Since its isolation from cats with opportunistic infections in 1987, feline immunodeficiency virus (FIV) has been shown to cause a slowly progressive immunologic deterioration in cats similar to that caused by HIV in human beings. 1–3 Five FIV subtypes have been identified, some of which cause more rapid immunologic deterioration and are associated with higher plasma virus loads. 4–8 Cats infected with FIV exhibit humoral and cell-mediated immune responses similar to those in human AIDS. 2 Because of the ability of lentiviruses to evade cellular and humoral immune control, traditional vaccine strategies have not produced protective immunity. 9–13 FIV provides a practical animal model for the study of lentiviral vaccine efficacy. 3,14 As with other lentiviruses, vaccination of cats with whole inactivated FIV has protected against challenge with low-dose homologous viruses; however, protection against high-dose challenge or heterologous strains has not been consistently observed, 15–18 although, recently, a killed whole-virus vaccine with cross-clade specificity has been described and licensed. 19 Subunit vaccines for FIV have been disappointing in providing protection against challenge and may enhance infection in some instances. 20–24
Serologic surveys of 27 nondomestic feline species have revealed a minimum of 17 species that contain antibodies cross-reactive with FIV antigens. 25–32 Genetic characterization of puma (Felis concolor), lion (Panthera leo), pallas cat (Felis manul), and bobcat (Lynx rufus) isolates has revealed that these viruses are distinct from each other and related to domestic cat FIV. 25,26,33–35 Substantial sequence divergence, broad geographic dispersal, and nearly complete species monophyly among the FIV-related lentiviruses of Felidae suggest that these viruses have been endemic within cat families for a long time and that cross-species transmission has been rare. 25,26,35 Puma lentivirus (PLV) and lion lentivirus (LLV) demonstrate substantial genetic divergence from FIV of domestic cats, which is greater than the divergence between the simian immunodeficiency viruses (SIVs) indigenous to African primates and HIV-1 and HIV-2. 26 The clinical effects of the FIVs indigenous to wild and captive nondomestic cat populations have not been well studied; however, it does not appear that these infections cause widespread disease in free-ranging populations. 25,26,36 Although frequent cross-species transmission of feline lentiviruses appears unlikely in nature, we have shown that FIV-like viruses from lions and pumas can infect domestic cat origin cells in vitro and in vivo. 37,38 Unlike the course of infection with FIV, domestic cats infected with either LLV or PLV developed no evidence of immunologic deterioration or clinical signs of illness during 6 months of monitoring. 37 Additionally, cats experiencing LLV or PLV infection were able to partially control virologic and immunopathologic effects of subsequent FIV challenge. 39
Similar to FIVs of nondomestic cats, African primates infected with indigenous SIVs do not experience immune deficiency, even though viral replication is not consistently downregulated in these animals. 40,41 Humoral and cellular immune response against SIVs in natural hosts varies and is not always correlated with either viral load or pathogenicity. 14,42–44 To characterize feline lentiviral host/virus adaptation further and to elucidate the relationship between host immune response and disease expression, we challenged cats with either LLV or PLV and examined viral and immunologic kinetics over a 4-month period as described below. Because divergence among LLV, PLV, and FIV is greater than that among the primate viruses, the demonstration of antiviral immune responses with broad lentiviral-neutralizing capabilities is accentuated in this model, lending insight into lentiviral immunity and vaccine strategies.
Cats and Viruses
Fifteen weanling cats (16 weeks old) from the Colorado State University specific-pathogen-free cat colony were housed by inoculation group in an AAALAC International Accredited animal facility, and all procedures were approved by the Colorado State University Animal Care and Use Committee prior to initiation of the study. Animals were weighed, and blood was collected at 0, 1, 2, 4, 6, 8, 9, 12, 16, and 17 weeks postinoculation (PI). Physical examinations were performed at these times and on days 1 through 6 and day 10. Complete blood cells counts (CBCs) and lymphocyte subset analyses (described below in more detail) were performed on all animals at all blood collection time points, except week 17.
