell below detection at week 8, showed evidence of TL-3 resistance. However, cats 222 and 231 were found to harbor virus in the plasma that encoded TL-3 resistant protease. In particular, a D105G mutant demonstrated a 5-fold resistance to TL-3 relative to wild type protease, which may indicate the onset of drug resistance development and explain the higher viral levels in some of the TL-3 treated cats. Preparation of isogenic virus containing the D105G point mutation will allow the direct determination of potential drug resistance.
Interestingly, TL-3 treated cats with highest viral loads (221 and 229) developed severe disease syndromes, which suggests that TL-3 efficacy was limited to a specific viral threshold in this study. Once the threshold has been crossed, TL-3 may not be able to overcome the full-blown, acute, viral infection, resulting in rapid onset of immune suppression.
We were unable to show any correlation between humoral antibody responses and clinical outcome (data not shown). However, a consistent observation was a marked and progressive increase of CD8+ T cells in animals surviving the acute phase infection and a lack of such responses in animals that required euthanasia within the first 8 weeks following infection. Although not formally tested, the findings imply a strong cell-mediated response in the surviving animals contributed to controlling the viral infection.
Viral protease inhibitors are of paramount importance for HIV treatment and successful tempering of viral infection. However, drug resistant escape variants are an important consideration in treatment protocols [17, 18]. Although we previously failed to isolate TL-3-resistant FIV in vitro, the findings here suggest that in vivo, drug resistance to the compound may develop. The results were not unexpected in that we had been able to develop TL-3 resistant HIV variants  and it seemed unlikely that FIV would prove an exception. The finding of drug resistant mutants, in fact, strongly indicates that the feline/FIV model is valuable in the assessment of the ability of other protease drugs and drug cocktails to suppress virus infection and limit drug resistance development.
The findings indicate that the protease inhibitor TL-3, when given orally as a monotherapy, did not prevent viremia in cats infected with a high dose challenge with FIV-C and substantial virus loads were evident in circulation throughout the acute phase (between 2–6 weeks post infection) in all infected animals, regardless of drug regimen. Average peak viral loads in the acute phase were lower in TL-3 treated animals, but variability was such that the numbers did not reach statistical significance. However, of six animals that required euthanasia, four were from the untreated cohort and two were from the TL-3 treated group. Additionally, at eight weeks post infection, half the surviving TL-3 treated animals had viral loads below the detection limits, whereas only one of six untreated animals had markedly reduced viral loads. Thus, therapeutic benefit was noted with TL-3 treatment, even in the face of an aggressive FIV infection.
The findings also show clear differences in the lymphocyte responses of animals that succumb to acute phase illness versus those that survive to the asymptomatic phase. The most pronounced difference was in the lack of an increase in CD8+ cell numbers starting around three weeks post infection in animals that eventually required humane euthanasia versus a pronounced and significant increase in CD8+ T cell numbers in animals that survived the acute phase.
Certain animals that received TL-3 had higher than average viral loads after the acute phase. Analyses of the protease genes of FIV quasi-species prevalent in these animals revealed sequence variations relative to protease of wild type FIV-C. One particular protease, cloned and expressed from two TL-3-treated animals, contained the mutation D105G, which imparted 5-fold resistance against TL-3 relative to wild type protease. This may represent the initial stages of drug resistance development and preparation of this mutation in the context of isogenic virus will address this issue. The findings suggest that the cat model will serve as a valuable animal model for study of resistance development against lentivirus infections.
22 female purpose-bred 8–9 week old kittens purchased from Liberty Laboratories were inspected upon arrival for signs of illness, examined by a veterinarian and weighed. Animals were maintained in a 2-week quarantine and observed for any signs of illness prior to the beginning of the study. IACUC number ARC 61 JAN 3.
Plasma samples (105 RNA copies/ml) from a cat, that had died from an acute infection with CABCpady00C (FIV-C), were kindly provided by Dr. E. Hoover, of Colorado State University. Cats were injected I.V. with either 0.1 ml (105 RNA copies/ml) or 0.5 ml of plasma.
