Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Pittsburgh, PA 15261, USA1
Tulane Regional Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA2
Author for correspondence: Michael Murphey-Corb. Fax +1 412 648 1448. e-mail mcorb{at}pitt.edu
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Abstract |
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Introduction |
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As for HIV-infected humans (Haynes et al., 1996 ), SIV-infected macaques have been classified into three groups: fast progressors, progressors and slow/nonprogressors (Dykhuizen et al., 1998
; Kindt et al., 1992
; Letvin & King, 1990
; Zhang et al., 1988
). SIV-infected rapid progressors have a persistent viraemia with little or no virus-specific antibody response. Survival of these animals is approximately 23 months. Progressors have detectable viraemia, but also have a strong antibody response and survive for 13 years. Slow/nonprogressors remain persistently infected because viral sequences can be detected by PCR, but virus is difficult to recover from peripheral blood. These animals can survive for 5 or more years.
We have determined that cultured peripheral blood mononuclear cells (PBMC) from different naïve animals vary widely in their ability to support virus replication in vitro. To determine whether a relationship exists between this phenomenon and the variable survival noted in vivo following experimental infection, data from a cohort of 59 control animals used over the last decade were evaluated. These data demonstrate that there is a statistical relationship between the ability of an animals PBMC to produce virus in vitro and the rate of progression to disease and death following experimental infection. Furthermore, comparative evaluation of virus production in purified T cell cultures produced similar results, a finding that demonstrates that this phenotype is a property of the CD4+ T cell itself.
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Methods |
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Animal manipulations.
Rhesus macaques (Macaca mulatta) of either sex were obtained from the Tulane Regional Primate Center breeding colony. Animals were infected by inoculation of 1 ml of 10100 monkey infectious doses into the saphenous vein using a 23 gauge butterfly needle. Each injection was chased with 12 ml sterile saline to assure accurate delivery of the inoculum. Physical examinations were performed at biweekly intervals, and animals were provided full supportive care until they were deemed moribund by the attending veterinarian. Blood was drawn at these times for measurements of antigenaemia and for quantification of T lymphocytes. Antigenaemia was determined by an SIV core antigen ELISA (Coulter) according to the manufacturers instructions. Flow cytometric determination of lymphocyte subsets was performed as described (Martin et al., 1993 ). Complete necropsies were performed following humane sacrifice.
Analysis of virus replication in vitro.
PBMC were obtained as described above. Cell pellets containing 1x107 PBMC were infected by incubation with 1 ml of a cryopreserved stock of SIV/DeltaB670 containing 1000100000 tissue culture infectious doses for 12 h at 37 °C. No difference in the hierarchy with respect to virus production was observed when inocula within this range of infectivity were used (data not shown). After infection, cells were washed twice in complete medium (RPMI 1640 supplemented with 15% FBS, L-glutamine, and penicillinstreptomycin at concentrations indicated above). Twenty-four hours after infection, 10 µg/ml of phytohaemagglutinin (PHA) (GibcoBRL) was added. On day 4, the PHA was removed by washing the cells three times in complete medium. Cells were cultured in complete medium supplemented with human IL-2 (40 U/ml) for the entire experiment (21 days) and maintained at a concentration of 1x106 cells/ml. On days 7, 10, 14, 18 and 21, 1 ml of supernatant was removed and stored at -70 °C until analysis. The amount of virus in culture supernatants was determined by measuring reverse transcriptase (RT) activity in cell culture supernatants as described below. Alternatively, virus production was measured using an SIV Core antigen ELISA (Coulter), as per the manufacturers instructions. Measurement of RT activity or p27 antigen yielded similar results.
Measurement of RT activity.