LLV (strain Ple 458) is an expanded stock of reverse transcription (RT)-positive supernatant obtained from a seropositive Serengeti lion 25 and expanded in cat peripheral blood mononuclear cells (PBMCs). PLV-1695 stock virus was obtained by cocultivating PBMCs from a British Columbia cougar with domestic cat PBMCs. Viral RNA was isolated from RT-positive supernatants and amplified by RT polymerase chain reaction (PCR; using primers as described below); resultant fragments were sequenced to determine virus fidelity. Cats were inoculated with 1.5 (PLV) or 2.0 (LLV) mL of supernatant from stock supernatant from naive domestic cat PBMC cultures. All inoculations were performed intravenously while cats were anesthetized with ketamine hydrochloride.
Blood was collected from 5 age-matched FIV-infected control cats at a single time point for immunologic analysis. These animals had been intravenously infected with a plasma pool containing FIV-B-2542 for 16 weeks as part of a different study. Cells were processed as described below.
Viral Culture/Reverse Transcription Assay
Previous studies indicated that both LLV and PLV readily infected 3201 cells, and cell-free virus could be detected by RT assay. 38 Whole blood was collected from inoculated cats at the time points indicated previously. Plasma and PBMCs were isolated by standard methods. 45 Plasma (0.25 mL) and 106 PBMCs were cocultivated with 106 naive 3201 cells at weeks 0, 1, 2, 4, 6, 8, and 12. Supernatant was collected biweekly for 4 weeks. The final collection was analyzed for magnesium-dependent RT activity using a modified 32 P detection assay. 46,47 Results were recorded using autoradiography and β emissions of samples bound to DEAE-81 paper. On week 12, dilutions of PBMCs from 1 to 106 cells were cultured with 3201 cells to titrate viral load. Positive cells per 106 PBMCs were determined by RT positive signal at day 28 of culture.
Reverse Transcription Polymerase Chain Reaction
RNA was isolated from 200 μL of RT-positive supernatant using QIAmp Blood Kit reagents (Qiagen) per product directions and was eluted into RNAse-free water. RNA samples were split in half and annealed with either LLV- or PLV-specific primers as described below at 65°C for 10 minutes. A 2-step DNA synthesis/PCR reaction was performed in 1.5 mM of MgCl2, 150 μM of dNTPs, 8 U of AmpliTaq DNA polymerase (Perkin-Elmer), 5 U of AMV RT (Promega), and each primer at 1 μM. The RT-PCR synthesis/PCR conditions were 45°C for 30 minutes and 94°C for 4 minutes, followed by 40 cycles of 94°C for 10 minutes, 45°C for 15 minutes, and 72°C for 20 minutes in a Perkin-Elmer 9600 Gene Amp PCR system. Primers were designed from the PLV env region (R: 5′CGGATTGTCCAGAGGAGAC3′, F: 5′GGCTTTTCTTCTTTCCCAAAC3′) and LLV pol region (L5-285R: 5′TTATCTCTAAAGGTTCAAAT3′, L5-55F: 5′AGAAGTACAGCTTGGATTGC3′) and amplified ∼250– and ∼230–base pair fragments, respectively. Previous studies indicated that these primers did not amplify the domestic cat lentivirus at these reaction conditions and that sequences obtained were consistent with published sequences for these viruses (data not shown). Amplification products were visualized on a 1.5% agarose gel containing ethidium bromide.
Feline T-lymphocyte immunophenotype labeling was performed with monoclonal antibodies to feline CD4+ and CD8+ as previously described. 48–49 Total CD4+ or CD8+ cell numbers were calculated from subset percentages of total leukocytes. When CD8+ mean intensive fluorescence changes were noted in some infected animals, a commercial antibody (Southern Biotech) against the feline CD8+β subunit 50 was used to confirm correlation with CD8α+βlo phenotype expression.
Western Blot Analysis
Crude LLV or PLV viral protein preparations were electrophoresed on 12% polyacrylamide and transferred to nitrocellulose similar to previously described methods. 6,38 Dilutions of serum (1:100) from each cat were incubated with the appropriate immunoblot strips. FIV-infected and naive cat sera were used as positive and negative controls. At week 16, sera were serially diluted from 1:100 to 1:6400 and tested for seropositivity at each concentration.