All procedures for care of cats during dosing as well as dosing procedures were mandated by TSRI's IACUC. Oral TL-3 (L-Iditol,1,2,5,6-tetradeoxy-1,6-diphenyl-2,5-bis [N-[(phenylmethoxy)carbonyl]-L-alanyl-L-valyl]amino])  treatment was initiated in 12 cats, three days prior to infection of ten of the twelve animals with FIV-C. All TL-3 treated animals received 20 mg TL-3 by capsules at eight hour intervals, for approx. the first 7.5 weeks of the experiment. Dosage was then doubled to 40 mg TL-3 per dose at eight hour intervals for an additional week for the two control animals and the eight surviving animals in the TL-3 treated, infected cohort. No adverse effects were noted in the uninfected, TL-3 treated controls.
Uninfected and FIV-infected animals were intermittently scheduled for analyses of evoked potentials in conjunction with the blood sampling, including testing for both auditory and visual evoked potentials as previously described . Once recordings were complete, a blood sample was collected and animals returned to the vivarium.
Animals were examined daily and in case of health concerns further therapy/diagnostics were initiated. Animals with abnormal weight, or on antibiotics were placed on supplemental feeding with moist food and Nutrical. Dehydrated animals received subcutaneous fluid therapy. More affected animals received supportive care and medications, consisting of BID administration of antibiotics (Baytril), BID subcutaneous fluid therapy, BID temperature evaluation, BID application of antibiotic ophthalmic ointment supportive care. Any animals that required extensive supportive care (TID fluid therapy, TID force feeding) were euthanized. Euthanasia of research animals was conducted with strict adherence to NIH Office of Laboratory Animal Welfare protocols.
Blood Collection and Peripheral Blood Separation
Blood samples (1 ml/animal) were collected weekly during the first month of the study and then every two weeks thereafter, as described . Samples were placed in EDTA blood tubes (1 cc/tube) for further use.
Plasma was separated from blood by centrifugation at 3000 rpm for 5 minutes at room temperature. Blood cells were resuspended in 3 ml PBS and PBMC were separated from buffy coats by density gradient centrifugation using Ficoll-Hypaque Plus (Amersham Biosciences, Sweden). PBMC were washed once in PBS and twice in PBS/2% FBS for flow cytometry analyses.
Statistical p values for the FIV-C viral load were determined by the Student's two-tailed t-test (paired, two-tailed distribution between the treated and non-treated group and the symptomatic vs asymptomatic group). Statistical p values for the weekly total CD4 and CD8 cell counts were also determined by the Student's two-tailed t-test (paired, two-tailed distribution compared to base line levels at week 0).
Flow Cytometry Analysis
Two-color flow cytometry analysis was performed on cells stained with mouse α-feline CD4 FITC and mouse α-feline CD8 PE (Southern Biotech, Birmingham, AL). Anti-mouse IgG1κ FITC and PE (BD PharMingen, San Diego, CA) were used as isotype controls. Cells were fixed with 2% PFA prior to analysis performed on a FACScan flow cytometer (Beckton Dickenson Immunocytometry Systems) using the Cell Quest Software program.
RNA Isolation and Reverse Transcription
Plasma for weeks 0, 2, 4, 6 (terminal points for cats 215, 221, and 229), 6.5 (terminal points for cats 219, 220, and 234), and 8, were isolated from whole blood by centrifugation and stored at -20°C. Viral RNA was extracted using the QiaAmp Viral RNA Isolation Kit (Qiagen, Valencia, CA) according to manufacturer's instructions with slight modifications: Plasma samples (280 μl) were lysed in buffer AVL (1,120 μl) (Qiagen) for 10 minutes at room temperature in the presence of carrier RNA (10 μg/ml) and an external Kanamycin (KAN) RNA spike. Equal amounts of the external RNA spike (109 copies RNA /280 μl plasma), corresponding to the 1.2 kb KAN gene (Promega, Madison, WI), was used to normalize plasma volumes between samples and to correct for sample loss from viral RNA extraction and cDNA synthesis. An on-column DNase/ RNase free (Qiagen) incubation step for 10 minutes at room temperature was added to remove residual cellular DNA. Complimentary DNA (cDNA) was generated in a 20 μl total reaction using 13 μl of sample RNA, 0.5 μl (2 μM stock) each of KAN and FIV specific reverse primers (sequences below) and StrataScript reverse transcriptase, following the manufacturer's protocol (Stratagene, La Jolla, CA). After incubation, each cDNA sample was diluted in water to 30 μl and stored at -80°C for use in real-time PCR.