Cell-free culture fluid was collected in 1 ml aliquots and subjected to centrifugation at 12000 g for 45 min in a refrigerated microfuge. The supernatant was decanted, and the virus pellet was stored at -70 °C until needed. The pellet was thawed on ice and 50 µl of solubilization buffer (0·5% Triton X-100, 0·8 M NaCl, 0·5 mM PMSF, 20% glycerol and 50 mM TrisHCl, pH 7·8) was added to lyse the pelleted virus particles. The pellet was resuspended by vortexing, and the sample was retained on ice until incorporation into the assay. In each well of a 96-well flat-bottomed microtitre plate, 10 µl of the solubilized pellet was added to 90 µl of a solution containing 10 mM MgCl2, 5 mM dithiothreitol, 83 µg/ml dATP, 5 µg/ml poly(r-A)p(dT)1218 (Pharmacia), 52 µCi/ml [3H]TTP (New England Nuclear) and 52 mM TrisHCl, pH 7·8. The plates were then incubated at 37 °C for 2 h, and the reaction was stopped by the addition of 10 µl 25 mg/ml tRNA (GibcoBRL). To each well, 90 µl of cold 10% TCA+0·02% Na4P2O7 was then added and the reaction was allowed to sit for 30 min. Acid-precipitable radiolabelled nucleotides were harvested using a Skatron plate washer and the resulting radioactivity counted using a scintillation counter.
Purification of CD4+ T cells.
CD4+ T cells were purified from PBMC using MACS, a magnetic cell selection system (Miltenyi Biotec). After isolation from peripheral blood as described above, PBMC were washed twice in PBS and once in MACS buffer, which consisted of PBS containing 5 mM EDTA (Sigma), 0·5% BSA (Sigma) and 10 mM HEPES (GibcoBRL). Cells were incubated in 200 µl of buffer and 50 µl of CD4+ microbeads per 4x107 cells for 20 min at 4 °C. For all experiments, cells were run over two MS+ selection columns. Purity of selected cells was determined using flow cytometry and was >95% for all assays. Rhesus-reactive antibodies used for flow cytometry were: phycoerythrin-conjugated anti-CD4 clone MT477 (Pharmingen), fluorescein-conjugated anti-CD8 clone SK1 (Becton-Dickinson) and either phycoerythrin- or fluorescein-conjugated anti-CD3 clone SP34 (Pharmingen).
Statistics.
Data were analysed with Microsoft Excel 97 for data entry and coding. Statistical analysis was performed using SPSS Version 9.0 (SPSS Inc.). A P value of <0·05 was considered significant. KaplanMeier analysis was used to evaluate survival among groups. Statistical relationship of maximum RT and survival was analysed by one-way ANOVA.
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Results |
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Although the data from these 10 animals suggested a striking relationship between SIV production in vitro and rate of SIV-induced disease progression and time to death in vivo, larger numbers of animals were required to definitively establish this phenomenon. Since adequate cage space was available at the Center, the majority of animals enrolled in SIV studies spanning a decade of research were provided with supportive clinical care until they were moribund so that crucial data on survival in a large cohort of infected animals could be obtained. Furthermore, because we believed that the results of virus production in vitro were highly useful in the selection of animals for experimental groups, this analysis was performed on most of the animals used during this time period. Fifty-nine animals were chosen for further analysis. This cohort comprised all animals infected with SIV/DeltaB670 that were (1) inoculated intravenously, (2) received no other effective therapeutic or vaccine and (3) died of an AIDS-defining illness. However, different cryopreserved stocks (both human and monkey propagated) and different doses (1010000 animal infectious doses) were used over this time period. Maximum RT determined for each animal is presented graphically in Fig. 5(A). The overall mean peak RT was 101120±118764 c.p.m./ml. Animals were divided into three cohorts using natural breakpoints detected by direct visualization of the data. Each breakpoint is indicated by the vertical line in Fig. 5(A)
. These cohorts consisted of: (1) the upper 20th percentile, peak RT >160000 c.p.m./ml (mean=298119±128355 c.p.m./ml), 12 animals; (2) the lower 20th percentile, peak RT <15000 c.p.m./ml (mean=7838±4004 c.p.m./ml), 12 animals; (3) the remaining animals falling into the median range, 160000 >peak RT>15000 c.p.m./ml (mean=66548±32793 c.p.m./ml), 35 animals. The cause of death in these cohorts was consistent with their survival, with SIV-giant cell disease, pneumonia and colitis most prevalent in rapid progressors, and lymphoma, lymphoproliferative disease and amyloidosis being the predominant findings in slow progressors.
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The statistical relationship between peak RT and survival of the three cohorts is shown in Fig. 5(C). Mean survival differed markedly among the groups, with survival in high producers less than half that of low producers (168±123 vs 367±106). This difference was highly significant by one-way ANOVA (P=0·002). High producers also experienced reduced survival compared to that of the intermediate group (304±152, P=0·013). However, no significant difference in overall survival between intermediate and low producers was observed.