Crude antigen preparations were concentrated as described 37 from LLV-, PLV-, or FIV-containing supernatant. Antigen concentration was determined by the Biorad protein assay quantitation system. Antigen was diluted in tissue culture medium and subjected to 120 mJ of ultraviolet (UV) irradiation (UV Stratalinker, Stratagene). PBMCs were collected from whole blood at week 9 PI and plated in triplicate at 2 × 105 cells per well in a 96-well plate and exposed to 2, 10, or 25 μg/mL of either LLV, PLV, or FIV antigen. Media alone and with 5 μg/mL of Concanavalin A (Con A) served as negative and positive media controls, respectively. Five days after plating, cells were pulsed with 1.0 μCi of 3H-thymidine for 18 hours, harvested, and counted on a Wallac 1450 Microbeta Plus scintillation counter. Naive control cat PBMCs were exposed to FIV, LLV, and PLV antigens and served to measure nonspecific proliferation. LLV- or PLV-infected cat PBMCs were only exposed to the homologous virus and FIV antigens. Significant proliferation was noted using 10-μg/mL antigen concentrations; thus, for subsequent proliferation experiments (weeks 12 and 17), this was the only concentration used. In addition to controls listed previously, 4 age-matched FIV-B-2542–infected cat PBMCs (16 weeks PI) were exposed to LLV-, PLV-, and FIV-specific antigens as well as to Con A and media alone at the week 17 time point.
Cell Sorting and In Vitro Suppression Assay
On weeks 9, 12, and 16, PBMCs were harvested as described. 45
Cells (107) were washed 3 times, incubated for 30 minutes at 4°C with 1 μg of rat and mouse immunoglobulin (Pierce), washed 3 times, and then incubated with anticat CD8+ fluorescein isothiocyanate (FITC) monoclonal antibodies for 30 minutes at 4°C. After washing, cells were incubated with 20 μL of anti-FITC magnetic beads, washed, and passed over a magnetic column (Miltenyi Biotech). Both CD8+-depleted and -enriched fractions were collected, washed, and used in experiments as outlined below. Fractions were labeled and analyzed by flow cytometry as described previously; in general, CD8+ cells were 80% to 90% pure, and depleted fractions contained less than 5% CD8+ cells. Typically, 5 to 8 × 106 cells were recovered, and a subset of 2 cats per group was used for sorting.
Feline Immunodeficiency Virus Suppression
Twenty-four hours prior to PBMC harvest, Crandell feline kidney (CrFK) cells were exposed to 2 TCID100 of FIV-B 2542. On the day of the assay, cells were washed, split, and plated in 96-well plates to achieve an E/T ratio of 1:1. Cells were left undisturbed for 4 hours to allow adherence, and PBMC effector cells (whole, depleted, or enriched) were then added in triplicate to both naive and FIV-exposed CrFK. Supernatants were collected for analysis, and cultures were replenished with fresh media every 3 to 4 days for 2 to 4 weeks; samples were subjected to FIV p24 antigen capture enzyme-linked immunosorbent assay (ELISA), 51 which has been shown to recognize FIV but not LLV or PLV. 37
Lion Lentivirus/Puma Lentivirus Suppression
Naive 3201 cells were inoculated with 2 TCID100 of either LLV-458 or PLV-1695 supernatant 24 hours prior to suppression assay. On the day of the assay, cells were washed, resuspended, and plated in 96-well plates to achieve an E/T ratio of 1:1. PBMC effector cells (unfractionated, CD8+-depleted, or CD8+-enriched) were added in triplicate to either naive or LLV/PLV-exposed 3201. Supernatants were collected for analysis, and cultures were replenished every 3 to 4 days for 2 to 4 weeks. Samples were analyzed by RT microassay as described previously. 46,47
Clinical signs of LLV and PLV infection were mild to nonexistent. Growth rates of naive, LLV-infected, and PLV-infected animals were virtually identical (data not shown). Four of 5 cats in both the LLV-infected and PLV-infected groups experienced mild transient peripheral lymphadenopathy. One LLV-infected cat was febrile for 3 days 2 weeks PI. One control cat died of unrelated causes at week 4 and was replaced with an age- and sex-matched animal for the duration of the study. These findings were consistent with previous nondomestic cat lentivirus infection studies 38 and reaffirm the hypothesis that the more ancient and attenuated lion and puma viruses are less virulent pathogens than their domestic cat counterpart.