Real-Time Quantitative PCR
25 μl real-time PCR reactions were set up containing 2X Platinum Quantitative PCR SuperMix-UDG (12.5 μl) (Invitrogen, Carlsbad, CA), forward, reverse primers and probe mix (7.5 μl), and cDNA target (5 μl). The mixture was incubated at 50°C for 2 minutes, 95°C for 10 minutes, then cycled at 95°C for 15 seconds and 60°C for 60 seconds 55 times, using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Data were analyzed using the ABI 7700 Sequence Detection Software. Forward and reverse primers (10 nM, 100 nM, final concentration respectively) used in real-time PCR were made at IDT, (Coralville, IA), while the fluorescein-dabsyl Amplifluor UniPrimer (100 nM final concentration) was purchased from Serologicals (Norcross, GA). The primer sequences used for real-time PCR are as follows:
FIV reverse-transcriptase forward: 5'-ACTGAACCTGACCGTACAGATAAATTACAGGAA GAACCCCCATA-3'
FIV reverse-transcriptase reverse: 5'-TGTTAATGGATGTAATTCA TAACCCATC-3'
KAN forward: 5'-ACTGAACCTGACCGTACACGCTCAGGCGCAATCAC-3'
KAN reverse: 5'-CCAGCCATTACGCTCGTCAT-3'
Standard Curves and Background Detection
To determine the relative copy numbers of KAN and FIV from plasma samples, a linear standard curve was generated by plotting 10-fold dilutions (5 × 108 to 5 × 102 copies per well) of dsDNA plasmids of known copy number (log scale), against the cycle threshold (Ct) determined for that value. The pET28 vector (Novagen) was used as the target plasmid for the KAN gene, while a plasmid containing the molecular clone of FIV-C was used for the FIV reverse-transcriptase gene. Calculated values for each plasma sample represent relative copy numbers for the purposes of evaluation between individual samples.
Cloning, Purification and Analysis of Protease
Complementary DNA (cDNA) was synthesized from isolated plasma viral RNA of infected cats. The cDNA pool was used as a template for PCR reactions using 5' primer MFIVCPL5' (5'-GATTTATAAATCATATG GCATATAATAAAGTGGGTACCACTACAACATTAG-3'), which adds an NdeI restriction site, methionine, and alkaline to the N-terminal of the protease and 3' primer MFIVCPL33' (5'-CTGAGATCTGAGCAAGCTTTTACATTACTAATCT AATATTAAATTTAACCATG TTATC-3'), which adds a stop codon and a Hind III restriction site to the C-terminus of the protease. The amplified PCR product was gel purified and cloned into pCR-TOPOII vector (Invitrogen, Carlsbad, CA) for sequencing. Selected DNA of mutants, N55D, H72R, D105G, and M107R were digested with Nde I/Hind III and ligated into pET21a expression vector (Novagen). The mutant FIV-C proteases were expressed in Rosetta pLysS cells (Novagen) and purified as previously described .
Enzyme kinetics of FIV-C protease were assayed on the flourogenic substrate Arg-Ala-Leu-Thr-Lys(Abz)-Val-Gln/Phe(NO2)-Val-Gln-Ser-Lys-Gly-Arg-NH2. The concentration was determined by active-site titration with inhibitor TL-3. Inhibitor constant (Ki) of TL-3 against mutant FIV-C protease was analyzed as described .
The authors wish to thank Dr. Edward Hoover of Colorado State University for providing uncloned FIV-C used in these studies, Michael Carlson for technical assistance and Jackie Wold for administrative assistance. We also wish to thank the AIDS Resource and Reference Reagent Program for contracting the synthesis of TL-3 and providing the amount of compound required for these studies. D.A.S. was supported by the NIH/NINDS training grant 5 T32 NS41219-02. This research was supported by grants R01 AI40882 and R01 AI48411 of the Allergy and Infectious Diseases Institute of the National Institutes of Health and P30 MH6221 from the Mental Health Institute of the Institutes of Health.
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