The phenotypic differences observed between high and low producers were not due to infection with other viruses commonly encountered in macaques (e.g. simian retrovirus, simian T-lymphotropic virus-I, herpesvirus B) because differences in virus replicative capacity were observed in cells from monkeys that were PCR- and/or antibody-negative for these viruses (data not shown).
High/low producer phenotype is maintained in purified CD4+ T cell populations
To determine the relative contribution of other cell types in PBMC to virus production during the kinetics assay, cultures of purified CD4+ T cells and whole PBMC were run in parallel. CD4+ T cells were purified using anti-CD4+ antibodies coupled to magnetic beads according to the Miltenyi Biotec system. Flow cytometry was used to monitor the purity of the CD4+ T cell cultures, and in all cases, purity was equal to or greater than 95% (data not shown). The results from these assays are shown in Fig. 6. A comparison of virus production from whole PBMC cultures from high and low producer animals shows at least a log difference in the amount of virus produced, as measured by p27 antigen in the cell culture supernatants (Fig. 6A
, B
). When comparing virus production from purified CD4+ T cell cultures (Fig. 6C
, D
), there is still a difference of at least a log in virus production (notice difference in y-axes). This confirms that the CD4+ T cell itself is responsible for the differences in virus production in vitro.
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Discussion |
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These data support observations reported by Lifson et al. (1997) in which RNA plasma loads and survival in eight macaques correlated with the amount of virus required to infect macaque PBMC in vitro. These two studies thus clearly identify an innate property of individual macaques that controls virus replication both in vitro and in vivo. Further insight into this phenomenon was gained by determining that the hierarchy associated with high and low producer phenotypes is retained in cultures of infected purified CD4+ T lymphocytes. These findings indicate that this phenotype is an intrinsic property of the CD4+ T cell itself. Control of virus production could be at the level of virus entry [e.g. virus receptor(s)], or post-entry, with the differential production of either suppressor (in low producer) or enhancer (in high producer) proteins.
The intrinsic property of macaque PBMC to support the replication of SIV in vitro may be analogous to the differences in replicative capacity observed for human PBMC infected in vitro with HIV-1 (Williams & Cloyd, 1991 ). Together with the variable length of time from infection to death also observed in HIV humans (Buchbinder et al., 1994
; Cao et al., 1995
; Lifson et al., 1988
; Michael et al., 1997
; Munoz et al., 1995
; Operskalski et al., 1997
), the data reported here may be highly relevant to the pathogenesis of HIV as well. The experimental system described in this report should enable the identification of the specific host gene(s) responsible for this phenomenon as well as provide crucial information for the development of effective strategies for disease intervention.
The advantage of accurately predicting survival prior to selection of monkeys for SIV studies is obvious, particularly in experiments where both control and test groups are infected and more subtle parameters of efficacy are required. Since limited numbers of animals are often used in macaque trials due to the expense, optimal stratification of groups is crucial so that differences in delays in disease progression be appropriately identified. In this regard, it is important to note that in vitro determination of virus replicative capacity not only predicted overall survival in the animals reported here, but also correlated with other parameters such as antigenaemia and the rate of T cell decline (reported here) and plasma virus loads (Lifson et al., 1997 ), which are all used to determine the rate of disease progression before death.
We have successfully used this simple, in vitro assay in the design of many therapeutic and vaccine trials performed on SIV/DeltaB670-infected macaques at the Tulane Primate Center. Stratification of groups prior to the initiation of the study using this assay has enhanced the interpretation of these studies. For example, in a therapeutic trial evaluating the efficacy of treatment with cyclosporin during the acute viraemic episode (Martin et al., 1997 ), the parameters of survival, T cell changes and antigenaemia were used as indicators of efficacy. Stratification of the treatment and control groups with respect to maximum RT obtained form the in vitro assay permitted pairwise analysis of efficacy. This analysis identified differences in persistence of antigenaemia among the two groups that were statistically significant, even though the variability among the group as a whole precluded a significant outcome. The opposite effect can also occur. Differences in survival may be suggested when statistical analysis of small groups (n
4 animals) of randomly chosen animals is performed, but when the group size is doubled, the difference may no longer be significant. The inclusion of analysis of the in vitro replicative capacity of monkeys used in experimental trials should permit an appropriate interpretation of the outcome of these studies.