Cell-Associated and Plasma Viremia
Virus was cultured from the plasma of 2 LLV-infected cats and 1 PLV-infected cat at week 1 PI; all other plasma cultures were negative (Table 1). RT activity was detected in cocultures of 106 PBMCs from LLV- and PLV-inoculated cats at week 1, although some signals were weak (2–3 × negative control). All cocultures from LLV- and PLV-inoculated cats were strongly positive (generally >10 × negative control) by week 2 through week 8, at which time, LLV coculture RT values were again low (2–3 × negative control). At week 12, between 105 and 106 PBMCs were required to produce a positive RT signal in coculture (see Table 1). LLV- or PLV-specific RT-PCR amplified fragments from the coculture supernatants as expected (Fig. 1). These data demonstrate that despite the absence of significant clinical disease, LLV and PLV were able to infect domestic cats. Again, as shown in a previous study, 38 a sustained cell-associated viremia was documented, although plasma viremia was rarely detected by cocultivation. Additionally, the proviral load diminished over the 4-month observation period.
Hematology/Lymphocyte Subset Analysis
Lymphopenia and neutropenia were observed in LLV-infected cats from weeks 2 to 6. During this time CD4+ and CD8+ numbers were significantly lower than control values (Fig. 2). The CD4/CD8 ratio in LLV-infected cats was significantly lower than in controls between weeks 4 and 12, primarily because of expansion of CD8+ cells and the CD8α+βlo cell subset, which was observed at each time point in 60% to 80% (3of 5 cats or 4 of 5 cats, dependent on time point) of LLV-infected cats and in 20% to 40% (1of 5 cats or 2 of 5 cats, dependent on time point) of PLV-infected cats (data not shown). This change was greatest at week 6; by week 16, all cell subsets had returned to control levels. In PLV-infected cats, transient lymphopenia was only detected at week 2. Therefore, LLV and, to a lesser extent, PLV did alter lymphocyte subset kinetics similar to findings noted in domestic cat FIV infection, suggesting that these viruses are lymphotrophic in the domestic cat. An expansion of CD8+ T cells correlated with the appearance of CD8α+βlo cells has also been noted in FIV infection of domestic cats and has been correlated with T-cell suppression of viral infection. 52
PLV/LLV-specific antibodies detecting p26 Gag and Env glycoprotein were detected by Western blot analysis in all LLV- or PLV-inoculated cats by week 4. At week 16, titers were >1:6400 (Fig. 3).
Both LLV- and PLV-infected cats demonstrated lymphocyte proliferative responses to the homologous virus antigens at 9, 12, and 17 weeks PI; responses were significant (P < 0.05) at week 9 for LLV and PLV and at week 12 for PLV, and probability values for all time points were 0.12 or less. PBMCs from LLV-infected cats also demonstrated significant proliferative responses against FIV antigens at weeks 12 and 17; PLV-infected cats had significant proliferation at week 12. In contrast, cats infected with FIV did not demonstrate a significant proliferative response against FIV, PLV, or LLV antigens (Fig. 4). T helper –cell proliferative responses have been closely correlated with protection against HIV or SIV in vaccine studies and have been suggested to be important in FIV vaccine protection. 19,53 As observed here, cats infected with FIV do not consistently demonstrate proliferative responses against whole FIV or fractionated FIV antigens (Fig. 5C). In contrast, cats infected with LLV or PLV proliferated in response to either homologous whole-virus antigens or FIV (Fig. 5A, 5B). These responses were significant when compared with proliferative responses of PBMCs from control uninfected cats exposed to the same antigens, demonstrating that the results observed were specific and related to viral infection.
In Vitro Virus Suppression
Peripheral Blood Mononuclear Cells
Unfractionated PBMCs from LLV- or PLV-infected cats harvested at 12 weeks PI significantly inhibited FIV production in culture compared with PBMCs from naive cats (Fig. 5A). CrFK FIV production was inhibited by naive PBMCs relative to no cell control, suggesting that uninfected effector cells were able to suppress FIV nonspecifically; however, PBMC effectors from LLV- or PLV-infected cats exhibited significantly greater suppression activity than that of naive cats. This observation suggests that LLV or PLV infection served to either enhance the innate immune suppression present in naive cat PBMCs or to prime a specific immune response with enough heterogeneity to effectively downregulate FIV.