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Acknowledgments |
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We gratefully acknowledge the technical support of Calvin Lanclos, Eileen Deharo, Maury Duplantis, Gail Plauche and Robin Rodriquez and the professional staff in the Departments of Veterinary Sciences and Pathology at the Tulane Regional Primate Center.
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References |
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Cao, Y., Qin, L., Zhang, L., Safrit, J. & Ho, D. D. (1995). Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection.New England Journal of Medicine 322, 201-208.[Medline]
Dittmer, U., Luke, W., Stahl-Hennig, C., Coulibaly, C., Petry, H., Bodemer, W., Hunsmann, G. & Voss, G. (1994). Early helper T cell dysfunction in SIV-but not HIV-2-infected macaques.Journal of Medical Primatology 23, 298-303.[Medline]
Dykhuizen, M., Mitchen, J. L., Montefiori, D. C., Thomson, J., Acker, L., Lardy, H. & Pauza, C. D. (1998). Determinants of disease in the simian immunodeficiency virus-infected rhesus macaque: characterizing animals with low antibody responses and rapid progression.Journal of General Virology 79, 2461-2467.[Abstract]
Farzadegan, H., Henrard, D. R., Kleeberger, C. A., Schrager, L., Kirby, A. J., Saah, A. J., Rinaldo, C. R.Jr, OGorman, M., Detels, R., Taylor, E., Phair, J. P. & Margolick, J. B. (1996). Virologic and serologic markers of rapid progression to AIDS after HIV-1 seroconversion.Journal of Acquired Immune Deficiency Syndromes 13, 448-455.
Habis, A., Baskin, G. B., Murphey-Corb, M. & Levy, L. S. (1999). SAIDS-associated lymphoma (SAL) in rhesus and cynomolgus monkeys recapitulates the primary pathobiological features of AIDS-associated non-Hodgkins lymphoma (AAL).AIDS Research and Human Retroviruses 15, 1389-1398.[Medline]
Haynes, B. F., Pantaleo, G. & Fauci, A. S. (1996). Toward an understanding of the correlates of protective immunity to HIV infection.Science 271, 324-328.[Abstract]
Kindt, T. J., Hirsch, V. M., Johnson, P. R. & Sawasdikosol, S. (1992). Animal models for acquired immunodeficiency syndrome.Advances in Immunology 52, 425-474.[Medline]
Lackner, A. (1994). Pathology of simian immunodeficiency virus infection.Current Topics in Microbiology and Immunology 188, 35-64.[Medline]
Letvin, N. L. & King, N. W. (1990). Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques.Journal of Acquired Immune Deficiency Syndromes 3, 1023-1040.[Medline]
Lifson, A. R., Rutherford, G. W. & Jaffe, H. W. (1988). The natural history of human immunodeficiency virus infection.Journal of Infectious Diseases 158, 1360-1367.[Medline]
Lifson, J. D., Nowak, M. A., Goldstein, S., Rossio, J. L., Kinter, A., Vasquez, G., Wiltrout, T. A., Brown, C., Schneider, D., Wahl, L., Lloyd, A. L., Williams, J., Elkins, W. R., Fauci, A. S. & Hirsch, V. M. (1997). The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.Journal of Virology 71, 9508-9514.[Abstract]
Liu, S. L., Schacker, T., Musey, L., Shriner, D., McElrath, M. J., Corey, L. & Mullins, J. I. (1997). Divergent patterns of progression to AIDS after infection from the same source: human immunodeficiency virus type 1 evolution and antiviral responses.Journal of Virology71, 4284-4295.[Abstract]
Martin, L. N., Murphey-Corb, M., Soike, K. F., Davison-Fairburn, B. & Baskin, G. B. (1993). Effects of initiation of 3'-azido,3'-deoxythymidine (zidovudine) treatment at different times after infection of rhesus monkeys with simian immunodeficiency virus.Journal of Infectious Diseases168, 825-835.[Medline]
Martin, L. N., Soike, K. F., Murphey-Corb, M., Bohn, R. P., Roberts, E. D., Kakuk, T. J., Thaisrivongs, S., Vidmar, T. J., Ruwart, M. J., Davio, S. R. & Tarpley, W. G. (1994). Effects of U-75875, a peptidomimetic inhibitor of retroviral proteases, on simian immunodeficiency virus infection in rhesus monkeys.Antimicrobial Agents and Chemotherapy38, 1277-1283.[Abstract]
Martin, L. N., Murphey-Corb, M., Mack, P., Baskin, G. B., Pantaleo, G., Vaccarezza, M., Fox, C. H. & Fauci, A. S. (1997). Cyclosporin: a modulation of early virologic and immunologic events during primary simian immunodeficiency virus infection in rhesus monkeys.Journal of Infectious Diseases176, 374-383.[Medline]
Michael, N. L., Brown, A. E., Voigt, R. F., Frankel, S. S., Mascola, J. R., Brothers, K. S., Louder, M., Birx, D. L. & Cassol, S. A. (1997). Rapid disease progression without seroconversion following primary human immunodeficiency type 1 infection evidence for highly susceptible human hosts.Journal of Infectious Diseases174, 1352-1359.
Munoz, A., Wang, M. C., Bass, S., Taylor, J. M., Kingsley, L. A., Chmiel, J. S. & Polk, B. F. (1989). Acquired immunodeficiency syndrome (AIDS)-free time after human immunodeficiency virus type 1 (HIV-1) seroconversion in homosexual men.American Journal of Epidemiology130, 530-539.[Abstract]
Munoz, A., Kirby, A. J., He, Y. D., Margolick, J. B., Visscher, B. R., Rinaldo, C. R., Kaslow, R. A. & Phair, J. P. (1995). Long-term survivors with HIV-1 infection: incubation period and longitudinal patterns of CD4+ lymphocytes.Journal of Acquired Immune Deficiency Syndromes8, 496-505.
Murphey-Corb, M., Martin, L. N., Rangan, S. R. S., Baskin, G. B., Gormus, B. J., Wolf, R. H., Andes, W. A., West, M. & Montelaro, R. C. (1986). Isolation of an HTLV-III-related retrovirus from macaques with simian AIDS and its possible origin in asymptomatic mangabeys.Nature321, 435-437.[Medline]
Murphey-Corb, M., Martin, L. & Davison-Fairburn, B. (1989). The susceptibility of rhesus PBL to SIV/Delta infection in vitro is predictive of in vivo disease. 8th Annual Symposium on Non-human Primate Models for AIDS.
Nicholson, J. K., Spira, T. J., Aloisio, C. H., Jones, B. M., Kennedy, M. S., Holman, R. C. & McDougal, J. S. (1989). Serial determinations of HIV-1 titers in HIV-infected homosexual men: association of rising titers with CD4 T cell depletion and progression to AIDS.AIDS Research and Human Retroviruses5, 205-215.[Medline]
OBrien, T., Blattner, W. A., Waters, D., Eyster, E., Hilgartner, M. W., Cohen, A. R., Luban, N., Hatzakis, A., Aledort, L. M., Rosenberg, P. S., Miley, W. J., Kroner, B. L. & Goedert, J. J. (1996). Serum HIV-1 RNA levels and time to development of AIDS in the multicenter hemophilia cohort study.Journal of the American Medical Association276, 105-110.[Abstract]
Operskalski, E. A., Busch, M. P., Mosley, J. W., Stram, D. O. & Transfusion Study Safety Group (1997). Comparative rates of disease progression among persons infected with the same or different HIV-1 strains. Journal of Acquired Immune Deficiency Syndromes 15, 145150.
Williams, L. M. & Cloyd, M. W. (1991). Polymorphic human gene(s) determines differential susceptibility of CD4 lymphocytes to infection by certain HIV-1 isolates.Virology184, 723-728.[Medline]
Zhang, J. Y., Martin, L. N., Watson, E. A., Montelaro, R. C., West, M., Epstein, L. & Murphey-Corb, M. (1988). Simian immunodeficiency virus/delta-induced immunodeficiency disease in rhesus monkeys: relation of antibody response and antigenemia.Journal of Infectious Diseases158, 1277-1286.[Medline]
Received 3 May 2000;
accepted 16 June 2000.