CD8+ Suppression of Feline Immunodeficiency Virus
Unexpectedly, enrichment of CD8+ PBMC fractions from cats infected with PLV or LLV enhanced rather than further inhibited FIV infection in the CrFK FIV suppression system. Depletion of CD8+ cells produced either no effect or enhanced FIV production (see Fig. 5B, C). Similar results were obtained at 16 weeks PI, and contrast with results demonstrating suppression of FIV infection in vitro is associated with CD8+ cells. 52,54,55 Therefore, our results suggest that the whole PBMC suppression of nonhomologous FIV observed is not CD8+ dependent and may represent a noncytolytic innate mechanism for viral suppression.
CD8+ Suppression of Homologous Virus (Lion Lentivirus or Puma Lentivirus)
Unfractionated PBMCs from cats infected with LLV or PLV did not cause significant downregulation of viral infection in homologous virus-infected 3201 target cells (data not shown). Interpretation of these data was complicated by the fact that LLV- or PLV-infected effector cells produced virus during cocultivation that was indistinguishable from the virus expressed by preinfected target cells. When CD8+-depleted or -enriched PBMCs from LLV- or PLV-infected cats were used in homologous virus suppression studies, however, effector cell CD8+-enriched fractions did downmodulate production of LLV or PLV growth in 3201 target cells. Conversely, effector cell CD8+-depleted fractions were associated with enhanced viral expression from 3201 target cells (Fig. 6). Therefore, in contrast to the heterologous FIV viral suppression assays described previously (but as predicted by other lentiviral systems), viral suppression of LLV or PLV of the homologous virus was associated with CD8+ effector cell phenotype.
LLV and PLV were able to persistently and apathogenically infect domestic cats. In contrast to a previous study, all cats challenged with PLV became infected. The PLV isolate used in the present study (PLV-1695) was amplified by cocultivation of domestic cat PBMCs with infected puma PBMCs and therefore may have been better adapted to domestic cats than previous 3201-adapted PLV isolates used for challenge (PLV-14 and PcLV). 37 These results confirm that even though LLV and PLV are substantially divergent from FIV, 25,26,33–35 these more ancient viruses can nevertheless still replicate in the nonadapted domestic cat host. This is reminiscent of the ability of primate lentiviruses adapted in a specific host species to infect another; for example, SIVs derived from African primates are able to infect Asian macaques, and HIV-1 and HIV-2 are thought to have originated from different strains of SIV. 40,53,56,57
Many of the clinical and virologic parameters of LLV/PLV infection in domestic cats observed previously (eg, mild transient lymphadenopathy, persistent cell-associated and acute transient plasma viremia) were also noted in this study. In contrast to previous observations, 1 cat experienced a transient febrile episode early in infection, and cats infected with LLV had temporary leukocyte alterations (neutropenia, lymphopenia,CD4/CD8 inversion). 37 Clinical signs in domestic cats acutely infected with FIV include fever, lymphadenopathy, oral ulcerations, and lethargy. 2,3
CD8+ phenotype alterations from CD8α+β+ to CD8α+βlo were detected in LLV-infected animals. Domestic cats infected with FIV experience similar hematologic variations. 2,3 Gebhard et al 58 demonstrated that the CD8α+βloCD62L cell subset expanded after FIV infection and represents the cell population responsible for suppression of FIV replication. In contrast to the transient nature of the hematologic and CD8+ subset changes observed in LLV infection, in cats with virulent FIV infection, CD8 subset alterations persist for the duration of the asymptomatic phase of the disease. 54,55
The present study also demonstrated that a significant lymphocyte proliferative response developed in LLV- and PLV-infected versus FIV-infected domestic cats. Although this effect was more pronounced against homologous viral antigens (ie, LLV and PLV), LLV- and PLV-infected cats also demonstrated cross-reactive proliferative activity against domestic cat FIV. All LLV- and PLV-infected cats developed extremely high antibody titers against homologous viral antigens (>1:6400), consistent with previously reported findings. 37 These findings indicate that cats infected with LLV and PLV are able to mount a robust T helper–cell immune response during the acute and subacute stages of infection, a finding that has not been consistently demonstrated in FIV infection. 2
PBMCs from LLV- and PLV-infected animals suppressed FIV production in vitro, although this response did not appear to be mediated primarily by CD8+ cells as might be expected based on observations in HIV, SIV, and FIV systems. 14,52,54,55,58 Although naive cat PBMCs did restrict FIV growth in vitro, PBMCs from cats infected with LLV and PLV were significantly more inhibitory than naive PBMCs, suggesting enhanced capacity for viral suppression of these infected cells. In standard PBMC/CD8+ lentiviral suppression assays, effector cells produce the same virus as target cells, complicating interpretation of assay results. In the assays described in this study, target cell FIV was differentiated from effector cell LLV and PLV by the specificity of the capture ELISA for FIV. The suppressor mechanism observed in the heterologous virus suppression assay appears to result from a CD8+-independent mechanism and may represent an enhancement of innate immunity noted with naive PBMC effector cell activity. Alternatively, this phenomenon may represent a lentiviral-specific immune response with broad-based activity, which is also reactive against FIV. Although it is also possible that CD8+ effector cells obtained from LLV- or PLV-infected animals may have been efficient target cells for FIV production, thus augmenting the FIV load during cocultivation, this seems unlikely due to the E/T ratio used and the CrFK-adapted FIV strain used in this assay.
In contrast to non-CD8+ cell-dependent viral suppression detected in a heterologous FIV assay, downregulation of homologous LLV or PLV production in a T-cell line was correlated with the CD8+ cell fraction over multiple time points. This finding is consistent with other studies reporting FIV CD8+ suppression in individual animals. 55 Such observations are consistent with the production of soluble immune modulators secreted by CD8+ cells as associated with viral control of HIV. PBMCs from individuals with HIV infection can downregulate infection with macrophage or T-cell tropic strains in vitro. 59 In particular, PBMCs from HIV-2–infected individuals are more readily infected with T-cell tropic versus macrophage tropic CCR-5–utilizing strains of HIV-1 in vitro, 60 likely due to production of β-chemokines by CD8+ and other immune cells. 59,60 Other soluble factors that inhibit viral infection by targeting intracellular gene transcription have been detected. 61 Recent work has associated CD8+ soluble factor suppression with the production of α-defensins, although this work remains controversial. 62 Other possible nonimmune mechanisms that may contribute to prevention of superinfection include fewer numbers of target cells in animals with prior infection or inhibition of receptor-virus binding. 63–65 Although the readouts for virus suppression differed between heterologous FIV suppression (ELISA) and homologous LLV/PLV suppression (RT assay), both assays would be considered quantitative. Results were expressed as percentage of control values for either assay so as to enable relative comparison of viral production.
Our data suggest that at least 2 independent pathways exist for suppression of lentiviral-infected cells by PBMCs from infected animals: a virus-specific CD8+ response and a non-CD8+ restricted pathway with potentially broader antiviral capabilities. Vigorous humoral immunity, heterologous and homologous proliferative responses, and reversion of the CD8+βlo phenotype suggest that cats infected with a nonnative lentivirus respond with a more robust and effective immune response than cats infected with domestic cat FIV. It is possible that the more ancient nondomestic cat viruses have evolved to become more symbiotic with their host. We cannot, however, exclude the possibility that nonimmune mechanisms (eg, receptor affinity, host-virus protein interactions) may restrict viral replication of these viruses within the nonadapted domestic cat. This feline lentivirus system affords a unique opportunity to examine host and viral interactions mediating pathogenicity and resistance to virulent lentiviral infection.
The authors deeply appreciate technical assistance provided by Mary Jo Burkhard and Mary Tompkins at North Carolina State University in design of suppression assays. The authors thank Paul Avery and Kerry Sondgeroth for assistance with flow cytometric assays, Candace Mathiason for multifold technical and laboratory assistance, and Julie Terwee for manuscript review. They are grateful to Stephen J. O'Brien and Kenneth Langelier for providing lion and puma virus samples.
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Keywords:Copyright © 2003 Wolters Kluwer Health, Inc. All rights reserved.
feline immunodeficiency virus (FIV); animal model; protective immunity; viral suppression